**Distribution, Mechanisms, Impact and Management of Insecticide Resistance in Malaria Vectors: A Pragmatic Review**

Vincent Corbel and Raphael N'Guessan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56117

## **1. Introduction**

Malaria is still a major burden causing the death of nearly 655,000 people each year, mostly in children under the age of five, and affecting those living in the poorest countries [1]. Currently, the major obstacles to malaria control and elimination are the absence of a protective vaccine, the spread of parasite resistance to anti-malarial drugs and the mosquito resistance to insec‐ ticides [2]. Controlling mosquito vectors is fundamental to reduce mosquito-borne diseases by targeting vectorial capacity and hence the transmission. Vector control through the use of chemicals for mosquito bed nets and indoor residual spraying is still the cornerstone of malaria prevention [1]. Unfortunately, the extensive use of insecticides since the 1950s has led to the development of strong resistance worldwide hence representing a major public health problem where insecticidal vector control is implemented. Here, we propose to review the current level, distribution and mechanisms of insecticide résistance in malaria vectors and address their impact on the efficacy of vector control interventions. Strategies to prevent and/or delay the spread of insecticide resistance in natural mosquito populations are also discussed.

## **2. Definition of resistance**

According to the *World Health Organization* (WHO), resistance is defined as the ability of an insect to withstand the effects of an insecticide by becoming resistant to its toxic effects by means of natural selection and mutations [3]. This definition differs from that provided by the *Insecticide Resistance Action committee* (IRAC) (www.irac-online.org) that gathers independent scientists and experts belonging to Agrochemical Companies who define operational (field) resistance as a heritable change in the sensitivity of a pest population that is reflected in the

© 2013 Corbel and N'Guessan; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Corbel and N'Guessan; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species. The IRAC definition, although pragmatic, is less "sensitive" with the scope to implement early *Insecticide Resistance Management* (IRM) strategies in the field. In both cases however, appropriate tools (biological, biochemical and/or molecular) are needed to identify the mechanisms involved and to conduct surveillance at individual and/or population levels [4].

Resistance has been observed in more than 500 insect species worldwide among which more than 50 *Anopheles* species (Diptera: Culicidae) are responsible for the transmission of malaria parasites to humans [5]. Resistance is a heritable character that relies on a genetic basis. Resistance results from the selection of a genetic modification in one or several genes occurring by migration and/or mutation. For example, when a mosquito population is exposed to an insecticide A, the individuals having resistant genes to this insecticide A survive and reproduce until the resistant allele becomes almost fixed. The use of insecticides for agricultural purposes and more recently for public health has played pivotal step in the selection of resistance in malaria vectors [6]. Resistance can involve several physiological and/or behavioural changes. Changes in the insecticide target site that reduce its binding to insecticides (known as targetsite resistance) is the best understood type of resistance mechanism and molecular diagnostics to detect this resistance mechanism are now integrated into insecticide resistance monitoring strategies in malaria control programmes [7, 8]. Enhanced insecticide metabolism that lowers the amount of insecticide reaching the target site (known as metabolic resistance) is more complex but recent advances have identified key enzymes responsible for insecticide detoxi‐ fication, paving the way for the development of molecular markers for this type of resistance mechanism [9, 10]. Other physiological changes (e.g. reduce penetration through cuticular resistance) and/or behavioural changes in the mosquito population were identified but their impact on the efficacy of insecticides is still poorly understood.

lt is commonly accepted that the enhanced metabolism and target site modifications are responsible for high level of insecticide resistance in malaria vectors. To date, malaria vectors have developed resistance to the main chemical classes used in public health (i.e. pyrethroids, DDT, carbamates and organophosphates) (table 1) and the occurrence of cross-resistance1 and multiple resistance2 represent a serious threat to achieving the Millennium Development Goals for malaria control (i.e 75% reduction of global malaria cases by 2015). Surveillance and routine monitoring campaigns to assess the level and type of resistance are essential to help Malaria Control Programme (MCPs) to design more effective and sustainable malaria vector control strategies at an operational scale [4].

## **3. History of resistance to public health insecticides**

Since the humans used chemicals for crop protection and/or the prevention of vector borne diseases, cases of resistances have been reported [11, 12]. Insecticides used for malaria control

<sup>1</sup>**Cross resistance:** occurs when a resistance mechanism, which allows insects to resist one insecticide, also confers resistance to another insecticide. Cross resistance can occur between insecticides from different chemical classes.

<sup>2</sup>**Multiple resistance:** occurs when insects develop resistance to several compounds by expressing multiple resistance mechanisms. The different resistance mechanisms can combine to provide resistance to multiple classes of products.

have included organochlorine, organophosphorus, carbamate, and pyrethroid insecticides, with the latter now taking increasing market share for both indoor residual spraying and Long Lasting Insecticidal mosquito Nets (LLINs) programmes [13]. Resistance has naturally tended to follow the use and switches of these insecticides [5].

repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species. The IRAC definition, although pragmatic, is less "sensitive" with the scope to implement early *Insecticide Resistance Management* (IRM) strategies in the field. In both cases however, appropriate tools (biological, biochemical and/or molecular) are needed to identify the mechanisms involved and to conduct surveillance

Resistance has been observed in more than 500 insect species worldwide among which more than 50 *Anopheles* species (Diptera: Culicidae) are responsible for the transmission of malaria parasites to humans [5]. Resistance is a heritable character that relies on a genetic basis. Resistance results from the selection of a genetic modification in one or several genes occurring by migration and/or mutation. For example, when a mosquito population is exposed to an insecticide A, the individuals having resistant genes to this insecticide A survive and reproduce until the resistant allele becomes almost fixed. The use of insecticides for agricultural purposes and more recently for public health has played pivotal step in the selection of resistance in malaria vectors [6]. Resistance can involve several physiological and/or behavioural changes. Changes in the insecticide target site that reduce its binding to insecticides (known as targetsite resistance) is the best understood type of resistance mechanism and molecular diagnostics to detect this resistance mechanism are now integrated into insecticide resistance monitoring strategies in malaria control programmes [7, 8]. Enhanced insecticide metabolism that lowers the amount of insecticide reaching the target site (known as metabolic resistance) is more complex but recent advances have identified key enzymes responsible for insecticide detoxi‐ fication, paving the way for the development of molecular markers for this type of resistance mechanism [9, 10]. Other physiological changes (e.g. reduce penetration through cuticular resistance) and/or behavioural changes in the mosquito population were identified but their

lt is commonly accepted that the enhanced metabolism and target site modifications are responsible for high level of insecticide resistance in malaria vectors. To date, malaria vectors have developed resistance to the main chemical classes used in public health (i.e. pyrethroids, DDT, carbamates and organophosphates) (table 1) and the occurrence of cross-resistance1 and

for malaria control (i.e 75% reduction of global malaria cases by 2015). Surveillance and routine monitoring campaigns to assess the level and type of resistance are essential to help Malaria Control Programme (MCPs) to design more effective and sustainable malaria vector control

Since the humans used chemicals for crop protection and/or the prevention of vector borne diseases, cases of resistances have been reported [11, 12]. Insecticides used for malaria control

1**Cross resistance:** occurs when a resistance mechanism, which allows insects to resist one insecticide, also confers resistance to another insecticide. Cross resistance can occur between insecticides from different chemical classes. 2**Multiple resistance:** occurs when insects develop resistance to several compounds by expressing multiple resistance mechanisms. The different resistance mechanisms can combine to provide resistance to multiple classes of products.

represent a serious threat to achieving the Millennium Development Goals

at individual and/or population levels [4].

580 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

impact on the efficacy of insecticides is still poorly understood.

**3. History of resistance to public health insecticides**

multiple resistance2

strategies at an operational scale [4].


**Table 1.** Mechanisms of insect resistance to the main insecticide families of public health interest

Historically, DDT was first introduced for mosquito control and malaria eradication pro‐ gramme in 1946. The first case of DDT resistance was reported in *An. sacharovi* in Greece in 1953 and was followed by dieldrin resistance in 1954 [15]. Onset of resistance was marked by deterioration in malaria control that has continued for more than 30 years with sporadic epidemics of disease [16]. Resistance in *An. sacharovi* has been later reported in Bulgaria, Lebanon, Iran, Iraq, and Syria [12]. Pronounced DDT resistance appeared in *An. stephensi* in Iran and Iraq when full scale house spraying operations began in 1957 and dieldrin resistance appeared three years later. In India, house-spraying of DDT and Lindane (HCH) under the public health programme was introduced in the 1950s. Resistance of the main malaria vector *An. culicifacies* to dieldrin developed in 1958 [17] and resistance to DDT in 1959 [18], but the malaria control programme continued until 1965-1966 when both DDT and HCH failed to control outbreaks of malaria [19]. As a result, malathion was introduced in some areas in 1969 with some success but *An*. *culicifacies* rapidly developed resistance by 1973 [20]. Malathion resistance resulted in colossal epidemics of malaria in 1975 with 4 million cases reported as compared with 125,000 in 1965. The experience in Pakistan was similar with DDT resistance appearing in 1963. The importance of the resistance was not recognized until outbreaks of malaria began in 1969 and neither DDT nor HCH was effective. By 1975, malaria cases were reported in Pakistan to number 100 million as compared to 9,500 in 1961 [12]. DDT resistance in *An. culicifacies* was reported in Sri Lanka in 1968 resulting in a severe epidemic of malaria [21]. This vector is now resistant to DDT, dieldrin, organophosphates, carbamates and pyrethroids [11].

Similar trend was noted in Central America and the Caribbean. Dieldrin spraying against *An. albimanus* begun in 1956 and widespread resistance appeared in 1958 [12]. A return to DDT spraying produced generalized resistance by 1960 [18]. The carbamate propoxur was em‐ ployed in El Salvador, Guatemala, Honduras and Nicaragua in 1970 and resistance developed by 1974. *An albimanus* now exhibits multiple resistances to DDT, dieldrin, lindane and other chemical recently used in public health [22].

Much less information is however available for South East Asian malaria vectors, most probably because resistance monitoring was not carried out in routine before the 80s. In Vietnam, DDT resistance was found in 1989 in *An. epiroticus* of the Sundaicus Complex and is still occurring [23]. From 1990 till 2000, pyrethroid resistance was almost absent in all tested species except in some populations of *An. vagus* and *An. minimus s.l*. [24]. In Thailand, no evidence of insecticide resistance in malaria vectors was present before 1985 [25]. In 1986, development of physiological resistance to DDT was detected in *An. aconitus* from the north where DDT was commonly used for malaria control. One year later, DDT resistance was found in field collected mosquitoes of *An. philippinenis*, *An. nivipes* and *An. aconitus* from the same northern region. Between 1990 and 1997, DDT resistance has been detected in the three primary malaria vectors *An. dirus s.l*., *An. minimus s.l*. and *An. maculatus s.l.* and permethrin resistance was suggested in a population of *An. minimus s.l*. from northern Thailand, based however on a lower discriminative dosage (0.25%) of permethrin than that used today [25].

In Africa, resistance was initially found in *An. gambiae* in Bobo Dioulasso by 1967 (Burkina Faso), hence less than 7 years after the end of DDT use for malaria control [12]. DDT resistance was found in neighbouring countries including Cote d'Ivoire, Nigeria and Mali [26] and was then reported in most of Central and East African countries [27]. Strong association was observed between the level of DDT resistance in malaria vectors and the amount of DDT use for cotton protection [28]. Regarding BHC/dieldrin, the first cases of resistance were reported in Nigeria in 1954 hence only few months after the introduction of this molecule for malaria control. Initially found in very limited geographical areas, dieldrin resistance has spread in areas free of any insecticide treatments [29]. Few years later, resistance was reported in Bobo-Dioulasso and Cote d'Ivoire [30]. Today, resistance to BHC/dieldrin is still widespread in wild field anopheline populations despite its abandon in public health for many decades [31]. As for DDT, dieldrin resistance in malaria vectors arose and persisted from intensive use of pesticide for agricultural practices and in some specific settings due to public health pro‐ grammes [32, 33].

After the 80s, DDT has been more or less abandoned worldwide and replaced by organophos‐ phate (OP), pyrethroids and, to lesser extent, carbamates. However, insecticide resistance continued to be a problem, and vector control operations were affected, particularly in India, Africa and Latin America, by extensive use of agricultural pesticides. OP resistance, either in the form of broad-spectrum OP resistance or malathion-specific resistance was found in the major malaria vector species worldwide [12]. Pyrethroids were introduced in late 70s in public health and increasingly used in the 90s; however, cases of resistance were rapidly reported in

the main malaria vectors worldwide including *An. albimanus* [34], *An. darlingi* [35], *An. culicifacies* [36], *An. stephensi* [37], *An. gambiae* [38], *An. funestus* [39] and *An minimus* [40]. Despite a sporadic use (compared to DDT and pyrethroids), resistance to carbamates was earlier reported in several mosquito species including *An. albimanus* [41], *An. atroparvus* [42], *An. sacharovi*[5] and *An. gambiae*[43]. Carbamate resistance is now spreading in malaria vectors especially in West Africa where it has been reported in Cote d'Ivoire [44], Burkina Faso [45, 46], Benin [47] and Nigeria [48]. Increased level of carbamate resistance in African mosquito populations is worrying for malaria control because these chemicals are increasingly used in replacement to pyrethroids for Indoor Residual Spraying (IRS) [49].

It is obvious that insecticide resistance in malaria vectors is increasing worldwide due to the increasing selection pressure on mosquito populations caused by the presence of urban, domestic and/or agricultural pollutants in the environment [50]. Transversal and longitudinal monitoring surveys are essential to address the spatio-temporal changes in resistance (dy‐ namic) and to design appropriate strategies for a better control of resistant malaria vector populations worldwide.

## **4. Resistance mechanisms**

[21]. This vector is now resistant to DDT, dieldrin, organophosphates, carbamates and

Similar trend was noted in Central America and the Caribbean. Dieldrin spraying against *An. albimanus* begun in 1956 and widespread resistance appeared in 1958 [12]. A return to DDT spraying produced generalized resistance by 1960 [18]. The carbamate propoxur was em‐ ployed in El Salvador, Guatemala, Honduras and Nicaragua in 1970 and resistance developed by 1974. *An albimanus* now exhibits multiple resistances to DDT, dieldrin, lindane and other

Much less information is however available for South East Asian malaria vectors, most probably because resistance monitoring was not carried out in routine before the 80s. In Vietnam, DDT resistance was found in 1989 in *An. epiroticus* of the Sundaicus Complex and is still occurring [23]. From 1990 till 2000, pyrethroid resistance was almost absent in all tested species except in some populations of *An. vagus* and *An. minimus s.l*. [24]. In Thailand, no evidence of insecticide resistance in malaria vectors was present before 1985 [25]. In 1986, development of physiological resistance to DDT was detected in *An. aconitus* from the north where DDT was commonly used for malaria control. One year later, DDT resistance was found in field collected mosquitoes of *An. philippinenis*, *An. nivipes* and *An. aconitus* from the same northern region. Between 1990 and 1997, DDT resistance has been detected in the three primary malaria vectors *An. dirus s.l*., *An. minimus s.l*. and *An. maculatus s.l.* and permethrin resistance was suggested in a population of *An. minimus s.l*. from northern Thailand, based however on

a lower discriminative dosage (0.25%) of permethrin than that used today [25].

In Africa, resistance was initially found in *An. gambiae* in Bobo Dioulasso by 1967 (Burkina Faso), hence less than 7 years after the end of DDT use for malaria control [12]. DDT resistance was found in neighbouring countries including Cote d'Ivoire, Nigeria and Mali [26] and was then reported in most of Central and East African countries [27]. Strong association was observed between the level of DDT resistance in malaria vectors and the amount of DDT use for cotton protection [28]. Regarding BHC/dieldrin, the first cases of resistance were reported in Nigeria in 1954 hence only few months after the introduction of this molecule for malaria control. Initially found in very limited geographical areas, dieldrin resistance has spread in areas free of any insecticide treatments [29]. Few years later, resistance was reported in Bobo-Dioulasso and Cote d'Ivoire [30]. Today, resistance to BHC/dieldrin is still widespread in wild field anopheline populations despite its abandon in public health for many decades [31]. As for DDT, dieldrin resistance in malaria vectors arose and persisted from intensive use of pesticide for agricultural practices and in some specific settings due to public health pro‐

After the 80s, DDT has been more or less abandoned worldwide and replaced by organophos‐ phate (OP), pyrethroids and, to lesser extent, carbamates. However, insecticide resistance continued to be a problem, and vector control operations were affected, particularly in India, Africa and Latin America, by extensive use of agricultural pesticides. OP resistance, either in the form of broad-spectrum OP resistance or malathion-specific resistance was found in the major malaria vector species worldwide [12]. Pyrethroids were introduced in late 70s in public health and increasingly used in the 90s; however, cases of resistance were rapidly reported in

pyrethroids [11].

grammes [32, 33].

chemical recently used in public health [22].

582 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

The various mechanisms that enable insects to resist the action of insecticides can be grouped into four distinct categories including metabolic resistance, target-site resistance, reduce penetration and behavioral avoidance. These mechanisms that are shown in the figure 1 are briefly described in the following sections.

#### **4.1. Metabolic resistance**

Metabolic resistance is the most common resistance mechanism that occurs in insects. This mechanism is based on the enzyme systems which all insects possess to help them to detoxify naturally occurring xenobiotics/insecticides. It is commonly accepted that insect detoxification systems derived from the plant-insect evolutionary arm race and several insect detoxification enzymes have been associated to the detoxification of plant toxins and all types of chemicals, including insecticides [51]. Over-expression of enzymes capable of detoxifying insecticides or amino acid substitutions within these enzymes, which alter the affinity of the enzyme for the insecticide, can result in high levels of insecticide resistance (see [52] for review). Increased expression of the genes encoding the major xenobiotic metabolizing enzymes is the most common cause of insecticide resistance in mosquitoes. Over expression of detoxyfing enzymes can occur as the result of gene amplification (e.g. duplication) or due to changes in either transacting regulator elements or in the promoter region of the gene [5, 53, 54]. The consequence is a significant increase of enzyme production in resistant insects that enables them to metabolize or degrade insecticides before they are able to exert a toxic effect. Three categories of enzymes, namely esterases, P450s and glutathione-S-transferases are known to confer resistance to insecticides in insect pest such as malaria vectors. These large enzyme families contain multiple enzymes with broad overlapping substrate specificities, and one member of the family might

**Figure 1.** Scheme of potential behavioral and physiological changes associated with insecticide resistance in malaria vectors; (a) susceptible insect; (b) resistant insect *(*source *; see [14])*

be capable of metabolizing limited number of insecticides. Similarly, the level of resistance conferred can vary from low to very high and may differ from compound to compound. Metabolic resistance mechanisms have been identified in mosquito populations for all major classes of insecticides currently used for vector control, including organochlorine, organo‐ phosphates, carbamates, and pyrethroids.

*Esterase*s. One of the most common metabolic resistance mechanisms is that of elevated levels, or activity, of esterase enzymes which hydrolyze ester bonds or sequester insecticides. A striking example comes from studies on *Culex quinquefasciatus* that resist to a broad range of OP insecticides. In this species, multiple copies of EST-genes was already found hence enabling it to overproduce this type of enzyme [55]. In contrast to the situation in *Culex,* a number of *Anopheles* species (ie *An. culicifacies*, *An. stephensi* and *An. arabiensis*) have a non-elevated esterase mechanism that confers resistance specifically to malathion through increased rates of metabolism. Malathion resistance in *Anopheles* spp was associated with an altered form of esterase that specifically metabolizes the molecule at a much faster rate than that in susceptible counterparts [56, 57]. Although Carboxylesterase (CCEs) have been mostly associated to organophosphate resistance in mosquitoes, their role in pyrethroid resistance is probable. Indeed, the ability of esterases to metabolize pyrethroids has been suggested in mosquitoes [58, 59] even if no specific mosquito CCE has yet been validated as a pyrethroid metabolizer [50]. Clearly much more information is needed on the esterase-mediated resistance in malaria vectors.

*P450s.* Cytochrome P450-dependent monooxygenases are an important and diverse family of enzymes involved in the metabolism of numerous endogenous and exogenous com‐ pounds. P450 belong to six families and increased transcription of genes belonging to the CYP4, CYP6, and CYP9 has been observed in various insecticide-resistant species from different taxa [60]. There is increasing number of reports demonstrating elevated P450 monooxygenase activities in insecticide-resistant mosquitoes, frequently in conjunction with altered activities of other enzymes. In most cases where a link between insecticide resist‐ ance and elevated P450 activity has been shown, the CYP gene belongs to the CYP6 family. Since the publication of the *An. gambiae* genome [61], P450s were extensively studied in the primary malaria vector in Africa. A total of 111 P450 enzymes were identified [62] and, as in other insects, only a small number of these enzymes are capable of detoxifying insecticides. However, higher activity of enzymes and/or expression of detoxification genes in insecti‐ cide resistant colonies do not necessarily correlate with insecticide resistance. For example, some authors have shown elevated transcript levels of an adult-specific CYP6 P450 gene, CYP6Z1, in pyrethroid-resistant strain *of An. gambiae* [63, 64] and *An. funestus* [65]. Further validation studies conducted in *An. gambiae* showed that cyp6z1 was however not capable to metabolize pyrethroids but was capable to metabolize DDT [66]. Another study showed that CYP6z2 displays broad substrate specificity, which may be associated with xenobiotics metabolism and detoxification [67]. Despite, CYP6Z2 being able to bind to permethrin and cypermethrin, this gene does not metabolise any of these insecticides. Microarray-based approaches have lately identified three new "candidate" P450 genes that were found to be repeatedly over-produced in pyrethroid resistant populations of *An. gambiae*: CYP6M2, CYP6P3 and CYP6Z2 [68-70]. All of these genes encode for enzymes that are able to bind to type I and type II pyrethroids but only CYP6P3 and CYP6M2 showed to metabolize the insecticides [10, 71]. More recently, some authors demonstrated that CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in *An. gambiae*, hence demonstrating the first evidence for a metabolic cross-resistance in malaria vectors [9]. Interestingly the putative ortholog of *An. gambiae* CYP6P3, CYP6P9, as being the prime candidate for conferring pyrethroid resistance, have been identified in *An. funestus* [72, 73] but only the CYP6P9 showed to metabolize types I and II pyrethroids [74]. Recent works showed that overproduction of CYP6P9 in *An. funestus* result from gene duplication [72]. In *An. minimus* mosquito, CYP6AA3 and CYP6P7 were up-regulated in pyrethroid-resistance population of Thailand [75] and seem to possess activities toward pyrethroid degradation [76, 77].

be capable of metabolizing limited number of insecticides. Similarly, the level of resistance conferred can vary from low to very high and may differ from compound to compound. Metabolic resistance mechanisms have been identified in mosquito populations for all major classes of insecticides currently used for vector control, including organochlorine, organo‐

**Figure 1.** Scheme of potential behavioral and physiological changes associated with insecticide resistance in malaria

*Esterase*s. One of the most common metabolic resistance mechanisms is that of elevated levels, or activity, of esterase enzymes which hydrolyze ester bonds or sequester insecticides. A striking example comes from studies on *Culex quinquefasciatus* that resist to a broad range of OP insecticides. In this species, multiple copies of EST-genes was already found hence enabling it to overproduce this type of enzyme [55]. In contrast to the situation in *Culex,* a number of *Anopheles* species (ie *An. culicifacies*, *An. stephensi* and *An. arabiensis*) have a non-elevated esterase mechanism that confers resistance specifically to malathion through increased rates of metabolism. Malathion resistance in *Anopheles* spp was associated with an altered form of esterase that specifically metabolizes the molecule at a much faster rate than that in susceptible counterparts [56, 57]. Although Carboxylesterase (CCEs) have been mostly associated to organophosphate resistance in mosquitoes, their role in pyrethroid resistance is probable. Indeed, the ability of esterases to metabolize pyrethroids has been suggested in mosquitoes [58, 59] even if no specific mosquito CCE has yet been validated as a pyrethroid metabolizer

phosphates, carbamates, and pyrethroids.

584 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

vectors; (a) susceptible insect; (b) resistant insect *(*source *; see [14])*

Glutathione S-transferases (GSTs). Glutathione transferases (GSTs) are multifunctional enzymes involved in the detoxification of many endogenous and xenobiotic compounds. Conjugation of Glutathione (GSH) to such organic molecules enhances solubility, thus facilitating their eventual elimination [78]. Elevated GST activity has been implicated in resistance to at least four classes of insecticides in insects. Higher enzyme activity is usually due to an increase in the amount of one or more GST enzymes, either as a result of gene amplification or more commonly through increases in transcriptional rate, rather than qualitative changes in individual enzymes [52]. At least six classes of insect GSTs have been identified in *An. gambiae* [79], found in several large clusters on all three chromosomes. The Delta and Epsilon classes found exclusively in insects are the largest classes of insect GSTs. Members of both classes have been implicated in resistance to all the major classes of insecti‐ cide. The primary role of GSTs in mosquito insecticide resistance is in the metabolism of DDT to DDE (non toxic products), although they also have a secondary role in organophosphate resistance [80]. GST-based DDT resistance is common in a number of anopheline species including *An. gambiae* [81-83], reflecting the heavy use of this insecticide for malaria control over several decades. Molecular biology and *in vitro* expression studies showed that *aggst3-2* was over expressed in resistant strain of *An. gambiae* and that recombinant *aggst3-2* was very efficient at metabolizing DDT [84]. Most studies of GSTs suggested that regulation occurs at the transcriptional level. Several regulatory elements have been identified in the promoter regions of GSTs that may mediate their induction but the significance of these findings is unclear. Genetic mapping of the major genes controlling GST-based DDT resistance in *An. gambiae* provided however evidence for a trans-acting regulator [84], although in this species, mutations in promoter elements of the Epsilon GST cluster are also associated with resistance [81]. It has been suggested that GSTs may play a role in pyrethroid resistance by detoxifying lipid peroxidation products induced by pyrethroids and/or by protecting from insecticide exposure induced oxidative stress [85]. Furthermore, GST might confer secondary role in pyrethroid resistance by sequestering the insecticide hence reducing the total *in vivo* concen‐ tration of insecticide [86].

Despite the great advance obtained recently in the identification of the role of detoxifying enzymes in insecticide resistance, force is to note that the function of >90% of metabolic genes is still unknown. Although only a limited number of resistance mechanisms have been implicated to date, the diversity within enzyme families involved in metabolic resistance is likely to contribute substantially to resistance to many insecticide classes. Further functional genomics and post-genomic technology are needed to reveal the contributions of hitherto unsuspected enzymes in insecticide metabolism and/or sequestration and to identify the causal mutations associated with metabolic resistance in mosquitoes. The contribution that these enzymes make towards various insecticide resistance phenotypes in malaria vectors is yet to be elucidated.

#### **4.2. Target-site resistance**

The second most common resistance mechanism encountered in insects is target-site resist‐ ance. Insecticides generally act at a specific site within the insect, typically within the nervous system (e.g. OP, carbamate, DDT and pyrethroid insecticides). The site of action can be modified in resistant strains of insects such that the insecticide no longer binds effectively. Reduce sensitivity of the target receptors to insecticide results from non-silent point mutations in the gene encoding the protein. For example, the target site for OP and carbamate insecticides is acetylcholinesterase (AChE) in the nerve cell synapses. Several mutations in the gene encoding for an acetylcholinesterase have been found in insects" [87] which result in reduced sensitivity to inhibition of the enzyme by these insecticides [88, 89]. In malaria vectors, the G119S mutation (i.e. glycine to serine substitution at position 119) responsible for carbamate and OP resistance has been reported in *An. gambiae* and *An. albimanus,* essentially at the heterozygous state [90]. Recent sequence analysis of some resistant mosquitoes collected in Benin revealed the presence of a duplication of the *ace-1* gene in both A\*n\*. *A. gambiae* M and S forms [91]. In addition, mutations at a single codon (position 302) in the Rdl (resistance to dieldrin) gene encoding one receptor subunit, from an alanine residue to a serine (or more rarely to a glycine), have been documented in dieldrin-resistant insect species [92] including the malaria vectors *An. stephensi* [93], *An. gambiae s.l*. [94] and *An. funestus* [31]. Similarly, mutations in the amino acid sequence in the voltage-gated sodium channels of nerve cell membranes leads to a reduction in the sensitivity of the channels to the binding of DDT and pyrethroid insecticides [95]. Alterations in the target site that cause resistance to insecticides are often referred to as knockdown resistance (*kdr*) in reference to the ability of insects with these alleles to withstand prolonged exposure to insecticides without being 'knocked-down" [96]. One of the most common amino acid replacements associated with pyrethroid resistance in malaria vectors is a substitution of the leucine residue found at codon 1014 with either phenylalanine (1014F) [97] or serine (1014S) [98] in the Voltage-Gated Sodium Channel (VGSC). Interestingly, residue 1014 does not appear to interact directly with the insecticide but is predicted to alter channel activation kinetics [99]. Note that a *de novo* mutation (N1575Y) recently emerged within domains III-IV of voltage gate sodium channel in pyrethroid resistant populations of *An. gambiae* and seems to occurs only in a single long-range haplotype, also bearing 1014F allele [100]. It has been suggested that the N1575Y mutation may compensates for deleterious fitness effects of 1014F and/or confers additional resistance to pyrethroid insecticides.

#### **4.3. Reduced penetration**

qualitative changes in individual enzymes [52]. At least six classes of insect GSTs have been identified in *An. gambiae* [79], found in several large clusters on all three chromosomes. The Delta and Epsilon classes found exclusively in insects are the largest classes of insect GSTs. Members of both classes have been implicated in resistance to all the major classes of insecti‐ cide. The primary role of GSTs in mosquito insecticide resistance is in the metabolism of DDT to DDE (non toxic products), although they also have a secondary role in organophosphate resistance [80]. GST-based DDT resistance is common in a number of anopheline species including *An. gambiae* [81-83], reflecting the heavy use of this insecticide for malaria control over several decades. Molecular biology and *in vitro* expression studies showed that *aggst3-2* was over expressed in resistant strain of *An. gambiae* and that recombinant *aggst3-2* was very efficient at metabolizing DDT [84]. Most studies of GSTs suggested that regulation occurs at the transcriptional level. Several regulatory elements have been identified in the promoter regions of GSTs that may mediate their induction but the significance of these findings is unclear. Genetic mapping of the major genes controlling GST-based DDT resistance in *An. gambiae* provided however evidence for a trans-acting regulator [84], although in this species, mutations in promoter elements of the Epsilon GST cluster are also associated with resistance [81]. It has been suggested that GSTs may play a role in pyrethroid resistance by detoxifying lipid peroxidation products induced by pyrethroids and/or by protecting from insecticide exposure induced oxidative stress [85]. Furthermore, GST might confer secondary role in pyrethroid resistance by sequestering the insecticide hence reducing the total *in vivo* concen‐

Despite the great advance obtained recently in the identification of the role of detoxifying enzymes in insecticide resistance, force is to note that the function of >90% of metabolic genes is still unknown. Although only a limited number of resistance mechanisms have been implicated to date, the diversity within enzyme families involved in metabolic resistance is likely to contribute substantially to resistance to many insecticide classes. Further functional genomics and post-genomic technology are needed to reveal the contributions of hitherto unsuspected enzymes in insecticide metabolism and/or sequestration and to identify the causal mutations associated with metabolic resistance in mosquitoes. The contribution that these enzymes make towards various insecticide resistance phenotypes in malaria vectors is yet to

The second most common resistance mechanism encountered in insects is target-site resist‐ ance. Insecticides generally act at a specific site within the insect, typically within the nervous system (e.g. OP, carbamate, DDT and pyrethroid insecticides). The site of action can be modified in resistant strains of insects such that the insecticide no longer binds effectively. Reduce sensitivity of the target receptors to insecticide results from non-silent point mutations in the gene encoding the protein. For example, the target site for OP and carbamate insecticides is acetylcholinesterase (AChE) in the nerve cell synapses. Several mutations in the gene encoding for an acetylcholinesterase have been found in insects" [87] which result in reduced sensitivity to inhibition of the enzyme by these insecticides [88, 89]. In malaria vectors, the

tration of insecticide [86].

586 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

be elucidated.

**4.2. Target-site resistance**

Modifications in the insect cuticle or digestive tract linings that prevent or slow the absorption or penetration of insecticides can be found is some resistant insects. This resistance mechanism is not specific and can affect a broad range of insecticides. Reduced uptake of insecticide, often referred to as cuticular resistance, is frequently described as a minor resistance mechanism. Certainly for pests where the major route of insecticide delivery is via ingestion, this is likely to be the case. However, for malaria control, where insecticides are typically delivered on bed nets or on wall surfaces, uptake of insecticides is primarily through the appendages. An increase in the thickness of the tarsal cuticle, or a reduction in its permeability to lipophilic insecticides, could have a major impact on the bioavailability of an insecticide *in vivo*. Examples of reduced-penetration mechanisms are however limited; cuticular resistance was reported for the domestic Fly *Musca domestica* [101] and the lymphatic filariasis vector *Culex quinque‐ fasciatus*[102]. In *Anopheles*, microarrays studies have recently identified two genes, cplcg3 and cplcg4, encoding cuticular proteins that were upregulated in pyrethroid resistant strains from four populations and two different species (i.e. *An. gambiae* and *An. stephensi*) ([69, 103, 104]. Recently, measures of mean cuticle thickness in a laboratory strain of *An. funestus* using scanning electron microscopy (SEM) showed that the mean cuticle thickness was significantly greater in pyrethroid tolerant mosquitoes than their susceptible counterparts [105]. Clearly much more work is required in order to identify the significance of cuticular resistance in phenotypic resistance.

#### **4.4. Behavioural resistance**

Insecticide resistance in mosquitoes is not always based on biochemical mechanisms such as metabolic detoxification or target site mutations, but may also be conferred by behavioural changes in response to prolonged exposure to an insecticide. Behavioural resistance does not have the same "importance" as physiological resistance but may be considered to be a contributing factor, leading to the avoidance of lethal doses of an insecticide [106, 107]. For example, the first study on the irritant effect of DDT residual deposits was conducted using *Anopheles quadrimaculatus* where females were found to be irritated shortly after making contact with the treated surfaces resulting in a rapid escape response from a treated house prior to taking a blood meal [108]. This type of response can be further divided into direct contact excitation (sometimes referred to as 'irritancy') and non-contact spatial repellency that is used when insects move away from the insecticide-treated area before making direct contact [106, 109]. Examples of behavioral resistance or avoidance are few. Change in vector compo‐ sition (i.e. switch from *An. minimus* to *An. harrisoni*) has been observed following implemen‐ tation of ITNs in a village form central Vietnam [110]. With regard to *An. funestus*, recent findings showed a shift from indoor to outdoor biting preferences in Tanzania in relation to increasing coverage of pyrethroid-impregnated net [111]. Significant changes in the hostseeking behavior of the *An. funestus* population was confirmed in Benin (West Africa) where scaling up of LLINs at community level induced a change from night biting to early-morning biting behaviour [112]. It is unclear however whether adaptation of malaria vectors species to insecticidal based vector control interventions such as LLIN may result from a phenotypic plasticity or from selected behavioral traits (see Durnez & Coosemans for details).

#### **5. Method to detect insecticide resistance**

Currently most resistance monitoring is dependent on bioassays, using fixed insecticide concentrations and exposure times, and the data is reported as percentage mortality and/or Knock Down (KD) effect. The World Health Organisation (WHO) has defined diagnostic doses (i.e. twice the dosage that killed 100% susceptible mosquitoes of a given species) for most insecticides used in malaria control and produces susceptibility test kits consisting of exposure chambers and insecticide treated filter papers [113-115]. Although simple to perform, these diagnostic dose assays provide limited information and several alternative methods for detecting resistance are available (Table 2). These alternative assays generally detect specific resistance mechanisms, and should always be performed as an addition, not a substitute, to bioassays, to avoid the risk that unknown resistance mechanisms go undetected. It should be noted that none of the current methods listed in Table 2 are suitable for detecting cuticular and/or behavioural resistance. Regular monitoring for insecticide resistance is essential in order to react proactively to prevent insecticide resistance from compromising control. If the frequency of resistance alleles is going to build up unchecked, resistance may eventually become 'fixed' in the populations. Once resistance reaches very high levels, strategies to restore susceptibility are unlikely to be effective.


**Table 2.** Methods for detecting insecticide resistance (source: see [6])

#### **5.1. Bioassays**

much more work is required in order to identify the significance of cuticular resistance in

Insecticide resistance in mosquitoes is not always based on biochemical mechanisms such as metabolic detoxification or target site mutations, but may also be conferred by behavioural changes in response to prolonged exposure to an insecticide. Behavioural resistance does not have the same "importance" as physiological resistance but may be considered to be a contributing factor, leading to the avoidance of lethal doses of an insecticide [106, 107]. For example, the first study on the irritant effect of DDT residual deposits was conducted using *Anopheles quadrimaculatus* where females were found to be irritated shortly after making contact with the treated surfaces resulting in a rapid escape response from a treated house prior to taking a blood meal [108]. This type of response can be further divided into direct contact excitation (sometimes referred to as 'irritancy') and non-contact spatial repellency that is used when insects move away from the insecticide-treated area before making direct contact [106, 109]. Examples of behavioral resistance or avoidance are few. Change in vector compo‐ sition (i.e. switch from *An. minimus* to *An. harrisoni*) has been observed following implemen‐ tation of ITNs in a village form central Vietnam [110]. With regard to *An. funestus*, recent findings showed a shift from indoor to outdoor biting preferences in Tanzania in relation to increasing coverage of pyrethroid-impregnated net [111]. Significant changes in the hostseeking behavior of the *An. funestus* population was confirmed in Benin (West Africa) where scaling up of LLINs at community level induced a change from night biting to early-morning biting behaviour [112]. It is unclear however whether adaptation of malaria vectors species to insecticidal based vector control interventions such as LLIN may result from a phenotypic

plasticity or from selected behavioral traits (see Durnez & Coosemans for details).

Currently most resistance monitoring is dependent on bioassays, using fixed insecticide concentrations and exposure times, and the data is reported as percentage mortality and/or Knock Down (KD) effect. The World Health Organisation (WHO) has defined diagnostic doses (i.e. twice the dosage that killed 100% susceptible mosquitoes of a given species) for most insecticides used in malaria control and produces susceptibility test kits consisting of exposure chambers and insecticide treated filter papers [113-115]. Although simple to perform, these diagnostic dose assays provide limited information and several alternative methods for detecting resistance are available (Table 2). These alternative assays generally detect specific resistance mechanisms, and should always be performed as an addition, not a substitute, to bioassays, to avoid the risk that unknown resistance mechanisms go undetected. It should be noted that none of the current methods listed in Table 2 are suitable for detecting cuticular and/or behavioural resistance. Regular monitoring for insecticide resistance is essential in order to react proactively to prevent insecticide resistance from compromising control. If the frequency of resistance alleles is going to build up unchecked, resistance may eventually

**5. Method to detect insecticide resistance**

phenotypic resistance.

**4.4. Behavioural resistance**

588 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Guidelines for test procedures and interpretation of results are available from the WHOPES3 (see http://www.who.int/whopes/resistance/en/). It is important that the mosquitoes used for the bioassays are standardized for age, sex and physiological status as all of these can affect the outcome of the tests. Typically either adults raised from isofemale lines or F1 progeny from field collected blood fed females are used. The limitations and advantages of these two alternatives have recently been discussed [116].

These diagnostic dose assays are simple to perform and provide standardized data sets that, assuming the guidelines are followed, can be readily compared to identify temporal and/or geographical variations in the resistant status of malaria vector populations. However, it is important to recognize some of the limitations of these susceptibility tests. As only a single concentration of insecticide is used, the results do not provide any information about the level of resistance in a population. For example if 50 % of population A and 20 % of population B were killed after exposure to the diagnostic dose of permethrin, it cannot be concluded that population B is more resistant than population A. The results only indicate that both popula‐ tions are resistant (according to WHO definitions if there is < 80 % mortality, the population is defined as resistant) and that, subject to tests of significance, there is a higher frequency of

<sup>3</sup> World Health Organization Pesticide Evaluation Scheme

resistant individuals in population B than in A. Dose response assays would be needed to compare the levels of resistance in two populations (e.g. by measuring the Resistant Ratios and their 95% confidence intervals). For pyrethroids, median knock down time (MKDT) is also a useful quantifiable variable [117]. Similarly, the results of these tests cannot be used to compare the levels of resistance to two different insecticides. If 50 % mortality was observed after exposure to the diagnostic dose of permethrin (0.75 %) whereas mortality was 70% after exposure to the diagnostic dose of deltamethrin (0.05%), it is not correct to state that the population is more resistant to permethrin than deltamethrin. Again, all that can be stated is that the population is resistant to both insecticides.

Partly due to the limitations of the diagnostic dose assays described above and partly due to the difficulties that are sometimes incurred in obtaining a regular supply of the insecticide impregnated papers from WHO, an alternative bioassay methodology has been developed [118] and is being adopted by some monitoring programmes. This method, known as the CDC bottle bioassay, uses glass bottles coated with a known concentration of insecticide. As these test kits are assembled in the users own laboratory, the concentration of insecticide can be readily adjusted enabling dose response curves to be developed to compare two or more strains. A caveat to this is that the flexibility, and the potential variation in the insecticide grade used in the tests, impairs comparison of results between two separate studies.

Both WHO diagnostic doses and CDC bottle bioassays can be modified to incorporate synergists. Synergists such as piperonyl butoxide, that block the activity of two major detox‐ ification enzyme families, can be used to explore the role of different resistance mechanisms. If resistance is due to increased metabolism, exposure to an appropriate synergist prior to insecticide bioassays should increase the level of mortality observed.

#### **5.2. Biochemical tests**

Biochemical tests to detect alterations in activities of enzyme families associated with insecti‐ cide resistance have been available for over two decades and are sometimes used in combi‐ nation with insecticide bioassays [119]. These assays employ model substrates to record the overall activity of glutathione transferases, carboxylesterases or cytochrome P450s in individ‐ ual insects. Biochemical assays are also available to detect target site resistance to organo‐ phosphate and carbamate insecticides caused by insensitive acetylcholinesterase (AChE). The enzymatic reaction produces a colour change that is generally visible to the naked eye and hence these assays do not require access to expensive equipment (spectrophotometer is appropriate). However, it is important that the mosquitoes are kept on ice from the point of collection to the performance of the assay and this can often pose logistical challenges. Furthermore, there are sensitivity and specificity issues that limit the utility of some of these assays. For example, with over 100 different cytochrome P450 enzymes in malaria vectors, an assay that measures the total level or activity of this enzyme family may not have the sensitivity to detect over production of the single or small number of P450 enzymes that are thought to be involved in pyrethroid metabolism. This may explain the lack of significant correlation observed in many studies between cytochrome P450 activity and bioassay mortality results [120, 121]. In addition not all members of the enzyme family will have the same affinity for the model substrates used in these assays (e.g. CDNB (1-chloro 2-4, dinitrobenzene) is the substrate typically used to assess glutathione transferase activity but the Epsilon class of GSTs which are responsible for DDT resistance have relatively low activity with this substrate). In order to incorporate data from resistance monitoring into evidence based decisions on appropriate insecticide based interventions for malaria control, it is clearly essential that the data is both reliable and accessible. Although guidelines for conducting the various assays exist, there is little consensus on the number of sites and frequency with which resistance monitoring should occur [122]. It is clear that resistance is a dynamic trait, and wide fluctuations in resistance levels throughout the malaria transmission season have been reported [116, 123, 124]. Resist‐ ance can also be very focal, particularly when vector composition differs between sites [125], hence a minimum number of sampling sites should be established, taking into account patterns of vector distribution and insecticide usage. The WHO/AFRO African Network for Vector Resistance was established in 2000 and amongst its objectives was the important goal of improving the dissemination of resistance data. Accordingly a database was established to store the results of resistance monitoring activities by the African Network for Vector Resist‐ ance (ANVR) members but until recently, this database was not readily accessible by outside users. The recent establishment of new data base (see section 6), as an online centralized resource for collating data on insecticide resistance in disease vectors and the integration of this with the ANVR database, will hopefully ensure that both published and unpublished data on resistance in malaria vectors are more readily available to all interested parties.

#### **5.3. Molecular tests**

resistant individuals in population B than in A. Dose response assays would be needed to compare the levels of resistance in two populations (e.g. by measuring the Resistant Ratios and their 95% confidence intervals). For pyrethroids, median knock down time (MKDT) is also a useful quantifiable variable [117]. Similarly, the results of these tests cannot be used to compare the levels of resistance to two different insecticides. If 50 % mortality was observed after exposure to the diagnostic dose of permethrin (0.75 %) whereas mortality was 70% after exposure to the diagnostic dose of deltamethrin (0.05%), it is not correct to state that the population is more resistant to permethrin than deltamethrin. Again, all that can be stated is

Partly due to the limitations of the diagnostic dose assays described above and partly due to the difficulties that are sometimes incurred in obtaining a regular supply of the insecticide impregnated papers from WHO, an alternative bioassay methodology has been developed [118] and is being adopted by some monitoring programmes. This method, known as the CDC bottle bioassay, uses glass bottles coated with a known concentration of insecticide. As these test kits are assembled in the users own laboratory, the concentration of insecticide can be readily adjusted enabling dose response curves to be developed to compare two or more strains. A caveat to this is that the flexibility, and the potential variation in the insecticide grade

Both WHO diagnostic doses and CDC bottle bioassays can be modified to incorporate synergists. Synergists such as piperonyl butoxide, that block the activity of two major detox‐ ification enzyme families, can be used to explore the role of different resistance mechanisms. If resistance is due to increased metabolism, exposure to an appropriate synergist prior to

Biochemical tests to detect alterations in activities of enzyme families associated with insecti‐ cide resistance have been available for over two decades and are sometimes used in combi‐ nation with insecticide bioassays [119]. These assays employ model substrates to record the overall activity of glutathione transferases, carboxylesterases or cytochrome P450s in individ‐ ual insects. Biochemical assays are also available to detect target site resistance to organo‐ phosphate and carbamate insecticides caused by insensitive acetylcholinesterase (AChE). The enzymatic reaction produces a colour change that is generally visible to the naked eye and hence these assays do not require access to expensive equipment (spectrophotometer is appropriate). However, it is important that the mosquitoes are kept on ice from the point of collection to the performance of the assay and this can often pose logistical challenges. Furthermore, there are sensitivity and specificity issues that limit the utility of some of these assays. For example, with over 100 different cytochrome P450 enzymes in malaria vectors, an assay that measures the total level or activity of this enzyme family may not have the sensitivity to detect over production of the single or small number of P450 enzymes that are thought to be involved in pyrethroid metabolism. This may explain the lack of significant correlation observed in many studies between cytochrome P450 activity and bioassay mortality results [120, 121]. In addition not all members of the enzyme family will have the same affinity for the model substrates used in these assays (e.g. CDNB (1-chloro 2-4, dinitrobenzene) is the substrate

used in the tests, impairs comparison of results between two separate studies.

insecticide bioassays should increase the level of mortality observed.

**5.2. Biochemical tests**

that the population is resistant to both insecticides.

590 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

A multitude of molecular assays have been developed to detect *kdr* alleles in malaria mosqui‐ toes, several of which were recently compared in a study by Bass et al (see [126]). These are routinely used by research laboratories monitoring for insecticide resistance and are gradually being incorporated into some national malaria control resistance monitoring programmes. Unfortunately, despite the recent identification of the key enzymes responsible for metabolic resistance to pyrethroids in *An gambiae* and *An funestus*, there are currently no simple DNA based assays to detect these resistance mechanisms. Detection of these genes is presently dependent on RNA based approaches using relatively sophisticated equipment (e.g. RTqPCR). Assays to detect the genetic mutation(s) responsible for the resistance phenotype in individual insects can provide an early warning of the emergence of resistance which may not have been detectable by bioassays that can only record the population response. The presence of a single individual with an allele known to confer resistance should be cause for concern as experience dictates that resistance can spread very rapidly in a population unless the selection pressure is eased and/or the genetic cost associated with the resistant allele is high. Conversely, a negative result from a molecular assay should not lead to complacency. As discussed above, molecular assays are presently only available for target site resistance and the failure to detect *kdr* clearly cannot be interpreted as an absence of resistance in a population. Hence molecular assays should be seen as a complement rather than a substitute for bioassays.

## **6. Current distribution of insecticide resistance**

Insecticide resistance has been reported in the main malaria vectors worldwide. Resistance is however not uniformly distributed among vector species and can greatly differ from one village, province, country, region and continent to another. Unfortunately, the highest levels of insecticide resistance were reported in Africa where malaria burden is still the highest in the world [1]. Resistance to pyrethroids, the gold standard insecticides used for LLIN and IRS will be extensively discussed in the present chapter as it remains a real and ever-present danger to future success of malaria vector control. Note that more information on the distribution of insecticide resistance in malaria vectors can be found in *Anobase,* http://anobase.vector‐ base.org/ir/; *MARA* http://www.mara.org.za; *Arthropod Pesticide Resistance Database*, http:// www.pesticideresistance.org.; and IR mapper, http://www.irmapper.com.

#### **6.1. Africa**

Although the occurrence of insecticide resistance in malaria vectors in Africa is not a "new" event (see section 2.), the speed at which pyrethroid-resistance recently evolved in field populations is worrying as it may jeopardize the current malaria vector control initiatives carried out in the continent. As shown in figure 2, pyrethroid resistance in *Anopheles* sp. is widespread but not uniformly distributed among the different countries. In the 49 African countries that have been investigated (see [6] for details), 15 did not report any data on resistance in the last 10 years i.e. Algeria, Botswana, Democratic Republic of Congo, Djibouti, Sierra Leone, Lesotho, Liberia, Libya, Maurice, Mauritania, Namibia, Rwanda, Somalia, Swaziland, Tunisia. If a lot of data has been generated in West Africa (as far as *An. gambiae s.l.* is concerned), a lack of information is globally observed in Central, Eastern and Austral Africa. It is obvious that the frequent conflicts that has occurred in the last decades in some African countries has rendered difficult the conduct of routine monitoring surveys by NMCP, International Organisation (WHO/ANVR) and/or research institutions.

Globally, pyrethroid resistance is high in *An. gambiae s.l.* in West Africa including Benin [127], Burkina Faso [128], Guinea Konakry ([129], Ghana [130], Mali [131], Niger [132], Nigeria [133] and Cote d'Ivoire [134]). In this region, pyrethroid resistance is predominant in *An. gambiae s.s*., compared to *An. arabiensis*. Surprisingly, susceptibility to pyrethroids (permethrin and/or deltamethrin) was reported in *An. gambiae* s.l. in Guinea Bissau [135] despite the presence of the L1014F mutation. In Central Africa, pyrethroid resistance/tolerance is widespread in *An. gambiae s.l* in Cameroon [136-138], Chad [116, 139], Gabon [140, 141], Equatorial Guinea [8] and Sudan [142, 143]. In Chad, North Cameroon and Sudan, pyrethroid resistance is present essentially in *An. arabiensis,* which is consistent with the higher prevalence of this mosquito species in more arid areas with higher mean annual temperatures [144]. In East and Austral Africa, *An. gambiae* and *An. arabiensis* populations are mostly susceptible to pyrethroids in Tanzania [145, 146], Mozambique [147] and Madagascar [148], but highly resistant in eastern Uganda [149, 150], Ethiopia [151], Kenya [152, 153], Zambia [154], South Africa [155] and the Gwave Region of Zimbabwe [120]. Regarding *An. funestus*, most of the literature reporting pyrethroid resistance comes from South Africa [39, 156] and Mozambique [157-159], most probably because *An. funestus* is the main malaria vector in these countries. The data available Distribution, Mechanisms, Impact and Management of Insecticide Resistance in Malaria Vectors: A Pragmatic Review http://dx.doi.org/10.5772/56117 593

**6. Current distribution of insecticide resistance**

592 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**6.1. Africa**

www.pesticideresistance.org.; and IR mapper, http://www.irmapper.com.

International Organisation (WHO/ANVR) and/or research institutions.

Insecticide resistance has been reported in the main malaria vectors worldwide. Resistance is however not uniformly distributed among vector species and can greatly differ from one village, province, country, region and continent to another. Unfortunately, the highest levels of insecticide resistance were reported in Africa where malaria burden is still the highest in the world [1]. Resistance to pyrethroids, the gold standard insecticides used for LLIN and IRS will be extensively discussed in the present chapter as it remains a real and ever-present danger to future success of malaria vector control. Note that more information on the distribution of insecticide resistance in malaria vectors can be found in *Anobase,* http://anobase.vector‐ base.org/ir/; *MARA* http://www.mara.org.za; *Arthropod Pesticide Resistance Database*, http://

Although the occurrence of insecticide resistance in malaria vectors in Africa is not a "new" event (see section 2.), the speed at which pyrethroid-resistance recently evolved in field populations is worrying as it may jeopardize the current malaria vector control initiatives carried out in the continent. As shown in figure 2, pyrethroid resistance in *Anopheles* sp. is widespread but not uniformly distributed among the different countries. In the 49 African countries that have been investigated (see [6] for details), 15 did not report any data on resistance in the last 10 years i.e. Algeria, Botswana, Democratic Republic of Congo, Djibouti, Sierra Leone, Lesotho, Liberia, Libya, Maurice, Mauritania, Namibia, Rwanda, Somalia, Swaziland, Tunisia. If a lot of data has been generated in West Africa (as far as *An. gambiae s.l.* is concerned), a lack of information is globally observed in Central, Eastern and Austral Africa. It is obvious that the frequent conflicts that has occurred in the last decades in some African countries has rendered difficult the conduct of routine monitoring surveys by NMCP,

Globally, pyrethroid resistance is high in *An. gambiae s.l.* in West Africa including Benin [127], Burkina Faso [128], Guinea Konakry ([129], Ghana [130], Mali [131], Niger [132], Nigeria [133] and Cote d'Ivoire [134]). In this region, pyrethroid resistance is predominant in *An. gambiae s.s*., compared to *An. arabiensis*. Surprisingly, susceptibility to pyrethroids (permethrin and/or deltamethrin) was reported in *An. gambiae* s.l. in Guinea Bissau [135] despite the presence of the L1014F mutation. In Central Africa, pyrethroid resistance/tolerance is widespread in *An. gambiae s.l* in Cameroon [136-138], Chad [116, 139], Gabon [140, 141], Equatorial Guinea [8] and Sudan [142, 143]. In Chad, North Cameroon and Sudan, pyrethroid resistance is present essentially in *An. arabiensis,* which is consistent with the higher prevalence of this mosquito species in more arid areas with higher mean annual temperatures [144]. In East and Austral Africa, *An. gambiae* and *An. arabiensis* populations are mostly susceptible to pyrethroids in Tanzania [145, 146], Mozambique [147] and Madagascar [148], but highly resistant in eastern Uganda [149, 150], Ethiopia [151], Kenya [152, 153], Zambia [154], South Africa [155] and the Gwave Region of Zimbabwe [120]. Regarding *An. funestus*, most of the literature reporting pyrethroid resistance comes from South Africa [39, 156] and Mozambique [157-159], most probably because *An. funestus* is the main malaria vector in these countries. The data available

**Figure 2.** Maps showing the distribution of pyrethroid-resistance in African malaria vectors; A) status of pyrethroid resistance according to WHO criteria ; B) Target site (*kdr*) and metabolic resistance reported for a given mosquito spe‐ cies (Source; see [6]).

in other African countries is very limited in partly due to the difficulty to colonize *An. funestus* species in laboratory. Pyrethroid resistance/tolerance was detected in Malawi [160, 161] and suspected in Obusi and Kassena-Nankana Regions from Ghana [121, 162] and Benin [163] whereas full susceptibility to permethrin and deltamethrin was found in Burkina Faso [164] and Tanzania [145]. There is a lack of information on secondary vectors e.g. *An. mouche‐ ti* and *An. nili* which can play important role in malaria transmission in specific settings (e.g. Cameroon, Congo, Côte d'Ivoire). Regarding other vectors species, full susceptibility to pyrethroids has been reported in *An. labranchiae* in Morocco [165] and in *An. pharoensis* in Egypt [166] and Ethiopia [167].

In Africa, the L1014F mutation is widespread (figure 2) and predominant in the molecular S form compared to the M form, except in Benin [168], Guinea Equatorial [8] and Niger [132]. Some authors suggested that the *kdr* alleles may have arisen from at least four independent mutation events in the *An. gambiae* S-form [169]. Regarding the M form, it is not clear whether the *kdr* mutation resulted from an introgression from the S form only [170, 171] and/or from independent mutation events, has recently suggested for Bioko Island [172]. The second mutation, a leucine–serine substitution at the same codon (L1014S), was identified first in a colony of *An. gambiae s.l.* from Kenya [98]. This substitution has been lately reported in Burundi [173], Cameroon [136, 138], Gabon [174], Equatorial Guinea [175], Uganda [176], Republic of Congo [177] and Angola [140], mainly in co-occurrence with the 1014F *kdr* allele. Although some authors have reported that the 1014S allele may confer lower level of pyrethroid resistance than the 1014F allele [178], its spread from eastern to central Africa and more recently to West Africa [124, 179] suggest a survival advantage of mosquitoes sharing this mutation in presence of pyrethroids. So far, the L1014S substitution has always been detected in the S molecular form [180] but recent findings showed the occurrence of the 1014S allele in the M form in Equatorial Guinea [181] and Cameroon [182]. In these two countries, the 1014S allele was present at very low frequencies, alone or associated with the L1014F allele. It is currently impossible to know whether the *kdr* alleles have arisen first in Cameroon or Equatorial Guinea. The higher frequency of the 1014S allele in the S form compared with the M form could either be attributed to an introgression from the S taxon or to a *de novo* mutation. Regarding the sister taxa *An. arabiensis*, both of these mutations were reported in Western [124], Central [183] and Eastern Africa [184]. Interestingly, a new *kdr* mutation (N1575Y) occurring within domains III-IV of voltage gate sodium channel was found in both S and M molecular forms of *An. gam‐ biae* and occurs upon a 1014F haplotypic background only [100]. Additive resistance of *1575Y* was demonstrated for permethrin and DDT in both molecular forms of *An. gambiae*. The prevalence of the 1575Y mutation has increased in West Africa in the last years hence indicating that the 1014F-1575Y haplotype is under strong selection pressure (Djégbé pers. com). It is possible that besides the 1014F/1014S *kdr* mutation, other mutations in the para-type sodium channel gene might be needed for mosquitoes to survive after exposure to a discriminating concentration of an insecticide. Further investigation is needed to better address the distribu‐ tion and the role of the N1575Y mutation in pyrethroid resistance as well as to assess the fitness benefits conferred by this allele on the L1014F mutation in malaria vectors.

Beyond the spread of *kdr* alleles, metabolic-based resistance due to detoxifying enzymes namely oxidase, the GST (epsilon) and CCE families have expanded in African malaria vectors. In *An. gambiae s.l.* metabolic resistance involving increased levels of P450 has been reported at least in Kenya [185], Cameroon [186], Benin [69], Nigeria [69], Ghana [70], Mozambique [147], South Africa [187] and Zimbabwe [120]. Up to now, only genes encoding CYP6P3 and CYP6M2 P450 enzymes have been clearly involved in cellular mechanisms known to metabolize deltamethrin and permethrin [10, 71].These genes were found over-expressed in pyrethroidresistant *An. gambiae* populations from Benin, Nigeria and Ghana [69, 70], mainly in coassociation with the *kdr* L1014F allele. In *An. funestus*, pyrethroid resistance involving increased activity of P450 monooxygenase and/or GST was demonstrated in South Africa [157], Mo‐ zambique [188] and Malawi [161].

To conclude, the immense challenge in Africa will be not to manage and control *kdr*-resistant mosquitoes only but to deal with the development of "multiple resistant" populations that could resist to different class of insecticides used in public health. One other issue is the occurrence and development of carbamate resistance in some countries (eg Benin, Nigeria) where this chemical class is in use for IRS through the PMI programme [47, 48]. The spread of carbamate resistance in malaria vectors in Africa is worrying for insecticide resistance management and alternative insecticides, and innovative strategies are urgently needed to better reduce the vectorial capacity of mosquitoes and hence effectively reduce the burden of malaria in the region. Resistance management strategy for malaria control is discussed in section 8.

#### **6.2. South-East Asia and India**

in other African countries is very limited in partly due to the difficulty to colonize *An. funestus* species in laboratory. Pyrethroid resistance/tolerance was detected in Malawi [160, 161] and suspected in Obusi and Kassena-Nankana Regions from Ghana [121, 162] and Benin [163] whereas full susceptibility to permethrin and deltamethrin was found in Burkina Faso [164] and Tanzania [145]. There is a lack of information on secondary vectors e.g. *An. mouche‐ ti* and *An. nili* which can play important role in malaria transmission in specific settings (e.g. Cameroon, Congo, Côte d'Ivoire). Regarding other vectors species, full susceptibility to pyrethroids has been reported in *An. labranchiae* in Morocco [165] and in *An. pharoensis* in Egypt

In Africa, the L1014F mutation is widespread (figure 2) and predominant in the molecular S form compared to the M form, except in Benin [168], Guinea Equatorial [8] and Niger [132]. Some authors suggested that the *kdr* alleles may have arisen from at least four independent mutation events in the *An. gambiae* S-form [169]. Regarding the M form, it is not clear whether the *kdr* mutation resulted from an introgression from the S form only [170, 171] and/or from independent mutation events, has recently suggested for Bioko Island [172]. The second mutation, a leucine–serine substitution at the same codon (L1014S), was identified first in a colony of *An. gambiae s.l.* from Kenya [98]. This substitution has been lately reported in Burundi [173], Cameroon [136, 138], Gabon [174], Equatorial Guinea [175], Uganda [176], Republic of Congo [177] and Angola [140], mainly in co-occurrence with the 1014F *kdr* allele. Although some authors have reported that the 1014S allele may confer lower level of pyrethroid resistance than the 1014F allele [178], its spread from eastern to central Africa and more recently to West Africa [124, 179] suggest a survival advantage of mosquitoes sharing this mutation in presence of pyrethroids. So far, the L1014S substitution has always been detected in the S molecular form [180] but recent findings showed the occurrence of the 1014S allele in the M form in Equatorial Guinea [181] and Cameroon [182]. In these two countries, the 1014S allele was present at very low frequencies, alone or associated with the L1014F allele. It is currently impossible to know whether the *kdr* alleles have arisen first in Cameroon or Equatorial Guinea. The higher frequency of the 1014S allele in the S form compared with the M form could either be attributed to an introgression from the S taxon or to a *de novo* mutation. Regarding the sister taxa *An. arabiensis*, both of these mutations were reported in Western [124], Central [183] and Eastern Africa [184]. Interestingly, a new *kdr* mutation (N1575Y) occurring within domains III-IV of voltage gate sodium channel was found in both S and M molecular forms of *An. gam‐ biae* and occurs upon a 1014F haplotypic background only [100]. Additive resistance of *1575Y* was demonstrated for permethrin and DDT in both molecular forms of *An. gambiae*. The prevalence of the 1575Y mutation has increased in West Africa in the last years hence indicating that the 1014F-1575Y haplotype is under strong selection pressure (Djégbé pers. com). It is possible that besides the 1014F/1014S *kdr* mutation, other mutations in the para-type sodium channel gene might be needed for mosquitoes to survive after exposure to a discriminating concentration of an insecticide. Further investigation is needed to better address the distribu‐ tion and the role of the N1575Y mutation in pyrethroid resistance as well as to assess the fitness

benefits conferred by this allele on the L1014F mutation in malaria vectors.

Beyond the spread of *kdr* alleles, metabolic-based resistance due to detoxifying enzymes namely oxidase, the GST (epsilon) and CCE families have expanded in African malaria vectors.

[166] and Ethiopia [167].

594 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

The South East Asia Region (SEAR) that account for 13% of the total malaria cases worldwide (2nd position after Africa) [1] is not spare of insecticide resistance in the main malaria vector species.

In the Mekong region, cross-country monitoring of insecticide resistance has been conducted through the MALVECASIA network (http://www.itg.be/malvecasia/) to help MCPs in the choice of insecticide to use at regional level. Large differences in insecticide resistance status were observed among species and countries. *Anopheles dirus s.s*., the main vector in forested malaria foci, was mainly susceptible to permethrin except in central Vietnam where it showed possible resistance to type II pyrethroids [23]. *Anopheles minimus* s.l. populations were found resistant / tolerant in Vietnam and northern Thailand [189] but almost susceptible in Cambodia and Laos. No *kdr* mutation has been observed so far in these species [190] and pyrethroid resistance seems to result from increased detoxification by esterases and/or P450 monooxy‐ genases [191]. Indeed, increased mRNA expression of two P450 genes, *CYP6P7* and *CYP6AA3*, suspected to metabolize some pyrethroids [76] have been reported in a deltameth‐ rin-resistant population of *An. minimus* in Thailand [75, 192].

*Anopheles epiroticus* of the Sundaicus Complex showed to be highly resistant to all pyrethroids in the Mekong Delta [23] but susceptible to DDT, except near Ho Chi Minh City. DDT and pyrethroid-resistant populations of *An. subpictus* were reported in Vietnam and Cambodia. Biochemical assays suggest an esterase-mediated pyrethroid detoxification in both *An. epiroticus* and *An. subpictus* whereas DDT resistance in *An. subpictus* might be conferred to a higher GST activity. In Vietnam and Cambodia, *An. vagus* and *An sinensis* showed various levels of pyrethroid resistance and sequence-analysis of the DIIS6 region of the VGSC revealed the presence of the 1014S *kdr* allele [193]. Pyrethroid resistant populations of *An. sinensis* were also reported in the Republic of Korea (ROK) [194] and in China [195]. In China, cypermethrin resistance in *An. sinensis* was associated with the presence of both 1014F and L1014C substi‐ tutions, whereas only 1014F and 1014S mutations were found in the ROK and Vietnam, respectively. In Indonesia, molecular analysis carried out in field mosquito samples revealed the presence of the 1014F allele in the four main malaria vectors i.e. *An. sundaicus*, *An. aconi‐ tus*, *An. subpictus* and *An. vagus* [196]. At the present time, it is difficult to speculate on the relative contribution of the *kdr* mutations *versus* metabolic detoxification on pyrethroid and DDT resistance in malaria vectors from the SEA region and more work are needed to establish a clear trend.

Insecticide resistance is known to be widespread in other part of Asia such as India. In this country, resistance has a long history (see section 2) and it represents a big challenge for malaria vector control. Among the *Anopheles* species, *An. culicifacies s.l*., the major vector of malaria in most parts of the country, has developed strong resistance to pyrethroids [36], DDT [197, 198], dieldrin/HCH [199], and malathion [198]. The 1014F mutation, which generates the *kdr* phenotype was detected in pyrethroid and DDT resistant *An. culicifacies s.l.* populations sometimes in co-occurrence with the 1014S mutation [197]. Note that a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC was recently identified in Indian *An. culicifacies* and was tightly linked to 1014S substitution [197]. Elevated activities of GST seem to play also an important role in DDT-resistance in this mosquito species [82]. Similarly, strong level of pyrethroid resistance due to the presence of both 1014F and 1014S mutations was found in the urban malaria vector *An. stephensi* particularly in the Rajasthan District [200]. Other vectors that are reported to be resistant to pyrethroid, DDT and/or dieldrin/HCH in India are *An. annularis*, *An. subpictus* and *An. philippinensis* [201]. In contrast, *An. minimus* has still not showed pyrethroid and DDT resistance [202].

The same trend was noted in Sri Lanka where the main malaria vectors species, i.e. *An. culificifacies s.l.* and *An. subpictus* have developed DDT, pyrethroid and malathion resistance in several districts [203, 204]. However, the main mechanisms associated with DDT and malathion resistance in *An. culicifacies s.l.* and *An. subpictus* are primarily metabolic and involve carboxylesterases (malathion) or monooxygenases and GSTs (for DDT) [205, 206]. An altered acetylcholinesterase conferring organophosphate resistance has been suspected in both vector species [205].

In the delta region of Bangladesh, the *An. sundaicus* malaria vector is fully susceptible to DDT but other malaria vectors such as *An. philippinensis*, *An. maculatus s.l.,* and *An. aconitus* have all developed resistance to DDT [207]. *Anopheles aconitus,* additionally, has been reported to be resistant to dieldrin/HCH. Bhutan records *An. maculatus s.l.* as resistant to DDT, but there is no record of its resistance to any other insecticides [207]. Two vectors of malaria in Nepal, *An. maculatus s.l.* and *An. aconitus*, also have developed resistance to DDT whereas only malathion resistance was reported in *An. stephensi* in Pakistan [208]. Finally, in Iran and Turkey, *An. stephensi* and *An. sacharovi* showed resistance to DDT and dieldrin but both species are mostly susceptible to pyrethroids [209-211].

### **6.3. Latin America**

levels of pyrethroid resistance and sequence-analysis of the DIIS6 region of the VGSC revealed the presence of the 1014S *kdr* allele [193]. Pyrethroid resistant populations of *An. sinensis* were also reported in the Republic of Korea (ROK) [194] and in China [195]. In China, cypermethrin resistance in *An. sinensis* was associated with the presence of both 1014F and L1014C substi‐ tutions, whereas only 1014F and 1014S mutations were found in the ROK and Vietnam, respectively. In Indonesia, molecular analysis carried out in field mosquito samples revealed the presence of the 1014F allele in the four main malaria vectors i.e. *An. sundaicus*, *An. aconi‐ tus*, *An. subpictus* and *An. vagus* [196]. At the present time, it is difficult to speculate on the relative contribution of the *kdr* mutations *versus* metabolic detoxification on pyrethroid and DDT resistance in malaria vectors from the SEA region and more work are needed to establish

Insecticide resistance is known to be widespread in other part of Asia such as India. In this country, resistance has a long history (see section 2) and it represents a big challenge for malaria vector control. Among the *Anopheles* species, *An. culicifacies s.l*., the major vector of malaria in most parts of the country, has developed strong resistance to pyrethroids [36], DDT [197, 198], dieldrin/HCH [199], and malathion [198]. The 1014F mutation, which generates the *kdr* phenotype was detected in pyrethroid and DDT resistant *An. culicifacies s.l.* populations sometimes in co-occurrence with the 1014S mutation [197]. Note that a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC was recently identified in Indian *An. culicifacies* and was tightly linked to 1014S substitution [197]. Elevated activities of GST seem to play also an important role in DDT-resistance in this mosquito species [82]. Similarly, strong level of pyrethroid resistance due to the presence of both 1014F and 1014S mutations was found in the urban malaria vector *An. stephensi* particularly in the Rajasthan District [200]. Other vectors that are reported to be resistant to pyrethroid, DDT and/or dieldrin/HCH in India are *An. annularis*, *An. subpictus* and *An. philippinensis* [201]. In contrast, *An. minimus* has still not

The same trend was noted in Sri Lanka where the main malaria vectors species, i.e. *An. culificifacies s.l.* and *An. subpictus* have developed DDT, pyrethroid and malathion resistance in several districts [203, 204]. However, the main mechanisms associated with DDT and malathion resistance in *An. culicifacies s.l.* and *An. subpictus* are primarily metabolic and involve carboxylesterases (malathion) or monooxygenases and GSTs (for DDT) [205, 206]. An altered acetylcholinesterase conferring organophosphate resistance has been suspected in both vector

In the delta region of Bangladesh, the *An. sundaicus* malaria vector is fully susceptible to DDT but other malaria vectors such as *An. philippinensis*, *An. maculatus s.l.,* and *An. aconitus* have all developed resistance to DDT [207]. *Anopheles aconitus,* additionally, has been reported to be resistant to dieldrin/HCH. Bhutan records *An. maculatus s.l.* as resistant to DDT, but there is no record of its resistance to any other insecticides [207]. Two vectors of malaria in Nepal, *An. maculatus s.l.* and *An. aconitus*, also have developed resistance to DDT whereas only malathion resistance was reported in *An. stephensi* in Pakistan [208]. Finally, in Iran and Turkey, *An. stephensi* and *An. sacharovi* showed resistance to DDT and dieldrin but both species are mostly

a clear trend.

species [205].

showed pyrethroid and DDT resistance [202].

596 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

susceptible to pyrethroids [209-211].

The countries of the Amazon Basin (Bolivia, Brazil, Colombia, Ecuador, Guyana, Peru, Surinam and Venezuela) carry the greatest burden of malaria in the Americas. The primary vectors of this disease in the Amazon basin are *An. darlingi* and *An. albimanus*. Surprisingly, much less data on insecticide resistance is available for these two mosquito species comparatively to African and/or Asian malaria vector species [212].

In Colombia, DDT resistance was reported in the late 80's in some populations of *An. dar‐ lingi* in the districts of Quibdó and close to the Atrato River [213, 214]. Successive insecticide susceptibility evaluations revealed resistance to pyrethroids in both *An. darlingi* and *An. albimanus* mainly in the Chocó State [215]. In *An. darlingi*, increased levels of both Multi function Oxidase (MFO) and Non specific Esterase (NSE) were reported in a deltamethrin and DDTresistant population, hence suggesting a possible involvement of these detoxifying enzymes in cross resistance to DDT and deltamethrin [35]. Note that various levels of resistance to organophosphate and pyrethroids were also reported in the secondary malaria vector *An. nuneztovari* [216].

In neighboring countries, DDT, permethrin and deltamethrin resistance was found in labora‐ tory colonized populations of *An. albimanus* from Guatemala, whereas full susceptibility was noted in field populations from El Salvador and Belize [217, 218]. The colonies from Guatemala showed significant increase in the specific activity of esterase and/or oxidase as measured by spectrophotometer suggesting their potential involvement in pyrethroid-resistance [34, 217]. In Peru, monitoring campaigns carried out since 2000 showed that *An. albimanus* was the only Anopheline species to exhibit pyrethroid-resistance [219].

In Mexico, high level of DDT resistance and low levels of resistance to organophosphate, carbamate and pyrethroid insecticides were detected in field populations of *An. albimanus* in Chiapas, prior to a large-scale resistance management project [220]. Biochemical assays revealed that the DDT resistance was caused by elevated levels of GST activity leading to increased rates of metabolism of DDT to DDE [22], whereas carbamate resistance was attrib‐ uted to an altered acetylcholinesterase (AChE). More recent studies conducted in the southern Yucatan Peninsula showed high levels of DDT, deltamethrin and pirimiphos-methyl resistance in the *An. albimanus* populations tested [221]. Biochemical tests revealed elevated levels of GST, P450 and esterases activities that could be involved in DDT and pyrethroid-resistance. As for carbamate, pirimiphos-methyl resistance was strongly correlated with the presence of an insensitive acetylcholinesterase.

To our knowledge, it is the main "published" information available on the distribution, levels and mechanisms of resistance (i.e. accessible through Medline and pub med) in malaria vectors in Latin America. It is then essential to strengthen the capacity of all Latin America countries that suffering from malaria to make insecticide monitoring in routine to obtain much accurate information on the insecticide resistance situation in the malaria vectors. This will provide stake holders with useful information for the implementation of more effective and sustainable malaria control programmes in the region.

## **7. Impact of pyrethroid-resistance on programmatic malaria control**

Few operational reports exist that measure the impact of pyrethroid resistance on epidemio‐ logical outcomes of malaria, owing to body of factors that mislead the attributable component of resistance. Where tentative evidence is provided in most cases, the design of the study has been observational and the effect of confounding factors can never be excluded with confi‐ dence, making difficult the interpretation of data.

Most probably, the only clearest evidence of control failure being directly linked to pyrethroid resistance was reported from the borders of Mozambique and South Africa. In 1996, the malaria control programme in KwaZulu Natal switched from using DDT to deltamethrin for indoor spraying. Within four years, notified malaria cases had increased about four fold, *An. funes‐ tus* had re-appeared and was observable emerging alive from pyrethroid sprayed houses. Bioassays showed that this species was resistant to pyrethroids but susceptible to DDT [39]. A decision was taken to switch back to DDT spraying and, within the two years after this switch was made, *An. funestus* was no longer observed emerging alive from insecticide sprayed houses. The combination of DDT and antimalarial drugs in KwaZulu-Natal has resulted in a 91% decline in the malaria incidence rate [222, 223]. There is no doubt that that the emergence of pyrethroid resistance and the avoidance of its effects by switching to DDT, has been of major operational importance [224].

Additional evidence was brought on the island of Bioko on the West African Coast. A malaria control strategy based on IRS with lambdacyhalothrin was launched by the Bioko Island Malaria Control Project (BIMCP) funded by the Government of Equatorial Guinea and a consortium of private donors led by Marathon Oil Corporation. One round of IRS using the pyrethroid deltamethrin (K-Orthrine WP50, Bayer Crop Sciences, Isando, South Africa) failed to curtail an increase in the population density of *An. gambiae* M form because of evidence in the rise of the knock-down resistance (*kdr*) gene in this species [8]. The programme switched to carbamate insecticide before a substantial decline in the mosquito population, transmission index and malaria prevalence in children was seen. Nevertheless, in an observational study such as this, the possible contribution of other confounding factors to the failure of pyrethroid IRS cannot be overlooked so the direct consequence of the *kdr* frequency is unclear.

Another programmatic study was conducted in the highland provinces of Burundi. Between 2002 and 2005, a well targeted vector control programme (conducted in foot of valleys only) combining IRS with pyrethroids and/or PermaNet 1.0 LLINs was initiated in one of the most affected island provinces, Karuzi [225]. Initially, one round per year of pyrethroid-IRS was carried out in all human dwellings and cattle sheds before the seasonal increase in transmis‐ sion. LLIN distribution preceded the first IRS round in the same year. The S-form of *An. gambiae* was the predominant vector species in Karuzi District and showed resistance to pyrethroids due to the *kdr* mutation. The entomological data showed that the intervention, overall, effectively reduced *Anopheles* density by 82% and malaria transmission was decreased by 90% despite high frequencies of the L1014S allele in the local *An. gambiae* population [173].

In a more recent observational study conducted in Malawi, the impact of pyrethroid resist‐ ance on operational malaria control has been assessed with more controversial evidence of resistance impacting pyrethroid-based vector control [161]. In this trial, pyrethroid-LLINs were distributed to communities in 2007 followed by a pilot campaign of IRS with lambda‐ cyhalothrin supported by the President's Malaria Initiatives between 2008-2010 within dis‐ tricts. A series of sentinel sites were established during these periods to track the effect of the increase in pyrethroid resistance in the local malaria vectors (*An. gambiae* and *An. funes‐ tus*) and assess any impact on malaria transmission and prevalence of infection. Pyrethroid resistance had been selected over the 3 years of the programme in these two major malaria vectors with the resistance in the later vector (i.e; *An. funestus*) being metabolically-mediated and involving the up-regulation of two duplicated P450s. The selection of resistance over 3 years had however not triggered a major increase in parasite prevalence in Malawian chil‐ dren, but it may have reduced the benefit of introducing IRS alone in several districts [161]. The impact of this pyrethroid resistance on the ability of LLIN and IRS to reduce malaria infection in Malawi needs to be further elucidated.

**7. Impact of pyrethroid-resistance on programmatic malaria control**

dence, making difficult the interpretation of data.

598 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

operational importance [224].

Few operational reports exist that measure the impact of pyrethroid resistance on epidemio‐ logical outcomes of malaria, owing to body of factors that mislead the attributable component of resistance. Where tentative evidence is provided in most cases, the design of the study has been observational and the effect of confounding factors can never be excluded with confi‐

Most probably, the only clearest evidence of control failure being directly linked to pyrethroid resistance was reported from the borders of Mozambique and South Africa. In 1996, the malaria control programme in KwaZulu Natal switched from using DDT to deltamethrin for indoor spraying. Within four years, notified malaria cases had increased about four fold, *An. funes‐ tus* had re-appeared and was observable emerging alive from pyrethroid sprayed houses. Bioassays showed that this species was resistant to pyrethroids but susceptible to DDT [39]. A decision was taken to switch back to DDT spraying and, within the two years after this switch was made, *An. funestus* was no longer observed emerging alive from insecticide sprayed houses. The combination of DDT and antimalarial drugs in KwaZulu-Natal has resulted in a 91% decline in the malaria incidence rate [222, 223]. There is no doubt that that the emergence of pyrethroid resistance and the avoidance of its effects by switching to DDT, has been of major

Additional evidence was brought on the island of Bioko on the West African Coast. A malaria control strategy based on IRS with lambdacyhalothrin was launched by the Bioko Island Malaria Control Project (BIMCP) funded by the Government of Equatorial Guinea and a consortium of private donors led by Marathon Oil Corporation. One round of IRS using the pyrethroid deltamethrin (K-Orthrine WP50, Bayer Crop Sciences, Isando, South Africa) failed to curtail an increase in the population density of *An. gambiae* M form because of evidence in the rise of the knock-down resistance (*kdr*) gene in this species [8]. The programme switched to carbamate insecticide before a substantial decline in the mosquito population, transmission index and malaria prevalence in children was seen. Nevertheless, in an observational study such as this, the possible contribution of other confounding factors to the failure of pyrethroid

IRS cannot be overlooked so the direct consequence of the *kdr* frequency is unclear.

Another programmatic study was conducted in the highland provinces of Burundi. Between 2002 and 2005, a well targeted vector control programme (conducted in foot of valleys only) combining IRS with pyrethroids and/or PermaNet 1.0 LLINs was initiated in one of the most affected island provinces, Karuzi [225]. Initially, one round per year of pyrethroid-IRS was carried out in all human dwellings and cattle sheds before the seasonal increase in transmis‐ sion. LLIN distribution preceded the first IRS round in the same year. The S-form of *An. gambiae* was the predominant vector species in Karuzi District and showed resistance to pyrethroids due to the *kdr* mutation. The entomological data showed that the intervention, overall, effectively reduced *Anopheles* density by 82% and malaria transmission was decreased by 90% despite high frequencies of the L1014S allele in the local *An. gambiae* population [173]. In a more recent observational study conducted in Malawi, the impact of pyrethroid resist‐ ance on operational malaria control has been assessed with more controversial evidence of

Similarly, in the Dielmo Village of Senegal, a longitudinal study of inhabitants was carried out between January, 2007, and December, 2010 [226]. In July, 2008, deltamethrin-LLINs were provided to all villagers and asymptomatic carriage of malaria parasites was assessed from cross-sectional surveys. Overall, the incidence density of malaria attacks decreased from 5.45 per 100 person-months before LLINs distribution in 2007 to 0.41 by August 2010, but increased sharply back to 4.57 between September and December, 2010, i.e, in less than 3 years after the distribution of LLINs. Within the same time frame, the malaria vector became gradually resistant to pyrethroids and the prevalence of the 1014F *kdr* resistance allele increased from scratch, i.e. 8% in 2007 to 48% in 2010. Once again, these results should be considered with caution as the study was conducted in an unique village and the conclusions drawn could not be extrapolated or extended to Senegal or other areas of Western Africa. Moreover, the link between the slight rise of pyrethroid resistance and the rebound in malaria cases cannot be established with accuracy and such rebound could be due to other sources of factors totally independent of resistance.

Another recent study reports the presence of pyrethroid-resistance in malaria vectors *versus* the gain in current efforts to control malaria in the Zambia [154]. In line with the Global trend to improve malaria control efforts, a country wide campaign of Olyset Nets and PermaNets (LLIN) distribution was initiated in 1999 and indoor residual spraying with DDT or pyreth‐ roids was reintroduced in 2000 in the country by the NMCP. In 2006, these efforts were strengthened by the PMI. Both major malaria vectors*, An. gambiae* and *An. funestus* were controlled effectively with the ITN and IRS programme in Zambia, maintaining a reduced disease transmission and burden, despite the discovery of DDT and pyrethroid resistance in the country.

There have been extensive randomized controlled trials (RCTs) (phase III) in part of Africa aiming at investigating the efficacy of ITNs for malaria prevention [227], but very few have assessed how pyrethroid resistance might affect the effectiveness of such intervention. RCTs entail a set of communities randomly divided into groups, one that receives the novel form of vector control intervention, and comparison arms that often receive the old form of vector control tools or nothing. The key difficulty is that it is impossible to address the question to whether vector control would produce a smaller reduction in malaria if the vector mosquitoes are resistant than it would have done if they were susceptible, using RCT methods. This is simply because resistance is not an easy factor that can be allocated randomly to some communities and not to others. The distribution of resistance is patchy and its severity seems to differ from one location (village) to another. Moreover there may be more resistance or survival trend of mosquitoes in some villages than others because of variations in the quality of vector control operations, or in mosquito behavior [228, 229]. This is important to mention, because many health scientists regard evidence from randomized-controlled studies as the only reliable basis for decision-making in public health.

The first RCT that investigated the impact of pyrethroid-resistance on LLIN efficacy was conducted in the Korhogo area in the north of Côte d'Ivoire. The trial encompassed multiple villages where the 1014F *kdr* allele frequency was >90% [28] and malaria was endemic. The regular use of conventionally lambdacyhalothrin-treated nets had a significant impact on the entomological inoculation rate (55% reduction) and on malaria incidence in children < 5 (56% reduction of clinical attacks) compared to a control group having no nets [230]. This was the first clear-cut evidence of ITNs continuing to provide effective personal protection against malaria in an area with a very high frequency of *kdr* in the vector population. However, as reported in Ranson et al. [6], the absence of a physical barrier in the control group may have overestimated the impact of pyrethroid treated nets against *kdr* mosquitoes in this study.

More recently, another RCT of LLINs and/or IRS was conducted in 28 villages in southern Benin, from 2007 to 2010 [231]. The objective of the study was to examine whether carbamate-IRS applied every 8 months, as practiced by the PMI programme in Benin provided additional benefit over LLINs (ie Permanet 2.0) in term of malaria prevention and management of pyrethroid resistance in malaria vectors. Results showed that combination of LLINs and IRS did not reduce malaria transmission and morbidity compared to LLIN alone in an area of pyrethroid resistance [124]. Significant increase of 1014F *kdr* frequency was observed in the reference and treated arms only 18 months post intervention hence indicating that LLIN and IRS failed to reduce the spread of the 1014F allele in malaria vectors. The authors suggested that the increase in pyrethroid resistance might have accounted for the reduction of LLIN efficacy at a community level. Clearly, further investigation is needed to assess whether pyrethroid-resistance can reduce efficiency of LLINs and IRS for malaria prevention in Africa.

Given the many obstacles for evaluating the epidemiological impact of resistance, other alternative methods to measure operational impact has been to measure proxy entomological outcomes, such as the relative mortality and feeding success of resistant and susceptible vectors in experimental huts [232, 233]. Although such results can be remarkably clear, and definitively linked to resistance, experimental hut methods have their own limitations owing to the controlled hut structures that differ in many ways to normal houses in rural African context.

An early experimental hut trial of ITNs was conducted in the western African country of Benin. In southern Benin (Ladji), pyrethroid resistance has evolved in the M form of *An. gambiae* mosquitoes that appear to combine the knockdown resistance (*kdr*) gene with oxidase mech‐ anisms [127, 234]. In Ladji, carrier mosquitoes of this resistance were not controlled by pyrethroid treatments in experimental hut trials of ITNs or the leading brands of LLINs, PermaNet 2.0 (Vestergaard Frandsen SA, Aarhus, Denmark) and Olyset (Sumitomo Chemi‐ cals, Osaka, Japan) [235]), compared to Malanville in the north where the vector was largely susceptible to pyrethroids [127]. Further household randomized trial conducted in northern susceptible and southern resistance areas demonstrated that lambdacyalothrin-ITNs (regard‐ less the physical condition) lose their capacity to confer personal protection against pyrethroidresistant A*n. gambiae* [236].

One of the problems associated with many of these studies is that, due to the lack of molecular markers for alternative resistance mechanisms (i.e. metabolic or even cuticular and behavio‐ ral), the frequency of *kdr* alleles is frequently used as a proxy for resistance. It has been recently demonstrated in Southern Benin that *kdr* by itself in *An. gambiae* does not seem to bear more malaria parasites than in a susceptible [237] but this conception can be misleading when metabolic or other resistance mechanisms are predominant or combine with *kdr* to confer resistance. There is an urgent need for properly controlled large-scale trials to assess the impact of pyrethroid resistance on IRS and ITNs in Africa but also in different regions affected by malaria (e.g. Asia and Latin America). Such studies should use both entomological and epidemiological indices and should be conducted in areas where alternative resistance mechanisms are known to be responsible for pyrethroid resistance. Furthermore, these studies must consider the possibility of behavioural resistance as recently suggested in Benin [238] and Tanzania [111] and monitor for changes in key traits such as location of resting and feeding which may impact on the efficacy of current insecticide based interventions.

## **8. Resistance management strategies**

whether vector control would produce a smaller reduction in malaria if the vector mosquitoes are resistant than it would have done if they were susceptible, using RCT methods. This is simply because resistance is not an easy factor that can be allocated randomly to some communities and not to others. The distribution of resistance is patchy and its severity seems to differ from one location (village) to another. Moreover there may be more resistance or survival trend of mosquitoes in some villages than others because of variations in the quality of vector control operations, or in mosquito behavior [228, 229]. This is important to mention, because many health scientists regard evidence from randomized-controlled studies as the

The first RCT that investigated the impact of pyrethroid-resistance on LLIN efficacy was conducted in the Korhogo area in the north of Côte d'Ivoire. The trial encompassed multiple villages where the 1014F *kdr* allele frequency was >90% [28] and malaria was endemic. The regular use of conventionally lambdacyhalothrin-treated nets had a significant impact on the entomological inoculation rate (55% reduction) and on malaria incidence in children < 5 (56% reduction of clinical attacks) compared to a control group having no nets [230]. This was the first clear-cut evidence of ITNs continuing to provide effective personal protection against malaria in an area with a very high frequency of *kdr* in the vector population. However, as reported in Ranson et al. [6], the absence of a physical barrier in the control group may have overestimated the impact of pyrethroid treated nets against *kdr* mosquitoes in this study.

More recently, another RCT of LLINs and/or IRS was conducted in 28 villages in southern Benin, from 2007 to 2010 [231]. The objective of the study was to examine whether carbamate-IRS applied every 8 months, as practiced by the PMI programme in Benin provided additional benefit over LLINs (ie Permanet 2.0) in term of malaria prevention and management of pyrethroid resistance in malaria vectors. Results showed that combination of LLINs and IRS did not reduce malaria transmission and morbidity compared to LLIN alone in an area of pyrethroid resistance [124]. Significant increase of 1014F *kdr* frequency was observed in the reference and treated arms only 18 months post intervention hence indicating that LLIN and IRS failed to reduce the spread of the 1014F allele in malaria vectors. The authors suggested that the increase in pyrethroid resistance might have accounted for the reduction of LLIN efficacy at a community level. Clearly, further investigation is needed to assess whether pyrethroid-resistance can reduce efficiency of LLINs and IRS for malaria prevention in Africa. Given the many obstacles for evaluating the epidemiological impact of resistance, other alternative methods to measure operational impact has been to measure proxy entomological outcomes, such as the relative mortality and feeding success of resistant and susceptible vectors in experimental huts [232, 233]. Although such results can be remarkably clear, and definitively linked to resistance, experimental hut methods have their own limitations owing to the controlled hut structures that differ in many ways to normal houses in rural African context. An early experimental hut trial of ITNs was conducted in the western African country of Benin. In southern Benin (Ladji), pyrethroid resistance has evolved in the M form of *An. gambiae* mosquitoes that appear to combine the knockdown resistance (*kdr*) gene with oxidase mech‐ anisms [127, 234]. In Ladji, carrier mosquitoes of this resistance were not controlled by pyrethroid treatments in experimental hut trials of ITNs or the leading brands of LLINs, PermaNet 2.0 (Vestergaard Frandsen SA, Aarhus, Denmark) and Olyset (Sumitomo Chemi‐

only reliable basis for decision-making in public health.

600 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

As a general statement, the use of insecticides does not create resistance by itself but select small proportion of individuals having a genetic mutation that allow them to resist and survive the effects of the insecticide. If this advantage is maintained by constant use of the same insecticide, the resistant insects will reproduce and the genetic changes that confer resistance will be transferred to offspring so that they become more prevalent within the population (figure 3). This selection process will take longer time to occur if the gene conferring resistance is rare or present at a low prevalence. Resistance should not be confused with "induction" that can occur after sub-lethal (or low dose) exposure to any insecticide and/or xenobiotic and is not passed on to offspring [239].

#### **8.1. Main factors influencing resistance development**

The evolution of insecticide resistance is complex and depends on several genetic, biological and operational factors [240-242]. The biological factors relate by the life cycle of the insect (e.g. rate of reproduction, number of generation/offspring, rate of migration and isolation, etc), while the genetic factors include the intrinsic characteristics of the resistant genes (e.g. mono *versus* polygenic resistance, dominance, fitness cost and gene interaction). Operational factors concern the treatment itself including the method and frequency of application, dosage and residual activity of the insecticides as well as insecticide coverage.

**Figure 3.** Possible scenario for resistance development in a mosquito population (source; [240])

#### *8.1.1. Biological factors*

#### *Rate of reproduction*

Insect species that have a short life cycle and high rates of reproduction are likely to develop resistance more rapidly than species that have a lower rate of reproduction, as any resistance genes can rapidly spread throughout the population. Because mosquitoes can produce high number of offspring (i.e. females can lay several hundred eggs during their reproductive life) they are much likely to develop resistance to insecticides than other species.

#### *Population migration / isolation*

With mosquitoes, the goal is to eliminate all or the majority of the population, however the greater the selection pressure that is put on a population, the faster susceptibility may be lost. Immigration of individuals possessing susceptible genes from untreated areas can beneficially dilute and compete with the resistance genes in the overall population. An early step in a malaria vector control programme should therefore be to estimate the susceptibility status of vector populations (see section 5 for details) and estimate potential immigration of untreated insects. This can be achieved by using genetic markers to estimate the gene flow (migrants) and genetic structure between populations. For example, an isolated area (e.g. island) where the entire area is treated would have a higher risk of developing resistance as few "susceptible" genotypes would join the treated population. The risk of insecticide resistance developing should be considered when planning a resistance management strategy. Awareness of and coordination with neighboring vector control programmes and agricultural activities should be encouraged, so that the regional and potential "side effect" on the target population is considered.

#### *8.1.2. Genetics factor*

#### *Dominance*

Resistance genes can range from dominant through semi-dominant to recessive. If dominant or semi-dominant, only one parent needs to possess the characteristic to be fully or partially expressed in the offspring. If recessive, both parents must possess the trait. Fortunately, most resistance mechanisms (e.g. *kdr*) are controlled by recessive or semi-recessive genes, which slows their spread within the population at early stage of resistance development when most individuals are present at heterozygous state. In contrast, when the resistance is genetically dominant, e.g. the *Ace.1R* gene conferring cross-resistance to carbamates and OPs [243], it can rapidly become established within the population and will be difficult to manage. Fortunately, strong genetic cost is often associated with dominant resistant gene that can compensate the effect of the dominance and slow down the increase of resistance gene frequency in natural populations [244].

#### *Gene interactions*

*8.1.1. Biological factors*

602 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Population migration / isolation*

Insect species that have a short life cycle and high rates of reproduction are likely to develop resistance more rapidly than species that have a lower rate of reproduction, as any resistance genes can rapidly spread throughout the population. Because mosquitoes can produce high number of offspring (i.e. females can lay several hundred eggs during their reproductive life)

With mosquitoes, the goal is to eliminate all or the majority of the population, however the greater the selection pressure that is put on a population, the faster susceptibility may be lost. Immigration of individuals possessing susceptible genes from untreated areas can beneficially dilute and compete with the resistance genes in the overall population. An early step in a malaria vector control programme should therefore be to estimate the susceptibility status of vector populations (see section 5 for details) and estimate potential immigration of untreated insects. This can be achieved by using genetic markers to estimate the gene flow (migrants) and genetic structure between populations. For example, an isolated area (e.g. island) where the entire area is treated would have a higher risk of developing resistance as few "susceptible"

they are much likely to develop resistance to insecticides than other species.

**Figure 3.** Possible scenario for resistance development in a mosquito population (source; [240])

*Rate of reproduction*

Epistasis is the non-additive interaction (synergistic *versus* antagonistic) between different loci which contribute to a phenotype [245]. Epistasis between independent loci conferring insec‐ ticide resistance is important to investigate as this phenomenon can shape the rate at which resistance evolves and can dictate the level of resistance in the field. Epistasis can be measured in laboratory studies on susceptible and resistant colonies, but without these data, it is generally impossible to predict whether or not it will occur when two genes are being evaluated. Studies of the interactions between resistance loci have been most commonly conducted in house flies [246-248]. Generally, a greater than additive interaction was observed between two loci that were both homozygous resistant, whereas additively (i.e. lack of epistasis) occurs between two loci that were both heterozygous. In mosquitoes, Harstone and colleagues [249] showed multiplicative interactions between *kdr* and P450 detoxification in *Culex pipiens quinquefasciatus* whether the resistance alleles were homozygous or heterozyges. For example, resistance ratio 50 (i.e. LC50 resistant strain ⁄ LC50 susceptible strain) to perme‐ thrin in the double homozygote mosquitoes (RR50 of 30,000) was much higher than that expected (RR50 of 1,400) by simple additive effect of the two loci. Overall, interactions between independent resistant genes are complex. It is therefore important to better understand the interactions between resistant loci as well as to address how the fitness costs ⁄ benefits of the mechanisms can manipulate the observed interactions.

#### *Fitness cost*

Populations of insects that have never been exposed to insecticides are usually fully suscepti‐ ble, and resistance genes within those populations are very rare. This usually occurs through a "fitness cost", which means that insects sharing the resistance allele lack some other attribute or "quality" such that it gives an advantage to the susceptible insects in an insecticide free environment [250]. For example, resistant insects may have lower mating success, be more susceptible to natural enemies [251], or more prone to mortality during over-wintering [252]. Increased production of metabolic enzymes generally shows lower associated fitness cost than those associated with alterations in the structural genes most probably because the primary function of the enzyme is not disrupted [253]. There is good laboratory and field evidence to suggest that the deficit of insecticide selection pressure, in most cases, selects for susceptible genotypes. For example, the absence of homozygote's resistant genotypes in *An. gambiae* populations in West Africa is most probably due to the strong genetic cost associated with the carbamate-resistance allele a*ce.1R* (G119S) [254]. In addition, once resistance in the field has been selected it often rapidly reverts once the insecticide treatment regime is changed. A good example of this occurred in *An. arabiensis* in Sudan, where malathion specific insecticide resistance was selected in the early 1980s through antimalarial house spraying. The develop‐ ment of resistance prompted a switch of insecticide treatment to fenitrothion and the malathion resistance rapidly reverted in the following years. However, reversion rates are variable and may be very slow, particularly when an insecticide has been used for many years. For example, DDT was used extensively for malaria control over a 20 year period up to the 1960s in Sri Lanka to control *An. culicifacies s.l.* and *An. subpictus*. DDT was replaced by malathion in Sri Lanka in the early 1970s when a total and effective ban on DDT use was implemented. Subsequent regular monitoring has shown that DDT resistance has reverted very slowly towards suscept‐ ibility; around 80% of the adult mosquito population was resistant in the 1970s compared to about 50% in the 1990s. The same is true with the R*dl* gene that was maintained in field mosquito populations despite the abandon of cyclodiene for mosquito control for more than 30 years [33]. Rate of reversion is an important parameter to consider before implementing any resistance management strategy in the field.

#### *8.1.3. Operational factors*

In practice, only operational factors such as the insecticide(s) used, the area of coverage (for example for IRS or LLIN), and the timing, rate, and method of application can be manipulated directly to reduce the selection pressure for resistance. Operational factors influence selection by determining the overall fraction of a population exposed (larvae/adults) to a selecting agent and the degree of contact and pick-up of toxicant by exposed pests at what has been termed the "interface between insects and insecticides" [242]. At both stages, operational and intrinsic factors interact in complex ways to establish the net effect of a control treatment on both genetic composition and total population size. Management of resistance therefore entails resolving these interactions to anticipate with some confidence both the suppressive and selective effects of potential control strategies.

#### *Frequency of application, dosage and persistence of effect*

How often an insecticide is used is one of the most important factors that influence resistance development [240]. With each use, an advantage is given to the resistant insects within a population. The rate of increase of resistance on any population will generally be faster in the presence of a lower fitness cost and high reproductive and short life cycles producing several generations per season. The length of time that an insecticide remains effective, also called its persistence, is dependent upon the physical chemistry of the insecticide, the type of formula‐ tion, the application rate and the substrate. Products which provide a persistent effect provide continual selection pressure in a similar manner to multiple treatments. For example, a space spray will persist for a very short time and will select only against a single generation of mosquitoes. In contrast, a residual wall application (IRS) or Insecticide Treated nets treatment (especially Long Lasting Nets) will persist for months or years providing a selection pressure against many generations of the same insect. For example, repeated application of DDT for indoor residual spraying has contributed to increase the number of DDT-resistant malaria vector species in various geographical settings [255]. Several studies showed however that the use of insecticides in agriculture play a key role in the selection of resistance in mosquitoes [256, 257]. Indeed, most insecticides used in agriculture are of the same chemical classes and have the same targets and modes of action as those used in public health programme. In practice, VC programmes cannot influence the choice of the pesticide used for crop protection and the only thing that can be done is to appropriately select the most judicious insecticide for mosquito control. However, there is more published evidence that public health insecticides can contribute to select for pyrethroid resistance alleles (see section 7 for details). It is obvious that we can expect enhanced selection pressure on resistance genes through the scaling up of LLIN and/or IRS for malaria elimination.

#### *Choice of the insecticide*

a "fitness cost", which means that insects sharing the resistance allele lack some other attribute or "quality" such that it gives an advantage to the susceptible insects in an insecticide free environment [250]. For example, resistant insects may have lower mating success, be more susceptible to natural enemies [251], or more prone to mortality during over-wintering [252]. Increased production of metabolic enzymes generally shows lower associated fitness cost than those associated with alterations in the structural genes most probably because the primary function of the enzyme is not disrupted [253]. There is good laboratory and field evidence to suggest that the deficit of insecticide selection pressure, in most cases, selects for susceptible genotypes. For example, the absence of homozygote's resistant genotypes in *An. gambiae* populations in West Africa is most probably due to the strong genetic cost associated with the

been selected it often rapidly reverts once the insecticide treatment regime is changed. A good example of this occurred in *An. arabiensis* in Sudan, where malathion specific insecticide resistance was selected in the early 1980s through antimalarial house spraying. The develop‐ ment of resistance prompted a switch of insecticide treatment to fenitrothion and the malathion resistance rapidly reverted in the following years. However, reversion rates are variable and may be very slow, particularly when an insecticide has been used for many years. For example, DDT was used extensively for malaria control over a 20 year period up to the 1960s in Sri Lanka to control *An. culicifacies s.l.* and *An. subpictus*. DDT was replaced by malathion in Sri Lanka in the early 1970s when a total and effective ban on DDT use was implemented. Subsequent regular monitoring has shown that DDT resistance has reverted very slowly towards suscept‐ ibility; around 80% of the adult mosquito population was resistant in the 1970s compared to about 50% in the 1990s. The same is true with the R*dl* gene that was maintained in field mosquito populations despite the abandon of cyclodiene for mosquito control for more than 30 years [33]. Rate of reversion is an important parameter to consider before implementing

In practice, only operational factors such as the insecticide(s) used, the area of coverage (for example for IRS or LLIN), and the timing, rate, and method of application can be manipulated directly to reduce the selection pressure for resistance. Operational factors influence selection by determining the overall fraction of a population exposed (larvae/adults) to a selecting agent and the degree of contact and pick-up of toxicant by exposed pests at what has been termed the "interface between insects and insecticides" [242]. At both stages, operational and intrinsic factors interact in complex ways to establish the net effect of a control treatment on both genetic composition and total population size. Management of resistance therefore entails resolving these interactions to anticipate with some confidence both the suppressive and selective effects

How often an insecticide is used is one of the most important factors that influence resistance development [240]. With each use, an advantage is given to the resistant insects within a population. The rate of increase of resistance on any population will generally be faster in the

(G119S) [254]. In addition, once resistance in the field has

carbamate-resistance allele a*ce.1R*

604 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

any resistance management strategy in the field.

*Frequency of application, dosage and persistence of effect*

*8.1.3. Operational factors*

of potential control strategies.

The speed at which an insecticide effectively kills an insect can also influence the evolution of resistance. All current insecticides approved for ITNs or IRS kill extremely rapidly after contact. While fast-acting conventional insecticides can produce even more effective initial control, they impose enormous selection for resistance by killing young female adults. The consequence is that spectacular initial mosquito control can last as little as a few years, thus providing very poor medium- to long-term disease control [258]. Some authors recently suggest that Late Acting insecticides (e.g. entomofungus) may be a more tactical strategy to manage resistance if female mosquitoes are killed after 2 or more gonotrophic cycles [259]. Indeed, the less the insecticide impact on mosquito fitness, the less the strength of selection, especially if the resistance allele is associated with a strong genetic cost. In theory, it would be possible to create an insecticide that would provide effective malaria control yet never be undermined by the evolution of resistant mosquitoes. However, further studies are required as "proof of principle" i.e. to demonstrate that this strategy can be effective for vector man‐ agement and malaria prevention in a real setting.

#### **8.2. Resistance management — Strategies and tactics**

Historically, the practice of using an insecticide until resistance becomes a limiting factor has rapidly eroded the number of suitable/available insecticides for vector control. Rotations, mosaics, and mixtures have all been proposed as resistance management tools [260, 261] but there are very few "success stories" in public health. Numerous mathematical models have been produced to estimate how these tools could be optimally used [262-264] but these models have rarely been tested under field conditions due to the practical difficulties in estimating changes in resistance gene frequencies (especially for metabolic resistance) in large samples of insects [220]. With the advent of different molecular techniques for resistance-gene frequency estimation, field trials of resistance management strategies have now become more feasible.

#### *8.2.1. Approaches to resistance management*

Ideally insecticide resistance management should be undertaken using insecticide based approaches in conjunction with other non-insecticidal vector control methods, i.e. as part of Integrated Vector Management (IVM4 ) [265]. The insecticides used have to be safe to humans and comply with WHO specifications. In practice, most of IVM programmes work well in experimental trials but become challenging when programmes are scaling up into long-term (operational) control. Operationally, the simplest form of resistance management is likely to be "insecticide" based, and this could take several forms.

#### *Rotations*

Rotational strategies are based on the rotation over time of two or preferably more insecticide classes with different modes of action. This approach assumes that if resistance to each insecticide is rare, then multiple resistances will be extremely rare [266]. Rotation allows any resistance developed to the first insecticide to decline over time when the second insecticide class is introduced. As for other strategy, rotations are particularly effective if the resistance gene has an associated fitness cost. The timeframe for rotation needs to be sufficiently short to prevent significant levels of resistance to develop to any one rotation partner. Rotations have been successful in many applications in agriculture and are considered to be effective in slowing the evolution of resistance (see [240] for details*)*. The most striking example of "success story" using this strategy was within the framework of the Onchocerciasis Control Programme (OCP) carried in West Africa 40 years ago. Indeed, weekly application of unrelated larvicides in rivers was successful to kill the larvae of the blackfly vector and mitigate the spread of temephos resistance over the 17 years of its implementation [267]. However, the rotation was introduced at early stage of the OCP, as soon as the operators faced temephos resistance problems in pilot localities. As for all IRM strategies, the status of resistance of the insecticide used in the rotation must be known when implementing rotations and the chemicals used should not present any (known) cross-resistance. For LLIN, it is difficult to implement this method knowing that only pyrethroids are recommended so far by WHO for the impregnation [268]. For IRS, the pragmatic approach would be to rotate insecticides annually. Indeed, changing insecticides more than once a year (which could be the case in areas where two spray rounds are conducted each year) is not recommended, because of procurement and other financial and logistical challenges (see [4]). Despite higher cost of implementing rotation than single spray (as available alternatives to pyrethroids —the carbamates organophosphates, insect growth regulators, pyroles —are currently more expensive), this is probably the price to pay to preserve the arsenal of cost-effective insecticides for malaria vector control.

<sup>4</sup>*Integrated Vector Management* can be defined as "a rational decision making process for the optimal use of resources for vector control". IRM is therefore an integral part of IVM, as only through the active management of insecticide resistance can the available resources be optimally and sustainably used.

#### *Mosaics*

have rarely been tested under field conditions due to the practical difficulties in estimating changes in resistance gene frequencies (especially for metabolic resistance) in large samples of insects [220]. With the advent of different molecular techniques for resistance-gene frequency estimation, field trials of resistance management strategies have now become more feasible.

Ideally insecticide resistance management should be undertaken using insecticide based approaches in conjunction with other non-insecticidal vector control methods, i.e. as part of

and comply with WHO specifications. In practice, most of IVM programmes work well in experimental trials but become challenging when programmes are scaling up into long-term (operational) control. Operationally, the simplest form of resistance management is likely to

Rotational strategies are based on the rotation over time of two or preferably more insecticide classes with different modes of action. This approach assumes that if resistance to each insecticide is rare, then multiple resistances will be extremely rare [266]. Rotation allows any resistance developed to the first insecticide to decline over time when the second insecticide class is introduced. As for other strategy, rotations are particularly effective if the resistance gene has an associated fitness cost. The timeframe for rotation needs to be sufficiently short to prevent significant levels of resistance to develop to any one rotation partner. Rotations have been successful in many applications in agriculture and are considered to be effective in slowing the evolution of resistance (see [240] for details*)*. The most striking example of "success story" using this strategy was within the framework of the Onchocerciasis Control Programme (OCP) carried in West Africa 40 years ago. Indeed, weekly application of unrelated larvicides in rivers was successful to kill the larvae of the blackfly vector and mitigate the spread of temephos resistance over the 17 years of its implementation [267]. However, the rotation was introduced at early stage of the OCP, as soon as the operators faced temephos resistance problems in pilot localities. As for all IRM strategies, the status of resistance of the insecticide used in the rotation must be known when implementing rotations and the chemicals used should not present any (known) cross-resistance. For LLIN, it is difficult to implement this method knowing that only pyrethroids are recommended so far by WHO for the impregnation [268]. For IRS, the pragmatic approach would be to rotate insecticides annually. Indeed, changing insecticides more than once a year (which could be the case in areas where two spray rounds are conducted each year) is not recommended, because of procurement and other financial and logistical challenges (see [4]). Despite higher cost of implementing rotation than single spray (as available alternatives to pyrethroids —the carbamates organophosphates, insect growth regulators, pyroles —are currently more expensive), this is probably the price

to pay to preserve the arsenal of cost-effective insecticides for malaria vector control.

can the available resources be optimally and sustainably used.

4*Integrated Vector Management* can be defined as "a rational decision making process for the optimal use of resources for vector control". IRM is therefore an integral part of IVM, as only through the active management of insecticide resistance

) [265]. The insecticides used have to be safe to humans

*8.2.1. Approaches to resistance management*

606 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Integrated Vector Management (IVM4

*Rotations*

be "insecticide" based, and this could take several forms.

Spatially separated applications of different compounds against the same insect constitute a "mosaic" approach to resistance management [240]. Fine scale mosaics can be achieved in malaria vector control programmes, for example, by using two insecticides in different dwellings within the same village. The aim of this strategy is to preserve susceptibility by spatial restriction of insecticides [4]. If such a fine scale mosaic is to be used, careful records of which insecticide was used in each house are essential. Larger scale mosaics have been shown to be effective for the management of pyrethroid resistance in *An. albimanus* in Mexico [22]. Indeed, pyrethroid resistance rose more rapidly in the areas under pyrethroid treatment alone than in the mosaic areas using OP, Pyrethroid and carbamate [240]. Whilst there are some practical difficulties implementing a mosaic in a vector control programme (eg spray with different insecticides, dosages, apparatus, etc), it may offer the advantages of a mixture strategy with lower insecticide inputs and hence cost. The scale at which a mosaic needs to be applied has not been clearly established. In South Africa for example, different insecticides have been used in different types of houses within the same community and this is considered by some to be a mosaic-like strategy [240]. Similarly, mosquito bed nets from panels treated with different insecticides achieve a similar mosaic effect to treating houses with different com‐ pounds but on a much finer scale. Industry has recently developed mosaic LLINs containing a pyrethroid insecticide and a synergist (Piperonyl butoxide or PBO an oxidase inhibitor) on the roof to increase efficacy against pyrethroid-resistant malaria vectors. Small scale field trial [235, 269] and mathematical exercises [270] suggested that mosaic LLIN may provide better insecticidal effect against resistant mosquitoes and enhanced community-level protection against malaria compared to "classical" LLIN in area of pyrethroid resistance. Clearly further operational research is required to establish the applicability and effectiveness of mosaics approaches for malaria control.

#### *Mixtures*

A mixture is defined by the simultaneous use of two or more insecticides of unrelated mode of action. If two insecticides A and B, with independent resistance mechanisms, are applied together in a mixture, and if resistance to A and resistance to B are both rare, then we expect doubly resistant insects to be extremely rare, and almost all insects resistant to A will be killed by B, and vice versa [266]. This system of "redundant killing" means that resistance to the two insecticides will evolve much more slowly than if either had been used on its own [271]. This approach may be not successful if resistance to one of the components used is already present at a detectable level and/or if linkage disequilibrium is present in the targeted population [4]. Unlike rotations,the effectiveness of mixtures is not directly related to the degree offitness cost. Rather the mixture aims to overpower resistance instead of preserving susceptibility. Howev‐ er, for mixtures to work well in practice both insecticides need to be used at their full applica‐ tion rate in order that the efficacy and persistence of the two insecticides would be broadly similar (same decay rate). Further, theoretical models suggest that mixtures might delay resistance longer than rotations or broad mosaics [271, 272]. However, mixtures of products were rarely adopted in malaria vector control programmes on grounds of cost, logistics, and safety issue and because of the limited number ofrecommended compounds available for both

IRS and LLIN. It is not yet clear however how much the addition of a second active ingredi‐ ent will add to the total cost of manufacturing since the cost of additional insecticide can greatly vary according to the strategy, ie. cost for LLINs would be much lower than that for IRS. For LLIN, previous laboratory and field trials showed interesting prospects for reducing mosqui‐ tosurvival andbitingrateswiththeuseofinsecticidemixtures appliedonmosquitonets against *kdr*-resistant *An. gambiae* in Africa [273, 274]. Other chemicals, such as insect growth regula‐ tors (IGR), represent also promising alternative to be included in mixture formulations as they may impact on mosquito longevity, fertility and fecundity [275, 276]. With the development of next-generation of LLIN, combined use of non-pyrethroids and pyrethroids on bed nets is technically achievable and has the potential to provide better control of malaria and prevent further development of pyrethroid-resistance in malaria vectors. Risk assessment and accepta‐ bility of such new tools should be however carefully investigated before any trial being implemented at operational level.

#### *Combinations*

In this context, combinations expose the vector population to two vector control tools, such that a mosquito that survives contact with one (e.g. LLIN) is exposed to the other one (e.g. IRS), or vice versa. In practice, exposure to two insecticides is not guaranteed but there is some evidence to indicate that this is likely [277]. The effectiveness of combinations in IRM does not depend on the ability to reduce the level of resistance, but on the ability to kill the vector despite the existence of resistance, through the use of another insecticide or intervention, which compensates for resistance [231]. As for other strategy, the combination should not contain insecticides with same mode of action (e.g. avoid pyrethroids for both IRS and LLINs), as this would increase selection pressure rather than reducing it. As combinations require doubling of interventions, cost would be significantly higher than rotations and mosaics. This might nevertheless be warranted in some circumstances, for example where malaria transmission is very high and/or where targeted IRS can help overcome identified resistance to pyrethroids in areas with high LLIN coverage. In practice, combinations would be more easily implement‐ ed in countries having sufficient human and financial resources allocated to public health programmes. So far, a small number of observational studies [278-280] and mathematical modeling exercises [263, 264] suggest that VC combination has an added benefit for reduction of the risk of infection because the people not protected by one of the interventions are protected by the other. A recent cluster randomized controlled trial carried out in Benin showed however that neither clinical malaria in children younger than 6 years nor transmis‐ sion intensity differ between LLIN and carbamate-IRS or Carbamate Treated Plastic Sheeting and the reference group (LLIN alone) and the insecticide combinations did not slow down the evolution of the *kdr* allele in *An gambiae s.s.* compared with LLIN [231]. It was concluded from this study that IRS should be timely implemented (i.e. using appropriate insecticide, dosage and time interval) to ensure optimum efficacy of the IRS intervention over LLIN. Clearly, costeffectiveness of combined vector control interventions need to be carefully considered to ensure that increased efforts and cost dedicated to combinations effectively contribute to better control and management of pyrethroid-resistant malaria vectors.

## **9. Conclusions**

IRS and LLIN. It is not yet clear however how much the addition of a second active ingredi‐ ent will add to the total cost of manufacturing since the cost of additional insecticide can greatly vary according to the strategy, ie. cost for LLINs would be much lower than that for IRS. For LLIN, previous laboratory and field trials showed interesting prospects for reducing mosqui‐ tosurvival andbitingrateswiththeuseofinsecticidemixtures appliedonmosquitonets against *kdr*-resistant *An. gambiae* in Africa [273, 274]. Other chemicals, such as insect growth regula‐ tors (IGR), represent also promising alternative to be included in mixture formulations as they may impact on mosquito longevity, fertility and fecundity [275, 276]. With the development of next-generation of LLIN, combined use of non-pyrethroids and pyrethroids on bed nets is technically achievable and has the potential to provide better control of malaria and prevent further development of pyrethroid-resistance in malaria vectors. Risk assessment and accepta‐ bility of such new tools should be however carefully investigated before any trial being

In this context, combinations expose the vector population to two vector control tools, such that a mosquito that survives contact with one (e.g. LLIN) is exposed to the other one (e.g. IRS), or vice versa. In practice, exposure to two insecticides is not guaranteed but there is some evidence to indicate that this is likely [277]. The effectiveness of combinations in IRM does not depend on the ability to reduce the level of resistance, but on the ability to kill the vector despite the existence of resistance, through the use of another insecticide or intervention, which compensates for resistance [231]. As for other strategy, the combination should not contain insecticides with same mode of action (e.g. avoid pyrethroids for both IRS and LLINs), as this would increase selection pressure rather than reducing it. As combinations require doubling of interventions, cost would be significantly higher than rotations and mosaics. This might nevertheless be warranted in some circumstances, for example where malaria transmission is very high and/or where targeted IRS can help overcome identified resistance to pyrethroids in areas with high LLIN coverage. In practice, combinations would be more easily implement‐ ed in countries having sufficient human and financial resources allocated to public health programmes. So far, a small number of observational studies [278-280] and mathematical modeling exercises [263, 264] suggest that VC combination has an added benefit for reduction of the risk of infection because the people not protected by one of the interventions are protected by the other. A recent cluster randomized controlled trial carried out in Benin showed however that neither clinical malaria in children younger than 6 years nor transmis‐ sion intensity differ between LLIN and carbamate-IRS or Carbamate Treated Plastic Sheeting and the reference group (LLIN alone) and the insecticide combinations did not slow down the evolution of the *kdr* allele in *An gambiae s.s.* compared with LLIN [231]. It was concluded from this study that IRS should be timely implemented (i.e. using appropriate insecticide, dosage and time interval) to ensure optimum efficacy of the IRS intervention over LLIN. Clearly, costeffectiveness of combined vector control interventions need to be carefully considered to ensure that increased efforts and cost dedicated to combinations effectively contribute to better

control and management of pyrethroid-resistant malaria vectors.

implemented at operational level.

608 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Combinations*

Insecticide resistance develops in an insect population when individuals carrying genes that allow them to survive exposure to the insecticide pass these genes on. Thus, any activities that control the individuals with the resistance trait will delay the spread of the resistance genes in the population. IRM should then be seen in the context of IVM and should therefore also include activities such as habitat management, community education, and/or larval source management (e.g. biological control). In order to successfully develop and implement any resistance management strategies based on rotations, mosaics, mixtures or combinations, knowledge of the mode of action, chemical properties, and residual life of the available insecticide products is essential. Although insecticides with novel modes of action have recently been introduced in public health (neonicotinoids, pyroles, oxadiazin, etc) few of them appear to have the optimum biological and/or physical properties required for residual wall spray and/or mosquito net. Unfortunately, the exorbitant costs associated with developing and registering new insecticides (see [281] for details) mean that products appear in the more profitable agricultural markets before consideration is given to their public health potential. We have then no other option than to make an appropriate and judicious use of the current insecticides if we want to avoid any disillusion with pyrethroids as we faced before with DDT or dieldrin. The philosopher George Santayana said *"those who cannot remember the past are condemned to repeat it."* Hope it's not too late for malaria vector control.

## **Author details**

Vincent Corbel1,2\* and Raphael N'Guessan3,4

\*Address all correspondence to: vincent.corbel@ird.fr

1 Institut de Recherche pour le Développement, Maladies Infectieuses et Vecteurs, Ecologie, Génétique, Evolution et Contrôle (IRD 224-CNRS 5290 UM1-UM2), Benin

2 Department of Entomology, Kasetsart University, Bangkok, Thailand

3 London School of Hygiene & Tropical Medicine, London, UK

4 Centre de Recherches Entomologiques de Cotonou, Cotonou, Benin

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## **Perspectives on Barriers to Control of** *Anopheles* **Mosquitoes and Malaria**

Donald R. Roberts, Richard Tren and Kimberly Hess

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55466

## **1. Introduction**

Though mankind has struggled against malaria for countless generations, it remains a major global health problem. The malaria parasite and the *Anopheles* mosquito have evolved and developed with mankind since earliest recorded history, but there is nothing inevitable about the disease. Although thousands of children die from malaria every year, the disease is preventable and entirely curable, and the history of malaria control in the 20th century demonstrates that with the right tools and funding, malaria can be controlled, or even eradicated. The key, of course, is the cost-effective use of the right tools.

## **2. Statement of the problem**

This chapter will examine arguably the most important tool for malaria control – public health insecticides (PHIs). Insecticide opponents often mischaracterize the public health use of insecticides, to include how they are used and consequences of their use in public health programs. Common inferences are that public health use of insecticides results in broad-scale environmental contamination and harm to wildlife. It is important for the reader to understand that there are internationally accepted guidelines for public health use of insecticides and that public health use is very different from how insecticides are used for agriculture. Optimum public health use of PHIs is to spray small quantities on inside walls of houses. In the case of DDT, it is approved only for use in public health programs. Applying it to inside walls leverages DDT's powerful repellent actions, giving continual protection from malaria-infected mosquitoes, for months on end, to those living inside the sprayed house. It should be obvious that a small amount of an insecticide on house walls is a far cry from spraying insecticides on

© 2013 Roberts et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Roberts et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

vast acreages of cropland, as one might envisage for insecticides used in agriculture. Thus we emphasize that the subject of this chapter is public health use of insecticides, with no conno‐ tations whatsoever for the use of insecticides in agriculture.

We will summarize, with specific examples, the way that modern PHIs, and DDT in particular, have saved millions of lives since the 1940s. Despite this remarkable achievement, popular campaigns by activists, some scientists and even United Nations (UN) agencies, have stigma‐ tized and often demonized PHIs. Instead of regarding insecticides in the same light as medicines and diagnostics, essential elements of a malaria control program, insecticide opponents have mounted vocal campaigns to halt their use. Frequently these campaigns avoid or ignore the scientific process and rely on the flimsiest of evidence to make great claims about human health or ecological effects of PHIs. We will characterize examples of studies and claims against PHIs used by the activist communities and we will describe the major failings of each as they relate to the use of PHIs.

The claims by those who oppose PHIs, as we will explain and demonstrate with specific examples, do not comply with even the most basic epidemiologic criteria to prove a cause and effect relationship – yet those claims drive public opinion and policy. We will also document how UN bureaucrats have made outrageous claims that malaria can be controlled without PHIs. At the same time, the UN has set grand goals of achieving near-zero deaths from malaria by 2015. There is a valid debate to be had about whether or not this goal can be met, or even properly defined and measured; however, what is clear, is that progress against malaria cannot be achieved and sustained without access to PHIs. For access to be secured, the malaria community, including program managers, researchers, advocates and others, must defend PHIs rigorously and emphatically. The overarching goal of this chapter is to help with that defense. Without it, the lives of men, women and children living at risk of malaria will be greatly imperiled. However, for proper defense of PHIs, there must be a clear understanding about how insecticide opponents have succeeded in past anti-insecticide campaigns, and that influential groups and UN organizations actively oppose the use of PHIs. As anti-insecticide campaigners employ distinct strategies and tactics, it is important to know what they are and how they are used.

## **3. Malaria control today versus the early years of PHI use**

Today there is great enthusiasm and substantial funding to advance global efforts to control and, in some regions, eradicate malaria. Indeed, and as suggested by recent outcomes of control programs, we are beginning to see promising results [1,2]. The necessary change for refocusing efforts to control malaria started in 1998, when, faced with mounting evidence that the global burden of malaria was increasing, and had been for some time, the World Health Organization (WHO) formed a new malaria control partnership, Roll Back Malaria (RBM). The RBM Partnership is made up of WHO and several UN agencies, such as UNICEF and UNDP, and development agencies, such as the World Bank and the US Agency for International Devel‐ opment (USAID), along with the private sector and NGOs. RBM's stated goal in 1998 was to halve the burden of malaria by 2010 [3].

RBM began with limited funding and an apparent disdain for scientific evidence. The early efforts were disappointing. Far from achieving any reduction in malaria cases, by 2004 there was evidence that malaria cases were in fact increasing. RBM was described in a stinging editorial in the *British Medical Journal* as a 'failing public health campaign [4].' One of the main reasons for this was the Partnership's dogged support for the use of insecticide treated bednets (ITNs) over other vector control interventions, e.g., indoor residual spraying (IRS) with insecticides such as DDT. The limited and controlled spraying of insecticides inside houses has long been known to rapidly reduce malaria cases and deaths, yet in the early years of the RBM Partnership was roundly ignored. In addition RBM's Partners failed to support any change in treatment policy away from failing drug therapies to the new artemisinin-based combination therapies (ACTs).

vast acreages of cropland, as one might envisage for insecticides used in agriculture. Thus we emphasize that the subject of this chapter is public health use of insecticides, with no conno‐

We will summarize, with specific examples, the way that modern PHIs, and DDT in particular, have saved millions of lives since the 1940s. Despite this remarkable achievement, popular campaigns by activists, some scientists and even United Nations (UN) agencies, have stigma‐ tized and often demonized PHIs. Instead of regarding insecticides in the same light as medicines and diagnostics, essential elements of a malaria control program, insecticide opponents have mounted vocal campaigns to halt their use. Frequently these campaigns avoid or ignore the scientific process and rely on the flimsiest of evidence to make great claims about human health or ecological effects of PHIs. We will characterize examples of studies and claims against PHIs used by the activist communities and we will describe the major failings of each

The claims by those who oppose PHIs, as we will explain and demonstrate with specific examples, do not comply with even the most basic epidemiologic criteria to prove a cause and effect relationship – yet those claims drive public opinion and policy. We will also document how UN bureaucrats have made outrageous claims that malaria can be controlled without PHIs. At the same time, the UN has set grand goals of achieving near-zero deaths from malaria by 2015. There is a valid debate to be had about whether or not this goal can be met, or even properly defined and measured; however, what is clear, is that progress against malaria cannot be achieved and sustained without access to PHIs. For access to be secured, the malaria community, including program managers, researchers, advocates and others, must defend PHIs rigorously and emphatically. The overarching goal of this chapter is to help with that defense. Without it, the lives of men, women and children living at risk of malaria will be greatly imperiled. However, for proper defense of PHIs, there must be a clear understanding about how insecticide opponents have succeeded in past anti-insecticide campaigns, and that influential groups and UN organizations actively oppose the use of PHIs. As anti-insecticide campaigners employ distinct strategies and tactics, it is important to know what they are and

**3. Malaria control today versus the early years of PHI use**

Today there is great enthusiasm and substantial funding to advance global efforts to control and, in some regions, eradicate malaria. Indeed, and as suggested by recent outcomes of control programs, we are beginning to see promising results [1,2]. The necessary change for refocusing efforts to control malaria started in 1998, when, faced with mounting evidence that the global burden of malaria was increasing, and had been for some time, the World Health Organization (WHO) formed a new malaria control partnership, Roll Back Malaria (RBM). The RBM Partnership is made up of WHO and several UN agencies, such as UNICEF and UNDP, and development agencies, such as the World Bank and the US Agency for International Devel‐ opment (USAID), along with the private sector and NGOs. RBM's stated goal in 1998 was to

tations whatsoever for the use of insecticides in agriculture.

636 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

as they relate to the use of PHIs.

how they are used.

halve the burden of malaria by 2010 [3].

It was not until 2006 that progress against malaria finally started to be made. To its credit RBM acknowledged some of the problems it faced and set about restructuring and reforming. Much of impetus for these reforms came from a newly appointed head of the WHO's Global Malaria Program, Dr. Arata Kochi. Dr. Kochi had little history in malaria control and perhaps because of this had no need to defend any misguided previous policy decisions. One of Kochi's first acts was to re-issue WHO's treatment guidelines, recommending ACTs.

Shortly thereafter Kochi re-addressed WHO's policy on both DDT and IRS, and in a public and, for WHO, aggressive gesture issued a statement strongly endorsing the use of DDT. At the same time the US global malaria control program run by USAID underwent a major reform, creating the President's Malaria Initiative (PMI). A distinguishing feature of the PMI, which sets it apart from other major bi-lateral donor funded malaria control programs, is its support for IRS and its willingness to pay for use of DDT [5].

Together these reforms marked a change in global malaria control and as a result, malaria cases began to decline. As described below, malaria funding increased by more than 20 fold in a decade and malaria deaths, according to WHO modeling data, have fallen.

Malaria funding for the PMI and the Global Fund to Fight AIDS, TB and Malaria (Global Fund) through 2011 is estimated at \$1,858,370,500 for the PMI [6], and \$6,156,000,000 in malaria grants through 2011 for the Global Fund (based on \$22.8b value of grant portfolio as of December 31, 2011, of which 27% is for malaria) [7].

International funding for malaria control has gone from less than \$100 million in 2000 to \$2 billion in 2011 [8]. Likewise, the estimated changes in global malaria burden since 2000 are compliant with improved funding of control efforts after 2005. For example, estimated numbers of malaria cases and malaria deaths in 2000 were 223 million and 755,000 respectively. In 2005 the values were 237 million cases and 801,000 deaths, whereas in 2011, the values were 216 million cases and 655,000 deaths [8].

Clearly progress is being made in the renewed focus on malaria. The positive changes with regard to funding IRS and DDT's place in malaria control are obviously welcomed. However these advances can be reversed at any time and as we explain in this chapter, the forces opposing the careful and effective use of PHIs are well-funded, organized, and aggressive. The malaria control community should remember, and learn from history, that we have been at this stage before. We can get a sense of this by looking back to what was happening in 1959. At this time DDT was used widely in agriculture and for pest management around the world. Aerial spraying of DDT was common as farmers sought to protect their crops, but in malaria control DDT use was entirely different. Most malaria vectors enter houses in search of blood meals, and so protecting people while they are at home, often asleep, is crucial. Soon after the Allied forces first used DDT during World War II, scientists discovered that DDT acts primarily as a spatial repellent. In other words, if the interior of a house is sprayed with DDT, mosquitoes are driven away and are unlikely to enter. DDT will also act as a contact irritant, so if a mosquito lands on a sprayed surface, it is likely to exit the house rapidly, often before feeding. Of course DDT will also act as a toxicant, killing the mosquito. However it is a relatively weak toxicant and its spatial repellency is the insecticide's most important mode of action by far. Widespread area spraying of DDT would have been pointless for malaria control.

In 1959 malaria was in rapid retreat in many endemic countries as a consequence of effective DDT use. The global malaria eradication program was just barely underway. By that time, the malaria control community had already used DDT to free 300 million people from the burdens of endemic disease. By the program's end in 1969, the lives of almost one billion people would be equally improved. In 1959 there was a wealth of malaria control expertise, substantial funding, and programmatic emphasis on malaria prevention; there were powerful and successful national programs, goal-oriented malaria control policies, and great enthusiasm for the goals of the global program. We suggest that few, if any workers of that time could, in their wildest imaginings, have predicted what was to come. In just 20 years from that auspicious beginning most highly effective national control programs would begin grinding to a halt. Their malaria control expertise would be frittered away, their funding would be gone, the price of DDT would be up and its availability down, and the international policies for malaria control would be changed from disease prevention to case detection and treatment. The declining population of malaria control workers would begin seeing the disease they had worked so hard to control expanding back into malaria-free areas. Malaria would once again be inflicting ever-greater harm on the people they had tried to help. We should pause and consider how that happened, how our community failed to recognize the threat, and why it failed to respond.

The answers to these questions are perhaps more simple than one might think. During the 1960s, and into the 1970s, our community was committed, and had its nose to the grindstone, so to speak. From the initial use of DDT in the mid-1940s, our community had been in a position to observe any adverse effects from insecticides, if they were to occur. The community had close and continuous contact with the populations living in sprayed houses, and they saw no meaningful adverse effects. In brief, it had no evidence of any problems that appeared suddenly or gradually with the public health use of insecticides. Simultaneously the com‐ munity saw great improvements in health when DDT was used to prevent the diseases it sought to eliminate. It was, perhaps, beyond the community's ability to think that anyone would work against a worthy and effective public health program; but the community was wrong. Additionally, the community had not focused on diverging malaria control interests of developed and developing countries. Divergences occurred because the developed coun‐ tries had used DDT to eliminate malaria and no longer needed it. Meanwhile the developing countries still needed DDT to help with their disease control problems. Last but not least, the community had no prior experience with the ruthless and scientifically indefensible fear tactics that were being unleashed against its disease control programs.

this stage before. We can get a sense of this by looking back to what was happening in 1959. At this time DDT was used widely in agriculture and for pest management around the world. Aerial spraying of DDT was common as farmers sought to protect their crops, but in malaria control DDT use was entirely different. Most malaria vectors enter houses in search of blood meals, and so protecting people while they are at home, often asleep, is crucial. Soon after the Allied forces first used DDT during World War II, scientists discovered that DDT acts primarily as a spatial repellent. In other words, if the interior of a house is sprayed with DDT, mosquitoes are driven away and are unlikely to enter. DDT will also act as a contact irritant, so if a mosquito lands on a sprayed surface, it is likely to exit the house rapidly, often before feeding. Of course DDT will also act as a toxicant, killing the mosquito. However it is a relatively weak toxicant and its spatial repellency is the insecticide's most important mode of action by far. Widespread

In 1959 malaria was in rapid retreat in many endemic countries as a consequence of effective DDT use. The global malaria eradication program was just barely underway. By that time, the malaria control community had already used DDT to free 300 million people from the burdens of endemic disease. By the program's end in 1969, the lives of almost one billion people would be equally improved. In 1959 there was a wealth of malaria control expertise, substantial funding, and programmatic emphasis on malaria prevention; there were powerful and successful national programs, goal-oriented malaria control policies, and great enthusiasm for the goals of the global program. We suggest that few, if any workers of that time could, in their wildest imaginings, have predicted what was to come. In just 20 years from that auspicious beginning most highly effective national control programs would begin grinding to a halt. Their malaria control expertise would be frittered away, their funding would be gone, the price of DDT would be up and its availability down, and the international policies for malaria control would be changed from disease prevention to case detection and treatment. The declining population of malaria control workers would begin seeing the disease they had worked so hard to control expanding back into malaria-free areas. Malaria would once again be inflicting ever-greater harm on the people they had tried to help. We should pause and consider how that happened, how our community failed to recognize the threat, and why it failed to respond.

The answers to these questions are perhaps more simple than one might think. During the 1960s, and into the 1970s, our community was committed, and had its nose to the grindstone, so to speak. From the initial use of DDT in the mid-1940s, our community had been in a position to observe any adverse effects from insecticides, if they were to occur. The community had close and continuous contact with the populations living in sprayed houses, and they saw no meaningful adverse effects. In brief, it had no evidence of any problems that appeared suddenly or gradually with the public health use of insecticides. Simultaneously the com‐ munity saw great improvements in health when DDT was used to prevent the diseases it sought to eliminate. It was, perhaps, beyond the community's ability to think that anyone would work against a worthy and effective public health program; but the community was wrong. Additionally, the community had not focused on diverging malaria control interests of developed and developing countries. Divergences occurred because the developed coun‐ tries had used DDT to eliminate malaria and no longer needed it. Meanwhile the developing

area spraying of DDT would have been pointless for malaria control.

638 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Threats to the old malaria eradication effort evolved from two ideologies within the environ‐ mental movement. One was that there are too many people on planet earth and malaria elimination allowed excessive population growth of poor people in developing countries. The second theme was that man-made chemicals endangered wildlife and human health. In 1970, George Woodwell, a prominent and entrenched anti-insecticide campaigner, captured the two ideologies in a paper he published in *Science* magazine. He concluded that the answer to the problem of environmental pollution was "Fewer people, unpopular but increasing restrictions on technology (making it more and more expensive) [9]." His concluding comment captured the thinking of major stakeholders within the environmental movement at that time. Through the careful use of fear tactics, global campaigns grew up around each ideology. Eventually the ideologies became established at the highest levels of the UN and national governments of developed countries. Those campaigns eventually destroyed effective disease control pro‐ grams. The campaigns against PHIs achieved success through misrepresentations of science, by dragging companies and public organizations into courts in order to grab headlines for their fear-invoking claims, by using smear tactics against those who spoke in defense of insecticides, and, lastly, through extremely well-funded anti-insecticide advocacy. Through it all, anti-insecticide campaigners were supported by a popular press that fed off the fear invoked by the movement's predictions of insecticides causing catastrophic harm to wildlife and human health.

Naysayers will claim this is an exaggeration and that the old disease eradication programs were eliminated for a slew of reasons not mentioned here. Indeed there were other factors; but the overwhelming factors, as documented in annual proceedings of the WHO's Executive Board, discussions of the World Health Assembly (WHA), internal documents of UNICEF, and other published and unpublished reports, were those delineated above. Those who choose to believe current programs are not at risk of a similar fate may venture the opinion that regardless of past events, circumstances are entirely different now. They might even conclude movements that brought down the old programs are no longer active. For certain, the people, the claims, and the organizations have changed; but the themes and the scare tactics are the same. Nevertheless we will concede one point. The circumstances facing disease control programs today are entirely different from those that confronted the old disease eradication programs. Chief among the differences are that the old programs were not confronted by:


## **4. Environmentalism over public health policies**

With an annual caseload estimated at 216 million and 655,000 deaths, malaria continues as one of the most important insect-borne diseases [10]. Yet, it is just one of many insect-borne diseases that collectively claim millions of lives and stifle economic growth and development in disease endemic countries. PHIs and other public health chemicals are vital to the global struggle to control these diseases. Where PHIs are removed or their use restricted, disease rates increase. For example, two large eradication programs that were based almost entirely on public health use of DDT, freed Bolivia of malaria, dengue fever, and risk of urban yellow fever from the 1950s to the mid-1970s. The WHO acknowledges the importance of one program as follows: "Historically, mosquito control campaigns [that employed DDT] successfully eliminated *Aedes aegypti*, the urban yellow fever vector, from most mainland countries of central and South America. However, this mosquito species has re-colonized urban areas [with cessation of the *Aedes aegypti* eradication program] in the region and poses a renewed risk of urban yellow fever [11]." In spite of marvelous improvements in human health that were achieved by use of PHIs, international anti-insecticide pressures were brought to bear on those programs.

Bolivia abandoned *Aedes aegypti* eradication in the 1970s. This occurred because Bolivia, as with many countries of the Americas, ramped down eradication efforts once the US buckled to anti-DDT pressures in 1969 and ended use of DDT for *Aedes aegypti* eradication. Almost all countries of the Americas followed the US example in the 1970s. Years later Bolivia abandoned use of DDT for malaria control. As a consequence, malaria and threats of urban yellow fever are once again commonplace in Bolivia [12], and in 2009 Bolivia was savaged by a major dengue epidemic.

India is another case study. In the early 1950s, India had an estimated 75 million malaria infections, with roughly 800,000 deaths each year. Spraying DDT brought numbers of cases down to 49,151 by 1961. Today, the number of malaria cases each year is in doubt. What seems certain however is that the number of cases is huge and the number of deaths is on an order of hundreds of thousands. Estimates for cases vary from a few million to tens of millions of cases per year [13].

Despite the considerable human and economic toll caused by past increases in diseases like malaria and dengue, the current arsenal of PHIs for spraying on house walls is limited to just 12 compounds from four chemical classes, namely pyrethroids, organophosphates, carba‐ mates and organochlorines. Most PHIs are pyrethroids. DDT, the only organochlorine permitted for use, is one of the 12 approved compounds.

**•** Large national and international bureaucracies for regulatory control of insecticides,

**•** Billions of dollars for regulatory control and research against insecticides,

**•** A declining arsenal of insecticides for malaria control, and

**4. Environmentalism over public health policies**

research institutes around the world,

640 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

PHIs.

epidemic.

cases per year [13].

**•** A vast, and largely anti-insecticide, research establishment functioning in universities and

**•** Regulatory controls that are major impediments to the research and development of new

With an annual caseload estimated at 216 million and 655,000 deaths, malaria continues as one of the most important insect-borne diseases [10]. Yet, it is just one of many insect-borne diseases that collectively claim millions of lives and stifle economic growth and development in disease endemic countries. PHIs and other public health chemicals are vital to the global struggle to control these diseases. Where PHIs are removed or their use restricted, disease rates increase. For example, two large eradication programs that were based almost entirely on public health use of DDT, freed Bolivia of malaria, dengue fever, and risk of urban yellow fever from the 1950s to the mid-1970s. The WHO acknowledges the importance of one program as follows: "Historically, mosquito control campaigns [that employed DDT] successfully eliminated *Aedes aegypti*, the urban yellow fever vector, from most mainland countries of central and South America. However, this mosquito species has re-colonized urban areas [with cessation of the *Aedes aegypti* eradication program] in the region and poses a renewed risk of urban yellow fever [11]." In spite of marvelous improvements in human health that were achieved by use of PHIs, international anti-insecticide pressures were brought to bear on those programs.

Bolivia abandoned *Aedes aegypti* eradication in the 1970s. This occurred because Bolivia, as with many countries of the Americas, ramped down eradication efforts once the US buckled to anti-DDT pressures in 1969 and ended use of DDT for *Aedes aegypti* eradication. Almost all countries of the Americas followed the US example in the 1970s. Years later Bolivia abandoned use of DDT for malaria control. As a consequence, malaria and threats of urban yellow fever are once again commonplace in Bolivia [12], and in 2009 Bolivia was savaged by a major dengue

India is another case study. In the early 1950s, India had an estimated 75 million malaria infections, with roughly 800,000 deaths each year. Spraying DDT brought numbers of cases down to 49,151 by 1961. Today, the number of malaria cases each year is in doubt. What seems certain however is that the number of cases is huge and the number of deaths is on an order of hundreds of thousands. Estimates for cases vary from a few million to tens of millions of

Despite the considerable human and economic toll caused by past increases in diseases like malaria and dengue, the current arsenal of PHIs for spraying on house walls is limited to just Even though production and use of DDT has declined continuously during the last four decades, DDT has grown as a convenient target of environmental science research. A recent PubMed search (in early 2011) for research papers on insecticides uncovered almost 60,000 papers, and about one sixth (9,459) were on DDT. These are remarkable statistics considering that DDT is hardly in use anymore. The decline in usage was sudden and corresponds to precipitous drops in human body burdens of DDT residues. Today, for example, the amount of DDT in human breast milk, based on serial surveys in many countries, is an infinitesimal fraction of what it was in the 1960s—and even those exceedingly low levels are declining [14]. Along with precipitous reductions in DDT use, one could reasonably expect that research on DDT would decline. However, as revealed in Figure 1, the numbers of published papers on DDT have actually increased, and more so in recent years than in the past. Furthermore, papers on DDT and malaria account for only a minor proportion (2.6 to 14.8% per year) of those published papers. So, why is the research effort on DDT increasing even as the use of DDT fades to inconsequential levels? To answer this question we will delve more into the modern themes of environmental research and anti-insecticide advocacy.

**Figure 1.** Average number of papers published per year on DDT or DDE. Data based on PubMed searches on key words--DDT and/or DDE. Counts summed for five-year intervals of 1987-1991, 1997-2001, and 2007-2011.

## **5. Why increased research on insecticides?**

A 2005 paper by Dr. Stephen Safe, a Distinguished Professor and recipient of the Distinguished Lifetime Toxicology Scholar Award from the Society of Toxicology, explains much about the modern trend of increased funding and research on DDT [15]. Professor Safe is a professor at Texas A&M and is a specialist in toxicology and molecular biology of estrogenic and antiestrogenic compounds. To summarize introductory comments in his 2005 paper, modern emphasis on DDT is linked to a series of 1990 papers and the concept of the precautionary principle. The papers proposed that endocrine disrupting chemicals (EDCs), which include both man-made (synthetic) and naturally occurring chemicals, were contributing to diverse health problems worldwide. The diverse harms include decreased male sperm counts, increased birth defects, decreased fertility, increased incidence of breast and testicular cancers, etc. As Dr. Safe states, the role of synthetic EDCs as a cause of diverse health problems has been subjected to multiple challenges, to include a lack of biological plausibility for some responses and failure to consider that people are more heavily exposed to natural or dietary EDCs compared to relatively low exposures to the synthetic EDCs. Additionally, the natural compounds are often far more potent endocrine disruptors than synthetic EDCs.

The 1990s papers and the concept of the precautionary principle resulted in new funding and renewed interests in insecticides. As described by Dr. Safe, "Regulatory and research funding agencies have taken the endocrine disruptor hypothesis seriously [15]." Funds for research grew and, as a result, "... numerous laboratory animal and clinical studies have been initiated to test the validity of the hypothesis and to determine the association between health problems and exposure to EDCs [15]." This, in large part, seems to explain the huge growth in research and numbers of publications about potential harms from DDT and other insecticides. It is worth noting that extremely sensitive assays are available for DDT and other synthetic EDCs; but assays are often not available for more abundant and more diverse populations of natural EDCs. Thus it seems that the selection of DDT as a research topic is more closely related to availability and familiarity with quantitative assays opposed to some understanding of what the real threats are from synthetic versus natural EDCs.

In his 2005 paper Dr. Safe reviews many recent studies, and we refer the reader to his paper for more in-depth analyses. He comments on the synthetic EDCs as casual agents in breast cancer and male reproductive track anomalies. For the former, he reviews several studies, to include a meta-analysis, and concludes that the evidence does not support the hypothesis that DDE causes breast cancer. He concludes further that "If organochlorines do not significantly impact on this disease [breast cancer], it is now time to generate new hypotheses and focus on identifying other etiological factors that are linked to the high incidence of sporadic breast cancer in women [15]."

Dr. Safe reviewed numerous studies on DDT and other synthetic organochlorines (OCs) reportedly causing diseases of the male reproductive tract. The claim that sperm counts are declining is central to the thesis of many alarmists who propose that synthetic OCs are causing declining male sexual function. Dr. Safe reviews past reports and concludes, "results from various clinics are not sufficient to support a global decrease or increase [15]" in sperm counts. He also concludes "the hypothesized role of *in utero* exposure to estrogens as a factor in regulating sperm count in adult males is also questionable [15]." Dr. Safe goes on to review studies on possible associations between levels of synthetic EDCs with urogenital birth defects and increasing trends of testicular cancer. For the former, he found that both the evidence of increasing rate of birth defects and the hypothetical associations between those rates and exposures to synthetic EDCs were not persuasive. Additionally, evidence of multiple studies did not support the hypothesis that synthetic EDCs were a cause of testicular cancer.

Texas A&M and is a specialist in toxicology and molecular biology of estrogenic and antiestrogenic compounds. To summarize introductory comments in his 2005 paper, modern emphasis on DDT is linked to a series of 1990 papers and the concept of the precautionary principle. The papers proposed that endocrine disrupting chemicals (EDCs), which include both man-made (synthetic) and naturally occurring chemicals, were contributing to diverse health problems worldwide. The diverse harms include decreased male sperm counts, increased birth defects, decreased fertility, increased incidence of breast and testicular cancers, etc. As Dr. Safe states, the role of synthetic EDCs as a cause of diverse health problems has been subjected to multiple challenges, to include a lack of biological plausibility for some responses and failure to consider that people are more heavily exposed to natural or dietary EDCs compared to relatively low exposures to the synthetic EDCs. Additionally, the natural

compounds are often far more potent endocrine disruptors than synthetic EDCs.

the real threats are from synthetic versus natural EDCs.

642 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

cancer in women [15]."

The 1990s papers and the concept of the precautionary principle resulted in new funding and renewed interests in insecticides. As described by Dr. Safe, "Regulatory and research funding agencies have taken the endocrine disruptor hypothesis seriously [15]." Funds for research grew and, as a result, "... numerous laboratory animal and clinical studies have been initiated to test the validity of the hypothesis and to determine the association between health problems and exposure to EDCs [15]." This, in large part, seems to explain the huge growth in research and numbers of publications about potential harms from DDT and other insecticides. It is worth noting that extremely sensitive assays are available for DDT and other synthetic EDCs; but assays are often not available for more abundant and more diverse populations of natural EDCs. Thus it seems that the selection of DDT as a research topic is more closely related to availability and familiarity with quantitative assays opposed to some understanding of what

In his 2005 paper Dr. Safe reviews many recent studies, and we refer the reader to his paper for more in-depth analyses. He comments on the synthetic EDCs as casual agents in breast cancer and male reproductive track anomalies. For the former, he reviews several studies, to include a meta-analysis, and concludes that the evidence does not support the hypothesis that DDE causes breast cancer. He concludes further that "If organochlorines do not significantly impact on this disease [breast cancer], it is now time to generate new hypotheses and focus on identifying other etiological factors that are linked to the high incidence of sporadic breast

Dr. Safe reviewed numerous studies on DDT and other synthetic organochlorines (OCs) reportedly causing diseases of the male reproductive tract. The claim that sperm counts are declining is central to the thesis of many alarmists who propose that synthetic OCs are causing declining male sexual function. Dr. Safe reviews past reports and concludes, "results from various clinics are not sufficient to support a global decrease or increase [15]" in sperm counts. He also concludes "the hypothesized role of *in utero* exposure to estrogens as a factor in regulating sperm count in adult males is also questionable [15]." Dr. Safe goes on to review studies on possible associations between levels of synthetic EDCs with urogenital birth defects and increasing trends of testicular cancer. For the former, he found that both the evidence of increasing rate of birth defects and the hypothetical associations between those rates and In this brief section we have described the major themes of research that will be the source of future claims against PHIs. Dr. Safe sounded a warning in his comments about EDCs and breast cancer. He pointed out that our abilities to detect EDCs and a wealth of other variables (for example, biomarkers, genotypes, and a wealth of other biological, biochemical, environ‐ mental, and sociological variables) "increases the probability of 'chance' correlations, and there are several examples of these associations that are not consistent across all studies [15]." So, it seems clear that we should expect a greater frequency of claims against PHIs in the future. That said, anti-insecticide advocacy more so than research poses the greatest threat to the future of effective disease control programs. As we observed in the negotiations for the Stockholm Convention on Persistent Organic Pollutants (POPs) described below, well-funded anti-insecticide advocacy is the operational arm of the environmental movement. But unlike the careful deliberations of most environmental scientists, anti-insecticide groups are not constrained by subtle considerations of consistent and meaningful evidence and other criteria for cause-effect relationships, or by considerations of harm versus benefits of insecticide use.

## **6. Renewed malaria control programs beset by opposition to PHIs**

As stated in an earlier section, today there is great enthusiasm and considerable funding to advance the goals of global control of malaria. We arrive at this period of enthusiasm only because we lived through many years of almost no hope at all.

The steady increase in malaria cases that led to RBM's formation had several underlying causes. Among them was the spread of drug resistance around the world. Since the 1940s chloroquine had been a mainstay of malaria treatment programs, but resistance by the *Plasmodium falciparum* parasite to the drug first appeared in the 1950s and slowly spread worldwide. Chloroquine was duly replaced by sulphadoxine-pyrimethamine (SP) in the 1980s, but resistance soon emerged to this drug as well.

Another cause of the growing burden of malaria was the lack of interest in malaria control by major donor agencies and malarial country governments. Enthusiasm for malaria dissipated when the great push against malaria - the global malaria eradication campaign of the 1950s and 60s – was called off. Malaria control is expensive, requiring the employment of trained personnel, logistics specialists, scientists and large quantities of drugs and vector control products. Continuing to pay for malaria control year in and year out when it was clear that global eradication was not feasible was a tough sell. Concurrently the focus for many devel‐ opment agencies was away from disease control and towards population control, as we touch on in this chapter and explain in more detail in *The Excellent Powder, DDT's Political and Scientific History* [16]. Few newly independent and highly malarial African countries sustained malaria control programs that had been run by colonial rulers. In Zambia, for instance, malaria control programs that had been set up when the country was ruled by Great Britain as Northern Rhodesia collapsed along with the Zambian economy in the 1980s.

However, as illustrated in the examples of disease control history in Bolivia and India, arguably one of the greatest obstacles to sustained malaria control was the growing campaign against PHIs, and DDT in particular. DDT had been used in malaria control since World War II. The effectiveness of this insecticide in controlling malaria was unprecedented. As we explain above, DDT, when sprayed on the inside walls of houses, acts to repel mosquitoes, but it will also irritate mosquitoes so they exit houses sooner than they otherwise would and will kill mosquitoes that rest on a sprayed surface long enough.

Through these multiple modes of action, and thanks to the dedicated work of thousands of hard working malaria control program officers, DDT saved around one billion people from malaria during the eradication era. But what some people heralded as a great savior, others decried as a harbinger of doom. Chief among the anti-DDT crusaders was Rachel Carson whose 1962 book, *Silent Spring*, is a florid and grossly exaggerated attack on the chemical for its supposed impact on wildlife and human health [17]. There were, and are, no shortages of Carson acolytes who have joined in with their own attacks on DDT, as we explain later in this chapter.

Following the banning of DDT for most uses in the US and Western Europe in the 1970s, production fell dramatically. Although DDT was still permitted for use in disease control, supplies dwindled and predictably the cost began to rise. It mattered little that the WHO's malaria control advisers still supported the use of DDT, when the reality was that fewer countries could obtain it. In 1969, Scandinavian countries, Canada and the US started to place 'severe' restrictions on the use of DDT [18]. Thus, it was no coincidence that global malaria eradication and the United State's *Aedes aegypti* eradication programs were both stopped in 1969—just as it was no coincidence that both relied on use of DDT [16]. Unsurprisingly, within just a few years, malarial countries were complaining to the WHO of their inability to obtain the chemical and use it to save lives [19]. Along with the growing campaigns against DDT, donor agencies like USAID, under pressure of legal actions, began to withdraw funding for DDT and malaria control in the 1970s.

In the following section we will detail, with a specific example, how the bio-politics of environmental activism against DDT and other PHIs translated into real world harm to human health. For this example we have chosen a country that has a strong tradition in science and a long and proud history of combating malaria.

## **7. Public health insecticides and malaria**

The value of PHIs in controlling malaria is best evidenced by historical data on DDT sprayed houses. Brazil, as with other countries with territory within the Amazon Basin, struggles with difficult malaria control issues. The Amazon Basin is the most enduring environment in the Americas for the persistence of endemic malaria. Populated with many rural, poorly housed and mobile inhabitants, the Amazon Basin covers a vast geographical area of warm, humid environments. More importantly, it is populated with the Hemisphere's most dangerous vector of human malaria, *Anopheles darlingi*. In the absence of this species or in regions of the Americas where it is less common, the chain of malaria transmission is weaker and more easily interrupted. For this reason, malaria often declines to low levels in the face of organized control programs in regions outside the Amazon Basin. In contrast, within the Amazon Basin, malaria exhibited some refractoriness to control measures even during years of the global malaria eradication program. As a consequence, eradication was not achieved. Nevertheless the spraying of DDT on house walls greatly reduced malaria infections and lifted a large part of the burden of malaria from the backs of people in the Amazon Basin.

However, as illustrated in the examples of disease control history in Bolivia and India, arguably one of the greatest obstacles to sustained malaria control was the growing campaign against PHIs, and DDT in particular. DDT had been used in malaria control since World War II. The effectiveness of this insecticide in controlling malaria was unprecedented. As we explain above, DDT, when sprayed on the inside walls of houses, acts to repel mosquitoes, but it will also irritate mosquitoes so they exit houses sooner than they otherwise would and will kill

Through these multiple modes of action, and thanks to the dedicated work of thousands of hard working malaria control program officers, DDT saved around one billion people from malaria during the eradication era. But what some people heralded as a great savior, others decried as a harbinger of doom. Chief among the anti-DDT crusaders was Rachel Carson whose 1962 book, *Silent Spring*, is a florid and grossly exaggerated attack on the chemical for its supposed impact on wildlife and human health [17]. There were, and are, no shortages of Carson acolytes who have joined in with their own attacks on DDT, as we explain later in this

Following the banning of DDT for most uses in the US and Western Europe in the 1970s, production fell dramatically. Although DDT was still permitted for use in disease control, supplies dwindled and predictably the cost began to rise. It mattered little that the WHO's malaria control advisers still supported the use of DDT, when the reality was that fewer countries could obtain it. In 1969, Scandinavian countries, Canada and the US started to place 'severe' restrictions on the use of DDT [18]. Thus, it was no coincidence that global malaria eradication and the United State's *Aedes aegypti* eradication programs were both stopped in 1969—just as it was no coincidence that both relied on use of DDT [16]. Unsurprisingly, within just a few years, malarial countries were complaining to the WHO of their inability to obtain the chemical and use it to save lives [19]. Along with the growing campaigns against DDT, donor agencies like USAID, under pressure of legal actions, began to withdraw funding for

In the following section we will detail, with a specific example, how the bio-politics of environmental activism against DDT and other PHIs translated into real world harm to human health. For this example we have chosen a country that has a strong tradition in science and a

The value of PHIs in controlling malaria is best evidenced by historical data on DDT sprayed houses. Brazil, as with other countries with territory within the Amazon Basin, struggles with difficult malaria control issues. The Amazon Basin is the most enduring environment in the Americas for the persistence of endemic malaria. Populated with many rural, poorly housed and mobile inhabitants, the Amazon Basin covers a vast geographical area of warm, humid environments. More importantly, it is populated with the Hemisphere's most dangerous vector of human malaria, *Anopheles darlingi*. In the absence of this species or in regions of the

mosquitoes that rest on a sprayed surface long enough.

644 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

DDT and malaria control in the 1970s.

long and proud history of combating malaria.

**7. Public health insecticides and malaria**

chapter.

Successful malaria control by spraying DDT was maintained for many years. Yet, the succes‐ sion of bio-political events described in the previous section and elsewhere eventually destroyed Brazil's well-orchestrated malaria control program. Malaria cases began to increase when the numbers of houses being sprayed were progressively reduced in the 1980s. The many years of successful control followed by years when the spray program withered away are detailed in Figure 2.

**Figure 2.** Number of houses sprayed per 1000 population versus the annual parasite index (cases per 1000 popula‐ tion) in Brazil during the years 1962 to 1993. Data for these years were collected under uniform data collection meth‐ ods (see Roberts et al. 1997. for data sources [34]).

The graph presents annual parasite indices (APIs) and house spray rates (HSRs) from 1962 to 1993. Two clusters of data points are identified. One group represents the years from 1962 to 1981 when house spray rates were high and malaria indices were low. The API is a standard malaria control index, calculated as the annual number of diagnosed malaria cases X 1000/ population size. The HSR represents the number of houses sprayed per 1000 population. As shown in this graph, APIs in years after 1981 increased in response to reductions in numbers of houses being sprayed.

To bring Brazil's story up to date, Figure 3 presents statistics on malaria cases through 2010. As described in the previous section, there has been a global renewal in efforts to control malaria. Thus, in recent years, Brazil expanded its malaria control efforts. But even with increased financial support and availability of new malaria controltechnologies (e.g., case treatment with the new and effective ACTs, insecticide treated nets and so-called long-lasting nets), the accomplishments of recent years are less than what is needed and certainly far less than what was achieved and sustained during 20 years of spraying houses with DDT. As demonstrated in Figure 3, there was an average of 100,000 cases per year during those 20 years of major reli‐ ance on DDT. As DDT use declined in the 1980s, the average number of cases/year increased to 450,000. In the next decade, DDT use was abandoned completely and cases increased to over 500,000 per year. Today, even with an expanded program of control, the average number of cases per year is well over 400,000. The differences in results of the last 30 years over what was achieved with DDT roughly sums to 10.5 million cases that might have been prevented if DDT had not been abandoned. While population growth as an independent variable might account for some growth in numbers of cases, the increased number of cases corresponds, over time, to changes in slide positivity rates. The slide positivity rate is neutral in terms of population size. As a reminder, the estimate of 10.5 million excess malaria cases is for Brazil alone.

**Numbers of Malaria Cases In Brazil**

**Figure 3.** Average number of cases per year in Brazil across defined blocks of years (x-axis). Data for these years were collected under uniform data collection methods (see Roberts et al. 1997 for data sources [34] and PAHO malaria data [57]).

Clearly the great reductions of malaria from 1962 to 1981 compared to later blocks of years reveals the enormous benefit of DDT and other insecticides.

One of the most compelling examples of the usefulness of DDT in malaria control comes from recent experience in South Africa. This country had successfully used DDT in malaria control since the late 1940s and in so doing had dramatically reduced the malarial areas to the regions bordering Mozambique to the east and Zimbabwe to the north. In 1996 South Africa's Malaria Advisory Group (MAG) advised the national malaria control program to begin phasing out DDT. This advice was based on two main factors. First, DDT is best applied to the mud and dung walls of traditional African houses rather than on the plastered and painted walls of western style houses where the DDT can stain the walls. Given the staining, homeowners were often reluctant to allow the spray teams to enter their houses. As the rural areas of South Africa have developed and become wealthier, more and more people have built western style houses, requiring alternative insecticides. Second, the MAG had taken note of the political pressure against the use of DDT and anticipating greater restrictions on the use of DDT, decided to transition over to other chemicals. In the late 1990s therefore the provincial malaria programs began replacing DDT with pyrethroids. The first province to do so was KwaZulu Natal, which borders Mozambique and at the time was the most malarial of the countries three malarial provinces [16].

To bring Brazil's story up to date, Figure 3 presents statistics on malaria cases through 2010. As described in the previous section, there has been a global renewal in efforts to control malaria. Thus, in recent years, Brazil expanded its malaria control efforts. But even with increased financial support and availability of new malaria controltechnologies (e.g., case treatment with the new and effective ACTs, insecticide treated nets and so-called long-lasting nets), the accomplishments of recent years are less than what is needed and certainly far less than what was achieved and sustained during 20 years of spraying houses with DDT. As demonstrated in Figure 3, there was an average of 100,000 cases per year during those 20 years of major reli‐ ance on DDT. As DDT use declined in the 1980s, the average number of cases/year increased to 450,000. In the next decade, DDT use was abandoned completely and cases increased to over 500,000 per year. Today, even with an expanded program of control, the average number of cases per year is well over 400,000. The differences in results of the last 30 years over what was achieved with DDT roughly sums to 10.5 million cases that might have been prevented if DDT had not been abandoned. While population growth as an independent variable might account for some growth in numbers of cases, the increased number of cases corresponds, over time, to changes in slide positivity rates. The slide positivity rate is neutral in terms of population size.

As a reminder, the estimate of 10.5 million excess malaria cases is for Brazil alone.

**Numbers of Malaria Cases In Brazil**

1962-81 1982-91 1992-01 2001-10

**Intervals of Time**

**Figure 3.** Average number of cases per year in Brazil across defined blocks of years (x-axis). Data for these years were collected under uniform data collection methods (see Roberts et al. 1997 for data sources [34] and PAHO

Clearly the great reductions of malaria from 1962 to 1981 compared to later blocks of years

One of the most compelling examples of the usefulness of DDT in malaria control comes from recent experience in South Africa. This country had successfully used DDT in malaria control

0

reveals the enormous benefit of DDT and other insecticides.

100000

200000

300000

**Average number of cases/year**

malaria data [57]).

400000

500000

600000

646 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Almost as soon as the KwaZulu Natal malaria control program changed over to pyrethroids, malaria cases started to rise. By 2000, malaria cases had increased five fold from just over 8,500 cases to almost 42,000 cases. Malaria deaths increased from just 22 in 1996 to 320 in 2000 as malaria patients overwhelmed clinics and hospitals [20].

Research showed that a major driver of the epidemic was resistance to pyrethroid insecticides. In addition, evidence was rising that malaria parasite resistance had grown to SP, or Fansidar. The Department of Health took the decision to reintroduce DDT and change treatment regimen from Fansidar to the newly-available ACT, artemether-lumefantrine, or Coartem. Within a year malaria cases plummeted by around 80 percent [21]. The combination of a proven and effective PHI along with effective treatment reduced malaria transmission so dramatically that within just a few years, malaria elimination was within sight.

Given the benefit and usefulness of DDT and other PHIs in the control of malaria, as described above, how is it possible that PHIs have been so effectively demonized? In the next section we will describe strategies and tactics that have been employed to paralyze malaria control programs in countries around the world. As an aside, it is worth noting that those who ruthlessly campaign against DDT and other PHIs shamelessly deny any responsibility whatsoever for the increasing burdens of disease that inevitably occur when their campaigns succeed.

## **8. Goals, strategies, and tactics of anti-insecticide campaigns**

The goal for environmental campaigns is to reduce or eliminate use of PHIs for the presumed but ambiguous purpose of better environmental health. Another goal, at least for some, appears to be stopping the use of chemicals that protect health and save lives in order to slow growth of human populations.

In the 1960s, the goal of halting or reducing the use of man-made insecticides was laid out in Rachel Carson's unscientific writings in *Silent Spring*. In 1968, the Malthusian rantings of Paul Ehrlich in *The Population Bomb* focused attention on the contributions of DDT to growth of human populations in malaria-endemic countries. The goal of reducing human populations was never silenced; and it is once again a topic of heated debate, with some claiming billions of people must be eliminated [22].

The goal of today's anti-insecticide activists is still to reduce or eliminate synthetic insecticides. Achieving such a goal requires strategies and tactics. There are three visible strategies for achieving the goal of reducing or eliminating PHIs. The first is to convince people that PHIs are harmful. The second is to claim the chemicals are not needed in order to control diseases. The third strategy is to predict that grave harm will occur if the PHIs continue to be used. In this section we will give background information and three examples of the first strategy. In most cases we will focus on issues of DDT, but the same strategies and tactics are employed against other PHIs.

In a historical context, anti-insecticide advocates used propaganda and emotional arguments to convince people insecticides were dangerous and their use should be stopped. They were helped by science writers of the popular press and their efforts led to public health programs being abandoned around the world – and a resurgence of malaria infections. We have already presented one example of such an outcome (see Figures 2 and 3).

Anti-insecticide activism is an even stronger force today, and anti-insecticide advocates are even more determined to deny developing countries the protections from disease and death that only insecticides can provide. Because of environmental and anti-insecticide advocacy, the WHA adopted a resolution (WHA 50.13) in May 1997 that calls on countries to reduce reliance on use of insecticides for disease control [23]. Then, in 1998, the United Nations Environment Programme (UNEP) began negotiations for a POPs treaty targeting DDT and 11 other chemicals for global elimination [24]. The beginning of those negotiations stimulated malaria scientists and other public health professionals to mount a global campaign to defend the use of DDT in disease control programs. The public health campaign was successful and DDT was listed on Annex B of the Stockholm Convention on Persistent Organic Pollutants, which allowed its continued use. Yet, and despite the public health campaign's success, anti-DDT and anti-insecticide advocacy is unabated in UNEP, the US Environmental Protection Agency, the European Union, and, to lesser extent, in public agencies financing disease control programs. As a result, DDT factories closed their doors. Today, only one in India is still in operation. Also, environmental campaigners have erected formidable international barriers to the purchase and supply of DDT. Countries are under continual pressure from anti-DDT advocacy groups, and they are being enticed by financial mechanisms of Global Environment Facility (GEF) to stop using DDT.

WHA resolution 50.13 and the Stockholm Convention on Persistent Organic Pollutants, described above, are only the most recent in 50 years of efforts to eliminate DDT and other PHIs. Success in anti-PHI campaigns has been achieved by scaring people with false claims. Anti-DDT propaganda typically claims DDT causes all manner of harm to human health. Readily embraced and trumpeted by the popular press, the claims, in reality, never satisfy even the most minimal cause-effect criteria [25]. These internationally accepted criteria are:

**•** Strength of the association. The stronger an observed association appears over a series of different studies, the less likely this association is spurious because of bias.

Ehrlich in *The Population Bomb* focused attention on the contributions of DDT to growth of human populations in malaria-endemic countries. The goal of reducing human populations was never silenced; and it is once again a topic of heated debate, with some claiming billions

The goal of today's anti-insecticide activists is still to reduce or eliminate synthetic insecticides. Achieving such a goal requires strategies and tactics. There are three visible strategies for achieving the goal of reducing or eliminating PHIs. The first is to convince people that PHIs are harmful. The second is to claim the chemicals are not needed in order to control diseases. The third strategy is to predict that grave harm will occur if the PHIs continue to be used. In this section we will give background information and three examples of the first strategy. In most cases we will focus on issues of DDT, but the same strategies and tactics are employed

In a historical context, anti-insecticide advocates used propaganda and emotional arguments to convince people insecticides were dangerous and their use should be stopped. They were helped by science writers of the popular press and their efforts led to public health programs being abandoned around the world – and a resurgence of malaria infections. We have already

Anti-insecticide activism is an even stronger force today, and anti-insecticide advocates are even more determined to deny developing countries the protections from disease and death that only insecticides can provide. Because of environmental and anti-insecticide advocacy, the WHA adopted a resolution (WHA 50.13) in May 1997 that calls on countries to reduce reliance on use of insecticides for disease control [23]. Then, in 1998, the United Nations Environment Programme (UNEP) began negotiations for a POPs treaty targeting DDT and 11 other chemicals for global elimination [24]. The beginning of those negotiations stimulated malaria scientists and other public health professionals to mount a global campaign to defend the use of DDT in disease control programs. The public health campaign was successful and DDT was listed on Annex B of the Stockholm Convention on Persistent Organic Pollutants, which allowed its continued use. Yet, and despite the public health campaign's success, anti-DDT and anti-insecticide advocacy is unabated in UNEP, the US Environmental Protection Agency, the European Union, and, to lesser extent, in public agencies financing disease control programs. As a result, DDT factories closed their doors. Today, only one in India is still in operation. Also, environmental campaigners have erected formidable international barriers to the purchase and supply of DDT. Countries are under continual pressure from anti-DDT advocacy groups, and they are being enticed by financial mechanisms of Global Environment

WHA resolution 50.13 and the Stockholm Convention on Persistent Organic Pollutants, described above, are only the most recent in 50 years of efforts to eliminate DDT and other PHIs. Success in anti-PHI campaigns has been achieved by scaring people with false claims. Anti-DDT propaganda typically claims DDT causes all manner of harm to human health. Readily embraced and trumpeted by the popular press, the claims, in reality, never satisfy even the most minimal cause-effect criteria [25]. These internationally accepted criteria are:

presented one example of such an outcome (see Figures 2 and 3).

of people must be eliminated [22].

648 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Facility (GEF) to stop using DDT.

against other PHIs.


In the case of a true cause-effect relationship we can reasonably expect measurable levels of harm as a result of human exposures. Levels of harm will be proportional to harmfulness of the agent and to durations and characteristics of exposures. The more harmful an agent, the more likely it is to produce obvious levels of harm. Harm from weaker agents, on the other hand, will probably not be obvious and be definable only through population-based statistics. Regardless, ending use of a weak, but truly harmful, agent will reduce exposure to the chemical, reduce chemical concentration in the environment, and reduce the levels of harm. This is true even if the chemical is characterized as persistent, as is DDT. Persistence does not mean the chemical does not degrade. It just means that in certain compartments of the environment or living organisms it will degrade or be eliminated more slowly. Levels of DDT in the environment generally decline rapidly after its use is stopped. It is precisely because DDT does degrade that house walls are re-sprayed once or twice a year in order to achieve effective levels of malaria control.

Here, with the example of cigarette smoke and cancer, we illustrate application of cause-effect criteria. The link between smoking and human cancer has been validated through experimen‐ tation and vital statistics. In general, the argument that cigarette smoke caused cancer was convincing because patterns of low or high cancer rates consistently correlated with patterns of low or high smoking rates and duration of smoking. Furthermore, as people stopped smoking their risk of cancer actually declined. Consistent and persuasive evidence of causeeffect relationships between cigarette smoking and cancers formed the basis of public health campaigns to reduce or stop cigarette smoking. Unlike those public health campaigns, however, the environmental campaigns against PHIs are not based on persuasive and, certainly not, consistent, scientific evidence. The occasional observational study that suggests use of a public health insecticide harms health is countered by many other studies that suggest otherwise. Nevertheless, and as illustrated below, environmental campaigners readily ignore essential criteria for establishing a cause-effect relationship and greedily grab any new study that suggests some association between PHIs and human disease. The activist community has shown itself to be highly adept at getting such studies widespread national and international media coverage, often with headlines and messages designed to strike fear into people's hearts. These headlines are also very useful in advancing careers and ensuring ongoing research funding. We will describe three examples of how environmental advocates, and in some cases the environmental scientists themselves, ignore the criteria for establishing cause-effect relationships and use preliminary studies to push their anti-PHI agenda, or, more selfishly, their personal research agenda. The three examples are illustrations of the first strategy to convince people that DDT is a public health threat.

#### **Example 1:**

Mary Wolff and co-authors (1993) published a paper in which they claimed a statistical association of DDE (a major DDT metabolite) with breast cancer [26]. DDT opponents then used this paper to gain public attention and convince people that DDT caused breast cancer. To be specific, we are talking about anti-insecticide activists, not Dr. Wolff. Years later, with completion of many other studies, and without fanfare or wide publicity, researchers con‐ cluded DDE was not a cause of breast cancer. The WHO reassessment of DDT exposures from indoor spray programs states, "Overall, the association between DDT and breast cancer is inconclusive [27]." Regardless, for many years, anti-DDT activists heralded the 1993 paper as final proof of DDT harm and used it to generate funds and recruit new members to campaigns for DDT elimination [28].

#### **Example 2:**

Following a different thread of research, Rogan and coauthors reported that DDE was associated with reduced duration of lactation [29,30]. As with the reported association of DDT and breast cancer, this claim was grabbed by the WWF in 1998 and used in the propaganda campaign leading up to the Stockholm Convention on Persistent Organic Pollutants. The stated goal of the WWF campaign was a phase out of DDT by 2007 [28]. In their coverage of this topic, the WWF stated that studies "showed that the duration of lactation was inversely related to the concentration of DDE in milk." Separate from the WWF's use of these claims, the claims were, in part, also the basis for two high-profile publications by Rogan and coauthors in the journals, *Emerging Infectious Diseases* [31] and *The Lancet* [32]. They proposed that the benefits of spraying DDT on house walls to control malaria in Africa would be cancelled out by lowered child survival due to reduced durations of lactation and potential increases in premature births. The claims were used in campaigns against DDT and used to justify more research support.

Once published, the claims became tools for anti-DDT advocacy. For example, the claim is part of a 2005 Physicians for Social Responsibility (PSR) document about DDT and its use in Kenya. The PSR author states, "DDT may have a substantial impact on infant mortality, by increasing the risk of pre-term birth and by decreasing the duration of breast-feeding after birth. In this paper, Chen and Rogan conclude that DDT may cause comparable increase in infant mortality through these mechanisms compared to the decrease in infant mortality it causes by killing mosquitoes and thus reducing malaria cases [33]."

Without doubt the papers had great value for the anti-DDT advocacy community, yet the background studies for those claims did not fulfill the criteria for establishing DDT as the cause of reduced lactation or of pre-term births. In fact, even Chen and Rogan [31] stated the reported associations did not prove DDT caused any of the illnesses they discussed. Regardless, the claims were used as if they proved, beyond any doubt, that DDT was the cause of harm. This was illustrated in an exchange of letters to the Editor of the journal, *Emerging Infectious Diseases*. The exchange was between Roberts [34,35] and the WWF (written by Matteson) [36]. Matteson stated in her letter, "DDT also is associated with reduced lactation, premature births…" Naturally, Matteson used those reported associations to demonize DDT as part of WWF's push for global elimination of DDT by 2007. Misuse of those claims is further illustrated by an article defending Rachel Carson by the Rachel Carson Council. As with the PSR author, this writer used both claims plus the assertions included in the two papers by Rogan and coauthors about the benefits of DDT being canceled out by increased deaths of newborns in Africa. As stated in this very recent online article: "…significant shortening of the lactation cycle-time that human mothers can produce milk for their babies linked to DDT exposure. Based on reports for both premature births and reduced lactation cycles, scientists have predicted that regular DDT exposure could increase the possibility of higher levels of infant mortality for women in Africa who live in treated environments [37]."

There are many other examples of how these claims have been used and continue to be used in anti-insecticide propaganda. As stated in a 2006 article advocating against the use of DDT by the Pesticide Action Network in the UK, "Other studies have linked DDT to reduced breastmilk production, premature delivery and reduced infant birthweights [sic] [38]." Last but not least, Wikipedia includes the following statement:

Human epidemiological studies suggest that exposure is a risk factor for premature birth and low birth weight, and may harm a mother's ability to breast feed. Some 21st-century research‐ ers argue that these effects may increase infant deaths, offsetting any anti-malarial benefits. A 2008 study, however, failed to confirm the association between exposure and difficulty breastfeeding [39].

Mention of the 2008 study is perhaps helpful; but it is not sufficient. Given that DDT produces great benefit in control of malaria, Wikipedia contributors should be careful in comments about DDT lest their written assessments inflict grave harm on poor people in malaria endemic countries. Point of fact, the Wikipedia assessment leaves the reader thinking that DDT causes premature births and reduced duration of lactation, when the weight of scientific evidence shows it does not.

#### **Example 3:**

shown itself to be highly adept at getting such studies widespread national and international media coverage, often with headlines and messages designed to strike fear into people's hearts. These headlines are also very useful in advancing careers and ensuring ongoing research funding. We will describe three examples of how environmental advocates, and in some cases the environmental scientists themselves, ignore the criteria for establishing cause-effect relationships and use preliminary studies to push their anti-PHI agenda, or, more selfishly, their personal research agenda. The three examples are illustrations of the first strategy to

Mary Wolff and co-authors (1993) published a paper in which they claimed a statistical association of DDE (a major DDT metabolite) with breast cancer [26]. DDT opponents then used this paper to gain public attention and convince people that DDT caused breast cancer. To be specific, we are talking about anti-insecticide activists, not Dr. Wolff. Years later, with completion of many other studies, and without fanfare or wide publicity, researchers con‐ cluded DDE was not a cause of breast cancer. The WHO reassessment of DDT exposures from indoor spray programs states, "Overall, the association between DDT and breast cancer is inconclusive [27]." Regardless, for many years, anti-DDT activists heralded the 1993 paper as final proof of DDT harm and used it to generate funds and recruit new members to campaigns

Following a different thread of research, Rogan and coauthors reported that DDE was associated with reduced duration of lactation [29,30]. As with the reported association of DDT and breast cancer, this claim was grabbed by the WWF in 1998 and used in the propaganda campaign leading up to the Stockholm Convention on Persistent Organic Pollutants. The stated goal of the WWF campaign was a phase out of DDT by 2007 [28]. In their coverage of this topic, the WWF stated that studies "showed that the duration of lactation was inversely related to the concentration of DDE in milk." Separate from the WWF's use of these claims, the claims were, in part, also the basis for two high-profile publications by Rogan and coauthors in the journals, *Emerging Infectious Diseases* [31] and *The Lancet* [32]. They proposed that the benefits of spraying DDT on house walls to control malaria in Africa would be cancelled out by lowered child survival due to reduced durations of lactation and potential increases in premature births. The claims were used in campaigns against DDT and used to justify more research

Once published, the claims became tools for anti-DDT advocacy. For example, the claim is part of a 2005 Physicians for Social Responsibility (PSR) document about DDT and its use in Kenya. The PSR author states, "DDT may have a substantial impact on infant mortality, by increasing the risk of pre-term birth and by decreasing the duration of breast-feeding after birth. In this paper, Chen and Rogan conclude that DDT may cause comparable increase in infant mortality through these mechanisms compared to the decrease in infant mortality it causes by killing

convince people that DDT is a public health threat.

650 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

mosquitoes and thus reducing malaria cases [33]."

**Example 1:**

**Example 2:**

support.

for DDT elimination [28].

Unfortunately, the false claims against DDT are unabated. One of the more recent and truly tragic examples of a false public image for PHIs occurred in 2009 when researchers in South Africa reported DDT was associated with urogenital birth defects in boys in a region where houses are sprayed with DDT to control malaria [40]. Although the authors, led by Prof. Riana Bornman of the University of Pretoria, suggest that DDT may not have caused the birth defects, the authors still state people should be informed about risks of birth defects if DDT is used. Their interpretations and claims were aired broadly in the print and electronic media in South Africa. The public's concern over the researcher's claims created difficulties for the malaria control program. DDT, through decades of use in South Africa, had already proven its disease preventing capabilities. Given its proven record of performance, it is hardly reasonable to alarm people unless DDT is proven to be seriously harmful. In this case, the weaknesses of the researcher's claims had been addressed in the journal where the paper was published. Richard Grady addressed this issue in the editorial comment that accompanied the Bornman *et al*. paper [40]. Grady stated that issues of association and causality could not be distinguished in the paper. Grady was right; Bornman and coauthor's claims that DDT caused birth defects did not fulfill criteria for establishing a cause-effect relationship. As point of published fact, there were no statistically significant differences in the proportions of malformed genitalia among boys in sprayed and unsprayed villages. Given this fundamental failing, their pronouncements should not have been published and certainly should not have been used to scare the public away from having their houses sprayed. However, attempts in South Africa to scare people about DDT continue even now.

One of the researchers behind the urogenital birth defects claims recently reported on the levels of DDT in breast milk in sprayed villages in South Africa compared to results of an unsprayed village [41]. During the 70+ years of DDT use, many studies of DDT in breast milk have been performed. Based on those reports, it is expected that residents of DDT sprayed houses will have higher quantities of DDT in breast milk than residents of unsprayed villages. It is expected that intake by some infants will exceed the Provisional Tolerable Daily Intake (PTDI) and, in some cases, the residue levels will exceed the Maximum Residue Limit (MRL). In order to exaggerate the importance of their study, the authors emphasized the outlier measurements beyond confidence limits of mean values, e.g., in the abstract they report their statistics include "the highest ΣDDT level ever reported for breast milk from South Africa." Their control village was not sprayed and had no history of ever being sprayed. Yet the authors fail to mention that mean values of residues were at or above the MRL in the unsprayed village. They fail to mention that outlier data points in the control village, as with sprayed villages, exceeded the PTDI. They fail to mention that confidence limits for measurements from the control village overlap those of some sprayed villages. Authors emphasize gender differences in infants and associated levels of DDT in breast milk even though the differences were not statistically significant. They suggest the results require further research. Additionally, authors [41] report that mean levels of DDT had no impact on duration of lactation.

In press coverage of this paper the headlines read, "Researchers measure highest DDT levels in breast milk from South African nursing mothers [42]." In fact, outlier data points can result from erroneous dilutions, tests, conversions, or other parts of the experimental process, or just uncommon natural variation. For these reasons most researchers give outlier data points little weight. Yet the authors of this study used an outlier data point as a hook for grabbing headlines in the popular media. Media coverage went on to state, "In the region where the measurements were carried out, malformed genitalia among boys was significantly more common in areas treated with DDT compared with untreated areas." The assertion that DDT affects male urogenital development is mentioned in the paper, e.g., referring to the 2009 study they state, "Research…identified DDT-associated effects on male urogenital parameters…[41]." Howev‐ er, the statement is misleading because, as described above, there were no statistically significant differences in the proportions of malformed genitalia among boys in sprayed and unsprayed villages.

Their interpretations and claims were aired broadly in the print and electronic media in South Africa. The public's concern over the researcher's claims created difficulties for the malaria control program. DDT, through decades of use in South Africa, had already proven its disease preventing capabilities. Given its proven record of performance, it is hardly reasonable to alarm people unless DDT is proven to be seriously harmful. In this case, the weaknesses of the researcher's claims had been addressed in the journal where the paper was published. Richard Grady addressed this issue in the editorial comment that accompanied the Bornman *et al*. paper [40]. Grady stated that issues of association and causality could not be distinguished in the paper. Grady was right; Bornman and coauthor's claims that DDT caused birth defects did not fulfill criteria for establishing a cause-effect relationship. As point of published fact, there were no statistically significant differences in the proportions of malformed genitalia among boys in sprayed and unsprayed villages. Given this fundamental failing, their pronouncements should not have been published and certainly should not have been used to scare the public away from having their houses sprayed. However, attempts in South Africa to scare people

One of the researchers behind the urogenital birth defects claims recently reported on the levels of DDT in breast milk in sprayed villages in South Africa compared to results of an unsprayed village [41]. During the 70+ years of DDT use, many studies of DDT in breast milk have been performed. Based on those reports, it is expected that residents of DDT sprayed houses will have higher quantities of DDT in breast milk than residents of unsprayed villages. It is expected that intake by some infants will exceed the Provisional Tolerable Daily Intake (PTDI) and, in some cases, the residue levels will exceed the Maximum Residue Limit (MRL). In order to exaggerate the importance of their study, the authors emphasized the outlier measurements beyond confidence limits of mean values, e.g., in the abstract they report their statistics include "the highest ΣDDT level ever reported for breast milk from South Africa." Their control village was not sprayed and had no history of ever being sprayed. Yet the authors fail to mention that mean values of residues were at or above the MRL in the unsprayed village. They fail to mention that outlier data points in the control village, as with sprayed villages, exceeded the PTDI. They fail to mention that confidence limits for measurements from the control village overlap those of some sprayed villages. Authors emphasize gender differences in infants and associated levels of DDT in breast milk even though the differences were not statistically significant. They suggest the results require further research. Additionally, authors [41] report

In press coverage of this paper the headlines read, "Researchers measure highest DDT levels in breast milk from South African nursing mothers [42]." In fact, outlier data points can result from erroneous dilutions, tests, conversions, or other parts of the experimental process, or just uncommon natural variation. For these reasons most researchers give outlier data points little weight. Yet the authors of this study used an outlier data point as a hook for grabbing headlines in the popular media. Media coverage went on to state, "In the region where the measurements were carried out, malformed genitalia among boys was significantly more common in areas treated with DDT compared with untreated areas." The assertion that DDT affects male urogenital development is mentioned in the paper, e.g., referring to the 2009 study they state,

that mean levels of DDT had no impact on duration of lactation.

about DDT continue even now.

652 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Presented in three examples above is clear and unambiguous demonstration of orchestrat‐ ed and non-scientific campaigns against PHIs, and DDT in particular. Claims that DDT causes one sort of harm or another are repeated in anti-insecticide propaganda even after published studies show the claims are false, or published rebuttals draw attention to errors in data analyses or research interpretations. A common part of these campaigns is how activists use the term "association" or "associated" as meaning there is a cause-effect relationship between an exposure and disease. In fact, these terms relate only to a statistical association that is often an artifact of study design or a product of systematic bias. Such issues as bias are of particular concern, and are discussed at length in David Savitz's book *Interpreting epidemiologic evidence* [43].

In the history of efforts to preserve use of DDT for public health programs, this chain of events has been repeated over and over, with claims of causation eventually being disproven, but not before they were used to generate funds, recruit new members to anti-insecticide campaigns, and change public health policies. Last but not least, each change in disease control policy has weakened global capacities to control malaria and other diseases. Almost every change is a result of anti-insecticide propaganda that misrepresents the scientific process, as revealed for the three examples described above:

	- **◦** More common with higher DDT exposure and less common with lower exposure,
	- **◦** Less common prior to DDT exposure and appeared or increased in frequency with onset of DDT exposure, and
	- **◦** More common with DDT exposure and less common once DDT use was stopped.

dangers of DDT and to cast further attention on the results of weak studies. Sadly, the two studies are being used to scare people who live in malarious regions.

## **9. Dichotomies in patterns/trends of human disease with/without DDT**

Decades ago, developed countries used extraordinary quantities of DDT. The richer countries placed DDT in the human food chain through its heavy agricultural use at that time. More explicitly, DDT was used in the environment, around houses, and intensively inside homes. It is now 40 years since being banned for most uses in the US and other developed countries. Yet, recent claims of DDT causing disease or birth defects are not reflected in the historical medical reports and vital statistics for regions and years of broad and heavy DDT usage. The lack of proof that DDT caused harm to human health back in the days of intense exposures goes far in explaining why, to this day, there is no evidence that human health has been improved in any way by stopping public health uses of DDT.

There is a dichotomy in the huge benefit from use of DDT to prevent diseases and deaths versus no definable benefit from stopping its use. For slightly more than three decades (1945-1979) many malaria endemic countries maintained house spray programs. That era was followed by decades, from 1979 through to present time, when most of the same countries phased house spraying out of national programs. The result is a historical record of years when DDT and other insecticides were sprayed in houses followed by almost as many years when spraying was greatly decreased or stopped entirely. An even more drastic stoppage of DDT spraying occurred in agriculture. The dichotomies of outcomes are listed in Table 1.


**Table 1.** Grid of cause-effect relationships for public health outcomes during periods of use and non-use of DDT in public health programs.

As explained for smoking and human cancers, the relationship of declining risk with reduced exposure attests to a true and meaningful causal relationship. An inverse finding of increasing risk with increasing exposure to a causative agent also attests to a true and meaningful causal relationship. These indicators of causation make it all the more amazing that through decades of anti-insecticide advocacy, insecticide opponents have documented no obvious public health harm as a result of DDT residues on house walls. Likewise, they have documented no meaningful improvements in health or reduced deaths as a direct result of having eliminated DDT exposure by ending house spray programs. These failings suggest DDT opponents have not been challenged to balance an equation of measurable benefits from preventing the use of DDT and other public health insecticides versus the measurable increases in human deaths and diseases, like malaria, as consequence of stopping use of public health insecticides.

## **10. Models for modern advocacy against PHIs**

dangers of DDT and to cast further attention on the results of weak studies. Sadly, the two

**9. Dichotomies in patterns/trends of human disease with/without DDT**

Decades ago, developed countries used extraordinary quantities of DDT. The richer countries placed DDT in the human food chain through its heavy agricultural use at that time. More explicitly, DDT was used in the environment, around houses, and intensively inside homes. It is now 40 years since being banned for most uses in the US and other developed countries. Yet, recent claims of DDT causing disease or birth defects are not reflected in the historical medical reports and vital statistics for regions and years of broad and heavy DDT usage. The lack of proof that DDT caused harm to human health back in the days of intense exposures goes far in explaining why, to this day, there is no evidence that human health has been

There is a dichotomy in the huge benefit from use of DDT to prevent diseases and deaths versus no definable benefit from stopping its use. For slightly more than three decades (1945-1979) many malaria endemic countries maintained house spray programs. That era was followed by decades, from 1979 through to present time, when most of the same countries phased house spraying out of national programs. The result is a historical record of years when DDT and other insecticides were sprayed in houses followed by almost as many years when spraying was greatly decreased or stopped entirely. An even more drastic stoppage of DDT spraying

**1946-79 (period of DDT spraying**

from insecticide exposures in houses

Reductions in malaria infections and deaths as a consequence of DDT on

**Table 1.** Grid of cause-effect relationships for public health outcomes during periods of use and non-use of DDT in

As explained for smoking and human cancers, the relationship of declining risk with reduced exposure attests to a true and meaningful causal relationship. An inverse finding of increasing risk with increasing exposure to a causative agent also attests to a true and meaningful causal relationship. These indicators of causation make it all the more amazing that through decades of anti-insecticide advocacy, insecticide opponents have documented no obvious public health harm as a result of DDT residues on house walls. Likewise, they have documented no meaningful improvements in health or reduced deaths as a direct result of having eliminated

**1980-present (period when DDT spraying was reduced or stopped)**

Increases in malaria infections and deaths as house spraying of DDT is

Reductions in poisonings and deaths as house spraying is

eliminated

eliminated

studies are being used to scare people who live in malarious regions.

654 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

improved in any way by stopping public health uses of DDT.

**Benefits versus harms of public**

Benefits from using insecticides to control malaria and other diseases

**health insecticides**

public health programs.

occurred in agriculture. The dichotomies of outcomes are listed in Table 1.

**in houses)**

house walls

Harm from insecticide exposures Increases in poisonings and deaths

Now, on the fiftieth anniversary of *Silent Spring*, the goal of reducing or eliminating DDT and other PHIs is, and has been for decades, entrenched in environmental advocacy literature and in bureaucracies of the UN. In the case of DDT, this goal was clearly enunciated by UNEP in 2000:

*WHO and UNEP have joined forces to protect both human health and the environment by promoting strategies to reduce malaria*

*with reduced reliance on DDT. An important first step was taken in March 2000 through a WHO-convened Regional Consultation*

*to Prepare African Countries Towards Reduction of Reliance on DDT for Malaria Control, with UNEP support. [44]*

For UNEP bureaucrats, the statement codifies the environmentalist's belief that small quanti‐ ties of DDT sprayed on house walls harms the environment. Also it codifies the belief that DDT is not needed in malaria control programs. In both cases, the bureaucrats are wrong.

Information presented in Figures 2 and 3 illustrate the enormous danger of forcing countries to abandon DDT and other PHIs. Since the early 1980s over 10.5 million preventable malaria cases were recorded above and beyond what might have occurred if Brazil had not abandoned DDT. There were no DDT resistance issues that caused malaria program managers to abandon DDT, there were no important studies showing DDT repellent properties did not work, there were no malaria trend analyses showing a lack of efficacious control with DDT sprayed walls, and there were no cost-effective insecticides that could be used instead. DDT was abandoned in Brazil and in other countries of South America as a consequence of global environmental policies and anti-insecticide campaigns. DDT was not eliminated from Peru's malaria program until the late 1980s. Peru's malaria problems grew exponentially worse immediately after the country dispensed with DDT spraying. These disastrous outcomes were repeated in many countries.

With the beginning of the 21st Century and infused with renewed support and improved targeting in application of control efforts, malaria control programs are beginning to make some progress. But further progress is needed and malaria continues as a huge public health problem. Meanwhile, as in the 1960s, insecticide opponents are poised to counter the recent progress against malaria. We will now focus on specific tactics that are and will continue to be used in the anti-insecticide campaigns.

As we have described, the first strategy of insecticide opposition is to convince people that DDT or other PHIs are harmful. An important tactic for achieving success is to develop and broadcast widely and repeatedly a list of diverse claims of chemical harm. We have already described examples of how this tactic is implemented. A list of diverse sources of harm is not easy to counter. When an authoritative rebuttal of one claim occurs, the other claims are still in play. Additionally, a broad list of claims allows campaigners to tailor platforms for constit‐ uencies, advancing one set of claims with one constituency and a different combination for another. Another tactic is to focus on the most recent study hinting at some health impact of the chemical. It is easier to get the popular media interested in a study that can be presented as a new and sensational finding--a favorite theme of science writers. Regardless, a list of multiple claims of harm is hardly sufficient to achieve a ban of a truly useful PHI. Thus, the second strategy of convincing people the chemical is not needed becomes extraordinarily important. The tactic behind this goal is to argue that alternative chemicals or methods can be used as replacements. We will present two examples of tactics employed in support of this strategy. The third strategy is to predict that grave harm will occur if the chemical continues to be used.

The success of Rachel Carson's *Silent Spring* serves as a model for the three strategies. In *Silent Spring*, Rachel Carson used the strategies on her primary target, DDT. She described a very large list of potential adverse effects of insecticides, including human health and ecological effects. She argued that insecticides were not really needed because their use selected for super bugs that were resistant to the insecticides and that the chemicals only made problems worse. Last but not least, she described scary scenarios of severe harm with continued use of DDT and other insecticides.

Carson focused attention on examples of overuse or misuse of DDT and other insecticides and described the effects of their misuse. Nevertheless, the misuse of chemicals is not a valid reason for banning an insecticide. In the case of DDT, a successful campaign to eliminate it requires that even its proper use will cause a large and systematic adverse effect. However, the proper public health uses of DDT yield no large and systematic adverse effects. Absent such adverse actions, the activists must then rely on claims about insidious effects, particularly insidious effects that scientists will find difficult to prove one way or the other, and that activists can use to predict a future catastrophe.

Rachel Carson relied heavily on possible insidious chemical actions as a means of alarming and scaring the public. Many of those who joined the resulting campaign to ban DDT and other insecticides made extensive use of claims of insidious effects. In particular Carson alluded to insidious effects on reproduction. Her assertions were amplified by the popular press and became part of the public perception about insecticides. Although those perceptions are wrong, they are firmly entrenched in anti-insecticide propaganda.

The three strategies, while largely bogus in terms of their scientific underpinnings, were very effective in anti-insecticide campaigns. The strategies are still used today. Rogan and Chen used these strategies in their two papers against DDT [31,32]. The authors presented strategy number two in the form of a superficial review of the role of DDT in malaria control. They strove to cast doubt on DDT's value in modern malaria control programs. They admitted that DDT had been very effective in the past, but then argued that malaria control programs no longer needed it and alternative methods of control should be used. Rogan and Chen also employed the first strategy of environmentalism [32]. Their list of potential harms from DDT exposures included toxic effects, neurobehavioral effects, cancers, decrements in various facets of reproductive health, decrements in infant and child development, and immunology and DNA damage. To get the paper past reviewers they presented balanced coverage of their diverse claims of harm, and, as consequence, had to conclude they could not prove that DDT caused any harm at all. Amazingly, they promptly negated this honest conclusion by asserting that if DDT is used for malaria control then great harm might occur. So, while not proving DDT causes harm, the authors still predict severe harm if it is used.

As we have described, the first strategy of insecticide opposition is to convince people that DDT or other PHIs are harmful. An important tactic for achieving success is to develop and broadcast widely and repeatedly a list of diverse claims of chemical harm. We have already described examples of how this tactic is implemented. A list of diverse sources of harm is not easy to counter. When an authoritative rebuttal of one claim occurs, the other claims are still in play. Additionally, a broad list of claims allows campaigners to tailor platforms for constit‐ uencies, advancing one set of claims with one constituency and a different combination for another. Another tactic is to focus on the most recent study hinting at some health impact of the chemical. It is easier to get the popular media interested in a study that can be presented as a new and sensational finding--a favorite theme of science writers. Regardless, a list of multiple claims of harm is hardly sufficient to achieve a ban of a truly useful PHI. Thus, the second strategy of convincing people the chemical is not needed becomes extraordinarily important. The tactic behind this goal is to argue that alternative chemicals or methods can be used as replacements. We will present two examples of tactics employed in support of this strategy. The third strategy is to predict that grave harm will occur if the chemical continues

The success of Rachel Carson's *Silent Spring* serves as a model for the three strategies. In *Silent Spring*, Rachel Carson used the strategies on her primary target, DDT. She described a very large list of potential adverse effects of insecticides, including human health and ecological effects. She argued that insecticides were not really needed because their use selected for super bugs that were resistant to the insecticides and that the chemicals only made problems worse. Last but not least, she described scary scenarios of severe harm with continued use of DDT

Carson focused attention on examples of overuse or misuse of DDT and other insecticides and described the effects of their misuse. Nevertheless, the misuse of chemicals is not a valid reason for banning an insecticide. In the case of DDT, a successful campaign to eliminate it requires that even its proper use will cause a large and systematic adverse effect. However, the proper public health uses of DDT yield no large and systematic adverse effects. Absent such adverse actions, the activists must then rely on claims about insidious effects, particularly insidious effects that scientists will find difficult to prove one way or the other, and that activists can use

Rachel Carson relied heavily on possible insidious chemical actions as a means of alarming and scaring the public. Many of those who joined the resulting campaign to ban DDT and other insecticides made extensive use of claims of insidious effects. In particular Carson alluded to insidious effects on reproduction. Her assertions were amplified by the popular press and became part of the public perception about insecticides. Although those perceptions are wrong,

The three strategies, while largely bogus in terms of their scientific underpinnings, were very effective in anti-insecticide campaigns. The strategies are still used today. Rogan and Chen used these strategies in their two papers against DDT [31,32]. The authors presented strategy number two in the form of a superficial review of the role of DDT in malaria control. They strove to cast doubt on DDT's value in modern malaria control programs. They admitted that

to be used.

and other insecticides.

656 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

to predict a future catastrophe.

they are firmly entrenched in anti-insecticide propaganda.

Rogan and Chen end their paper with a call for more research. One could conclude that the intent of the whole paper is merely to lobby for research dollars to better define DDT harm, and what's the harm in that? Surely increasing knowledge is a fine goal. However, having engaged issues of malaria control and what should or should not be done to control the disease, specifying more research funds for research on potential harms of insecticide exposures is unjustified. Large numbers of children and pregnant women die from malaria every year, and the disease sickens hundreds of millions more. Yet, not one death or illness can be attributed to an exposure to the public health use of DDT. Figure 1 illustrates growth in DDT research, with numbers of published papers doubling from one decade to the next. Almost all papers are in environmental literature and many are on potential adverse effects of DDT. Only a small proportion of papers deal with malaria and DDT. It bears repeating that DDT is a spatial repellent, and hardly an insecticide at all, but a search on DDT and repellents will produce even fewer papers. This disparity represents an egregiously disproportionate emphasis on non-sources of harm compared to the enormous harm of malaria.

The US used DDT to eradicate malaria. After malaria disappeared as an endemic disease people in the US became richer. They built better and more enclosed houses. They screened their windows and doors. They air-conditioned their homes. Also, during those early years, the US developed an immense arsenal of mosquito control tools and chemicals. Today, when there is a risk of mosquito borne disease, urban and rural areas can bring this arsenal to bear and quickly eliminate risks. And, as illustrated by aerial spray missions in the aftermath of hurricane Katrina, they can afford to do so. Yet, those modern and very expensive chemicals are not what protect the US from introductions of the old diseases. Use of those chemicals can only respond to a threat; it cannot prevent the old diseases from being reintroduced. What protects US populations is their enclosed, screened, air-conditioned housing, the physical representation of their wealth. Their wealth and living standards stop dengue at the border with Mexico, not the use of insecticides. Stopping mosquitoes from entering and biting people inside their homes is critical in the prevention of malaria and many other insect-borne diseases. This is what DDT does for poor people in poor countries. It stops large proportions of mosquitoes from entering houses. It is, in fact, a form of chemical screening, and until people in disease endemic countries can afford properly enclosed houses and physical screening, or it is provided for them, chemical screening is the only kind they have.

DDT is a protective tool that has been taken away from countries around the world, mostly due to governments acceding to the whims of the anti-pesticide wing of environmentalism, but it is not only the anti-pesticide wing that lobbies against DDT. The activists have a sympathetic lobbying ally in the pesticide industry. DDT opposition was made clear in writings of those within the insecticide industry; a Bayer official stated:

*[I speak] Not only as the responsible manager for the vector control business in Bayer, being the market leader in vector control and pointing out by that we know what we are talking about and have decades of experiences in the evolution of this very particu‐ lar market. [but] Also as one of the private sector representatives in the RBM Partnership Board and being confronted with that discussion about DDT in the various WHO, RBM et al circles. So you can take it as a view from the field, from the operational commercial level - but our companies [sic] point of view. I know that all of my colleagues from other primary manufacturers and internationally operating companies are sharing my view. [45]*

#### The official goes on to say that,

*DDT use is for us a commercial threat (which is clear, but it is not that dramatical [sic] because of limited use), it is mainly a public image threat.*

However the most damming part of this message was the statement that,

*...we fully support EU to ban imports of agricultural products coming from countries using DDT...*

This email message from Bayer, one of the largest global manufacturers of alternatives to DDT, provides clear evidence of industry applying international and developed country pressures to stop poor countries from using DDT to control malaria. This message also shows the complicity of the insecticide industry in those internationally orchestrated efforts.

The environmental movement lobbied for a WHA resolution that required countries to move away from using insecticides in disease control altogether [23]. The WHA is the premier policysetting forum for all health issues and is the governing body of the WHO. At that time, 1997, there was no evidence that vector-borne diseases could be controlled without man-made insecticides. The same is true today. The resolution was adopted by the WHA in 1997. Essentially, the lobbying of environmental groups elevated politics and anti-insecticide sentiment above scientific evidence and left hundreds of millions at high risk of death and illness from entirely preventable diseases. As we will show in the next section, UNEP has a particularly odious history of elevating environmental politics over science.

## **11. UNEP's war against PHIs**

DDT is a protective tool that has been taken away from countries around the world, mostly due to governments acceding to the whims of the anti-pesticide wing of environmentalism, but it is not only the anti-pesticide wing that lobbies against DDT. The activists have a sympathetic lobbying ally in the pesticide industry. DDT opposition was made clear in

*[I speak] Not only as the responsible manager for the vector control business in Bayer, being the market leader in vector control and pointing out by that we know what we are talking about and have decades of experiences in the evolution of this very particu‐ lar market. [but] Also as one of the private sector representatives in the RBM Partnership Board and being confronted with that discussion about DDT in the various WHO, RBM et al circles. So you can take it as a view from the field, from the operational commercial level - but our companies [sic] point of view. I know that all of my colleagues from other primary manufacturers and*

*DDT use is for us a commercial threat (which is clear, but it is not that dramatical [sic] because of limited use), it is mainly a*

This email message from Bayer, one of the largest global manufacturers of alternatives to DDT, provides clear evidence of industry applying international and developed country pressures to stop poor countries from using DDT to control malaria. This message also shows the

The environmental movement lobbied for a WHA resolution that required countries to move away from using insecticides in disease control altogether [23]. The WHA is the premier policysetting forum for all health issues and is the governing body of the WHO. At that time, 1997, there was no evidence that vector-borne diseases could be controlled without man-made insecticides. The same is true today. The resolution was adopted by the WHA in 1997. Essentially, the lobbying of environmental groups elevated politics and anti-insecticide sentiment above scientific evidence and left hundreds of millions at high risk of death and illness from entirely preventable diseases. As we will show in the next section, UNEP has a

complicity of the insecticide industry in those internationally orchestrated efforts.

particularly odious history of elevating environmental politics over science.

However the most damming part of this message was the statement that,

*...we fully support EU to ban imports of agricultural products coming from countries using DDT...*

writings of those within the insecticide industry; a Bayer official stated:

*internationally operating companies are sharing my view. [45]*

658 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

The official goes on to say that,

*public image threat.*

The UN Stockholm Convention on POPs, which came into force in 2004, governs the use of DDT. DDT is the only chemical under the POPs Convention that is granted an exemption for use in public health. It is against this background that the Stockholm Convention Secretariat (the Secretariat) and the financial mechanism of the Convention, the GEF, the UNEP, and groups within the Pan American Health Organization (PAHO) and WHO, have engaged in scientific malfeasance to achieve political goals. UNEP's target goal in 2007, now removed from the UNEP website, was DDT elimination by 2020.1

The GEF was established in 1991 and is a partnership of 10 agencies, including the World Bank, which houses the GEF. The GEF has allocated over \$9bn in funds for projects with the aim of improving the environment and has raised over \$40bn from other partners for its projects. At stake is not only increased power over the use of chemicals for the control of diseases but also the reputational benefits of achieving a goal deemed desirable by environmental groups. In addition, one cannot discount the fact that many millions of dollars are programmed by numerous governments via the UN system to rid the world of POPs and find alternatives to DDT. Control over the use of insecticides for public health also gives agencies control over, and benefit from, these funds.

UNEP's and GEF's misrepresentations of scientific records against the use of DDT and other PHIs were exposed in a peer-reviewed paper in *Research and Reports in Tropical Medicine* [46]. The paper exposed the false claims about an insecticide-free malaria control project managed by UNEP and financed by GEF in Mexico and Central America (Mexico/CA). The project was designed to demonstrate successful control of malaria through use of "environmentally sound" methods without DDT and other insecticides. Almost inevitably, the projects' backers claimed it achieved this objective. A proper analysis of epidemiologic data, however, revealed no such success; reductions in malaria cases and deaths in the region were achieved primarily through pharmacosuppression (therapeutic and prophylactic use of anti-malarial drugs). Claims that UNEP's environmental interventions were effective were invalid.

The project, Regional Program of Action and Demonstration of Sustainable Alternatives to DDT for Malaria Vector Control in Mexico and Central America (Mexico/CA Project), was conducted in eight countries (Belize, Costa Rica, Guatemala, Honduras, Mexico, Nicaragua, Panama and El Salvador). It was executed by PAHO's Sustainable Development and Envi‐ ronmental Health Program and implemented by UNEP. It was co-financed by the GEF with additional support from the Commission for Environmental Cooperation of North America (CEC), PAHO, and participating country governments. The project's aim was to improve coordination and national capacity so that new, integrated disease vector (mosquito) control techniques could be implemented, thereby eliminating the need for DDT reintroduction [47]. The objectives of the project (as stated by UNEP) were to: "Demonstrate feasibility of integrated

<sup>1</sup> The Stockholm Convention is a UN Convention that arose from UN Environment Program efforts to control and/or ban the production and use of certain persistent organic pollutants. PAHO is an international public health agency and is the Regional Office for the Americas of the WHO and part of the UN.

and environment-friendly methods for malaria vector control without the use of DDT," and "assess the effects of these methods on malaria occurrence [48]."

According to UNEP, the key interventions in the project were as follows: 1) Reduction of contact between mosquitoes and people via treated bed nets; meshes on doors and windows; the planting of repellent trees like neem and oak; and the liming of households. 2) Control of breeding sites by clearing vegetation; draining stagnant water, ditches and channels; and the use of biological controls such as fish and bacteria in some countries. 3) Elimination of places near houses that attract and shelter mosquitoes through, for example, the cleaning and tidying up of areas in and around homes, alongside the promotion of personal hygiene [49].

The project's final evaluation, published in November 2009, mentions various pharmaceutical methods of prophylaxis and treatment within human populations [50]. However, those methods were ongoing components of malaria control in each country prior to the Mexico/CA Project, operating nationally in each country before and during the project. The available evidence suggests national malaria control programs (NMCPs) functioned regardless of the presence or absence of UNEP's project personnel. Thus, anti-malarial treatment (the major component of the NMCPs) in demonstration areas was not part of the epidemiological evaluation of the Mexico/CA Project [51]. Likewise, use of ITNs had no obvious definable role in the Mexico/CA Project. Project successes are therefore advertised as having been achieved without mention of the accompanying use of insecticides.

The project included demonstration areas, where the GEF environmental interventions would be implemented, as well as control areas within epidemiologically similar areas, where the interventions would be excluded, for proper comparisons [51]. As stated by Cesar Chelala, medical consultant affiliated with the Mexico/CA Project, demonstration areas were selected "based on the high incidence of transmission and the persistence of malaria in those places [52]."

An epidemiological evaluation identified 202 demonstration areas and 51 control areas [51]. The former included a total population of 159,018 and the latter 50,834.

The public statements regarding the Mexico/CA Project proclaimed dramatic and very impressive reductions in malaria cases for its environmentally benign interventions. The final report of the Mexico/CA Project, published by the environmental sector of PAHO in December 2008, claims "a 63% reduction in the number of people with the disease without using DDT or any other type of pesticide [53]."

These statistics and claims of success were repeated in an official press release issued by UNEP, WHO and GEF in May 2009 [54]. UNEP Executive Director, Achim Steiner, also repeated these claims and characterized the project as "calculated and tested science [49]." Similar claims have been made in the popular media [52] and used by anti-insecticide activist groups as evidence that malaria control is possible without insecticides [55].

Regrettably, the claims of malaria control through application of GEF interventions were incorrect and fundamentally misleading.

Countries in Latin America were forced away from using DDT in compliance with the North American Free Trade Agreement (NAFTA), wherein the CEC pressured Mexico in the mid-1990s to stop production and use of DDT [56]. Without DDT, countries used more expensive insecticides, which had to be sprayed more frequently, creating problems for malaria control [57]. Over time, the countries in Central America moved to greater use of pharmacosuppression. Malaria cases have fallen as a result of this widespread use of malaria treatments, but not through the environmental controls touted by the UN. Officials of GEF, UNEP and the Secretariat, however, ignored the use of pharmacosuppression in their discus‐ sion of successful malaria control in Mexico/CA. Furthermore, these officials falsely attribute changes in malaria burdens to GEF's environmental interventions. A separate epidemiological evaluation which was designed to measure any changes in disease rates, found no statistical differences in malaria rates in demonstration areas versus rates in control areas, and this was consistent across all eight countries [51]. Malaria rates in most countries were falling, but with no difference between the demonstration areas and controls, the decline cannot be attributed to the environmental interventions. But UNEP, GEF, the Secretariat and other officials ignored those findings. Furthermore, despite the fact that the control areas were a crucially important part of the project, they were not even mentioned in the 2008 final report [53]. Ultimately, the successful reduction of malaria was most likely entirely due to pharmacosuppression.

and environment-friendly methods for malaria vector control without the use of DDT," and

According to UNEP, the key interventions in the project were as follows: 1) Reduction of contact between mosquitoes and people via treated bed nets; meshes on doors and windows; the planting of repellent trees like neem and oak; and the liming of households. 2) Control of breeding sites by clearing vegetation; draining stagnant water, ditches and channels; and the use of biological controls such as fish and bacteria in some countries. 3) Elimination of places near houses that attract and shelter mosquitoes through, for example, the cleaning and tidying

The project's final evaluation, published in November 2009, mentions various pharmaceutical methods of prophylaxis and treatment within human populations [50]. However, those methods were ongoing components of malaria control in each country prior to the Mexico/CA Project, operating nationally in each country before and during the project. The available evidence suggests national malaria control programs (NMCPs) functioned regardless of the presence or absence of UNEP's project personnel. Thus, anti-malarial treatment (the major component of the NMCPs) in demonstration areas was not part of the epidemiological evaluation of the Mexico/CA Project [51]. Likewise, use of ITNs had no obvious definable role in the Mexico/CA Project. Project successes are therefore advertised as having been achieved

The project included demonstration areas, where the GEF environmental interventions would be implemented, as well as control areas within epidemiologically similar areas, where the interventions would be excluded, for proper comparisons [51]. As stated by Cesar Chelala, medical consultant affiliated with the Mexico/CA Project, demonstration areas were selected "based on the high incidence of transmission and the persistence of

An epidemiological evaluation identified 202 demonstration areas and 51 control areas [51].

The public statements regarding the Mexico/CA Project proclaimed dramatic and very impressive reductions in malaria cases for its environmentally benign interventions. The final report of the Mexico/CA Project, published by the environmental sector of PAHO in December 2008, claims "a 63% reduction in the number of people with the disease without using DDT or

These statistics and claims of success were repeated in an official press release issued by UNEP, WHO and GEF in May 2009 [54]. UNEP Executive Director, Achim Steiner, also repeated these claims and characterized the project as "calculated and tested science [49]." Similar claims have been made in the popular media [52] and used by anti-insecticide activist groups as evidence

Regrettably, the claims of malaria control through application of GEF interventions were

The former included a total population of 159,018 and the latter 50,834.

up of areas in and around homes, alongside the promotion of personal hygiene [49].

"assess the effects of these methods on malaria occurrence [48]."

660 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

without mention of the accompanying use of insecticides.

that malaria control is possible without insecticides [55].

malaria in those places [52]."

any other type of pesticide [53]."

incorrect and fundamentally misleading.

One might wonder why a control program would require insecticides and vector control if pharmacosuppression is such a powerful method of malaria control. This is a complex issue, but it is important to note that even though reductions in malaria cases have been achieved in Mexico and Central America, their model of widespread distribution of the anti-malarial drugs chloroquine and primaquine is not transferable elsewhere and may not be sustainable over the long-term. As a model for malaria control, it is not transferable for several reasons. First, widespread drug resistance to chloroquine in Africa and Southeast Asia would mean the intervention would be largely useless. Second, primaquine is a radical treatment for vivax malaria, whereas in Africa over 90 percent of malaria cases are caused by falciparum malaria, the more deadly form of the disease.2 Third, pharmacosuppression is expensive and requires more sophisticated health systems than exist in most of Africa, where the greatest burden of malaria lies. So even if UNEP, GEF and their partners were straightforward about the real reasons for the declines in malaria in the project areas, there would be no reasonable argument to claim that pharmacosuppression has any application in most other endemic areas.

Global malaria control policy gives scant notice to pharmacosuppression. In fact, it appears that global leaders are intent on ignoring how countries of the Americas are making use of pharmacosuppression. Yet, and as commonly observed in reports from South America, the only cost-effective insecticides (pyrethroids) they have must be sprayed so frequently as to be of limited value. Thus, countries of the Americas really have no viable cost-effective options for use of PHIs. In absence of an insecticidal solution then, pharmacosuppression becomes the best option for effectively reducing malaria caseloads.

<sup>2</sup> In addition, there are concerns about the side effects of using primaquine among people with G6PD deficiency. See Baird K. Eliminating malaria – all of them.Lancet 2010;376(9756): 1883-5. http://www.thelancet.com/journals/lancet/ article/PIIS0140-6736%2810%2961494-8/fulltext (accessed 19 September 2012).

If we assume there is a decision to keep quiet on how malaria is being controlled in absence of insecticides, then it is easier to understand why there is less transparency in malaria data for the Americas. Historically PAHO openly reported statistics on the numbers and types of curative treatments dispensed per year in each country. However, transparency of malaria control statistics is down from just two or three years ago. A visit to PAHO's website on interactive malaria control data for the Americas will reveal no data on numbers of treatments with chloroquine or primaquine. Indeed, the only data that is readily available is on use of ACTs for treating cases of falciparum malaria.

## **12. Conclusion**

We have described the systematic and often coordinated campaigns by activists, scientists and UN agencies against essential tools for disease control. We will conclude here with statements that bring our analyses full circle. Rachel Carson started broad scale unscientific attacks on DDT in 1962, with publication of her book, *Silent Spring*. The claims of harm by exposures to DDT, as we describe in this chapter, were not and are not true. In other words, the attributed harms are not caused by DDT exposures. Yet, presented in a 2012 article titled "Critisism [sic] of Carson over DDT unfounded" is a denial of any responsibility whatsoever for the reductions and eliminations of DDT in disease control programs as legacy of her book. In their article the Rachel Carson Council makes the following claims: "DDT has been associated with serious adverse effects in humans, including reduced sperm production in men, shorter lactation times and increasing numbers of pre-term births in women,… breast cancer...[58]."

We ask the reader to compare their claims with those we describe as not meeting even minimal criteria for cause-effect relationships. So the Rachel Carson Council denies responsibility for harm inflicted by Carson's anti-DDT rhetoric, while, at the same time, it continues to imple‐ ment her strategies for DDT elimination and employs her tactics of falsifying the scientific record to scare the public. Amazingly, when the false statements and fear tactics employed by anti-DDT campaigners succeed in stopping use of DDT to protect health and save lives, the anti-PHI advocacy community, as revealed in the Rachel Carson Council's denial of respon‐ sibilities, expects the public to think they had no role in such inhumanely disastrous changes in public health policies. As we have shown, they are, in fact, the very cause of those changes in policy.

We have shown that vast sums of money, mostly from taxpayers, have been spent over many decades undermining and often directly attacking the use of DDT in life-saving disease control programs. These vast expenditures have not delivered alternative strategies or tools to replace DDT. The few alternatives that disease control programs do have for some malaria-endemic regions pale in comparison to the powerful life-saving properties of DDT. It almost goes without saying that if the disease control tool in question were not DDT but were a vaccine or a medicine, there would be a sense of outrage in the general public along with well-funded advocacy to preserve and protect a tool that has the power to save lives. Yet such is the power of the environmental movement, that aside from a few outspoken scientists and individuals, there has been almost no response from the malaria community or the wider public health community. The strategies employed by anti-DDT activists are anti-science and rely on distortions, half-truths and sometimes outright lies. Ordinarily such behavior would be roundly criticized, yet because DDT is being attacked, such actions are given a free pass.

We are greatly concerned that the majority of private insecticide companies far from opposing the unscientific agenda of the anti-DDT campaigns, support them. These companies may be merely motivated to sell more of their own product, but this is surely one of the most shortsighted strategies imaginable. We already see a growing number of studies finding associa‐ tions between alternatives to DDT and possible human health harm. As with DDT, the antiinsecticide activists are starting to hype and spread fear about these associations. As the Stockholm Convention adds more and more chemicals to its list of banned or controlled substances, and as the UNEP flexes its regulatory muscles, we fully expect it will become more and more difficult to produce, trade, transport and use all PHIs. It is precisely because of such restrictions that countries of the Americas have had to adopt programs of mass drug distri‐ butions (pharmacosuppression) to control vivax malaria. Basically those countries have no cost-effective options for use of PHIs. Continuation of these anti-PHI practices, as we have learned from history, will inflict great harm on disease control efforts and eventually exact a heavy cost in lives from some of the poorest and most vulnerable communities on earth.

We hope this chapter has shed some light on the strategies and tactics of environmental groups, activists, scientists and UN agencies. Well-established patterns of behavior have been set with these groups and individuals and we hope that the malaria community and the wider public health community begin to recognize these patterns and begin to more effectively investigate and respond to claims against PHIs long before the claims become the basis for further restrictions on the efficacy of disease control programs.

## **List of the acronyms used in the text**

ACT-- artemisinin-based combination therapies

API—annual parasite index

If we assume there is a decision to keep quiet on how malaria is being controlled in absence of insecticides, then it is easier to understand why there is less transparency in malaria data for the Americas. Historically PAHO openly reported statistics on the numbers and types of curative treatments dispensed per year in each country. However, transparency of malaria control statistics is down from just two or three years ago. A visit to PAHO's website on interactive malaria control data for the Americas will reveal no data on numbers of treatments with chloroquine or primaquine. Indeed, the only data that is readily available is on use of

We have described the systematic and often coordinated campaigns by activists, scientists and UN agencies against essential tools for disease control. We will conclude here with statements that bring our analyses full circle. Rachel Carson started broad scale unscientific attacks on DDT in 1962, with publication of her book, *Silent Spring*. The claims of harm by exposures to DDT, as we describe in this chapter, were not and are not true. In other words, the attributed harms are not caused by DDT exposures. Yet, presented in a 2012 article titled "Critisism [sic] of Carson over DDT unfounded" is a denial of any responsibility whatsoever for the reductions and eliminations of DDT in disease control programs as legacy of her book. In their article the Rachel Carson Council makes the following claims: "DDT has been associated with serious adverse effects in humans, including reduced sperm production in men, shorter lactation times

We ask the reader to compare their claims with those we describe as not meeting even minimal criteria for cause-effect relationships. So the Rachel Carson Council denies responsibility for harm inflicted by Carson's anti-DDT rhetoric, while, at the same time, it continues to imple‐ ment her strategies for DDT elimination and employs her tactics of falsifying the scientific record to scare the public. Amazingly, when the false statements and fear tactics employed by anti-DDT campaigners succeed in stopping use of DDT to protect health and save lives, the anti-PHI advocacy community, as revealed in the Rachel Carson Council's denial of respon‐ sibilities, expects the public to think they had no role in such inhumanely disastrous changes in public health policies. As we have shown, they are, in fact, the very cause of those changes

We have shown that vast sums of money, mostly from taxpayers, have been spent over many decades undermining and often directly attacking the use of DDT in life-saving disease control programs. These vast expenditures have not delivered alternative strategies or tools to replace DDT. The few alternatives that disease control programs do have for some malaria-endemic regions pale in comparison to the powerful life-saving properties of DDT. It almost goes without saying that if the disease control tool in question were not DDT but were a vaccine or a medicine, there would be a sense of outrage in the general public along with well-funded advocacy to preserve and protect a tool that has the power to save lives. Yet such is the power of the environmental movement, that aside from a few outspoken scientists and individuals,

and increasing numbers of pre-term births in women,… breast cancer...[58]."

ACTs for treating cases of falciparum malaria.

662 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**12. Conclusion**

in policy.

CEC—Commission for Environmental Cooperation (The full title is North Americas Com‐ mission for Environmental Cooperation. Created as a side agreement of the North American Free Trade Agreement.)

DDT/DDE—Diethyl dichloro trichloroethelene. DDE is a metabolic product of DDT.

EDC-- endocrine disrupting chemicals

GEF—Global Environment Facility

HSR—house spray rate

IRS—indoor residual spray

ITN—insecticide treated net

MRL—maximum residue limit


#### **Author details**


## **References**

MRL—maximum residue limit

OC—organochlorine compound

PHI—public health insecticide

RBM—Roll Back Malaria

UN—United Nations

PMI—President's Malaria Initiative

POP—persistent organic pollutant

SP-- sulphadoxine-pyrimethamine

WHA—World Health Assembly

WWF—World Wildlife Fund

**Author details**

WHO—World Health Organization

Donald R. Roberts1,2\*, Richard Tren3

PTDI—provisional tolerable daily intake

UNDP—United Nations Development Programme

UNEP—United Nations Environment Programme

USAID—United States Agency for International Development

\*Address all correspondence to: drdonaldroberts42@gmail.com

2 Retired, Professor Emeritus, Clifton Forge, VA, USA

3 Africa Fighting Malaria, Washington, DC, USA

1 Uniformed Services University of the Health Sciences, Bethesda, MD, USA

and Kimberly Hess3

UNICEF—United Nations Children's Fund

NGO—Nongovernmental organization

664 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

NMCP—National Malaria Control Program

PAHO—Pan American Health Organization

NAFTA—North American Free Trade Agreement


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[16] Roberts D, Tren R, Bate R, Zambone J. The Excellent Powder, DDT's Political and Sci‐

[18] World Health Organization. Executive Board, 47th Session, Part II, Appendix 14. "The place of DDT in operations against malaria and other vector borne diseases."

[19] World Health Organization. Official Records, 1972, no. 198. Executive Board, 49th Session, Part II. Chapter II, page 23. Under section titled "Report on the Proposed

[21] Barnes KI, Durrheim DN, Little F, et al. Effect of artemether-lumefantrine policy and improved vector control on malaria burden in KwaZulu-Natal, South Africa. PLoS

[22] Navarro M. Breaking a long silence on population control. The New York Times 2011. http://www.nytimes.com/2011/11/01/science/earth/bringing-up-the-issue-of-

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[25] Hill AB. Principles of medical statistics. 9th edition. New York: Oxford University

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[28] World Wildlife Fund: Resolving the DDT Dilemma: Protecting Biodiversity and Hu‐ man Health. http://awsassets.panda.org/downloads/resolvingddt.pdf (accessed 19

[29] Rogan WJ, Gladen BC, McKinney JD, et al. Polychlorinated biphenyls (PCBs) and di‐ chlorodiphenyl dichloroethene (DDE) in human milk: effects on growth, morbidity,

and duration of lactation. Am J Public Health 1987;77(10) 1294-7.

residues and risk of breast cancer. J Natl Cancer Inst 1993;85(8) 648-52.

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Med 2005;2(11) e330.

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[46] Roberts DR, Tren R. International advocacy against DDT and other public health in‐ secticides for malaria control. Research and Reports in Tropical Medicine 2011;2011(2): 23-30. http://www.dovepress.com/international-advocacy-against-ddtand-other-public-health-insecticide-peer-reviewed-article-RRTM (accessed 19 Sep‐

[47] Global Environment Facility: Detail of GEF Project #1591: Regional Program of Ac‐ tion and Demonstration of Sustainable Alternatives to DDT for Malaria Vector Con‐ trol in Mexico and Central America. http://www.gefonline.org/

[48] Betlem J (UNEP), Neira M (WHO), Whyllie P (SSC). Demonstrating and Scaling-up of Sustainable Alternatives to DDT in Vector Management (DSSA - Global Pro‐ gramme). A program implemented by United Nations Environment Program- UNEP and executed by World Health Organization – WHO (Regional Offices) and the gov‐ ernments of participating countries. Approved by GEF Council on 23 April 2008. Pre‐

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[51] Arbeláez Montoya MP. Control de la malaria sin DDT en Mesoamérica: control focal‐ izado y manejo de criaderos como estrategias básicas Aspectos Epidemiológicos. Pro‐ grama Regional de Acción y Demostración de Alternativas de Control de Vectores de la Malaria sin el Uso de DDT (Proyecto DDT/PNUMA/GEF/OPS). Presentation on

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**Chapter 21**

## **Residual Transmission of Malaria: An Old Issue for New Approaches**

Lies Durnez and Marc Coosemans

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55925

## **1. Introduction**

Malaria is one of the most serious vector-borne diseases, affecting millions of people mainly in the tropics. Recently, a substantial decline in malaria incidence has been observed all over the world. Vector control is one of the key elements in achieving this world-wide malaria decline, with scaling up of Insecticide Treated Nets (ITNs) and the expansion of Indoor Residual Spraying (IRS) programmes contributing significantly. Besides the personal protec‐ tion, ITNs confer a community protection when wide coverage is assured, meaning that unprotected persons benefit from the large scale intervention [1]. IRS is only meaningful when applied at a large coverage. In the 2011 World Malaria Report [2], the percentage of households owning at least one ITN in sub-Saharan Africa is estimated to have risen from 3% in 2000 to 50% in 2011 while the percentage protected by indoor residual spraying (IRS) rose from less than 5% in 2005 to 11% in 2010. Household surveys indicate that 96% of persons with access to an ITN within the household actually use it [2]. Although these numbers might overestimate the real ITN use, they show that in recent years, several vector control measures were scaled up substantially. Despite these large increases in coverage, a widely held view is that with the currently available tools, namely vector control tools, intermittent preventive treatment, and early diagnosis and treatment, much greater gains could be achieved, including elimination from a number of countries and regions [3].

When considering vector control tools, even when hypothesizing a full coverage of ITNs and IRS, malaria transmission may still continue. Indeed, IRS only affects endophilic1 mosquitoes and ITNs only target night biting mosquitoes. Moreover both intervention methods will mainly affect anthropophilic2 mosquitoes that are endophagic3 . This leaves ample opportunity

<sup>1</sup> Endophily is the tendency for mosquitoes to prefer resting indoors

© 2013 Durnez and Coosemans; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Durnez and Coosemans; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

for more exophilic4 , zoophilic5 and/or exophagic6 vectors to escape from contact with insecti‐ cide treated surfaces and to maintain a certain level of transmission. Independently of the ITN and/or IRS coverage, outdoor and early malaria transmission occurs in many malaria endemic regions. In the west of Eritrea for example over a two year sampling period 36.4% of infective bites were acquired outdoors [4], in southern Tanzania this was 10% for non ITN users [5]. A study in northeastern Tanzania showed that 12% of the malaria transmission occurred before sleeping time [6]. In Uganda, in 6 sentinel sites throughout the country, up to 36% of indoor transmission and 49 % of outdoor transmission occurred before sleeping time, with the highest proportion of early in- and outdoor transmission in the suburban area of Jinja where *An. gambiae*<sup>7</sup> was the main vector [7]. In central Vietnam, where ITNs are used at large scale, 69% of the infective bites in forest plots were acquired before sleeping time [8]. In a study conducted in the east and west of Cambodia before widespread ITN use, 29% of the bites occurred before sleeping time in villages and forest plots [9]. In North-East India, 21% of the indoor infective bites occurred before 21h [10]. Also in Nicaragua, in an area with mainly Vivax malaria, 50% of the infective bites were acquired before sleeping time [11]. This part of the malaria trans‐ mission has the possibility to continue despite high coverage of ITNs and IRS, and is defined for the purpose of this review as 'residual transmission'.

Controlling residual transmission requires a different approach as compared to the currently used vector control measures. This is not new and was already perceived as a major obstacle in the previous malaria eradication era [12]. In 2007 malaria eradication was put as the ultimate goal [3] and renewed attention was given to residual transmission, with vector control models also incorporating outdoor and zoophilic malaria vectors. Recently, an established mathemat‐ ical model adjusted for human in- and outdoor movements was used to illustrate that even with 50% outdoor biting vectors, transmission suppression can be achieved by a large ITN coverage [13]. However the authors assumed a uniform exposure so that the ITN induced mortality affects equally in- and outdoor biting vectors. When assuming a uniform exposure all individuals of the vector population (belonging to the same or to different species), will exhibit at each gonotrophic cycle a random behaviour (e.g. exo- or endophily, exo-or endoph‐ agy, anthropo- or zoophily, early- or late-biting), so that all individual mosquitoes are equally affected by indoor-based vector control measures. In case of non-uniform exposure, two or more subpopulations of vectors (belonging to the same or to different species) are assumed, each exhibiting a specific behaviour. Therefore, each of these subpopulations is affected differently by indoor-based vector control measures [14]. As a result, a fraction of vectors will persist in the presence of these control measures and can be responsible for residual trans‐ mission. It was shown that pre-intervention variables reflecting behavior, such as the degree

<sup>2</sup> Anthropophily is the tendency for mosquitoes to prefer feeding on human hosts

<sup>3</sup> Endophagy is the tendency for mosquitoes to prefer biting indoors

<sup>4</sup> Exophily is the tendency for mosquitoes to prefer resting outdoors

<sup>5</sup> Zoophily is the tendency for mosquitoes to prefer feeding on animal hosts

<sup>6</sup> Exophagy is the tendency for mosquitoes to prefer biting outdoors

<sup>7</sup> In this paper, s.l. (sensu lato) is added to the species name when referred to the species complex *(An. gambiae s.l., An. minimus s.l., An. dirus s.l.).* In the absence of s.l., the species is concerned *(e.g. An. gambiae, An. minimus, An. dirus).*

of exophily, may predict the efficacy of a specific intervention [15,16]. Assuming non-uniform exposure, the exophagic fractions of vectors will be less exposed to ITNs, the probability of survival and the vectorial capacity of this subpopulation will be weakly affected, and malaria transmission cannot be reduced further. The model developed in [17] takes into account the non-uniform exposure of the different anopheline species, i.e., the anthropo-endophilic vector species *An. gambiae* and *An. funestus*, and the more zoo-exophilic vector *An. arabiensis*. As would be intuitively expected, this model predicts that even the combination of very effective ITN distribution, twice yearly mass screening and treatment campaigns, and IRS will not succeed in getting the parasite prevalence rate below the 1% threshold if the zoo-exophilic *An. arabiensis* is present. When only *An. gambiae* or *An. funestus* are present, the same combination of interventions are successful in this model [17]. Moreover even within a well-defined species different subpopulations may occur exhibiting different behavioural patterns, resulting in nonuniform exposure within a species.

Therefore, when designing and applying vector control strategies it would be essential to have a good knowledge of the vector behavioural traits particularly those relevant to the chosen control method. However, entomological findings for one region or one anopheline species do not necessarily hold true for the same or different anopheline species encountered in the same or different malaria-endemic regions. In this chapter we will show that even before widespread use of vector control measures, a heterogeneity in behaviour between and within species was present. Because of the heterogeneity in behaviour, mosquitoes have different opportunities to escape from the killing or excito-repellent actions of insecticides used in ITNs or IRS. We will give examples of species shifts, shifts to outdoor- or early biting, shifts to zoophily or to exophily from different malaria endemic regions linked to the use of ITNs and IRS. Although the causes and mechanisms behind these shifts are not yet well understood, we will argue that ITNs and IRS may select for vector populations that predominantly feed early or outdoors, rest outdoors, or that are able to change their behaviour in response to the presence of these insecticides. Therefore, residual transmission will be dominated by vectors that bite outdoors, early or on animals, and that rest outdoors. These vectors require different control strategies, which might also be based on reducing host-vector contact, or target other key environmental resources.

The concept of uniform versus non-uniform exposure is illustrated in Figure 1.

## **2. Heterogeneity in anopheline behaviour**

for more exophilic4

*gambiae*<sup>7</sup>

, zoophilic5

672 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

for the purpose of this review as 'residual transmission'.

2 Anthropophily is the tendency for mosquitoes to prefer feeding on human hosts

3 Endophagy is the tendency for mosquitoes to prefer biting indoors 4 Exophily is the tendency for mosquitoes to prefer resting outdoors 5 Zoophily is the tendency for mosquitoes to prefer feeding on animal hosts 6 Exophagy is the tendency for mosquitoes to prefer biting outdoors

and/or exophagic6 vectors to escape from contact with insecti‐

cide treated surfaces and to maintain a certain level of transmission. Independently of the ITN and/or IRS coverage, outdoor and early malaria transmission occurs in many malaria endemic regions. In the west of Eritrea for example over a two year sampling period 36.4% of infective bites were acquired outdoors [4], in southern Tanzania this was 10% for non ITN users [5]. A study in northeastern Tanzania showed that 12% of the malaria transmission occurred before sleeping time [6]. In Uganda, in 6 sentinel sites throughout the country, up to 36% of indoor transmission and 49 % of outdoor transmission occurred before sleeping time, with the highest proportion of early in- and outdoor transmission in the suburban area of Jinja where *An.*

was the main vector [7]. In central Vietnam, where ITNs are used at large scale, 69%

of the infective bites in forest plots were acquired before sleeping time [8]. In a study conducted in the east and west of Cambodia before widespread ITN use, 29% of the bites occurred before sleeping time in villages and forest plots [9]. In North-East India, 21% of the indoor infective bites occurred before 21h [10]. Also in Nicaragua, in an area with mainly Vivax malaria, 50% of the infective bites were acquired before sleeping time [11]. This part of the malaria trans‐ mission has the possibility to continue despite high coverage of ITNs and IRS, and is defined

Controlling residual transmission requires a different approach as compared to the currently used vector control measures. This is not new and was already perceived as a major obstacle in the previous malaria eradication era [12]. In 2007 malaria eradication was put as the ultimate goal [3] and renewed attention was given to residual transmission, with vector control models also incorporating outdoor and zoophilic malaria vectors. Recently, an established mathemat‐ ical model adjusted for human in- and outdoor movements was used to illustrate that even with 50% outdoor biting vectors, transmission suppression can be achieved by a large ITN coverage [13]. However the authors assumed a uniform exposure so that the ITN induced mortality affects equally in- and outdoor biting vectors. When assuming a uniform exposure all individuals of the vector population (belonging to the same or to different species), will exhibit at each gonotrophic cycle a random behaviour (e.g. exo- or endophily, exo-or endoph‐ agy, anthropo- or zoophily, early- or late-biting), so that all individual mosquitoes are equally affected by indoor-based vector control measures. In case of non-uniform exposure, two or more subpopulations of vectors (belonging to the same or to different species) are assumed, each exhibiting a specific behaviour. Therefore, each of these subpopulations is affected differently by indoor-based vector control measures [14]. As a result, a fraction of vectors will persist in the presence of these control measures and can be responsible for residual trans‐ mission. It was shown that pre-intervention variables reflecting behavior, such as the degree

7 In this paper, s.l. (sensu lato) is added to the species name when referred to the species complex *(An. gambiae s.l., An. minimus s.l., An. dirus s.l.).* In the absence of s.l., the species is concerned *(e.g. An. gambiae, An. minimus, An. dirus).*

Heterogeneity in behaviour of anopheline mosquitoes between and within species is present in all malaria endemic regions. In Africa, the two most efficient malaria vector species, *An. gambiae* and *An. funestus,* are very anthropophilic, endophilic, endophagic, and late-night biting [18]. In contrast, *An. arabiensis*, a species belonging to the same complex as *An. gambiae*, is more plastic in its behaviour, exhibiting more often zoophily, exophily, exophagy, and earlynight biting as compared to *An. gambiae* and *An. funestus*. However, different factors can influence the behaviour of the anophelines. Host availability for example plays an important

**Figure 1.** Effect of control measures on mosquito populations in the assumption of uniform exposure and non-uni‐ form exposure. The density of a uniform population (belonging to the same or to different species) A. before applying the control measure. B. after applying the control measure. The control measure reduces the density of the whole population by 80%. The density of a non-uniform population C. before applying the control measure. D. after apply‐ ing the control measure. The population consists of two subpopulations (Subpopulations 1 and 2, belonging to the same or to different species) each with a different behavioural tendency. Limited contact with the insecticide due to its behavioural tendency makes that Subpopulation 1 is reduced by 20% only, while Subpopulation 2 is reduced by 80% of its initial density. As a result, a fraction of vectors will persist in the presence of these control measures and can be responsible for malaria transmission.

factor in the final host choice of the vector. This has been shown for *An. gambiae* in several study sites. In Burkina Faso for example, a double choice experiment shows that 88% of the *An. gambiae* choose for a human odour baited trap and only 12 % for a cattle odour trap. In contrast, the human blood index of indoor-resting *An. gambiae* collected in the same locality was only 40% [19], showing that this population of *An. gambiae* will adapt its host choice in case of a lower availability of human hosts. *An. gambiae* in São Tomé feeds more on dogs and was observed to be extremely exophagic most probably due to a combination of preference and the ease to reach the dogs sleeping outside under pillar houses [20]. On the Bioko Island (Equatorial Guinea), *An. gambiae* was also observed to be partly exophagic and early-biting [21]. This means that when humans are not available inside, e.g. because of a high bed net use, some populations of *An. gambiae* are observed to feed outside or on animal hosts. In those cases, the frequency of human-vector contact will be lowered although humans will still be bitten in the evening. As a consequence, the longevity of these exophagic or zoophilic vectors will slightly, or not, be affected by ITNs, meaning that the vectorial capacity is not affected and malaria transmission continues.

Also in South-East Asia, heterogeneity of behaviour is observed for the primary and secondary vector species [22]: *An. dirus* is for example very anthropophilic, whereas *An. minimus*, depending on the geographical region, has both anthropophilic and zoophilic tendencies. *An. maculatus* has a high tendency for early biting as compared to *An. dirus* or *An. minimus*, but there are large differences between localities. Different populations of *An. minimus* observed in various localities also differ in their endophilic and endophagic tendencies [22]. Whereas *An. dirus* is generally observed to be very exophagic and exophilic, populations in Lao PDR have shown highly endophilic and endophagic trends [23]. Moreover, as reviewed in [24], *An. dirus* s.l. can even take blood-meals during daylight in the jungle.

In Latin-America, one of the most efficient vectors, *An. darlingi* is mainly anthropophilic, whereas the other dominant vectors, such as *An. albimanus*, *An. nuneztovari*, and *An. aquasalis* also have zoophilic tendencies or are more opportunistic. Most of the vectors in Latin America are mainly exophilic, but within each species, the degree of exophily can vary between geographical regions. *An. albimanus* for example is predominantly exophagic and exophilic, as observed in the Dominican Republic, Colombia, and Haiti. However, in Mexico and Central America, 80% of the *An. albimanus* was observed to have an endophilic resting behaviour [25]. Also the time and place of biting differs between sites for most of the species. In some localities for example, *An. darlingi* bites mostly during sleeping hours, or early in the morning [26], whereas in other localities, the main biting peak is early in the evening [27]. In French Guiana, *An. darlingi* was endo-exophagous with a clear predilection for biting outdoors [28].

## **3. How can the indoor use of insecticides select for exophilic, exophagic, zoophilic and/or early biting mosquito populations?**

factor in the final host choice of the vector. This has been shown for *An. gambiae* in several study sites. In Burkina Faso for example, a double choice experiment shows that 88% of the *An. gambiae* choose for a human odour baited trap and only 12 % for a cattle odour trap. In contrast, the human blood index of indoor-resting *An. gambiae* collected in the same locality was only 40% [19], showing that this population of *An. gambiae* will adapt its host choice in case of a lower availability of human hosts. *An. gambiae* in São Tomé feeds more on dogs and was observed to be extremely exophagic most probably due to a combination of preference and the ease to reach the dogs sleeping outside under pillar houses [20]. On the Bioko Island (Equatorial Guinea), *An. gambiae* was also observed to be partly exophagic and early-biting [21]. This means that when humans are not available inside, e.g. because of a high bed net use, some populations of *An. gambiae* are observed to feed outside or on animal hosts. In those cases, the frequency of human-vector contact will be lowered although humans will still be bitten in the evening. As a consequence, the longevity of these exophagic or zoophilic vectors will slightly, or not, be affected by ITNs, meaning that the vectorial capacity is not affected and

**Figure 1.** Effect of control measures on mosquito populations in the assumption of uniform exposure and non-uni‐ form exposure. The density of a uniform population (belonging to the same or to different species) A. before applying the control measure. B. after applying the control measure. The control measure reduces the density of the whole population by 80%. The density of a non-uniform population C. before applying the control measure. D. after apply‐ ing the control measure. The population consists of two subpopulations (Subpopulations 1 and 2, belonging to the same or to different species) each with a different behavioural tendency. Limited contact with the insecticide due to its behavioural tendency makes that Subpopulation 1 is reduced by 20% only, while Subpopulation 2 is reduced by 80% of its initial density. As a result, a fraction of vectors will persist in the presence of these control measures and can

Also in South-East Asia, heterogeneity of behaviour is observed for the primary and secondary vector species [22]: *An. dirus* is for example very anthropophilic, whereas *An. minimus*,

malaria transmission continues.

be responsible for malaria transmission.

674 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Insecticides can elicit different actions with different results on mosquitoes [29–31]. These various modes of action are important when talking about selection of 'insecticide avoiding' mosquitoes. Toxic or cidal actions result in knockdown or death after contact with the insecticide. Excito-repellent actions, including contact irritancy and non-contact repellency, result in above-normal levels of undirected movements coupled with loss of responsiveness to host cues. The insecticidal actions and their results depend among others on the insecticidal product used and on the mosquito species present. Large differences in actions of insecticides used in IRS have been observed: dieldrin for example only elicits a cidal action, while alpha‐ cypermethrin has both contact-irritant and killing actions, and DDT elicits mainly a repellent effect and secondarily a toxic action. [30]. Pyrethroids, the only family of insecticides used on ITNs, have well-documented excito-repellent actions [21] which are dose-dependent, but with for example higher toxic actions of alphacypermethrin as compared to deltamethrin and permethrin [31].

The general concepts of stress-induced variation in evolution [32] can be applied to the effect of insecticides on mosquito populations. Indoor use of insecticides will pose a stress on the female anopheline population, but only when the insecticides present a barrier for indoor feeding or indoor resting. At least three processes can be at the origin of perceived shifts in mosquito behaviour by insecticides:


## **4. Shifts observed in the presence of indoor insecticidal pressure**

In the following paragraphs we will review the shifts that were observed in the presence of IRS and ITNs. For the purpose of this review, a 'shift' means an observed change, including relative changes, with a reasonable link to the indoor use of insecticides (ITNs or IRS). A distinction is made between different kinds of shifts: species shifts describe changes in the species composition which can also be within species complexes, whereas shifts to early biting, exophagy, zoophily or exophily describe changes in biting time, biting place, host, or resting place within a species, or within a species complex if no species information was available. Because a large part of the shifts in literature are described in the Afrotropical region, this region will be handled separately.

## **5. Afrotropical region**

### **5.1. Species shifts**

**1.** A first protective mechanism can be behavioural plasticity in response to the presence of the insecticide. The ability to actively remove from the insecticide by either reloca‐ tion or avoidance requires an ability to detect (either by contact or non-contact) or anticipate the presence of the insecticide and the ability to exhibit insecticide avoid‐ ance strategies or adjustments [32]. The insecticide, or the unavailability of the host, can then trigger the expression of gene variants that have been accumulated, but were phenotypically neutral under a normal range of environments [32]. Many mosquitoes indeed naturally possess a high degree of irritability or repellency which is evident at the very first exposure of the population to residual insecticides [29]. Where this irritation is such that mosquitoes settling on the insecticide deposit are activated before they have absorbed a lethal dose of insecticide, and are able to avoid further contact and to escape unharmed, the term "protective avoidance" has been suggested. In the presence of a high coverage of IRS or ITNs, mosquitoes exhibiting this protective avoidance should then be able to redirect their behaviour to low-risk behaviour which also can lower their survival. For example, for a species that is normally endophilic changing its behaviour to resting outdoors, the external environment may be unfavourable to the survival of

**2.** A second protective mechanism for the mosquito is a consistent "protective behaviour" [29] such as exophily, exophagy, zoophily or early-biting resulting in a minimal contact with the insecticides used indoors. As mentioned above, some mosquito populations naturally exhibit this kind of protective behaviour, which is probably genetically deter‐ mined (see further). Also differences in responses to the insecticides can result in diverse exposure rates of different species or subpopulations to the insecticide. *An. minimus* for example, shows very strong repellency responses to several insecticides and would have a higher survival chance in the presence of insecticides as compared to *An. harrisoni* which shows a much lower repellency response [33]. In this case, insecticides will favour the (sub) populations of mosquitoes that have this innate preference for protective behaviour or for avoidant strategies by which they will escape the exposure to the insecticide. This is probably the mechanism that is occurring for many of the perceived species shifts that

**3.** Where these phenomena of protective avoidance or protective behaviour are not evident at the very first exposure of the population to the insecticides, but develop only gradually, perhaps over several years under continued insecticide pressure, the term "behaviouristic resistance" is employed [29]. The presence of the insecticide will in that case result in the selection of mutations and recombination that favour the survival of the mosquito in the presence of the insecticide, eventually leading to a directional selection. This can be compared to the development of insecticide resistance, although selections of many mutations will probably be required before an appropriate behavioural change may occur. Classification as "behaviouristic resistance" is only valid on the basis of accurate com‐ parisons made before and subsequent to the widespread use of residual insecticides in any particular area. As shown below, very few behaviour shifts observed so far, would

the species [12].

676 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

are illustrated below.

fit this definition of behaviouristic resistance.

An IRS campaign resulted in the elimination of *An. funestus* from the South Pare District (at the Tanzania-Kenya border), at the same time reducing the numbers of indoor-resting *An. gambiae* s.l. [34]. In the years immediately following this IRS campaign, populations of endophilic *An. gambiae* s.l. slowly regained their former levels, whereas gradual resurgence of *An. funestus* was not observed until almost 10 years after the campaign was abandoned. IRS campaigns in two Kenyan villages resulted in a large decrease (up to total disappearance) of *An. funestus*, with an increase in the more exophagic *An. rivulorum* [35] or *An. parensis* [36], both not considered as malaria vectors in the study sites. In Niger, nation-wide Long-lasting insecticidal net (LLIN) distribution caused a marked decrease of *An. funestus*, without effect on *An. gambiae* s.l. abundance[37].FollowinganIRScampaign,*An.gambiae*wascompletelyeliminatedfromPemba Island (Tanzania), leaving the salt-water breeding *An. merus*, an exophilic mosquito with a preference for cattle [38].In Kenya andTanzania, large scale ITN use significantlydecreasedthe proportion of indoor-resting *An. funestus* [39] and *An. gambiae* [39–42] while the proportion of *An. arabiensis* increased. The shift from *An. gambiae* to *An. arabiensis* was also observed in the larval collections [40,41]. As larvae of *An. gambiae* and *An. arabiensis* show no habitat segrega‐ tion, larval sampling reflects true proportions of the two species. The change from sub-popula‐ tions dominated by *An. gambiae* to those dominated by *An. arabiensis* took about a decade, as would be expected if caused by a constant ITN selection pressure [43].

In contrast, in Kenya and on the Bioko Island (Equatorial Guinea), the same species composi‐ tions were observed regardless of the use of ITNs or IRS [21,44]. Moreover, in the north-east of Tanzania, a species shift has been observed in the absence of insecticide selective pressure, in a region without organized vector control activities reported [45]: *An. gambiae*, the most dominant in the past, was replaced by *An. arabiensis* without any known reason.

#### **5.2. Shifts to early-evening or early-morning biting**

Studies have shown that widespread ITN use increases the proportion of early bites by *An. gambiae* [46] and *An. funestus* [42,46] in Tanzania. Such shift was not observed for *Culex* *quinquefasciatus* which is highly resistant against pyrethroids [46]. According to the authors [46], this suggests that for anophelines, where there is considerable killing by contact with ITNs, several years of selection has begun to produce an upward shift in the proportions of insects biting at a time when people are accessible. Also in southern Benin, a significant change in host seeking behaviour of *An. funestus* was observed after achieving a universal coverage of ITNs. The shift in biting time was here not to the early evening but to the early morning. Moreover in one locality about 26% of the *An. funestus* bites were observed after sunrise [47].

The use of ITNs resulted in a shift towards earlier biting of *An. gambiae* s.l. in Kenya [48] and Tanzania [42,49], possibly [48,49] or certainly [42] related to a species shift from *An. gambiae* to *An. arabiensis*.

In other studies however, no evidence for a shift in biting time after the introduction of ITNs or IRS was obtained for *An. gambiae* s.l. in Tanzania, Kenya, The Gambia and Nigeria [44,50–52], for *An. gambiae* the Bioko Island (Equatorial Guinea) [21], or for *An. funestus* in Kenya [44]. Widespreaduseofmostlyuntreatedbednetsdidnotresultinmoreearlybitingof*An.gambiae*[5].



a ITN: Insecticide treated nets; IRC: Indoor residual spraying; ITC: Insecticide treated curtains

b LLINs: Long lasting insecticidal nets

*quinquefasciatus* which is highly resistant against pyrethroids [46]. According to the authors [46], this suggests that for anophelines, where there is considerable killing by contact with ITNs, several years of selection has begun to produce an upward shift in the proportions of insects biting at a time when people are accessible. Also in southern Benin, a significant change in host seeking behaviour of *An. funestus* was observed after achieving a universal coverage of ITNs. The shift in biting time was here not to the early evening but to the early morning. Moreover in one locality about 26% of the *An. funestus* bites were observed after sunrise [47].

The use of ITNs resulted in a shift towards earlier biting of *An. gambiae* s.l. in Kenya [48] and Tanzania [42,49], possibly [48,49] or certainly [42] related to a species shift from *An. gambiae* to

In other studies however, no evidence for a shift in biting time after the introduction of ITNs or IRS was obtained for *An. gambiae* s.l. in Tanzania, Kenya, The Gambia and Nigeria [44,50–52], for *An. gambiae* the Bioko Island (Equatorial Guinea) [21], or for *An. funestus* in Kenya [44]. Widespreaduseofmostlyuntreatedbednetsdidnotresultinmoreearlybitingof*An.gambiae*[5].

> **Species shift d**

Not observed

Kenya ITN Permethrin IRC, ORC ND ND ND Yes [54]

Not observed

Kenya ITN Permethrin, IRC Yes ND ND ND [39]

**Shift to early-biting**

ND ND Not

Not observed

Not observed

IRC, LD Yes ND ND Not

**Shift to exophagy d**

ND Yes Yes ND [47]

observed

ND ND ND Yes [19]

Yes ND ND ND [35]

Yes ND ND ND [36]

ND Yes Yes Yes, but not

**Shift to zoophily d**

Not observed

Yes ND [21]

observed,

significant

ND ND [44]

observed

**Reference**

[53]

[34]

[48]

[41]

**d**

*An. arabiensis*.

**Country Vector**

Burkina Faso

Burkina Faso

Equatorial Guinea

**control measure a**

IRS ITN

Benin ITN Deltamethrin Indoor/

678 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Kenya IRS Dieldrin ORC, IRC,

Kenya IRS DDT Indoor/

Kenya ITN Permethrin IRC, indoor

Kenya ITN Permethrin WET, IRC,

alpha cypermethrin, Unspecified LLINs

Kenya ITN Permethrin,

ITC Permethrin Indoor/

ITN Unspecified IRC, Odour-

Deltamethrin, alpha cypermethrin, bendiocarb. Unspecified LLIN

**Insecticide b Collection**

**methods c**

outdoor HLC

baited traps

outdoor CDC LT

Indoor/ outdoor HLC

LD, HLC

outdoor HLC

and outdoor HLC

outdoor bed net traps

Kenya IRS Dieldrin IRC, ORC Yes ND ND Not

c IRS: Indoor resting collection; ORC: Outdoor resting collection; CDC LT: Center for Disease Control light trap; HLC: Human landing collection; WET: Window exit trap; LD: Larval dipping; CMR: Capture-Mark-Recapture d ND: Not done

**Table 1.** Review of the effect of insecticide based indoor vector control measures on malaria vectors in the Afrotropical region

### **5.3. Shifts to exophagy**

In Nigeria, IRS resulted in a threefold increase of the proportion of *An. gambiae* s.l. biting outdoors [14,52]. Several years of vector control by IRS and later ITNs in the Bioko Island, increased the trend for outdoor biting of *An. gambiae* [21] as compared to historical data in the same region of preferred behaviour for indoor biting. Also in Tanzania, high ITN-use resulted in an increased outdoor biting for *An. funestus* [42]. In the latter study the proportion of indoor contact with *An. funestus* bites had dropped to only half of the indoor contact before wide‐ spread ITN-use. In southern Benin as well, after achieving universal ITN coverage, a higher proportion of outdoor biting was observed for *An. funestus* [47], although this was only observed in one out of two localities that were studied.

Some studies have shown that distribution of ITNs in Niger, Kenya, and The Gambia decreased the endophagic rate of *An. gambiae* s.l. [37,48,55], and to a lesser extend of *An. funestus* [37]. However, as the species of the *An. gambiae* complex were not determined in these studies, a possible reason for this decrease would be a species shift from *An. gambiae* to *An. arabiensis*.

In other studies however, no evidence for a shift to outdoor biting of *An. gambiae* s.l. due to widespread IRS or ITNs use was found in Tanzania [42,50], Burkina Faso [53] and The Gambia [51]. Also widespread use of mostly untreated bed nets did not result in a higher outdoor biting rate of *An. gambiae* [5].

## **5.4. Shifts to zoophily**

In Kenya, ITN-use caused a shift in host selection of *An. gambiae* s.l. and *An. funestus* [54] from humans towards cattle or other animals. Similar observations were made in Burkina Faso with *An. gambiae* [19]. In other studies in Kenya and The Gambia, the use of ITNs caused only small and insignificant decreases in human blood index (HBI) for *An. gambiae* s.l. [40,48,51] and *An. funestus* [40].

The use of ITNs, IRS, or insecticide treated curtains caused no shift in host selection (or de‐ crease in HBI) for *An. arabiensis* in Zambia [56], for *An. gambiae* s.l. in Nigeria, Burkina Faso, The Gambia,TanzaniaandKenya[34,50,52,53,55],andfor*An.funestus*inTanzaniaandKenya[34,50].

### **5.5. Shifts to exophily**

As summarized in [57], different populations of *An. arabiensis*, e.g. in the Pare-Taveta malaria scheme, Mauritius, Madagascar, Zanzibar, Nigeria and other West African localities, became either completely exophilic or, at most, remained only partially endophilic after IRS cam‐ paigns. ITN distribution reduced the indoor resting fraction of *An. gambiae* s.l. in Niger and Kenya [37,48], and of *An. funestus* in Kenya [48]. No evidence for a resting place shift after introduction of ITNs or after IRS was observed in Tanzania [50].

## **6. Australasian, Oriental, and Neotropical Regions**

#### **6.1. Species shifts**

**5.3. Shifts to exophagy**

680 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

to *An. arabiensis*.

rate of *An. gambiae* [5].

**5.4. Shifts to zoophily**

**5.5. Shifts to exophily**

*funestus* [40].

observed in one out of two localities that were studied.

In Nigeria, IRS resulted in a threefold increase of the proportion of *An. gambiae* s.l. biting outdoors [14,52]. Several years of vector control by IRS and later ITNs in the Bioko Island, increased the trend for outdoor biting of *An. gambiae* [21] as compared to historical data in the same region of preferred behaviour for indoor biting. Also in Tanzania, high ITN-use resulted in an increased outdoor biting for *An. funestus* [42]. In the latter study the proportion of indoor contact with *An. funestus* bites had dropped to only half of the indoor contact before wide‐ spread ITN-use. In southern Benin as well, after achieving universal ITN coverage, a higher proportion of outdoor biting was observed for *An. funestus* [47], although this was only

Some studies have shown that distribution of ITNs in Niger, Kenya, and The Gambia decreased the endophagic rate of *An. gambiae* s.l. [37,48,55], and to a lesser extend of *An. funestus* [37]. However, as the species of the *An. gambiae* complex were not determined in these studies, a possible reason for this decrease would be a species shift from *An. gambiae*

In other studies however, no evidence for a shift to outdoor biting of *An. gambiae* s.l. due to widespread IRS or ITNs use was found in Tanzania [42,50], Burkina Faso [53] and The Gambia [51]. Also widespread use of mostly untreated bed nets did not result in a higher outdoor biting

In Kenya, ITN-use caused a shift in host selection of *An. gambiae* s.l. and *An. funestus* [54] from humans towards cattle or other animals. Similar observations were made in Burkina Faso with *An. gambiae* [19]. In other studies in Kenya and The Gambia, the use of ITNs caused only small and insignificant decreases in human blood index (HBI) for *An. gambiae* s.l. [40,48,51] and *An.*

The use of ITNs, IRS, or insecticide treated curtains caused no shift in host selection (or de‐ crease in HBI) for *An. arabiensis* in Zambia [56], for *An. gambiae* s.l. in Nigeria, Burkina Faso, The Gambia,TanzaniaandKenya[34,50,52,53,55],andfor*An.funestus*inTanzaniaandKenya[34,50].

As summarized in [57], different populations of *An. arabiensis*, e.g. in the Pare-Taveta malaria scheme, Mauritius, Madagascar, Zanzibar, Nigeria and other West African localities, became either completely exophilic or, at most, remained only partially endophilic after IRS cam‐ paigns. ITN distribution reduced the indoor resting fraction of *An. gambiae* s.l. in Niger and Kenya [37,48], and of *An. funestus* in Kenya [48]. No evidence for a resting place shift after

introduction of ITNs or after IRS was observed in Tanzania [50].

In the Solomon Islands, IRS in the 1960s has nearly eliminated the major malaria vectors *An. koliensis* and *An. punctulatus*, which are mainly endophagic and late-biters. The density of *An. farauti*, a more exophagic and early-biting malaria vector, remained quite high, particu‐ larly in outdoor man-biting situations [58]. The latter species is now the primary vector in the Solomon Islands, with the former major malaria vectors being totally absent. *An. hinesorum*, which is not considered a vector, has now occupied the breeding sites common‐ ly used by *An. koliensis* [59].

In the forested hilly areas of Thailand, IRS resulted in a higher proportional decrease of *An. dirus* s.l. as compared to *An. minimus* s.l. [60]. Widespread use of IRS resulted in a different behaviour of the *An. minimus* s.l. present [61], which probably reflects a species shift from *An. minimus* to *An. harrisoni*, as also observed in Vietnam as a result of widespread use of ITNs [62]. Residual spraying did effectively control indoor resting species in Nepal such as *An. annula‐ ris*, *An. culicifacies*, *An. splendidus* and *An. vagus*. The abundance of the partially outdoor resting species, *An. fluviatilis* s.l. and *An. maculatus* s.l. also decreased markedly after the spray application, but then rebounded rapidly within 1 or 2 months after treatment [63]. ITN use in China caused a higher decrease of the endophilic and anthropophilic *An. lesteri* (syn. *An. anthropophagus*) [64] and *An. minimus* s.l. [65] than of the exophagic and zoophilic *An. sinensis*.

In British Guiana, the primary malaria vector *An. darlingi* (both larvae and adults) was rapidly eliminated by IRS, whereas larvae and adults of a zoophilic species, *Anopheles aquasalis*, a possible malaria vector, were completely unaffected [66]. In Guatemala, *An. vestitipennis* decreased in abundance in communities with a wide distribution of ITNs, while *An. albima‐ nus* did not change. Whether this change was an effect of the ITNs could not be concluded as the study was not designed for answering that question [67].

#### **6.2. Shifts to early biting**

In Papua New Guinea, ITN distribution immediately changed the biting cycles of both *An. farauti* and *An. koliensis* from a post-midnight peak towards a pre-midnight peak [68]. Also on the Solomon Islands, intervention and longitudinal studies have shown that IRS, ITNs, or a combination of both, changed the biting cycle of *An. farauti* to an earlier biting peak [58,69,70].

IRS changed the indoor biting peak of *An. dirus* s.l. in the forested hilly areas of Thailand to one hour earlier. Outdoors, the peak remained the same, but a higher proportion bite earlier. Also for *An. minimus* s.l., a shift to earlier biting was observed [60]. In the foothills on the other hand, where *An. minimus* s.l. was the main vector, no effect of DDT was seen on the already early biting *An. minimus* s.l. population [71]. Also recent studies in Vietnam have shown that in the prolonged presence of impregnated bed nets, 45% of the *Anopheles* bites are acquired before sleeping time in the forest, and 64% before sleeping time in the village [8]. In Cambodia, in a period when ITN coverage was still low, already 29% of the *Anopheles* bites were acquired before sleeping time [9].

Although we have not encountered studies in Latin-America with evidence for shifts to earlier biting, some studies indicated that also in this region, early biting vectors can maintain residual transmission. In an area in Brazil covered by IRS for example, blood-feeding of *An. darlingi* started at sunset, remained high during the first half of the night, and decreased gradually until early morning [72]. Also in the Bolivian Amazon, in an area with high ITN use, peak outdoor biting of *An. darlingi* occurred between 19:00 and 21:00 hours, when 48% of the total night's biting took place, and 83% of the night's biting had occurred by 22:00 hours when most local people go to bed [73].

#### **6.3. Shifts to exophagy**

On different islands of the Solomon, proportional shifts to outdoor biting (from 47% to 67%) were observed for *An. farauti* after IRS [58]. Moreover, compared to *An. koliensis* and *An. punctulatus*, the exophagic *An. farauti* population recovered completely within nine months after the spraying campaign. However, in other intervention and longitudinal studies on the Solomon Islands, the shift to outdoor biting of *An. farauti* due to ITNs and/or IRS was not so obvious [59,69].

IRS increased the outdoor biting rate of *An. dirus* s.l. [60,74], and of *An. minimus* s.l. in forested and foothill regions in Thailand [60,61]. In contrast, in another foothill region of Thailand, an initial effect of DDT was seen on the malaria transmission, but this was not sustained for this already outdoor biting *An. minimus* s.l. population [71]. Also wide scale use of ITNs caused a higher decrease in the indoor biting populations as compared to the outdoor biting populations of *An. sinensis*, *An. lesteri* (syn. *An. anthropophagus*) and *An. minimus* s.l. in China [64,65]. In Vietnam, after prolonged ITNs distribution, outdoor biting densities of the main vectors, *An. dirus*, *An. maculatus* s.l. and *An. minimus* s.l. were significantly higher than indoor biting density [8]. In Laos, in contrast, the use of ITNs did not stop *An. dirus* from entering the houses [75].

In an IRS area in Brazil, *An. darlingi* fed more frequently outdoors, whereas in earlier years before IRS this species mainly fed indoors [72]. In contrast, in Colombia, IRS did not stop malaria vectors to bite both indoors and outdoors [76]. The combined use of ITNs and IRS has preceded the collapse of a mainly exophagic *An. darlingi* population in Suriname. However, this collapse can also be attributed to an unusual, extensive flooding which coincided with the onset of the control interventions [77].

#### **6.4. Shifts to zoophily**

A significant decrease in HBI of *An. farauti* was observed immediately after the distribution of ITNs in Papua New Guinea, although this shift could be due to a slightly changed sampling method [68].

In Thailand, in the prolonged presence of DDT use in IRS, *An. minimus* s.l. exhibited a marked zoophily, whereas in villages with lower DDT pressure, no preference was observed [61], although this apparent 'change in behaviour' could have been due to a species shift within the *An. minimus* complex as observed in Vietnam [62]. In an intervention study in India, the HBI of *An. culicifacies* was lower in areas with ITNs as compared to areas with untreated bed nets or no nets [78].

In Mexico, a much lower HBI was observed in areas where IRS was implemented as compared tohistoricaldata[79].AlsoinareascoveredbyIRSinBrazil,*An.darlingi*wasmostlyzoophilic[80].

#### **6.5. Shifts to exophily**

in a period when ITN coverage was still low, already 29% of the *Anopheles* bites were acquired

Although we have not encountered studies in Latin-America with evidence for shifts to earlier biting, some studies indicated that also in this region, early biting vectors can maintain residual transmission. In an area in Brazil covered by IRS for example, blood-feeding of *An. darlingi* started at sunset, remained high during the first half of the night, and decreased gradually until early morning [72]. Also in the Bolivian Amazon, in an area with high ITN use, peak outdoor biting of *An. darlingi* occurred between 19:00 and 21:00 hours, when 48% of the total night's biting took place, and 83% of the night's biting had occurred by 22:00 hours when most

On different islands of the Solomon, proportional shifts to outdoor biting (from 47% to 67%) were observed for *An. farauti* after IRS [58]. Moreover, compared to *An. koliensis* and *An. punctulatus*, the exophagic *An. farauti* population recovered completely within nine months after the spraying campaign. However, in other intervention and longitudinal studies on the Solomon Islands, the shift to outdoor biting of *An. farauti* due to ITNs and/or IRS was not so

IRS increased the outdoor biting rate of *An. dirus* s.l. [60,74], and of *An. minimus* s.l. in forested and foothill regions in Thailand [60,61]. In contrast, in another foothill region of Thailand, an initial effect of DDT was seen on the malaria transmission, but this was not sustained for this already outdoor biting *An. minimus* s.l. population [71]. Also wide scale use of ITNs caused a higher decrease in the indoor biting populations as compared to the outdoor biting populations of *An. sinensis*, *An. lesteri* (syn. *An. anthropophagus*) and *An. minimus* s.l. in China [64,65]. In Vietnam, after prolonged ITNs distribution, outdoor biting densities of the main vectors, *An. dirus*, *An. maculatus* s.l. and *An. minimus* s.l. were significantly higher than indoor biting density [8]. In Laos, in contrast, the use of ITNs did not stop *An. dirus* from entering

In an IRS area in Brazil, *An. darlingi* fed more frequently outdoors, whereas in earlier years before IRS this species mainly fed indoors [72]. In contrast, in Colombia, IRS did not stop malaria vectors to bite both indoors and outdoors [76]. The combined use of ITNs and IRS has preceded the collapse of a mainly exophagic *An. darlingi* population in Suriname. However, this collapse can also be attributed to an unusual, extensive flooding which coincided with the

A significant decrease in HBI of *An. farauti* was observed immediately after the distribution of ITNs in Papua New Guinea, although this shift could be due to a slightly changed sampling

In Thailand, in the prolonged presence of DDT use in IRS, *An. minimus* s.l. exhibited a marked zoophily, whereas in villages with lower DDT pressure, no preference was observed [61],

before sleeping time [9].

682 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

local people go to bed [73].

**6.3. Shifts to exophagy**

obvious [59,69].

the houses [75].

**6.4. Shifts to zoophily**

method [68].

onset of the control interventions [77].

A very low endophily rate was observed for *An. farauti* after several DDT spraying campaigns in the Solomon Islands [58].

IRS also significantly reduced the indoor resting abundance of all anopheline species except for *An. fluviatilis* s.l. in Nepal [63], and of *An. dirus* s.l. in Thailand [74]. In India, *An. culicifa‐ cies* s.l. has been observed to be highly exophilic in areas where residual spraying with DDT was widely used [81]. Also in areas with wide scale use of ITNs in India fewer *An. culicifacies* s.l. were collected indoors (resting collections) as compared to control areas. However, in this area more *An. culicifacies* s.l. were found indoor-resting in individual houses with untreated bed nets as compared to houses with ITNs, both located in the ITN-area [78]. This suggests that this mosquito population did not shift entirely to exophily, but that this behaviour mainly reflects the excito-repellent effect of the permethrin.

IRS has brought the disappearance of *An. darlingi* from the interior of houses in Brazil and French Guiana [28,80]. However, outdoor-resting still persists, either in the vicinity of the houses [80] or outside the peridomestic environment [28]. ITNs as well caused less indoorresting in an intervention trial in Guatemala [67]. In contrast, in Mexico, after prolonged use of DDT no deterrence was observed anymore for *An. pseudopunctipennis*, with as many mosquitoes seeking shelter in sprayed huts as in unsprayed huts [82].



a ITN: Insecticide treated nets; IRS: Indoor residual spraying; ITC: Insecticide treated curtains

b LLINs: Long lasting insecticidal nets

c IRC: Indoor resting collection; ORC: Outdoor resting collection; CDC LT: Center for Disease Control light trap; HLC: Human landing collection; WET: Window exit trap; LD: Larval dipping; CMR: Capture-Mark-Recapture d ND: Not done

**Table 2.** Review of the effect of insecticide based indoor vector control measures on malaria vectors in the Australasian, Oriental and Neotropical regions

## **7. Discussion**

**Country Vector** 

**control measure a**

India ITN Lambdacyhalot

Nepal IRS DDT,

Thailand IRS DDT,

Mexico IRS DDT,

Mexico IRS DDT (dieldrin

b LLINs: Long lasting insecticidal nets

Australasian, Oriental and Neotropical regions

Neotropical Region

British Guiana

a

c

d ND: Not done

China ITN Deltamethrin Indoor/

684 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

hrin

Thailand IRS DDT Indoor/

Thailand IRS DDT Indoor/

Thailand IRS DDT Indoor/

Vietnam ITN Permethrin Indoor/

Brazil IRS DDT IRC, ORC,

Brazil IRS DDT Indoor/

Guatemala ITN Permethrin Indoor/

bendiocarb

before)

bendiocarb, malathion

fenitrothion

**Insecticide b Collection** 

**methods c**

outdoor manbaited nets

IRC, Indoor HLC, Outdoor Cattle collection

Indoor/ outdoor HLC, IRC, ORC, cattle collections, LD

outdoor HLC

outdoor HLC

outdoor HLC, bovid-baited trap, IRC, ORC

outdoor HLC, IRC, CDC LT

animal baited trap,

outdoor HLC, outdoor animal baited trap

outdoor HLC, IRC, inspection of bed net surfaces, CMR

Entry traps, WET

Human landing collection; WET: Window exit trap; LD: Larval dipping; CMR: Capture-Mark-Recapture

**Table 2.** Review of the effect of insecticide based indoor vector control measures on malaria vectors in the

ITN: Insecticide treated nets; IRS: Indoor residual spraying; ITC: Insecticide treated curtains

Indoor/ outdoor HLC, IRC

**Species shift d**

China ITN Deltamethrin ? Yes ND Yes ND In [65]

Not observed

IRS DDT IRC, LD Yes ND ND ND [66]

IRC: Indoor resting collection; ORC: Outdoor resting collection; CDC LT: Center for Disease Control light trap; HLC:

Yes? ND Not

IRC, ORC ND ND ND Yes [79]

**Shift to earlybiting d**

Not observed **Shift to exophagy d**

Yes ND Yes ND [64]

ND ND ND Yes [78]

Yes ND ? ND [63]

Yes Yes Yes ND [60]

Not observed

Probably ND Yes Yes [61]

ND ND Yes ND [74]

Yes ND ND ND [62]

ND ND ND Yes? In [80]

ND ND Yes ND [72]

observed

ND ND ND ND [82]

**Shift to zoophily d**

ND [71]

ND [67]

**Reference**

#### **7.1. The importance of residual transmission by outdoor and early biting malaria vectors**

In this chapter we have shown that outdoor and early biting malaria vectors are widespread among malaria endemic countries and, as relative shifts to outdoor, early or animal-biting and outdoor resting vectors occur due to the use of IRS and ITNs, these vectors will increasingly contribute to malaria transmission in regions with a high coverage of ITNs and IRS. However the reported shifts are not always well documented: species identification of complexes are often missing, and confounding factors such as changes of the environment, habitat, human behaviour and occupation are not considered.

In Africa, most of the species shifts observed resulted in a large decrease of the important endophagic, endophilic and anthropophilic malaria vectors, *An. funestus* and *An. gambiae*, while the more exophagic, exophilic, and/or zoophilic species *An. arabiensis* persists. Reports on such species shift are recently increasing, with most of these shifts described in East-Africa. But also in the other geographical regions, shifts in species abundances have been observed. It is however important to note that the majority of shifts described are shifts in relative abundances, where the more endophagic, endophilic and/or anthropophilic species declines more (or is being eliminated) while the more exophagic, exophilic and/or zoophilic species maintains at the same density or declines less. Only in some cases, the density of the latter species actually increases (e.g. the non-vector species *An. rivulorum* [35] or *An. parensis* [36]), probably because they take over the breeding sites of the declining species. Moreover, as also mentioned in [83], the vectorial capacity of the species predominating after the intervention does not necessarily increase, but persisting species that are malaria vectors, such as *An. arabiensis*, will be responsible for the residual malaria transmission, while the role of e.g. *An. gambiae* or *An. funestus* decreases.

Therefore, one of the most plausible reasons for species shifts to occur in the presence of ITNs or IRS is the non-uniform exposure of the different species to the insecticides, as described above. This hypothesis is supported by a study in Kenya in which the persisting *An. arabien‐ sis* in an area with high ITN coverage had little to no pyrethroid resistance compared to the declining *An. gambiae*, with moderate to high levels of pyrethroid resistance [41,43]. Moreover, in experimental hut trials on northeast Tanzania, the mortality of *An. arabiensis* measured in experimental huts was consistently lower than that of *An. gambiae* and *An. funestus* [83], which probably is a major contributing factor to the species shifts observed in East Africa following scale up of ITNs. The authors state that, as cone tests on the nets prior to the trials produced rather similar levels of mortality among *An. gambiae* and *An. arabiensis*, the most likely explanation for lower *An. arabiensis* mortality was behavioural avoidance of treated net surfaces. As feeding inhibition in this experiment was similar for *An. arabiensis* and *An. gambiae*, outdoor blood-feeding would be the major mechanism to which *An. arabiensis* avoids contact with the ITN, as opposed to abandoning host-searching when confronted with ITNs.

Besides the species shifts, shifts to earlier-, outdoor-, and animal-biting have been observed for primary vectors such as *An. gambiae*, *An. funestus*, *An. farauti*, *An. koliensis*, *An. dirus* s.l.*, An.* *minimus* s.l., *An. culicifacies*, and *An. darlingi*. These shifts might also be linked to the nonrandom exposure of subpopulations of vectors to insecticide treated surfaces (ITNs or IRS). Several studies have indeed shown that the feeding and resting behaviour of anophelines is consistent in certain subpopulations and/or linked to certain genetic markers. Most of the studies on genetic determination of biting and resting behaviour are based on chromosomal inversions. Alleles captured within chromosome rearrangements are protected from recom‐ bination and can as such favour local adaptation by capturing sets of locally adapted genes which might lead to reproductive isolated entities or subpopulations [84]. In the Garki District in Nigeria, chromosomal arrangements in *An. arabiensis* and *An. gambiae* have been associated with exophagy and exophily [85,86] and with zoophily [87]. Exophagy and exophily were associated with the standard chromosomal arrangements 2R+a for *An. arabiensis* and 2R+b for *An. gambiae*, and the inverted arrangement 2Rbc for *An. arabiensis*. Moreover, the chromosome arrangements associated with indoor biting or resting are the ones adapted to drier environ‐ ments, while arrangements more frequent in outdoor collected specimens are those associated with more humid environments [85]. In the Zambesi valley, 2Rc *An. arabiensis* heterozygotes were associated with exophily and zoophily [57]. In Ethiopia *An. arabiensis* heterozygotes of the 2La and/or 2Rb chromosomal arrangements tended to bite later at night than the double homozygotes [88]. Also in laboratory experiments an association between chromosomal arrangements and circadian flight activity has been found [89]: female *An. stephensi* homozy‐ gotes for the 2Rb inversion showed more activity following light-on (corresponding to early morning) as compared to homozygous females for the standard 2R+b arrangement. Other fieldbased evidence on the existence of subpopulations showing consistent behaviour was obtained by studying behaviour of *An. balabacensis* in a capture-mark-recapture experiment in Borneo (Malaysia) [90]. This study revealed significant trends of *An. balabacensis* to be recaptured on the same host or resting site of the original capture. In contrast, a similar capture-markrecapture study on resting behaviour of *An. gambiae* s.l. in Tanzania showed no faithful tendencies of endo- or exophily [91]: the same individuals within the *An. gambiae* s.l. population mixed indoor and outdoor resting. More recent genetic studies are based on the frequencies of enzyme polymorphisms. In the Malaysian study [90], faithfully indoor and outdoor-resting populations showed significant differences in isozyme frequencies (loci *Est-3* and *Idh-3*). Also in Burundi, isozyme frequencies were significantly different between in- and out-door biting *An. arabiensis* (locus *Mdh-2*) and in- and out-door resting *An. gambiae* (*Mpi* and *Got-2* loci) [92]. Such differences were not observed for *An. gambiae* in Burkina Faso [93]. Moreover, mosquitoes carrying a specific genotype [93] or chromosome karyotypes [87] were found to be significantly more infected with sporozoites, suggesting the occurrence of subpopulations having different vector behaviours. These independent genetic studies, either based on karyotyping or on genotyping, provide evidence that active choice for the best place, time or host to bite, or the best place to rest can be associated with specific genotypes. This suggests the existence of subpopulations characterized by specific behavioural patterns which implies a non-uniform exposure to IRS or ITNs. Selection of specific behavioural patterns can then not be excluded.

However, other mechanisms can also explain these kinds of shifts. More early biting could occur as females that fail to obtain a blood meal during the previous night, might be more likely to commence host seeking in the early evening [44]. By disrupting the feeding behaviour, the ITNs would increase the length of the oviposition cycle of the overall population [68]. This mechanism could explain the immediate change in biting cycles of both *An. farauti* and *An. koliensis* after ITN distribution in Papua New Guinea. Both species shifted from a postmidnight biting peak towards a pre-midnight peak [68], with an extended oviposition cycle. Also in the Solomon Islands, the oviposition cycle was extended from 3 to 4 days due to ITN use, possibly explaining the higher tendency for early biting observed in the village with ITN use [69]. Shifts to outdoor biting by *An. farauti* also occurred immediately after DDT spraying [58]. This first effect would be caused by the deterrent effect of DDT, while only in second instance the endophilic fraction of *An. farauti* is being killed. Moreover, compared to *An. koliensis* and *An. punctulatus*, the *An. farauti* population recovered completely within nine months after the spraying campaign, indicating that this change of behaviour is due to a plastic response to the deterrent effect of DDT. Moreover, it has been shown that the occurrence of a shift in host selection does not necessarily reflect a selection of a more zoophilic vector subpopulation, but can also indicate plasticity in host selection. The *An. gambiae* population in Burkina Faso that showed a high proportion of cattle feeding (HBI of only 40%), had an innate preference for humans (88%) in a choice experiment using an odour-baited trap [19]. The weak accessibility of humans due to the use of ITNs, forces the mosquitoes to feed on cattle. According to the authors of the study, this suggests that in this area a plastic foraging strategy could provide greater benefits than a specialist strategy for this species.

*minimus* s.l., *An. culicifacies*, and *An. darlingi*. These shifts might also be linked to the nonrandom exposure of subpopulations of vectors to insecticide treated surfaces (ITNs or IRS). Several studies have indeed shown that the feeding and resting behaviour of anophelines is consistent in certain subpopulations and/or linked to certain genetic markers. Most of the studies on genetic determination of biting and resting behaviour are based on chromosomal inversions. Alleles captured within chromosome rearrangements are protected from recom‐ bination and can as such favour local adaptation by capturing sets of locally adapted genes which might lead to reproductive isolated entities or subpopulations [84]. In the Garki District in Nigeria, chromosomal arrangements in *An. arabiensis* and *An. gambiae* have been associated with exophagy and exophily [85,86] and with zoophily [87]. Exophagy and exophily were

*An. gambiae*, and the inverted arrangement 2Rbc for *An. arabiensis*. Moreover, the chromosome arrangements associated with indoor biting or resting are the ones adapted to drier environ‐ ments, while arrangements more frequent in outdoor collected specimens are those associated with more humid environments [85]. In the Zambesi valley, 2Rc *An. arabiensis* heterozygotes were associated with exophily and zoophily [57]. In Ethiopia *An. arabiensis* heterozygotes of the 2La and/or 2Rb chromosomal arrangements tended to bite later at night than the double homozygotes [88]. Also in laboratory experiments an association between chromosomal arrangements and circadian flight activity has been found [89]: female *An. stephensi* homozy‐ gotes for the 2Rb inversion showed more activity following light-on (corresponding to early morning) as compared to homozygous females for the standard 2R+b arrangement. Other fieldbased evidence on the existence of subpopulations showing consistent behaviour was obtained by studying behaviour of *An. balabacensis* in a capture-mark-recapture experiment in Borneo (Malaysia) [90]. This study revealed significant trends of *An. balabacensis* to be recaptured on the same host or resting site of the original capture. In contrast, a similar capture-markrecapture study on resting behaviour of *An. gambiae* s.l. in Tanzania showed no faithful tendencies of endo- or exophily [91]: the same individuals within the *An. gambiae* s.l. population mixed indoor and outdoor resting. More recent genetic studies are based on the frequencies of enzyme polymorphisms. In the Malaysian study [90], faithfully indoor and outdoor-resting populations showed significant differences in isozyme frequencies (loci *Est-3* and *Idh-3*). Also in Burundi, isozyme frequencies were significantly different between in- and out-door biting *An. arabiensis* (locus *Mdh-2*) and in- and out-door resting *An. gambiae* (*Mpi* and *Got-2* loci) [92]. Such differences were not observed for *An. gambiae* in Burkina Faso [93]. Moreover, mosquitoes carrying a specific genotype [93] or chromosome karyotypes [87] were found to be significantly more infected with sporozoites, suggesting the occurrence of subpopulations having different vector behaviours. These independent genetic studies, either based on karyotyping or on genotyping, provide evidence that active choice for the best place, time or host to bite, or the best place to rest can be associated with specific genotypes. This suggests the existence of subpopulations characterized by specific behavioural patterns which implies a non-uniform exposure to IRS or ITNs. Selection of specific behavioural patterns can then not be excluded.

However, other mechanisms can also explain these kinds of shifts. More early biting could occur as females that fail to obtain a blood meal during the previous night, might be more likely to commence host seeking in the early evening [44]. By disrupting the feeding behaviour,

for *An. arabiensis* and 2R+b

for

associated with the standard chromosomal arrangements 2R+a

686 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Regardless of the mechanism that causes these behavioural shifts, the case studies show that in several areas the proportion of outdoor-, early- and/or animal biting primary vectors are relatively increasing, which will then be responsible for residual transmission. Moreover, in a similar way, transmission by 'secondary' vectors that have outdoor or early biting behaviour might become more important than transmission by primary vectors in contexts of high coverage of ITNs and IRS. In a malaria endemic region of Thailand, one specimen of the Barbirostris Subgroup (*An. barbirostris/campestris*) was found to contain *Plasmodium* oocysts, in the prolonged absence of the main malaria vectors, showing that *An. barbirostris* s.l., an outdoor biting mosquito [94], might be responsible for maintaining malaria transmission in the absence of the main vectors [95]. As secondary vectors are often less anthropophilic, and might be more exophagic and early biting, planning of vector control should also take into account their behaviour. Moreover, as pointed out in [8], secondary vectors might be better vectors of *P. vivax* as compared to *P. falciparum*, as the extrinsic incubation period of *P. vivax* is shorter. In British Guiana, for example, *An. aquasalis*, a mostly zoophilic and exophilic mosquito species breeding in brackish water, was vector of several Vivax malaria outbreaks after *An. darlingi* was eliminated by DDT spraying [96]. Also more recently in Vietnam, *An. sawadwongporni*, a very early biting secondary vector, was found positive for *P. vivax* [8].

#### **7.2. ITNs and IRS are very effective, but additional measures are needed for reaching malaria elimination**

ITNs and IRS have been shown to have a large impact on malaria infection and disease [97,98]. Moreover, several entomological studies have also shown that where the vectors are mostly endophagic, endophilic and anthropophilic, ITNs and IRS are very effective in reducing their population density. This was for example shown for *An. minimus* in India [99] and for *An. dirus* in Laos [100], both of them being anthropophilic, indoor- and late-biting in the respective study sites. A recent study in Zambia also showed that even at a high coverage of ITNs and IRS, the highest probability for malaria transmission based on human and vector behaviour, still occurs indoors [101], making ITNs and IRS valuable tools.

ITNs can also have an effect on malaria transmitted by more zoophilic and exophagic mos‐ quitoes. In Sao Tomé for example, where *An. gambiae* is zoophilic and very exophagic, increased bed net use decreased the malaria prevalence in both bed net users and non-users [102]. The differences in prevalence between users and non-users were greatest in children under 5 years old, who are more likely to use the bed nets in the evening, showing that indeed the bed nets were the cause of the decrease. However, in older age groups, that are more likely to remain outside in the evening, no such difference was observed. Moreover, even at an almost 80% ITN coverage, still a 30% malaria prevalence was observed among bed net users. This means that, as expected, a part of transmission by these zoophilic and exophagic mosquitoes could not be prevented by ITNs [102]. Also in other parts of the world it has been shown that ITNs are less performing in areas with outdoor biting or resting vectors, for example in Peru and Nicaragua [11]. In the Garki District (Nigeria), the impact of the IRS campaign with propoxur was related to the prespraying ratio between the man-biting density and the indoor-resting density and to intraspecific cytogenetic variation [52]. Moreover, as reviewed in [103], even low levels of exophagy, exophily or zoophily may attenuate the impact of ITNs and IRS because this allows mosquitoes to obtain blood while avoiding fatal contact with insecticides.

As we have shown that outdoor-, animal- and early biting behaviour, as well as outdoor resting behaviour is widespread among malaria vectors all over the world and might be increasing as a result of widespread IRS or ITN use, there is an urgent need for additional control measures tackling malaria transmission by these vector populations [103–106]. In other words, there is a 'gap' in protection, not only before sleeping time, but also for people that remain outdoors during the night (Figure 2) and this gap needs to be tackled by additional vector control measures. There are many ways of additionally reducing host-vector contact, including the use of topical repellents, spatial repellents, insecticide treated clothing, long lasting insecticidal hammocks, etc. Recently much research is carried out on the effectiveness of these kind of tools. For example, in the Bolivian Amazon, where the primary vectors *An. darlingi* has a peak biting activity before sleeping time, a household based cluster randomized trial has shown that the combined use of a topical repellent (para-menthane-3,8-diol, PMD) and ITNs can reduce the incidence of malaria by 80%, which was only significant for *P. vivax* and not for *P. falciparum*, as compared to the use of ITNs alone [107]. DEET-based repellents also had an additional protective efficacy against malaria disease in a small scale community based trial in India [108], and DEET-based repellent soap against *P. falciparum* malaria in a household randomized trial in a refugee camp in Pakistan [109]. In an ongoing study in Cambodia, Picaridin based repellents are shown to provide a protection of more than 90% against the bites of the main malaria vectors *An. dirus* and *An. minimus* (MalaResT project led by ITM-Antwerp, preliminary results). Whether the mass use of this repellent will result in a decrease of malaria infection is currently under investigation using a cluster-randomized controlled trial in

population density. This was for example shown for *An. minimus* in India [99] and for *An. dirus* in Laos [100], both of them being anthropophilic, indoor- and late-biting in the respective study sites. A recent study in Zambia also showed that even at a high coverage of ITNs and IRS, the highest probability for malaria transmission based on human and vector behaviour,

ITNs can also have an effect on malaria transmitted by more zoophilic and exophagic mos‐ quitoes. In Sao Tomé for example, where *An. gambiae* is zoophilic and very exophagic, increased bed net use decreased the malaria prevalence in both bed net users and non-users [102]. The differences in prevalence between users and non-users were greatest in children under 5 years old, who are more likely to use the bed nets in the evening, showing that indeed the bed nets were the cause of the decrease. However, in older age groups, that are more likely to remain outside in the evening, no such difference was observed. Moreover, even at an almost 80% ITN coverage, still a 30% malaria prevalence was observed among bed net users. This means that, as expected, a part of transmission by these zoophilic and exophagic mosquitoes could not be prevented by ITNs [102]. Also in other parts of the world it has been shown that ITNs are less performing in areas with outdoor biting or resting vectors, for example in Peru and Nicaragua [11]. In the Garki District (Nigeria), the impact of the IRS campaign with propoxur was related to the prespraying ratio between the man-biting density and the indoor-resting density and to intraspecific cytogenetic variation [52]. Moreover, as reviewed in [103], even low levels of exophagy, exophily or zoophily may attenuate the impact of ITNs and IRS because this allows

As we have shown that outdoor-, animal- and early biting behaviour, as well as outdoor resting behaviour is widespread among malaria vectors all over the world and might be increasing as a result of widespread IRS or ITN use, there is an urgent need for additional control measures tackling malaria transmission by these vector populations [103–106]. In other words, there is a 'gap' in protection, not only before sleeping time, but also for people that remain outdoors during the night (Figure 2) and this gap needs to be tackled by additional vector control measures. There are many ways of additionally reducing host-vector contact, including the use of topical repellents, spatial repellents, insecticide treated clothing, long lasting insecticidal hammocks, etc. Recently much research is carried out on the effectiveness of these kind of tools. For example, in the Bolivian Amazon, where the primary vectors *An. darlingi* has a peak biting activity before sleeping time, a household based cluster randomized trial has shown that the combined use of a topical repellent (para-menthane-3,8-diol, PMD) and ITNs can reduce the incidence of malaria by 80%, which was only significant for *P. vivax* and not for *P. falciparum*, as compared to the use of ITNs alone [107]. DEET-based repellents also had an additional protective efficacy against malaria disease in a small scale community based trial in India [108], and DEET-based repellent soap against *P. falciparum* malaria in a household randomized trial in a refugee camp in Pakistan [109]. In an ongoing study in Cambodia, Picaridin based repellents are shown to provide a protection of more than 90% against the bites of the main malaria vectors *An. dirus* and *An. minimus* (MalaResT project led by ITM-Antwerp, preliminary results). Whether the mass use of this repellent will result in a decrease of malaria infection is currently under investigation using a cluster-randomized controlled trial in

still occurs indoors [101], making ITNs and IRS valuable tools.

688 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

mosquitoes to obtain blood while avoiding fatal contact with insecticides.

**Figure 2.** Protection 'gap' when only indoor insecticide-based vector control measures are applied. Anophelines gen‐ erally bite between 6pm and 6am. ITNs will only protect from infective bites that are acquired indoors, and during sleeping time. IRS only target mosquitoes that rest indoors. Therefore, there is a gap in protection both indoors and outdoors before and after people go to bed (A), but also for people conducting outdoor activities during the night (i.e. 'risk behaviour') (B).

Ratanakkiri province in Cambodia. In a refugee camp in Kenya, permethrin treated clothing and blankets reduced malaria infection significantly [110]. In Southeast Asia, long lasting insecticidal hammocks have been shown to be effective against malaria disease [111] and against *An. minimus* bites, but not *An. dirus* bites [112]. For zoophilic mosquitoes, intervening in the host-vector contact could be more efficient by focusing on its preferred hosts, e.g. by insecticide treatment of cattle. However, killing partly zoophilic mosquitoes in sufficient numbers to suppress malaria transmission would require high protective coverage of both human and animal blood sources [104]. Moreover, it has been observed in Ethiopia that more than 90% of the blood meals taken by zoophilic vectors were taken from the legs of cattle [113], which are more difficult to treat.

Alternative personal protection measures are also of interest for people that work or reside in the forest, a risk area of malaria transmission in Southeast Asia [114]. For temporary shelters in the forest, insecticide treated plastic sheeting could be useful as this has proven to be effective in protecting against malaria disease in emergency camps [115]. Their effectiveness will rely both on their repelling effect and their killing effect, and whether mosquitoes will rest on this sheeting. Alternatively, other more accepted insecticide treated bed net-designs (V-shaped


a E: Early evening & morning biting; N: Night biting; A: Antropophilic; Z: Zoophilic; I: Indoor; O: Outdoor

b Community protection can only be achieved if the coverage of the intervention is large enough.

\* Community protection is assumed or shown in a limited number of studies, but more evidence is required for confir‐ mation of community protection.

**Table 3.** Vector control tools and their targets.

nets, long lasting insecticidal hammocks, etc.), could provide protection for people staying in the forest during the night.

The more zoophilic, exophagic, or early biting a mosquito species or population, the more personal protection will act simply by blocking host-vector contact (through lethal or repellent effects). As shown by a mathematical model, malaria transmission involving zoophilic vectors (with 10% feeding on humans) can only be significantly decreased if the personal protection measures confer high levels of individual protection to users (80%) and be used by the majority of human population (80%) [116]. Therefore, the success of any intervention in this context will depend on its entomological efficacy, but also on the human behaviour, including acceptance and adherence to the preventive measures within the community. In São Tomé for example, many people watch communal television outdoors, posing them at risk for early-evening malaria transmission [117]. In Thailand, people do not take their ITN from the village to their farm plot [118]. Also in Vietnam, people often combine living in the village with a second home at their fields located in the forest [119], creating other malaria control needs, such as, for example, long lasting insecticidal hammocks. Taking into account human behaviour when adapting vector control strategies will then be crucial. In Bioko Island (Equatorial Guinea) for example, an increased trend of outdoor biting was observed for the main malaria vector *An. gambiae*[21]. However, the main malaria risk group, namely children under 15 years old, rarely stay outdoors when it is dark, and there is no evidence that children who report to stay outdoors during the night are at higher risk for malaria infection as compared to those who do not [120]. Implementing control measures that target outdoor biting mosquitoes in this age group would then provide no additional benefit and would be a waste of resources, as personal protection tools might be very expensive to implement.

Also other tools not relying on the host-vector contact can supplement ITNs and IRS as they are not specific for indoor biting and indoor resting mosquito populations [105,106]. Vector control tools could for example target key environmental resources such as the aquatic larval habitat, sugar sources, and resting behaviour. Very little is known about how to manipulate these environmental resources so that malaria transmission is interrupted [105]. Knowledge on vector ecology and behaviour therefore remains crucial. However, despite large knowledge gaps, several examples exist of malaria control by targeting non-blood meal related steps of the mosquito cycle. Larval source management has indeed shown to be effective where vectors breed in large water bodies [121]. However, when larval habitats are more dispersed and not permanent, this approach is considered less feasible. Renewed attention has been given to larval source management as complementary tool to ITNs as recent studies in Africa have shown that it provides substantial additional protection with a high cost-effectiveness in specific settings [122]. Moreover, other innovative ideas combined with knowledge on the vector behaviour can lead to successful vector control. Toxic sugar baits for example were successfully used in a targeted way for the control of the cistern dwelling malaria vector *An. claviger* in the desert oases of Israel [123]. Fungal biopesticides also have the potential to significantly reduce densities of malaria vectors [124] as well as associated malaria transmis‐ sion [125]. These fungi could be delivered through outdoor odour-baited stations, and in this way slowly eliminate a high proportion of outdoor-resting vectors [126].

## **8. Conclusion**

nets, long lasting insecticidal hammocks, etc.), could provide protection for people staying in

\* Community protection is assumed or shown in a limited number of studies, but more evidence is required for confir‐

**Mosquito behaviour that is targeted**

**Place of biting**

(E &) N A O O P & C

E & N A I & O I & O P & C\*

E & N A & Z I & O O C\*

E & N A & Z I & O O P

**Place of resting (I/O)a**

**(I/O)a**

**Time of biting (E/N)a**

**Host preference (A/Z)a**

ITNs N A I I P & C

Insecticide treatment of cattle E & N Z I & O I & O C\*

IRS E & N A & Z I & O I C Larval source management E & N A & Z I & O I & O C\* Toxic sugar baits E & N A & Z I & O I & O C\*

 E: Early evening & morning biting; N: Night biting; A: Antropophilic; Z: Zoophilic; I: Indoor; O: Outdoor b Community protection can only be achieved if the coverage of the intervention is large enough.

**Personal (P) or community (C)b protection**

The more zoophilic, exophagic, or early biting a mosquito species or population, the more personal protection will act simply by blocking host-vector contact (through lethal or repellent effects). As shown by a mathematical model, malaria transmission involving zoophilic vectors (with 10% feeding on humans) can only be significantly decreased if the personal protection measures confer high levels of individual protection to users (80%) and be used by the majority of human population (80%) [116]. Therefore, the success of any intervention in this context will depend on its entomological efficacy, but also on the human behaviour, including acceptance and adherence to the preventive measures within the community. In São Tomé for example, many people watch communal television outdoors, posing them at risk for early-evening malaria transmission [117]. In Thailand, people do not take their ITN from the village to their farm plot [118]. Also in Vietnam, people often combine living in the village with a second home at their fields located in the forest [119], creating other malaria control needs, such as, for example, long lasting insecticidal hammocks. Taking into account human behaviour when adapting vector control strategies will then be crucial. In Bioko Island (Equatorial Guinea) for example, an increased trend of outdoor biting was observed for the main malaria vector *An. gambiae*[21]. However, the main malaria risk group, namely children under 15 years old, rarely

the forest during the night.

mation of community protection.

**Tool**

690 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Long lasting insecticidal hammocks & other net designs adapted to outdoor conditions

Personal protection including Topical & spatial repellents, Insecticide treated clothing

Tools not relying on vector-host contact

Treatment of outdoor resting places, e.g. with

**Table 3.** Vector control tools and their targets.

Insecticide treated plastic sheeting for shelters

Tools relying on host-vector contact

in the forest

fungal biopesticides

a

For malaria eradication to succeed, all elements in the transmission cycle must be sufficiently targeted. With the current vector control tools, only indoor- and late-biting, and indoor-resting vectors are tackled. In this paper, we have shown that there is a 'gap' in protection, not only before sleeping time, but also for people that remain outdoors during the night. Moreover, by describing different shifts in vector species, and vector behaviour within species or species complexes, we have shown that the importance of this gap can increase as a result of wide‐ spread ITN or IRS use. Therefore, to eliminate residual malaria transmission, additional vector control tools will be needed. These new vector control tools should be designed to target outdoor and early feeding mosquitoes. Moreover, they should be accessible and acceptable for the populations at risk. A specific mosquito behaviour assuring its vectorial status is only relevant in relation to a specific human behaviour and the relation people have with their surrounding environment. Interrupting malaria transmission may than require different combinations of mosquito control methods addressing each mosquito behaviour at risk for transmission, but also taking into account possible changes in soil occupation, housing conditions, sleeping habits, and outdoor occupation. In conclusion, there is no 'silver bullet' in vector control and malaria prevention. New paradigms for controlling and/or interrupting malaria transmission should then be explored for their protective efficacy and adapted to the local context for a good efficiency. Although implementation of such new approaches might be very expensive, they will be crucial if malaria elimination is the final aim.

### **Acknowledgements**

This review was initiated under the impulse of the Roll Back Malaria - Vector Control Working Group – Work Stream Outdoor Malaria Transmission (http://www.rbm.who.int/mechanisms/ vcwgWorkstream2.html), the MalaResT research Project (B&M Gates Foundation OPP1032354) and the Third ITM-DGCD Framework Agreement Programme. We would like to thank Vincent Sluydts and Sylvie Manguin for their critical review of this paper.

## **Author details**

Lies Durnez1 and Marc Coosemans1,2\*

\*Address all correspondence to: mcoosemans@itg.be; ldurnez@itg.be

1 Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium

2 Department of Biomedical Sciences, Faculty of Pharmaceutical, Veterinary and Biomedical Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium

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in vector control and malaria prevention. New paradigms for controlling and/or interrupting malaria transmission should then be explored for their protective efficacy and adapted to the local context for a good efficiency. Although implementation of such new approaches might

This review was initiated under the impulse of the Roll Back Malaria - Vector Control Working Group – Work Stream Outdoor Malaria Transmission (http://www.rbm.who.int/mechanisms/ vcwgWorkstream2.html), the MalaResT research Project (B&M Gates Foundation OPP1032354) and the Third ITM-DGCD Framework Agreement Programme. We would like

to thank Vincent Sluydts and Sylvie Manguin for their critical review of this paper.

1 Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium

2 Department of Biomedical Sciences, Faculty of Pharmaceutical, Veterinary and Biomedical

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**Author details**

and Marc Coosemans1,2\*

\*Address all correspondence to: mcoosemans@itg.be; ldurnez@itg.be

Sciences, University of Antwerp, Antwerpen (Wilrijk), Belgium

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702 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors


## **Vector Control: Some New Paradigms and Approaches**

Claire Duchet, Richard Allan and Pierre Carnevale

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56258

## **1. Introduction**

#### **1.1. Context**

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1638-41.

The World Malaria Report 2012 [1] summarizes data received from 104 malaria-endemic countries and territories for 2011. Ninety-nine of these countries had on-going malaria transmission. According to the latest World Health Organization (WHO) estimates, there were about 219 million cases of malaria in 2010 and an estimated 660,000 deaths. Africa is the most affected continent: about 90% of all malaria deaths occur there.

Malaria surveillance systems detect now only around 10% of the estimated global number of cases. In 41 countries around the world, it is not possible to make a reliable assessment of malaria trends due to incompleteness or inconsistency of reporting over time.

Actually another estimation of mortality [2] gave the following figures of 1,238,000 (929,000-1,685,000) deaths in 2010. This "one to two" ratio for the same year is matter of concern when considering that the main target of RBM is to reduce by 50% the burden of malaria.

The Lives Saved Tool (LiST) was developed to provide national and regional estimates of cause-specific mortality based on the extent of intervention coverage scale-up in sub-Saharan Africa and it appeared that it "performed reasonably well at estimating the effect of vector control scale-up on child mortality when compared against measured data from studies across a range of malaria transmission settings and is a useful tool in estimating the potential mortality reduction achieved from scaling-up malaria control interventions" [3].

Three major issues deserve special attention: tools for vector control, resistance of mosquito to insecticides, of *Plasmodium* to drugs, of human population to change their behavior, and costs. To tackle these issues new paradigms must be developed with the objectives of efficacy, acceptability and cost-efficiency.

© 2013 Duchet et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Duchet et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Vector control remains the most generally effective measure to prevent malaria parasite transmission and therefore was one of the four basic technical elements of the Global Malaria Control Strategy [4]. Through the 1980s', vector control was mainly based upon Indoor Residual Spraying (IRS) and, in some circumstances, larval control, but an important break‐ through occurred with Insecticide Treated Nets (ITNs) then Long Lasting Insecticide treated Nets (LNs) (Figure 1) were introduced. The large scale implementation of ITN has, in several epidemiological settings, produced striking reductions in malaria transmission (-90%), incidence rate of malaria morbidity (-50%) and overall infant mortality (-17%) [5].

For WHO to achieve universal access to long-lasting insecticidal nets (LLINs), 780 million people at risk would need to have access to LLINs in sub-Saharan Africa, and approximately 150 million bed nets would need to be delivered each year. The number of LLINs delivered to endemic countries in sub-Saharan Africa dropped from a peak of 145 million in 2010 to an estimated 66 million in 2012 [1]. This will not be enough to fully replace the LLINs delivered 3 years earlier, indicating that total bed net coverage will decrease unless there is a massive scale-up in 2013. A decrease in LLIN coverage is likely to lead to major resurgences in the disease. In 2011, 153 million people were protected by indoor residual spraying (IRS) around the world, or 5% of the total global population at risk. In the WHO African Region, 77 million people, or 11% of the population at risk were protected through IRS in 2011.

Recent field observations have shown that LLINs may not be as durable as previously estimated and the majority of the most commonly distributed LLINs may have a shorter effective material life, which induce a higher than scheduled cost of global malaria control when LLIN have to be changed more frequently than expected. The problem of cost is a burning issue. International disbursements for malaria control rose steeply during the past eight years and were estimated to be US\$ 1.66 billion in 2011 and US\$ 1.84 billion in 2012. National government funding for malaria programmes has also been increasing in recent years, and stood at an estimated US\$ 625 million in 2011. However, the currently available funding for malaria prevention and control is far below the resources required to reach global malaria targets. An estimated US\$ 5.1 billion is needed every year between 2011 and 2020 to achieve universal access to malaria interventions. In 2011, only US\$ 2.3 billion was available, less than half of what is needed ([1] fact sheet).

In its 23rd meeting in Senegal, the RBM Partnership Board concluded with an urgent call to governments of malaria endemic countries and development partners to secure the US\$2.4 billion needed over the next two years to maintain high levels of coverage with life-saving malaria prevention and treatment interventions in eight African countries. This call follows a decade of success where *malaria deaths have fallen by over one-third in sub-Saharan Africa*.

Overall, out of a total of US\$6.8 billion required, US\$3.2 billion has been mobilized leaving a US\$3.6 billion gap to make sure all affected countries in Africa have enough insecticide treated nets, effective treatments and rapid diagnostic tests for all populations at risk of malaria to achieve the target of near-zero deaths by 2015.

In term of vector control several issues deserve special attention. The change in vector behavior from indoor to outdoor feeding under insecticide pressure may limit the impact of classical control interventions such as LNs and IRS which target indoor feeding and resting mosquitoes and new tools are obviously needed. On the other hand, species that naturally bite and spend most of their time outdoors such as *Anopheles dirus* in S.E. Asia are poorly controlled by these classical tools and new approaches are urgently needed.

Vector control remains the most generally effective measure to prevent malaria parasite transmission and therefore was one of the four basic technical elements of the Global Malaria Control Strategy [4]. Through the 1980s', vector control was mainly based upon Indoor Residual Spraying (IRS) and, in some circumstances, larval control, but an important break‐ through occurred with Insecticide Treated Nets (ITNs) then Long Lasting Insecticide treated Nets (LNs) (Figure 1) were introduced. The large scale implementation of ITN has, in several epidemiological settings, produced striking reductions in malaria transmission (-90%),

For WHO to achieve universal access to long-lasting insecticidal nets (LLINs), 780 million people at risk would need to have access to LLINs in sub-Saharan Africa, and approximately 150 million bed nets would need to be delivered each year. The number of LLINs delivered to endemic countries in sub-Saharan Africa dropped from a peak of 145 million in 2010 to an estimated 66 million in 2012 [1]. This will not be enough to fully replace the LLINs delivered 3 years earlier, indicating that total bed net coverage will decrease unless there is a massive scale-up in 2013. A decrease in LLIN coverage is likely to lead to major resurgences in the disease. In 2011, 153 million people were protected by indoor residual spraying (IRS) around the world, or 5% of the total global population at risk. In the WHO African Region, 77 million

Recent field observations have shown that LLINs may not be as durable as previously estimated and the majority of the most commonly distributed LLINs may have a shorter effective material life, which induce a higher than scheduled cost of global malaria control when LLIN have to be changed more frequently than expected. The problem of cost is a burning issue. International disbursements for malaria control rose steeply during the past eight years and were estimated to be US\$ 1.66 billion in 2011 and US\$ 1.84 billion in 2012. National government funding for malaria programmes has also been increasing in recent years, and stood at an estimated US\$ 625 million in 2011. However, the currently available funding for malaria prevention and control is far below the resources required to reach global malaria targets. An estimated US\$ 5.1 billion is needed every year between 2011 and 2020 to achieve universal access to malaria interventions. In 2011, only US\$ 2.3 billion was available,

In its 23rd meeting in Senegal, the RBM Partnership Board concluded with an urgent call to governments of malaria endemic countries and development partners to secure the US\$2.4 billion needed over the next two years to maintain high levels of coverage with life-saving malaria prevention and treatment interventions in eight African countries. This call follows a decade of success where *malaria deaths have fallen by over one-third in sub-Saharan Africa*.

Overall, out of a total of US\$6.8 billion required, US\$3.2 billion has been mobilized leaving a US\$3.6 billion gap to make sure all affected countries in Africa have enough insecticide treated nets, effective treatments and rapid diagnostic tests for all populations at risk of malaria to

In term of vector control several issues deserve special attention. The change in vector behavior from indoor to outdoor feeding under insecticide pressure may limit the impact of classical

incidence rate of malaria morbidity (-50%) and overall infant mortality (-17%) [5].

people, or 11% of the population at risk were protected through IRS in 2011.

less than half of what is needed ([1] fact sheet).

706 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

achieve the target of near-zero deaths by 2015.

Vector control is also threatened by *the development of insecticide resistance* [4-9]. The frequency of resistance, has risen sharply over the last decade and the relationship between current indicators of resistance and the impact of vector control interventions is still unclear according to the different mechanisms of resistance, though most scientists believe that at some point in the near future resistance will begin to compromise control efforts, and new active ingredients to replace the current ones are urgently needed. Mosquito resistance to at least one insecticide used for malaria control has been identified in 64 countries around the world. In May 2012, WHO and the Roll Back Malaria Partnership released the Global Plan for Insecticide Resistance Management in malaria vectors, a five-pillar strategy for managing the threat of insecticide resistance.

Overcoming insecticide resistance will require novel chemical modes of action or combined interventions, with multiple active agreements, used as part of an integrated vector manage‐ ment strategy or completely new tools to delay the emergence of resistance by reducing selection pressure (e.g. rotations), or kill resistant vectors by exposing them to multiple insecticides (e.g. mixtures, when they become available).

Thus, new paradigms and approaches to vector control will expand the range of species that can be controlled and the chemical modes of action that can be employed, as well as potentially reducing the costs and complications of delivering them.

### **1.2. Definitions (from Innovative Vector Control Consortium IVCC)**

A paradigm can be defined as a mean to deliver an active ingredient to the vector by targeting certain behaviors or ecologies. Paradigms can be associated with general chemical modes of action. Tools that target mosquito resting employ contact toxins. Those based on sugar feeding employ the so-called stomach poisons, etc. New paradigms open the door for exploitation of new chemical modes of action. An intervention paradigm (current examples: Insecticidal Nets or Indoor Residual Spray) is characterized by a primary mode of action (e.g. kills insect that land on the walls) and key characteristics such as the way it applied, its distribution process, economics, user, acceptability etc.

A paradigm may be served by several categories of products, each of which is described by a Target Product Profile (TPP) (e.g. ITNs *vs*. LLINs). The TPP will describe the primary func‐ tionality and characteristics that are required of a product to achieve a particular epidemio‐ logical outcome. Individual products within the category are defined by specifications.

Figure 1 illustrates the relationship among behaviors, paradigms and chemical mode of action. Where new paradigms do not exist in public health an example from agriculture or home and garden products is listed instead.

**Figure 1.** Relationship among behaviors, paradigms and chemical mode of action of insecticides.

## **2. New approaches to existing paradigms**

#### **2.1. New long lasting insecticide formulation for IRS**

A microencapsulated formulation (CS) of the organophosphate chlorpyrifos methyl has recently been developed as long lasting i.e., alternative to DDT. In experimental huts in South Benin, against pyrethroid resistant (*kdr* + metabolic resistance) *An. gambiae* M form (and *Cx. quinquefasciatus*), chlorpyrifos methyl (Figure 2) was used to treat mosquito nets, and for IRS, and was compared to other commonly used insecticides: DDT and lambdacyhalothrin [10].

On nets, for N'Guessan et al [10] "the percentage of mortality among *An. gambiae* was 45.2% with the chlorpyrifos methyl-treated net and only 29.8% with the lambacyhalothrin-treated net. Mortality rates among *Cx. quinquefasciatus* were lower than among *An. gambiae* and did not exceed 15% with either type of treated net". While "Mortality of pyrethroid resistant *An. gambiae* was 95.5% with chlorpyrifos methyl-IRS compared to 50.4% in the hut sprayed with DDT and 30.8% in the hut sprayed with lambdacyhalothrin. The mortality of *Cx. quinquefas‐*

**Figure 2.** Chemical formula of chlorpyrifos methyl

**Figure 1.** Relationship among behaviors, paradigms and chemical mode of action of insecticides.

A microencapsulated formulation (CS) of the organophosphate chlorpyrifos methyl has recently been developed as long lasting i.e., alternative to DDT. In experimental huts in South Benin, against pyrethroid resistant (*kdr* + metabolic resistance) *An. gambiae* M form (and *Cx. quinquefasciatus*), chlorpyrifos methyl (Figure 2) was used to treat mosquito nets, and for IRS, and was compared to other commonly used insecticides: DDT and lambdacyhalothrin [10]. On nets, for N'Guessan et al [10] "the percentage of mortality among *An. gambiae* was 45.2% with the chlorpyrifos methyl-treated net and only 29.8% with the lambacyhalothrin-treated net. Mortality rates among *Cx. quinquefasciatus* were lower than among *An. gambiae* and did not exceed 15% with either type of treated net". While "Mortality of pyrethroid resistant *An. gambiae* was 95.5% with chlorpyrifos methyl-IRS compared to 50.4% in the hut sprayed with DDT and 30.8% in the hut sprayed with lambdacyhalothrin. The mortality of *Cx. quinquefas‐*

**2. New approaches to existing paradigms**

708 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**2.1. New long lasting insecticide formulation for IRS**

*ciatus* in the chlorpyrifos methyl-IRS huts was 66.1% whereas in the DDT and lambdacyhalo‐ thrin-IRS huts it was only 14%". Therefore "chlorpyrifos methyl-IRS showed greater potential than DDT of lambdacyhalothrin-IRS for control of pyrethroid resistant *An. gambiae* M form and *Cx. quinquefasciatus* in areas of high *kdr* frequency" [11].

In terms of mortality the short residual activity of chlorpyrifos methyl on ITN is of great concern with a mortality rate decreasing from 100% to 9.7% within just one month while as IRS on cement it was observed "no loss of activity during the nine months of follow-up" compared to the fast decay of DDT and lambdacyhalothrin observed within the first month of spraying. A 9-month efficacy could be very valuable in many West and East African endemic countries with malaria transmission seasons lasting less than 8 months, and where IRS application of chlorpyrifos methyl each year could be adequate. In areas with developing pyrethroid resistance one might envisaged continued use of pyrethroid LLIN in combination with IRS, rotating the use of chlorfenapyr and CS long lasting chlorpyrifos methyl formulation.

#### **2.2. New insecticides paints combining several insecticides and an insect growing regulator for IRS**

Insecticide paints are new interesting paradigm for vector control with several advantages regarding classical IRS. It may provide future possibilities to combine several active ingredi‐ ents in one product and therefore be used to help manage insecticide resistance. Paints can be produced in different colors to fit with people's choice. They may also be potentially imple‐ mentable by households without the need for a specialized team to deliver the intervention, as is the case with IRS. This could improve community and household acceptance and uptake. Paints may also have the potential of being longer lasting than IRS. Insect growth regulator (IGR), a product usually used as larvicides, is also now being evaluated in Inesfly® 5A IGR™, a paint designed to target adult mosquitoes. Inesfly® 5A IGR™ is composed of two organo‐ phosphates (OPs), chlorpyriphos (1.5%), and diazinon (1.5%) and pyriproxyfen (0.063%) an IGR which was successfully used against *Triatoma infestans* [12]. The product is white vinyl paint with an aqueous base. Active ingredients reside within Ca CO3 + resin microcapsules. The formulation allows a gradual release of active ingredients, increasing its persistence.

In Benin the Inesfly® insecticide paint has been tested in laboratory [13] and in field [14] studies. In the laboratory study, the paint was tested against laboratory strains of the urban pest *Cx. quinquefasciatus* the susceptible (S-Lab) strain and the SR homozygote for the ace-1R resistant gene involved in the resistance to OPs and carbamates, with classical bioassay cones (tests on 30 min). Efficacy was measured not only in terms of induced mortality but also in terms of fecundity (number of eggs laid), fertility (% hatching) and larval development (% pupation and % emergence). Insecticidal paints were tested at different time points: T0, 6 (= 6 months), 9 (= 9 months) and 12 months after application on four different surfaces: softwood, hard plastic (non-porous materials), ready-mixed cement and ready-mixed stucco (porous materials) at two doses, 1kg/6 m2 (manufacturer's recommended dose to obtain surfaces completely white) and 1 kg/12 m2 . Female mosquitoes were given a blood meal 36 hours after standardized exposure to the painted surfaces. The study showed that the highest rates of mortality were obtained by both doses on susceptible as well as resistant strains even 12 months after treatment, on non-porous surfaces (softwood, plastic), whereas, on porous surfaces (cement, stucco) efficacy was much lower on resistant than on susceptible strain and it dropped to almost 0 at 6 and 12 months in both strains.

Thus long-term efficacy was an issue of porosity of materials rather than the pH of materials or the dose applied. It should be noted that 100% mortality was achieved on non-porous surface even against the OP resistant strain.

In terms of fecundity, fertility, and larval development, "a significant reduction in the number of eggs laid was shown at 0 and 9 months after treatment at either dose. A reduction in egg hatching was observed at T0, but not at T9. An increased mortality from the nymph to the adult stage was shown 9 months after treatment at the higher dose. No differences were found on the duration of the larval development. No IGR effect was observed 12 months after treatment". The percentage of emergence (i.e. adult emerging from pupa) dropped from 80% in control to #53% in samples from exposed females. Hence an adulticide could have impact not only on longevity of females exposed but also on their offspring which is a great advantage for mosquito population control.

Field trials were conducted in area where the local population of *An. gambiae* is composed of the M molecular form with resistance to pyrethroids and DDT, *kdr* is present at a high frequency, but is susceptible to OPs and carbamates, the ace-1R mutation was absent. *Cx. quinquefasciatus* shows high resistance to DDT, pyrethroids and carbosulfan with high *kdr* frequency and elevated levels of esterases and GST activity but the ace-1R mutation was absent [9]. In these trials, experimental huts were treated with either 1 or 2 layers of insecticide paint at one dose (6 kg/m2 ). Treatments were applied to either just walls, or to walls plus the ceiling. Unfed females of the lab-reared *An. gambiae* Kisumu strain (sensitive to all insecticides), were tested against local resistant wild strain *An. gambiae* and *Cx. quinquefasciatus*. The *An. gambiae* Kisumu strain mosquitoes were placed inside the huts at a distance of 1 m from two perpen‐ dicular walls, and left from 19:00 to 7:00 h [14]. The wild strains were tested using the standard WHO bioassay method.

Mortality of wild resistant *An. gambiae* was high with 83% even 9 months after treatment (2 paint layers on walls). Mortality of wild resistant *Cx. quinquefasciatus* was >50% even 9 months after treatment (2 paint layers on walls). No deterrent or excito-repellent effect was observed against *An. gambiae* nor *Cx. quinquefasciatus*. Mortality rates of exposed *An. gambiae* Kisumu strain in distance experiments in huts (1 m from two perpendicular walls; see above) with 2 layers were most striking, because even one year after treatment 100% of these sensitive mosquitoes were killed (Figure 3C).

Classical cone bioassay showed that in huts with 2 layers "twelve months after treatment mortality rates were of 70-80% against *An. gambiae* and *Cx. quinquefasciatus*". Release of insecticide susceptible unfed *An. gambiae* specimens in huts treated but without net (untreated) showed that 2-13% of females took their blood meal while 72% were well blood fed in control huts. Mortality rates observed in distance experiments were most striking, (Figures 3A & 3B) and even one year after treatment 100% of exposed *An. gambiae* Kisumu strain specimens were killed in huts with 2 layers (Figure 3C).

pupation and % emergence). Insecticidal paints were tested at different time points: T0, 6 (= 6 months), 9 (= 9 months) and 12 months after application on four different surfaces: softwood, hard plastic (non-porous materials), ready-mixed cement and ready-mixed stucco (porous

standardized exposure to the painted surfaces. The study showed that the highest rates of mortality were obtained by both doses on susceptible as well as resistant strains even 12 months after treatment, on non-porous surfaces (softwood, plastic), whereas, on porous surfaces (cement, stucco) efficacy was much lower on resistant than on susceptible strain and

Thus long-term efficacy was an issue of porosity of materials rather than the pH of materials or the dose applied. It should be noted that 100% mortality was achieved on non-porous surface

In terms of fecundity, fertility, and larval development, "a significant reduction in the number of eggs laid was shown at 0 and 9 months after treatment at either dose. A reduction in egg hatching was observed at T0, but not at T9. An increased mortality from the nymph to the adult stage was shown 9 months after treatment at the higher dose. No differences were found on the duration of the larval development. No IGR effect was observed 12 months after treatment". The percentage of emergence (i.e. adult emerging from pupa) dropped from 80% in control to #53% in samples from exposed females. Hence an adulticide could have impact not only on longevity of females exposed but also on their offspring which is a great advantage

Field trials were conducted in area where the local population of *An. gambiae* is composed of the M molecular form with resistance to pyrethroids and DDT, *kdr* is present at a high frequency, but is susceptible to OPs and carbamates, the ace-1R mutation was absent. *Cx. quinquefasciatus* shows high resistance to DDT, pyrethroids and carbosulfan with high *kdr* frequency and elevated levels of esterases and GST activity but the ace-1R mutation was absent [9]. In these trials, experimental huts were treated with either 1 or 2 layers of insecticide paint

Unfed females of the lab-reared *An. gambiae* Kisumu strain (sensitive to all insecticides), were tested against local resistant wild strain *An. gambiae* and *Cx. quinquefasciatus*. The *An. gambiae* Kisumu strain mosquitoes were placed inside the huts at a distance of 1 m from two perpen‐ dicular walls, and left from 19:00 to 7:00 h [14]. The wild strains were tested using the standard

Mortality of wild resistant *An. gambiae* was high with 83% even 9 months after treatment (2 paint layers on walls). Mortality of wild resistant *Cx. quinquefasciatus* was >50% even 9 months after treatment (2 paint layers on walls). No deterrent or excito-repellent effect was observed against *An. gambiae* nor *Cx. quinquefasciatus*. Mortality rates of exposed *An. gambiae* Kisumu strain in distance experiments in huts (1 m from two perpendicular walls; see above) with 2 layers were most striking, because even one year after treatment 100% of these sensitive

). Treatments were applied to either just walls, or to walls plus the ceiling.

(manufacturer's recommended dose to obtain surfaces

. Female mosquitoes were given a blood meal 36 hours after

materials) at two doses, 1kg/6 m2

710 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

even against the OP resistant strain.

for mosquito population control.

at one dose (6 kg/m2

WHO bioassay method.

mosquitoes were killed (Figure 3C).

it dropped to almost 0 at 6 and 12 months in both strains.

completely white) and 1 kg/12 m2

**Figure 3.** Mortality rates observed in distance experiments of exposed unfed *Anopheles gambiae* (A), unfed *Culex quinquefasciatus* (B), and *Anopheles gambiae* Kisumu strain (C) observed after 3 or 6 or 9 or 12 months after treat‐ ment (T3, T6, T9, T12 respectively).

These observations of "volume effect", "layer effect", "substrate effect", residual efficacy duration, and its efficacy against susceptible and resistant strains of the malaria vector *An. gambiae* and the nuisance insect *Culex quinquefasciatus*, are very encouraging. The paints ability to reduce mosquito fecundity and egg hatching opens up interesting new perspectives on malaria and mosquito control for urban settings where walls are commonly constructed with brick, concrete and plaster and provide suitable surfaces for paints, unlike classical mud made wall houses that characterize most rural communities. The paints ability to also reduce *Culex* mosquitoes is likely to increase community acceptance and maintenance of paint.

#### **2.3. New mode of action families for IRS usage: Neonicotinoids**

Neonicotinoid insecticides act on the central nervous system of insects by binding of agonist on postsynaptic nicotinic receptors [15]. Discovered in 1998, dinotefuran is a novel neonicoti‐ noid insecticide which belongs to the third-generation neonicotinoids (sub-class: furanicotinyl compounds) [16]. It is a neonicotinoid agonist of the nicotinoid acetylcholine receptor with no cross-resistance to other insecticides such as organochlorine (OC), organophosphate (OP), carbamates or pyrethroids. Its efficiency is not greatly diminished by the presence of resistance mechanisms such as *kdr* or ace-1R in mosquitoes.

In studies comparing the impact of dinotefuran, permethrin and propoxur on resistant strains of *Cx. quinquefasciatus*, dinotefuran was about 10 times more effective than permethrin on the BKPER strain, and 1000 times more effective than propoxur on resistant R-LAB strain [17]. If this product can be incorporated into material (e.g. LNs) or IRS applications then it should be useful in areas where resistance to pyrethroids and carbamates has developed.

The option of associating insecticides with different modes of action is one of the possible strategies for resistance management (as developed in another Chapter by Corbel & N'Gues‐ san). An interesting approach that has recently been studied, combined Piperonyl butoxide (PBO), organic compound used as pesticide synergist, and dinotefuran in an attempt to restore the efficacy of deltamethrin treated mosquito net against resistant *An. gambiae*[18]. Darriet and Chandre [18] have also conducted classical laboratory cone tests of nets treated with delta‐ methrin, PBO (the classical synergist, inhibitor of oxidases) and dinotefuran alone or in combination against susceptible ("KIS") and resistant '("VKPR") strains of laboratory reared *An. gambiae*. Results of these tests are summarized in Table 1.


**Table 1.** Effects of mosquito nets treated with deltamethrin, PBO and dinotefuran on susceptible ("KIS"), and resistant '("VKPR") strains of *Anopheles gambiae.*

WHO's minimum mortality level for insecticides is 80% and this provides a reasonable operational guideline for effectiveness. In this study PBO combined with deltamethrin increased significantly its efficacy (synergistic effect), but not to a level adequate for control against pyrethroid-resistant mosquitoes, "suggesting that the acetylcholine concentration within the synaptic gap probably also increased". Interestingly, PBO had an antagonistic effect when combined with dinotefuran, decreasing this insecticide's efficacy. However, when PBO and Dinotefuran were combined with deltamethrin, the combination resulted in 99% mortality against the pyrethroid resistant mosquito strain, comparable with deltamethrin treated nets (in terms of mortality and KD effect) on the fully susceptible mosquito strain. For Darriet and Chandre [18] "the concomitant action of enhanced acetylcholine concentration in the synaptic gap and inactivation of nicotinic receptors by dinotefuran probably explains the strong synergy observed after exposure to the three-compound mixture, which caused nearly 100% mortality in a pyrethroid-resistant strain of *An. gambiae".*

noid insecticide which belongs to the third-generation neonicotinoids (sub-class: furanicotinyl compounds) [16]. It is a neonicotinoid agonist of the nicotinoid acetylcholine receptor with no cross-resistance to other insecticides such as organochlorine (OC), organophosphate (OP), carbamates or pyrethroids. Its efficiency is not greatly diminished by the presence of resistance

In studies comparing the impact of dinotefuran, permethrin and propoxur on resistant strains of *Cx. quinquefasciatus*, dinotefuran was about 10 times more effective than permethrin on the BKPER strain, and 1000 times more effective than propoxur on resistant R-LAB strain [17]. If this product can be incorporated into material (e.g. LNs) or IRS applications then it should be

The option of associating insecticides with different modes of action is one of the possible strategies for resistance management (as developed in another Chapter by Corbel & N'Gues‐ san). An interesting approach that has recently been studied, combined Piperonyl butoxide (PBO), organic compound used as pesticide synergist, and dinotefuran in an attempt to restore the efficacy of deltamethrin treated mosquito net against resistant *An. gambiae*[18]. Darriet and Chandre [18] have also conducted classical laboratory cone tests of nets treated with delta‐ methrin, PBO (the classical synergist, inhibitor of oxidases) and dinotefuran alone or in combination against susceptible ("KIS") and resistant '("VKPR") strains of laboratory reared

mortality KDt50 KDt95 mortality KDT50 KDT95

58% 13' 36'

99% 10' 23'

useful in areas where resistance to pyrethroids and carbamates has developed.

*An. gambiae*. Results of these tests are summarized in Table 1.

Deltamethrin +PBO

PBO

Deltamethrin+ Dinotefuran +

'("VKPR") strains of *Anopheles gambiae.*

**Product/ strain KIS VKPR**

Deltamethrin 100% 8' 18' 7.5% 31' 194' Dinotefuran 39% No No PBO 4% No No

Dinotefuran + PBO 28% No No

**Table 1.** Effects of mosquito nets treated with deltamethrin, PBO and dinotefuran on susceptible ("KIS"), and resistant

WHO's minimum mortality level for insecticides is 80% and this provides a reasonable operational guideline for effectiveness. In this study PBO combined with deltamethrin increased significantly its efficacy (synergistic effect), but not to a level adequate for control against pyrethroid-resistant mosquitoes, "suggesting that the acetylcholine concentration within the synaptic gap probably also increased". Interestingly, PBO had an antagonistic effect when combined with dinotefuran, decreasing this insecticide's efficacy. However, when PBO

mechanisms such as *kdr* or ace-1R in mosquitoes.

712 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

## **2.4. New Insecticide Treated Plastic Sheeting (ITPS) and Durable Wall Linings (DL or WL)**

Insecticide Treated Plastic Sheeting (ITPS) was developed in 2001 to provide a dual purpose tool capable of providing effective shelter and malaria control to displaced families in human‐ itarian crises. Durable wall linings (DL), developed in 2005, follow similar principles to ITPS, but are designed to be applied to the surface of existing rural house walls. In both cases these tools were developed to overcome the operational complexities and short comings of IRS, increase user acceptance (as the materials are available in different colors), and to increase residual insecticide activity (from classical 3-6 months with IRS to multiple years with ITPS or DL), and to increase community participation with a tool that households can implement themselves, and finally to provide new tools and new insecticide delivery mechanisms within the framework of insecticide resistance management. To date all factories produced ITPS based on solid format of polyethylene treated with pyrethroid insecticide, either permethrin or deltamethrin. The first generation of DL is also a polyethylene, but in 50% shading material format (woven polyethylene threads, with equal sized spaces between the threads).

One study group [19] has used "plastic sheeting impregnated with carbamates combined with long-lasting insecticidal mosquito nets for the control of pyrethroid-resistant malaria vectors" but this version of ITPS is unlikely to be tested at Phase III level or commercialized due to significant toxicity and fire risk problems associated with carbamates in this format. Different commercial products have been developed with different deltamethrin surface concentrations such as "ZeroVector (DL)" (170 mg a.i./m2 ) or "Zero Fly" (360 mg a.i./m2 ). Zerofly ITPS have been studied (Phase II) in refugee's camps in Afghanistan [20], in Sierra Leone (Phase III) [21], as well as in India (in endemic area with *An. culicifacies* and *An. fluviatilis* vectors or laborer settlements with *An. culicifacies* and *An. stephensi* as vectors) [22-23].

In Angola, a Phase III field trial was implemented in rural area, 8 villages around Balombo which were paired and received LLIN PermaNet 2.0 (55 mg a.i./m2 ; Figures 3) or DL/WL ZeroVector or LLIN + ITPS "Zero Fly" or IRS with lambdacyhalothrin (25 mg a.i./m2 ) with comprehensive evaluation: entomology, parasitology and immunology; focus group and KAP surveys were also implemented to follow the household acceptability of the vector control methods introduced.

The main vector in these villages was *An. funestus*. Entomological and parasitological first studies results showed that deltamethrin treated DL ZeroVector alone gave same results as IRS (lambdacyhalothrin) or LLIN (PermaNet®) alone or both PermaNet + ITPS Zero Fly in reducing by 55% the *P. falciparum* prevalence and parasitic load in children 2-9 years old (Figure 5) [24].

**Figure 4.** LLIN PermaNet 2.0 inside a house in Caala village (A); Green DL/WL ZeroVector inside a house in Chisséquélé village (B); Silver DL/WL ZeroVector inside a house in Barragem village (C); LLIN PermaNet 2.0 + ITPS Zero Fly in a house of Capango village (D) (Photos by P. Carnevale).

Entomological data obtained by classical CDC light traps inside houses before/after imple‐ mentation of vector control measures were in line with the clinical results i.e. similar level of reduction of number of *Anopheles* in each village (Figure 6) such as 79.1% reduction all villages combined [24].

Immunological analysis of antibodies directed against saliva proteins of *Anopheles* [23] (Figure 7) confirmed the actual reduction of man/*Anopheles* contact with ITPS as well as IRS while association LLIN + ZF gave the best result.

A series of smaller Phase II DL/WL feasibility and acceptability studies, with entomological monitoring have also been conducted in Angola and Nigeria [25], Equatorial Guinea, Ghana, Mali, South Africa and Vietnam [26], and Papua New Guinea [27]. In each of these Phase II village studies, DL/WL acceptability data were collected using a standardized household survey used by each of the different study groups, with the conclusive result that DL/WL had an extremely high acceptance level amongst all cultures and communities in which it was

**Figure 5.** Regressive evolution of endemicity indice (plasmodic indices of 2 – 9 years old children) before/after imple‐ mentation of each one of the four vector control methods.

Entomological data obtained by classical CDC light traps inside houses before/after imple‐ mentation of vector control measures were in line with the clinical results i.e. similar level of reduction of number of *Anopheles* in each village (Figure 6) such as 79.1% reduction all villages

**Figure 4.** LLIN PermaNet 2.0 inside a house in Caala village (A); Green DL/WL ZeroVector inside a house in Chisséquélé village (B); Silver DL/WL ZeroVector inside a house in Barragem village (C); LLIN PermaNet 2.0 + ITPS Zero Fly in a

Immunological analysis of antibodies directed against saliva proteins of *Anopheles* [23] (Figure 7) confirmed the actual reduction of man/*Anopheles* contact with ITPS as well as IRS while

A series of smaller Phase II DL/WL feasibility and acceptability studies, with entomological monitoring have also been conducted in Angola and Nigeria [25], Equatorial Guinea, Ghana, Mali, South Africa and Vietnam [26], and Papua New Guinea [27]. In each of these Phase II village studies, DL/WL acceptability data were collected using a standardized household survey used by each of the different study groups, with the conclusive result that DL/WL had an extremely high acceptance level amongst all cultures and communities in which it was

combined [24].

association LLIN + ZF gave the best result.

house of Capango village (D) (Photos by P. Carnevale).

(A) (B)

714 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

(C) (D)

**Figure 6.** Reduction of number of *Anopheles* in CDC light trap sampling inside houses in villages before (2007-2008) and after (2009) vector control implementation [Caala and Cahata = LLIN alone; Canjala and Capango = LLIN + ZF; Barragem and Chisséquélé = WL alone; Candiero and Libata = IRS.

tested, and that when compared to IRS it was the preferred malaria prevention tool in every study. DL/WL proved feasible in every country study and in all house construction types tested, including brick, mud, wooden, and concrete walled rural houses. In each of these

**Figure 7.** Evolution of the median values of the IgG antibody response to *Anopheles* saliva for all 6 villages combined according to the survey period in 2008 and 2009 (VC: vector control methods implemented in December 2008) [24].

studies, samples of DL/WL were collected at 4 monthly time intervals and were examined for deltamethrin residual content and bioassay impact on vector mosquitoes. The different studies produced very similar results regardless of house construction type, and in all cases DL/WL retained full activity and achieved >90% mortality of vector mosquitoes for the full monitoring periods of each study. The minimum study monitoring period was 6 months and the maximum was 4 years.

In Sierra Leone, Burns et al [21] conducted a Phase III study of ITPS. They constructed two refugee camps, Largo and Tobanda, using ITPS in 50% of each camp for shelter construction. The remaining 50% of each camp had shelter constructed out of untreated plastic sheeting (UPS). In Largo Camp, ITPS/UPS was applied onto walls and the ceiling of each shelter. In Tobanda Camp, ITPS/UPS was used only on ceilings. In Largo, the *Plasmodium falciparum* incidence rate in children up to 3 years of age who were cleared of parasites and then monitored for 8 months, was 163/100 person-years under UPS and 63 under ITPS. In Tobanda, incidence rate was 157/100 person-years under UPS and 134 under ITPS. Protective efficacy was 61% under fully lined ITPS shelters, and 15% under roof lined ITPS alone. Anemia rates improved under ITPS in both camps. Burns et al [21] concluded that "this novel tool proved to be a convenient, safe, and long-lasting method of malaria control when used as a full shelter lining in an emergency setting". Of note Burns et al [21] observed great difference of ITPS on walls + ceiling *versus* ceiling only at *P. falciparum* incidence rate level. Diabate et al [28] found similarly significant entomological difference in experimental huts of Burkina Faso lined with perme‐ thrin treated plastic sheeting on walls only or walls + ceiling reporting that "ITPS had a major effect on the mortality of mosquitoes, the proportion killed being dependent upon the surface area covered" and "deterred entry of mosquitoes and inhibition of blood feeding were also correlated with surface area covered."

#### **2.5. New tools for LNs**

studies, samples of DL/WL were collected at 4 monthly time intervals and were examined for deltamethrin residual content and bioassay impact on vector mosquitoes. The different studies produced very similar results regardless of house construction type, and in all cases DL/WL retained full activity and achieved >90% mortality of vector mosquitoes for the full monitoring periods of each study. The minimum study monitoring period was 6 months and the maximum

**Figure 7.** Evolution of the median values of the IgG antibody response to *Anopheles* saliva for all 6 villages combined according to the survey period in 2008 and 2009 (VC: vector control methods implemented in December 2008) [24].

In Sierra Leone, Burns et al [21] conducted a Phase III study of ITPS. They constructed two refugee camps, Largo and Tobanda, using ITPS in 50% of each camp for shelter construction. The remaining 50% of each camp had shelter constructed out of untreated plastic sheeting (UPS). In Largo Camp, ITPS/UPS was applied onto walls and the ceiling of each shelter. In Tobanda Camp, ITPS/UPS was used only on ceilings. In Largo, the *Plasmodium falciparum* incidence rate in children up to 3 years of age who were cleared of parasites and then monitored for 8 months, was 163/100 person-years under UPS and 63 under ITPS. In Tobanda, incidence rate was 157/100 person-years under UPS and 134 under ITPS. Protective efficacy was 61% under fully lined ITPS shelters, and 15% under roof lined ITPS alone. Anemia rates improved under ITPS in both camps. Burns et al [21] concluded that "this novel tool proved to be a

was 4 years.

716 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

#### *2.5.1. Combined LN with PBO or two different class of insecticide*

Pyrethroid treated LNs are the principle tool upon which malaria control has relied for the last decade, however, the rapid ongoing spread of pyrethroid resistance in Africa, is likely to increasingly compromise their protective efficacy. This concern has highlighted the urgent need to develop alternative active ingredients for LNs. While a study on bitreated (OP or C + pyr) [29] nets showed positive results they have not been commercially developed or opera‐ tionalised due to safety concerns. To tackle the issue of pyrethroid resistance a new model of LLIN call "Permanet 3" (P3) was recently developed by Vestergaard Frandsen SA, Aarhus, Denmark [30] with *a top panel* made of monofilament polyethylene fabric incorporating deltamethrin (121mg/m2 ) and PBO (759mg/m2 ) plus side panels made of multifilament polyester fabric coated with a wash-resistant formulation of deltamethrin (85mg/m2 ) (while the usual concentration was 55 mg a.i./m2 in classical Permanet 2 and 25 mg a.i./m2 in former hand treated nets "ITN"). PBO is the synergist of pyrethrins and pyrethroids without intrinsic insecticidal activity. The action of the synergist PBO is due to inhibition of oxidative enzymes in the insect which can detoxify the insecticide (metabolic resistance). The inhibition or blocking of the detoxification enzyme significantly increases mortality of resistant insects. PBO is used in a ratio ranging generally from 3 to 8 with the active ingredient used, depending on the type of formulation and target insects. LLIN "Permanet 3" (P3) was recently tested in several countries of West, Central [31] and East Africa such as Tanzania [32] and Ethiopia [33].

In southern Benin, N'Guessan et al [11] tested LLIN Permanet 3 against *An. gambiae* M molecular form (highly resistant owing to knockdown resistance (*kdr*) site insensitivity and elevated oxidase and esterase metabolic mechanisms) and *Cx. quinquesfasciatus,* and showed that in experimental huts "the level of personal protection against *An. gambiae* biting from PermaNet 3.0 (50%) was similar to that from PermaNet 2.0 (47%)" and "protection fell significantly after 20 washes to 30% for PermaNet 3.0 and 33% for PermaNet 2.0".

In Côte d'Ivoire, in experimental huts of Yaokoffikro where *An. gambiae* population is mainly composed of S form (90%) *versus* M form (10%) and is strongly resistant with high *kdr*frequency (94%) and Cyt P 450 metabolic resistance, Permanet 3 (unwashed and washed 20x) were compared against the standard Permanet 2 (unwashed and washed 20x), and hand treated ITNs ("CTN") with K Otab® (washed 5x), with untreated nets as control [34]. It appeared that both unwashed and washed P3 reduced entry rate (- 60%) and increased exit rate as well as other treated nets. On the other hand "a significantly higher mortality rate of *An. gambiae* s.s was recorded for unwashed PermaNet® 3.0 (55%) than for unwashed PermaNet® 2.0. However, for washed nets, there was no statistical difference between the mortality rates of *An. gam‐ biae* s.s for washed PermaNet® 2.0, washed PermaNet® 3.0 and the CTN. Classical cone bioassays were conducted with the same nets (testing side panels and roofs) using either susceptible Kisumu strain of *An. gambiae* or local wild resistant population. Against Kisumu strain, all treatments including the washed CTN showed a mean KD rate over the threshold of 95% and a mean mortality rate >80%, (the official cut off).

Against pyrethroid-resistant wild caught *An. gambiae* s.s cone bioassays showed a mean KD rate < 95% and a mean mortality rate < 80% for all treatment arms, except with a mean KD of 94.3% and 98.6% and a mean mortality rate of 93.5% and 99.5%, respectively on side and roof showing a great efficacy even against polyresistant populations. The unwashed PermaNet® 3.0 gave the best results (KD 95.8% and mortality 97.0%)

In Tanzania, laboratory and experimental huts trial compared PermaNet 3.0 (P3), PermaNet 2.0 (P2) and a conventional deltamethrin treated net [32] against pyrethroid susceptible *An. gambiae* and pyrethroid resistant *Cx. quinquefasciatus*, (elevated oxidase and *kdr* mechanisms), Bioassays tests showed that against the susceptible *An. gambiae* P3 and P2 were still efficient after 20 washes while conventionally treated nets lost its efficacy. Against the pyrethroid resistant strain of *Cx. quinquefasciatus* Masimbani strain, it clearly appeared that the treated roof (with PBO) was much more efficient than sides (without PBO) of the LLIN. In experimental huts, general results of P3 and P2 (washed and unwashed) were comparable against pyrethroid susceptible *An. gambiae* and pyrethroid resistant *Cx. quinquefasciatus* and gave high similar personal protection. Mortality induced by unwashed P3 on resistant *Cx. quinquefasciatus* was higher than P2 (both washed and unwashed) and 20x washed P3, showing the increased efficacy achieved by PBO against pyrethroid resistant mosquitoes but this efficacy disappeared after 20 washes. Chemical concentration of the P3 roof decreased from 136 mg a.i./m2 to 132 mg a.i./m2 after 20 washes; whereas deltamethrin concentration of the P3 sides decreased from 103-109 mg a.i./m2 before washing to 53 mg a.i./m2 after 20 washes. The concentration of PBO decreased from 1142 mg/m2 before wash to 684 mg/m2 after 20 washes. Finally, chemical concentration of deltamethrin in P2 decreased from 61- 77 mg a.i./m2 to 25 77 mg a.i./m2 after the classical 20 washes.

Tungu et al [32] observed that "the tunnel tests demonstrated a synergistic interaction of PBO and deltamethrin on roof netting against susceptible *An. gambiae* and both susceptible and resistant *Cx. quinquefasciatus* relative to netting from side panels treated with deltamethrin alone. This synergy was manifested in higher mortality, reduced passage through the holes and reduced feeding rates with netting treated with PBO-deltamethrin. The synergy in tunnels against pyrethroid resistant *Cx. quinquefasciatus* was progressively lost over 10 washes and fully lost after 20 washes. Cone bioassays on resistant *Cx. quinquefasciatus* confirmed the loss of synergy over 20 wash".

Sumitomo have also recently released a new LLIN (Olyset Plus®) treated with a combination of permethrin and PBO, and they claim similar increased efficacy against resistant strains of mosquitoes. However, questions do remain about the efficacy of adding PBO and its impact on the development of resistance amongst mosquitoes [35].

#### *2.5.2. New kit: New formulation and binder for long lasting treating net*

other treated nets. On the other hand "a significantly higher mortality rate of *An. gambiae* s.s was recorded for unwashed PermaNet® 3.0 (55%) than for unwashed PermaNet® 2.0. However, for washed nets, there was no statistical difference between the mortality rates of *An. gam‐ biae* s.s for washed PermaNet® 2.0, washed PermaNet® 3.0 and the CTN. Classical cone bioassays were conducted with the same nets (testing side panels and roofs) using either susceptible Kisumu strain of *An. gambiae* or local wild resistant population. Against Kisumu strain, all treatments including the washed CTN showed a mean KD rate over the threshold

Against pyrethroid-resistant wild caught *An. gambiae* s.s cone bioassays showed a mean KD rate < 95% and a mean mortality rate < 80% for all treatment arms, except with a mean KD of 94.3% and 98.6% and a mean mortality rate of 93.5% and 99.5%, respectively on side and roof showing a great efficacy even against polyresistant populations. The unwashed PermaNet®

In Tanzania, laboratory and experimental huts trial compared PermaNet 3.0 (P3), PermaNet 2.0 (P2) and a conventional deltamethrin treated net [32] against pyrethroid susceptible *An. gambiae* and pyrethroid resistant *Cx. quinquefasciatus*, (elevated oxidase and *kdr* mechanisms), Bioassays tests showed that against the susceptible *An. gambiae* P3 and P2 were still efficient after 20 washes while conventionally treated nets lost its efficacy. Against the pyrethroid resistant strain of *Cx. quinquefasciatus* Masimbani strain, it clearly appeared that the treated roof (with PBO) was much more efficient than sides (without PBO) of the LLIN. In experimental huts, general results of P3 and P2 (washed and unwashed) were comparable against pyrethroid susceptible *An. gambiae* and pyrethroid resistant *Cx. quinquefasciatus* and gave high similar personal protection. Mortality induced by unwashed P3 on resistant *Cx. quinquefasciatus* was higher than P2 (both washed and unwashed) and 20x washed P3, showing the increased efficacy achieved by PBO against pyrethroid resistant mosquitoes but this efficacy disappeared after 20 washes. Chemical concentration of the P3 roof decreased from 136 mg a.i./m2

after 20 washes; whereas deltamethrin concentration of the P3 sides decreased from

103-109 mg a.i./m2 before washing to 53 mg a.i./m2 after 20 washes. The concentration of PBO

Tungu et al [32] observed that "the tunnel tests demonstrated a synergistic interaction of PBO and deltamethrin on roof netting against susceptible *An. gambiae* and both susceptible and resistant *Cx. quinquefasciatus* relative to netting from side panels treated with deltamethrin alone. This synergy was manifested in higher mortality, reduced passage through the holes and reduced feeding rates with netting treated with PBO-deltamethrin. The synergy in tunnels against pyrethroid resistant *Cx. quinquefasciatus* was progressively lost over 10 washes and fully lost after 20 washes. Cone bioassays on resistant *Cx. quinquefasciatus* confirmed the loss

Sumitomo have also recently released a new LLIN (Olyset Plus®) treated with a combination of permethrin and PBO, and they claim similar increased efficacy against resistant strains of

before wash to 684 mg/m2

concentration of deltamethrin in P2 decreased from 61- 77 mg a.i./m2

to 132

after

after 20 washes. Finally, chemical

to 25 77 mg a.i./m2

of 95% and a mean mortality rate >80%, (the official cut off).

718 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

3.0 gave the best results (KD 95.8% and mortality 97.0%)

mg a.i./m2

decreased from 1142 mg/m2

the classical 20 washes.

of synergy over 20 wash".

The efficacy of the long-lasting treatment kits ICON® Maxx (Syngenta) (slow release 10% capsule suspension formulation of lambdacyhalothrin + a polymer binding agent) was evaluated under laboratory conditions and in an experimental hut trial in various situa‐ tions [36].

Laboratory and field trials were recently implemented in central Côte d'Ivoire, where *Anopheles gambiae* s.s. are resistant to pyrethroid insecticides [37]. In laboratory studies, classical bioas‐ says were conducted on Kisumu SS susceptible *An. gambiae* strain, with polyester and poly‐ ethylene nets with up to 20 classical washes. Unwashed the treated polyester net resulted in 89% KD and 52% mortality while the polyethylene treated net achieved 98% KD and 46% mortality. Washing these nets had a serious negative impact on efficacy, in terms of both KD at 1 hour and mortality at 24 hours. After 20 washes, KD rates dropped to 59% with polyethy‐ lene and 55% for polyester net i.e. below the mean KD defined for LLINs by WHO Pesticide Evaluation Scheme (WHOPES) guideline (i.e. 95% after 20 washings). After 20 washes the mean mortality also decreased for both netting materials to around 20%, falling well below the WHOPES criteria for long-lasting nets (KD ≥ 95% and/ or mortality ≥ 80% for at least 20 standards WHO washes under laboratory conditions using an *An. gambiae* Kisumu-susceptible strain). Field evaluation of 2 ICON Maxx polyester treated nets and 2 untreated ones (= control) was carried out over one year in the experimental huts of M'bé. The wild *An. gambiae* population (mainly S form, 92%) used in these studies showed a high frequency of *kdr* (# 97% pyrethroid resistant heterozygotes) with 2 ICON Maxx polyester treated nets and 2 untreated one (= control). Blood feeding rate was reduced and mortality was significantly increased (70% for 8 months) in huts with treated nets even against the resistant wild *An. gambiae* population. It is worth noting this impact on insecticide resistant *An. gambiae* population and further epidemiological studies should be carried out.

#### **2.6. New non chemical approaches of larviciding**

#### *2.6.1. New formulations of entomopathogen fungus*

Laboratory and field bioassays have been implemented "to develop formulations that facilitate the application of *Metarhizium anisopliae* and *Beauveria bassiana* spores (to improve spreading) for the control of anopheline larvae [*An. gambiae* and *An. stephensi*], and also to improve their persistence under field conditions" [36]. These studies showed that the pathogenicity of dry *M. anisopliae* and *B. bassiana* spores against *An. stephensi* larvae is however too short (# 5 days) to have any application in control settings; with ShellSol T fungal spores only somewhat more persistent. In field bioassays (Western Kenya), the percentage of pupation observed in *An. gambiae* larvae treated with ShellSol T formulated spores was much lower than with unfor‐ mulated treatment: 43 to 49% with *M. anisopliae* and 39 to 50% with *B. bassiana* (at 10 mg and 20 mg respectively). Bukhari et al [38] suggest that "these formulated fungi can be utilized in the field, providing additional tools for biological control of malaria vectors".

#### *2.6.2. Another new class of product: Spinosad*

Spinosad has been considered as "a new larvicide against insecticide-resistant mosquito larvae" [39] representing a new class of insect control products [40] and it has been tested in several trials [41].

**Figure 8.** Two toxins of spinosad (Spinosyn A and Spinosyn D).

Spinosad is a fermented product derived from the mixture of two toxins (A and D spinosyns; Figure 8) secreted by soil based bacteria, *Saccharopolyspora spinosa*. It is traditionally used for crop protection [36] against pest insects. In the European Union, the active substance is included in Annex I to Directive 91/414/EEC by Directive 2007/6/EC and the rate of the pesticide residues in food is regulated in Europe. In France, the active substance is authorized for use in approved market products.

Spinosad acts on the nervous system of insects, by external contact or ingestion. It induces involuntary muscle contractions, prostration with tremors and paralysis. An insect stops feeding and paralysis may occur within minutes after ingestion of the product, death ensuing within one to three days. Spinosad has low toxicity to mammals, birds, fish and crustaceans but it is highly toxic to bees and aquatic invertebrates [42]. Spinosad (Group 5 insecticide) when used as a larvicide could be considered in rotation with another insecticide from a different class of pesticides.

Laboratory larval bioassays of spinosad on *Aedes aegypti*, *Cx. quinquefasciatus*, and *An. gam‐ biae* (specimens that were either susceptible or resistant to pyrethroids, carbamates, and organophosphates) have shown that this product has a lethal action (mortality after 24 h of exposure) regardless of the original status, susceptible or resistant, of the mosquito larvae and was significantly more effective against *An. gambiae* than against the two other species and more effective against *Cx. quinquefasciatus* than *Ae. aegypti* [39] (Table2).


**Table 2.** LC50 and LC100 of spinosad for *An. gambiae*, *Cx. quinquefasciatus*, and *Ae. aegypti* (SS, homozygote susceptible, RR: homozygote resistant).

Several other studies showed the potential of this bioinsecticide against different genera and species of mosquitoes [41, 43-44]. Different concentrations of spinosad were tested against larval instar and pupa of *An. stephensi* [45]. It was observed that "the reduction percentage of *Anopheles* larvae was 82.7%, 91.4% and 96.0% after 24, 48, 72 hours, respectively, while more than 80% reduction was observed after 3 weeks". A CS Spinosad formulation was tested in classical laboratory bioassays and successfully used for the control of *Ae. aegypti* and *An. albimanus* larvae in Mexico [46]. A spinosad shows an absence of cross resistance with insec‐ ticides commonly used in Public Health and it may be an interesting product to integrate into vector borne diseases control strategies where vectors are resistant to current insecticides.

## **3. Other new paradigms**

20 mg respectively). Bukhari et al [38] suggest that "these formulated fungi can be utilized in

Spinosad has been considered as "a new larvicide against insecticide-resistant mosquito larvae" [39] representing a new class of insect control products [40] and it has been tested in

Spinosad is a fermented product derived from the mixture of two toxins (A and D spinosyns; Figure 8) secreted by soil based bacteria, *Saccharopolyspora spinosa*. It is traditionally used for crop protection [36] against pest insects. In the European Union, the active substance is included in Annex I to Directive 91/414/EEC by Directive 2007/6/EC and the rate of the pesticide residues in food is regulated in Europe. In France, the active substance is authorized for use

Spinosad acts on the nervous system of insects, by external contact or ingestion. It induces involuntary muscle contractions, prostration with tremors and paralysis. An insect stops feeding and paralysis may occur within minutes after ingestion of the product, death ensuing within one to three days. Spinosad has low toxicity to mammals, birds, fish and crustaceans but it is highly toxic to bees and aquatic invertebrates [42]. Spinosad (Group 5 insecticide) when used as a larvicide could be considered in rotation with another insecticide from a different

Laboratory larval bioassays of spinosad on *Aedes aegypti*, *Cx. quinquefasciatus*, and *An. gam‐ biae* (specimens that were either susceptible or resistant to pyrethroids, carbamates, and organophosphates) have shown that this product has a lethal action (mortality after 24 h of exposure) regardless of the original status, susceptible or resistant, of the mosquito larvae and was significantly more effective against *An. gambiae* than against the two other species and

more effective against *Cx. quinquefasciatus* than *Ae. aegypti* [39] (Table2).

the field, providing additional tools for biological control of malaria vectors".

*2.6.2. Another new class of product: Spinosad*

720 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**Figure 8.** Two toxins of spinosad (Spinosyn A and Spinosyn D).

in approved market products.

class of pesticides.

several trials [41].

#### **3.1. Slow Acting Product (SAP) — Entomopathogens fungus**

A completely new paradigm in vector control would be *slow acting pr*oducts called «Late Life Acting products » [47]. As malaria parasite sporogonic development last at least 10 days, any product which kills mosquito vectors within that time frame will automatically reduce the number of infected vectors and therefore almost certainly also reduce *Plasmodium* inoculation rates.

Formulated as biopesticides, fungal entomopathogens may have a great potential for appli‐ cation in indoor residual spraying of house wall surfaces or other resting places in human or animal dwellings. Once infected the fungus physically proliferates within the insect and results in the production of various secondary metabolites that have negative impacts on insect physiology [48-49] and performance and eventual death [50]. Histopathological studies of tissues infected by fungus suggest that the insect dies due to the combination of nutrient depletion, mechanical damage, and toxicosis. These biopesticides, if they can be successfully applied, could be useful for malaria control [51-52] especially if they prove effective against insecticide-resistant mosquitoes [53-55].

#### *3.1.1. Entomopathogen fungus on clay*

In recent trials [56] adult females of *An. stephensi* mosquitoes were exposed with cone tests to clay tiles sprayed with an oil formulation of spores of the entomopathogenic fungus *Beauveria bassiana* using different concentrations or time of exposure. A mortality rate of 100% was observed in less than one week, even when no KD effect was observed.

In addition to reducing longevity, it was noticed that fungal infection also reduces feeding propensity and fecundity [56-57] which added to the reduction of longevity could have a significant impact on vectorial capacity and therefore also on malaria transmission. Blanford et al [56] showed that "fungal exposed mosquitoes showed a declining response to the feeding stimulus over time, with 77, 60 and 50% of mosquitoes initiating feeding behaviors on days 1, 2 and 3, respectively and no mosquitoes responding on day 4. Combining the proportion of mosquitoes alive with the proportion attempting to feed gives a measure of overall transmis‐ sion blocking (biting risk) on any given day. For treated mosquitoes, this combination of prelethal and lethal effects revealed reductions in biting risk of 36, 52, 72 and 100% on days 1–4, respectively. This represents complete transmission blocking within a feeding cycle".

Fungal infection was also observed to have a negative impact on flight performance which may be an important consideration for malaria control at focal level. Another very important character of entomopathogen fungus is its ability to control insecticide resistant mosquito strains. Exposure to the fungal biopesticide on clay tiles using the standard dose and a 30 minute-exposure period before classical bioassay (WHO cone test) of colonies of 3 species, *An. gambiae s.s., An. arabiensis* and *An. funestus,* (ranging from fully susceptible to resistant to DDT, and/or Bendiocarb, and/or Malathion, and/or Deltamethrin) showed 100% mortality by day 6 irrespective of mosquito species or the level of resistance to insecticides. Blanford et al. [56] who reported that "the *An. gambiae* colony ''TONGS'', which was fully resistant to all chemical classes, had an Median Lethal Time (MLT) of 4 (3.93–4.07) days and all individuals were dead by day 5 (± 0.0) which was not dissimilar to the fully susceptible *An. gambiae* colony ''SUA'' which had an MLT of 4 (3.82–4.18) days and were all dead by day 6 "(±0.25)". It clearly appeared that "insecticide resistance confers no cross resistance to fungal pathogens in the key African malaria vectors" and this point must be taken into account in the management of insecticide resistance. For Blanford et al [56] "what is striking here is that when the effects of blood feeding are added in, risk of malaria transmission is essentially reduced to zero within a day of fungal exposure and never recovers".

#### *3.1.2. Entomopathogen fungus on nets*

Howard et al [58] implemented several classical tube bioassays to compare the fungalsusceptibility of an insecticide-resistant (VKPER) and insecticide-susceptible strain (SKK) of *An. gambiae* and test the activity (and longevity) of *M. anisopliae* and *B. bassiana* conidia on white polyester netting (Table 3). It appeared that *M. anisopliae* and *B. bassiana* significantly increased mortality of both resistant and susceptible strains of *An. gambiae* exposed to 2 or 7 days after treatment of nets (Table 3). *B. bassiana* was significantly more pathogenic than *M. anisopliae* both for SKK and VKPER (Table 3). The insecticide-resistant mosquito strain VKPER was significantly more susceptible to fungal infection than the SKK strain after exposure to 2 or 7 days after treatment of nets (table) while other studies did not find any difference in efficacy of dry conidia of *B. bassiana* on resistant or susceptible strain. It is possible that the discrepancies in data could be due to the mode of formulation of conidia (dry or ShellSol T suspensions in this study). The mosquito pathogenicity was maintained seven days after net application, but the viability of the two fungal species after seven days at 27°C was low, 62% and 2% respec‐ tively, for *B. bassiana* and *M. anisopliae,* hampering their practical application in LLINs.


**Table 3.** Comparison of mortality rates of fungal-susceptibility (*M. anisopliae* and *B. bassiana*) between an insecticideresistant (VKPER) and insecticide-susceptible strain (SKK) of *Anopheles gambiae*.

Trials of entomopathogen fungus on mosquitoes have generated various results according to the protocol followed: formulation of fungus (dry/suspension); substrata (mud wall, cloth etc); field/lab trials; doses, exposure times; species of fungus; species/strain of mosquitoes, etc. Of note, Howard et al [58] successfully demonstrated the efficacy of nets treated with *B. bassi‐ ana* and tested against a resistant strain of *An. gambiae*. Even though the residual efficacy duration was short, the authors logically concluded that "Field trials over a longer trial period need to be carried out to see if wild insecticide-resistant mosquitoes are as susceptible as the colony strain used in this trial". Further studies, against resistant *An. gambiae* VKPER strain showed that "*B. bassiana* infection caused significantly increased mortality with the daily risk of dying being increased by 2.5 × for fungus-exposed mosquitoes compared to control mosquitoes. However, the virulence of the *B. bassiana* conidia decreased with increasing time spent exposed to the tropical field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the tropical climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%" [59].

#### *3.1.3. Influence of temperature*

*3.1.1. Entomopathogen fungus on clay*

722 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

exposure and never recovers".

*3.1.2. Entomopathogen fungus on nets*

In recent trials [56] adult females of *An. stephensi* mosquitoes were exposed with cone tests to clay tiles sprayed with an oil formulation of spores of the entomopathogenic fungus *Beauveria bassiana* using different concentrations or time of exposure. A mortality rate of 100% was

In addition to reducing longevity, it was noticed that fungal infection also reduces feeding propensity and fecundity [56-57] which added to the reduction of longevity could have a significant impact on vectorial capacity and therefore also on malaria transmission. Blanford et al [56] showed that "fungal exposed mosquitoes showed a declining response to the feeding stimulus over time, with 77, 60 and 50% of mosquitoes initiating feeding behaviors on days 1, 2 and 3, respectively and no mosquitoes responding on day 4. Combining the proportion of mosquitoes alive with the proportion attempting to feed gives a measure of overall transmis‐ sion blocking (biting risk) on any given day. For treated mosquitoes, this combination of prelethal and lethal effects revealed reductions in biting risk of 36, 52, 72 and 100% on days 1–4,

respectively. This represents complete transmission blocking within a feeding cycle".

Fungal infection was also observed to have a negative impact on flight performance which may be an important consideration for malaria control at focal level. Another very important character of entomopathogen fungus is its ability to control insecticide resistant mosquito strains. Exposure to the fungal biopesticide on clay tiles using the standard dose and a 30 minute-exposure period before classical bioassay (WHO cone test) of colonies of 3 species, *An. gambiae s.s., An. arabiensis* and *An. funestus,* (ranging from fully susceptible to resistant to DDT, and/or Bendiocarb, and/or Malathion, and/or Deltamethrin) showed 100% mortality by day 6 irrespective of mosquito species or the level of resistance to insecticides. Blanford et al. [56] who reported that "the *An. gambiae* colony ''TONGS'', which was fully resistant to all chemical classes, had an Median Lethal Time (MLT) of 4 (3.93–4.07) days and all individuals were dead by day 5 (± 0.0) which was not dissimilar to the fully susceptible *An. gambiae* colony ''SUA'' which had an MLT of 4 (3.82–4.18) days and were all dead by day 6 "(±0.25)". It clearly appeared that "insecticide resistance confers no cross resistance to fungal pathogens in the key African malaria vectors" and this point must be taken into account in the management of insecticide resistance. For Blanford et al [56] "what is striking here is that when the effects of blood feeding are added in, risk of malaria transmission is essentially reduced to zero within a day of fungal

Howard et al [58] implemented several classical tube bioassays to compare the fungalsusceptibility of an insecticide-resistant (VKPER) and insecticide-susceptible strain (SKK) of *An. gambiae* and test the activity (and longevity) of *M. anisopliae* and *B. bassiana* conidia on white polyester netting (Table 3). It appeared that *M. anisopliae* and *B. bassiana* significantly increased mortality of both resistant and susceptible strains of *An. gambiae* exposed to 2 or 7 days after treatment of nets (Table 3). *B. bassiana* was significantly more pathogenic than *M. anisopliae* both for SKK and VKPER (Table 3). The insecticide-resistant mosquito strain VKPER was significantly more susceptible to fungal infection than the SKK strain after exposure to 2 or 7

observed in less than one week, even when no KD effect was observed.

Kikankie et al [55] did several trials "to assess the susceptibility of insecticide-susceptible ("MBN") and resistant ("SENN") laboratory strains and wild-collected *An. arabiensis* to infection with the fungus *B. bassiana* under two different laboratory temperature regimes (21 ± 1°C or 25 ± 2°C)".

It appeared that exposure to dry *B. bassiana* spores resulted in significant reductions in longevity of the wild *An. arabiensis* mosquitoes and virulence was significantly higher at 25°C than 21°C, and exposure to *B. bassiana* spores resulted in significant reductions in longevity in all mosquito colonies regardless of their insecticide susceptibility levels and temperature regimes. Fungal susceptibility was not affected by resistance to insecticides.

It was also noted that "fungus-induced mortality rates were relatively rapid at 25°C, with 100% mortality taking 10-12 days post-fungus exposure in the baseline colonies (MBN and SENN) and field-collected mosquitoes" i.e. a lapse of time shorter than the duration of the sporogonic cycle of *P. falciparum* at this temperature, an important element for actual reduction of malaria transmission through vector control.

#### *3.1.4. Influence of physiological stage and age*

Mnyone et al [60] conducted bioassays using fed and unfed adult females of *An. gambiae* maintained in colony for several years with two fungal isolates: *M. anisopliae* and *B. bassiana* I93-825. Mosquitoes were exposed to conidia for 6 hours, with a follow up of 28 days. To study the effect of age, "three different age groups of female mosquitoes were exposed to both fungal isolates (2–4 days, 5–8 days, and 9–12 days post emergence), whereas to study the effect of physiological stage, five groups with differing blood-feeding status were exposed to both fungal isolates (non-fed, 3, 12, 36, or 72 h post-blood feeding). Results showed that, with both fungus, "older mosquitoes died relatively earlier than younger ones" and "blood-fed mos‐ quitoes had a lower risk of dying relative to unfed ones". Increased risk of death in older than younger individuals has also been reported elsewhere [61-62]. Mnyone et al [60] considered that "the fact that blood-fed mosquitoes are less susceptible to fungal infection could be beneficial in terms of evolution proofing against resistance development. Although fungal infection reduces the fecundity of female mosquitoes [57], they are still able to pass their genes to the subsequent generation reducing selection pressure on resistance against fungi [55]. Furthermore, fungal infections suppress the successful development of *Plasmodium* parasites in the vectors [51], and hence both effects (i.e., fungus-induced mortality and parasite resist‐ ance) lead to a significantly reduced parasite transmission risk".

#### **3.2. Attractive Toxic Sugar Bait (ATSB) methods**

Recent studies on sugar feeding behavior of *Anopheles* [63-73] have been conducted in order "to optimize strategies for malaria vector control in Africa using attractive toxic sugar bait methods" [74] and to develop a new approach for mosquito control [75-78]. Stone et al [79] developed "an effective indoor mesocosm for studying populations of *An. gambiae* in temper‐ ate climates" and used the mesocosm concept to "determine whether the sugar-or-blood meal choice of *An. gambiae* females one day after emergence is influenced by blood-host presence and accessibility, nectariferous plant abundance, and female size" [80].

Stone et al. [80] noted that with a sleeping human present in the mesocosm, the majority of one day-old females obtained a blood meal. This was the case even with treated mosquito net use. But when a blood host was not present, or access was restricted through the use of a net, sugar meals became more frequent. The feeding choices of female *An. gambiae* were determined to a great degree by the presence and accessibility of the blood host, and not by the abundance of potential nectar sources in the mesocosm. Concerning the use of sugar baits as a malaria vector control, the strong tendency to feed on blood, even at one day post-emergence, suggests that in areas where larval development sites are close to human habitations, the method may be useful mainly as a complement to mosquito nets. If larval development sites are located at considerable distance from humans, the dominance of blood feeding is a smaller issue. Though females are willing to feed on humans as early as 24 h after emergence, in nature they may not come into contact with humans that early, and attraction to sugar sources would be paramount. Males and small females are particularly likely to seek a sugar meal when access to blood hosts is restricted by mosquito nets, suggesting that a plant-based method may be an effective control tool for such endgame scenarios. The combination of sugar baits (for instance, placed indoors or near a house) and treated mosquito nets, is one of these options. Its feasibility will require bait substantially more attractive than the plant species used in this experiment, such as the one used in Mali [78].

It appeared that exposure to dry *B. bassiana* spores resulted in significant reductions in longevity of the wild *An. arabiensis* mosquitoes and virulence was significantly higher at 25°C than 21°C, and exposure to *B. bassiana* spores resulted in significant reductions in longevity in all mosquito colonies regardless of their insecticide susceptibility levels and temperature

It was also noted that "fungus-induced mortality rates were relatively rapid at 25°C, with 100% mortality taking 10-12 days post-fungus exposure in the baseline colonies (MBN and SENN) and field-collected mosquitoes" i.e. a lapse of time shorter than the duration of the sporogonic cycle of *P. falciparum* at this temperature, an important element for actual reduction of malaria

Mnyone et al [60] conducted bioassays using fed and unfed adult females of *An. gambiae* maintained in colony for several years with two fungal isolates: *M. anisopliae* and *B. bassiana* I93-825. Mosquitoes were exposed to conidia for 6 hours, with a follow up of 28 days. To study the effect of age, "three different age groups of female mosquitoes were exposed to both fungal isolates (2–4 days, 5–8 days, and 9–12 days post emergence), whereas to study the effect of physiological stage, five groups with differing blood-feeding status were exposed to both fungal isolates (non-fed, 3, 12, 36, or 72 h post-blood feeding). Results showed that, with both fungus, "older mosquitoes died relatively earlier than younger ones" and "blood-fed mos‐ quitoes had a lower risk of dying relative to unfed ones". Increased risk of death in older than younger individuals has also been reported elsewhere [61-62]. Mnyone et al [60] considered that "the fact that blood-fed mosquitoes are less susceptible to fungal infection could be beneficial in terms of evolution proofing against resistance development. Although fungal infection reduces the fecundity of female mosquitoes [57], they are still able to pass their genes to the subsequent generation reducing selection pressure on resistance against fungi [55]. Furthermore, fungal infections suppress the successful development of *Plasmodium* parasites in the vectors [51], and hence both effects (i.e., fungus-induced mortality and parasite resist‐

Recent studies on sugar feeding behavior of *Anopheles* [63-73] have been conducted in order "to optimize strategies for malaria vector control in Africa using attractive toxic sugar bait methods" [74] and to develop a new approach for mosquito control [75-78]. Stone et al [79] developed "an effective indoor mesocosm for studying populations of *An. gambiae* in temper‐ ate climates" and used the mesocosm concept to "determine whether the sugar-or-blood meal choice of *An. gambiae* females one day after emergence is influenced by blood-host presence

Stone et al. [80] noted that with a sleeping human present in the mesocosm, the majority of one day-old females obtained a blood meal. This was the case even with treated mosquito net use. But when a blood host was not present, or access was restricted through the use of a net,

regimes. Fungal susceptibility was not affected by resistance to insecticides.

ance) lead to a significantly reduced parasite transmission risk".

and accessibility, nectariferous plant abundance, and female size" [80].

**3.2. Attractive Toxic Sugar Bait (ATSB) methods**

transmission through vector control.

724 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*3.1.4. Influence of physiological stage and age*

Based on highly successful demonstrations in Israel [75-77, 81] that attractive toxic sugar bait (ATSB) methods can decimate local populations of mosquitoes, Muller et al [78] implemented a study "to determine the effectiveness of ATSB methods for malaria vector control in the semiarid Bandiagara District of Mali, West Africa". The *Anopheles* vector population was mainly composed of *An. gambiae* s.l. (mainly *An. gambiae* s.s. 86% and *An. arabiensis* 14%) and *An. funestus* [82]. The *Attractive Sugar Bait* (ASB) was composed, among other, by Guava (30%) (*Psidium guajava*) and honey melons (30%) (*Cucumis melo*) highly present in the area of the trial and known to be attractive for *An. gambiae s.l.* [83] while "ATSB was made by adding the boric acid [84-85] 1% (W/V) to ASB liquid". The ASB (in "control areas") and ATSB (in "treated areas") solutions were sprayed on the vegetation around the ponds and rice paddies and mosquitoes collected by CDC Light Traps at fixed positions between the ponds, during the 38 days of the trial, implemented at the end of the peak of malaria transmission period. It was observed that "ATSB treatment reduced densities of female and male *An. gambiae* s.l. by about 90%. After spraying ATSB in the treatment site, population densities of female and male *An. gambiae* s.l. declined rapidly over a week and then stabilized at low levels"; this impact on males is worth underlining as it could have an impact on decreased fertilized females and therefore on progeny. Furthermore, "ATSB treatment correspondingly affected the longevity of female *An. gambiae* s.l."

According to their data, Müller et al [78] considered that "ATSB methods differ from, and potentially complement, LLIN and IRS methods. In terms of malaria vector control in Africa, the ATSB methods when used operationally will likely reduce both total numbers of recently emerged female anophelines before they enter houses to feed on humans, and the proportion of females exiting houses to oviposit and then returning to houses to re-feed on humans. It is likely that ATSB approaches could soon be added as a major component of Integrated Vector Management (IVM) based malaria vector control programs" [86-88].

Along with their studies in Mali on the attractiveness of various local plants, fruits, flowers to mosquitoes *versus* human scents, Müller et al [83] noticed a very interesting "different rhythm of attractivity as plants showed peaks of *An. gambiae* s.l. attraction between 19:30-22:00 and 04:00-05:00, which differed considerably from the response to human odors, which peaked at around midnight". The well-known local *Acacia macrostachya* and *Acacia albida* (Fabaceae) appeared very attractive, and *Hyptis suaveolens* (Lamiaceae) appeared highly repellent.

It is clear that a great lot of questions still remain to be solved about ATSB such as, among others: What is the side effect of spraying vegetation on non-target fauna? What is the actual epidemiological efficacy in various epidemiological settings? Which attractant is the best in different ecological and entomological conditions? Which "toxin" is the most effective in various entomological conditions? And should it be used inside as well as outside and following which method and what about the acceptability and actual community participa‐ tion, etc?

Nevertheless ATSB is another interesting approach worth further study for potential use, in complement to other classical methods such as IRS and LLIN, to reduce the number and the longevity of vectors i.e. malaria transmission and hence incidence of parasite infection and malaria morbidity.

#### **3.3. New mathematical modeling of impacts of vector control**

Since Roos and Macdonald, many mathematical models have been developed [89-90] for example (Figure 9):


**•** to develop new approaches such as the idea of evolution-proof insecticide [99-100].

**Figure 9.** Schematic representation of model. The population of uninfected red blood cells (x) provides the source for the infected population (y). Level I immune effector (a) is stimulated by y. Level II immune effector (b) is stimulated by y interacting with a+b. M represents the number of merozoites, S represents an external source of inoculation

Mathematical models are useful in exposing what may otherwise be non-intuitive results, for example indoor residual spray (IRS) of insecticides in conjunction with mosquito nets can show antagonism, arising via interference of their modes of action while it is generally assumed that the two tools have synergistic benefits in reducing malaria transmission [101]. However, few have considered the spread of resistance in a variable selection pressure context [102]. A mathematical model [35] was recently developed to explore the effects on mosquito popula‐ tions of spatial heterogeneous deployment of insecticides, to predict changes in mosquito fitness and resistance allele frequency, to identify important parameters in the evolution of insecticide resistance, to examine the contribution of new generation long-lasting insecticidal mosquito nets, that incorporate a chemical synergist on the roof panel, in delaying insecticide resistance.

Four niches were considered:

of attractivity as plants showed peaks of *An. gambiae* s.l. attraction between 19:30-22:00 and 04:00-05:00, which differed considerably from the response to human odors, which peaked at around midnight". The well-known local *Acacia macrostachya* and *Acacia albida* (Fabaceae) appeared very attractive, and *Hyptis suaveolens* (Lamiaceae) appeared highly repellent.

It is clear that a great lot of questions still remain to be solved about ATSB such as, among others: What is the side effect of spraying vegetation on non-target fauna? What is the actual epidemiological efficacy in various epidemiological settings? Which attractant is the best in different ecological and entomological conditions? Which "toxin" is the most effective in various entomological conditions? And should it be used inside as well as outside and following which method and what about the acceptability and actual community participa‐

Nevertheless ATSB is another interesting approach worth further study for potential use, in complement to other classical methods such as IRS and LLIN, to reduce the number and the longevity of vectors i.e. malaria transmission and hence incidence of parasite infection and

Since Roos and Macdonald, many mathematical models have been developed [89-90] for

**•** to evaluate the influence of environmental variables (climate, rain, relative humidity etc)

**•** to facilitate the mathematicians to further develop suitable models and help the biologists and public health personnel to adopt better understanding of the modeling strategies to

**•** to evaluate the potential mortality impact achievable by different long lasting, insecticide-

**•** to develop "a novel, convenient and versatile method to model *Plasmodium falciparum* infection that accounts for the essential in-host processes: parasite replication and its

**•** to improve malaria elimination strategies in areas where data are still scarce or not fully

**•** to develop a flexible and user-friendly *website* with an online mathematical model of malaria elimination that is being developed interactively with end users [96]; the website can be accessed at http://www.tropmedres. ac/elimination (see Malaria Elimination Model. http:// elimination.tropmedres.ac and Internet Model of Malaria Elimination User Guide http://

**•** to inform resistance management practices [97] determining the impact of different mosquito control intervention strategies including the protection conferred by mosquito

**3.3. New mathematical modeling of impacts of vector control**

tion, etc?

malaria morbidity.

example (Figure 9):

reliable [95];

nets [98];

control the disease [92];

treated net delivery strategies [93];

726 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

regulation by innate and adaptive immunity" [94];

www.tropmedres.ac/images/modelling/userguide.pdf;

[91];


It appeared that resistance spreads slower in the presence of a synergist. The effect of synergist in males and females was not strictly comparable but was overall similar. The delay in the spread of resistance caused by the synergist was not very large; however, in approximately 10% of cases the rate of allele spread was higher when the synergist was fully effective. The predicted frequency of the resistance allele under different values of k at generation 70, the predicted frequency when the synergist is inefficient (*k* = 1), is 0.11 and when is fully effective (*k* = 0) is 0.26. The synergist has only a small impact in controlling the population, but even small values of *k* will help to recover the effect of the insecticide, and this is may be the main contribution of the synergist. Nevertheless adding synergists to mosquito nets does decrease the rate at which resistance spreads in about 90% of scenarios. If a fully effective synergist (*k* = 0) is present, the fitness of all genotypes inside the house will be zero (*k* affects the 3 genotypes equally, so all mosquitoes die irrespective of their genotype) and the next generation will be mostly composed by progeny of survivors from the niche outside the household where selection for resistance was high. One hypothesis is that in this particular case the synergist removes the refugia of weak selection in the house thereby magnifying the effects of selection for resistance outside the house.

According to Barbosa and Hastings [35], "The finding that a situation can arise in which having a fully effective synergist in place contributes to intensify the spread of resistance is the most interesting result of this work, a very important fact often overlooked in modeling resistance: that it is highly dangerous to consider selection in only a single niche, isolated from other selection pressures, and to then extrapolate the results from the single niche to the whole population. In this case it seems reasonable to conclude that adding effective synergists will reduce selection for resistance in the household niche because all three genotypes are killed. The level of impact that a fully effective synergist could have on disease transmission is a question that cannot be directly answered by the results presented here, because it is not clear how the genetic concept of fitness translates into the demographic factors, such as mosquito population size and longevity that determine the intensity of disease transmission. On the other hand, as noted above, if synergist throws most of the selection pressure onto another niche then overall the rate of selection for resistance may increase. Consequently the impact of the use of insecticide within the home (predominantly as wall sprays and/or mosquito nets) on mosquitoes cannot easily be isolated from other insecticide applications that mosquitoes may encounter during their lifetime. This suggests that the malaria community is correct in being alarmed at the often uncontrolled use of insecticides in applications such as agriculture".

Ghani et al [103] developed a very interesting model to consider the possibility that a large reduction in malaria transmission may result in a loss of immunity, and how useful integrated malaria control measures could be to counterbalance such an eventuality. They prepared "a mathematical model for malaria transmission which incorporates the acquisition and loss of both clinical and parasite immunity", to "explore the impact of the trade-off between reduction in exposure and decreased development of immunity on the dynamics of disease following a transmission-reducing intervention such as insecticide treated nets". It is worth noticing how their model "predicts that initially rapid reductions in clinical disease incidence will be observed as transmission is reduced in a highly immune population. However, these benefits in the first 5–10 years after the intervention may be offset by a greater burden of disease decades later as immunity at the population level is gradually lost. The negative impact of having fewer immune individuals in the population can be counterbalanced either by the implementation of highly-effective transmission-reducing interventions (such as the combined use of insecti‐ cide-treated nets and insecticide residual sprays) for an indefinite period, or the concurrent use of a pre-erythrocytic stage vaccine or prophylactic therapy in children to protect those at risk from disease as immunity is lost in the population".

One of the key issues is the still current lack of sound knowledge about "malaria immunity" called "premunition" which involves immunity against the parasite, and therefore against the disease. For Ghani et al [103] "Clinical immunity develops over time dependent on the force of infection in the population and reduces the probability that an individual will develop clinical disease. Parasite immunity develops as individuals' age, and reduces the amount of time spent in the asymptomatic patent infection state (mimicking a reduction in parasite density and hence onward infectiousness)". Their previous model "suggests that the loss of both clinical and parasite immunity occurs over a period of years rather than weeks or months" [104] and according to a study in Madagascar, it seems that "immunity" could be of long duration [105]. In their model, Ghani et al [103] "assume that clinical immunity is developed at a rate proportional to the EIR in each setting and has a half-life of approximately 7 years and that parasite-clearance immunity has a half-life of approximately 14 years". They consider that 3 phrases are crucial: sustain intervention/integrated measures/sustain financial support and "Sustaining both control interventions and effective case management for many years, possibly decades, should remain the primary goal of all intervention programmes and it is essential that these long-term goals are matched with financial commitments".

#### **3.4. New ecological care**

small values of *k* will help to recover the effect of the insecticide, and this is may be the main contribution of the synergist. Nevertheless adding synergists to mosquito nets does decrease the rate at which resistance spreads in about 90% of scenarios. If a fully effective synergist (*k* = 0) is present, the fitness of all genotypes inside the house will be zero (*k* affects the 3 genotypes equally, so all mosquitoes die irrespective of their genotype) and the next generation will be mostly composed by progeny of survivors from the niche outside the household where selection for resistance was high. One hypothesis is that in this particular case the synergist removes the refugia of weak selection in the house thereby magnifying the effects of selection

According to Barbosa and Hastings [35], "The finding that a situation can arise in which having a fully effective synergist in place contributes to intensify the spread of resistance is the most interesting result of this work, a very important fact often overlooked in modeling resistance: that it is highly dangerous to consider selection in only a single niche, isolated from other selection pressures, and to then extrapolate the results from the single niche to the whole population. In this case it seems reasonable to conclude that adding effective synergists will reduce selection for resistance in the household niche because all three genotypes are killed. The level of impact that a fully effective synergist could have on disease transmission is a question that cannot be directly answered by the results presented here, because it is not clear how the genetic concept of fitness translates into the demographic factors, such as mosquito population size and longevity that determine the intensity of disease transmission. On the other hand, as noted above, if synergist throws most of the selection pressure onto another niche then overall the rate of selection for resistance may increase. Consequently the impact of the use of insecticide within the home (predominantly as wall sprays and/or mosquito nets) on mosquitoes cannot easily be isolated from other insecticide applications that mosquitoes may encounter during their lifetime. This suggests that the malaria community is correct in being alarmed at the often uncontrolled use of insecticides in applications such as agriculture".

Ghani et al [103] developed a very interesting model to consider the possibility that a large reduction in malaria transmission may result in a loss of immunity, and how useful integrated malaria control measures could be to counterbalance such an eventuality. They prepared "a mathematical model for malaria transmission which incorporates the acquisition and loss of both clinical and parasite immunity", to "explore the impact of the trade-off between reduction in exposure and decreased development of immunity on the dynamics of disease following a transmission-reducing intervention such as insecticide treated nets". It is worth noticing how their model "predicts that initially rapid reductions in clinical disease incidence will be observed as transmission is reduced in a highly immune population. However, these benefits in the first 5–10 years after the intervention may be offset by a greater burden of disease decades later as immunity at the population level is gradually lost. The negative impact of having fewer immune individuals in the population can be counterbalanced either by the implementation of highly-effective transmission-reducing interventions (such as the combined use of insecti‐ cide-treated nets and insecticide residual sprays) for an indefinite period, or the concurrent use of a pre-erythrocytic stage vaccine or prophylactic therapy in children to protect those at

risk from disease as immunity is lost in the population".

for resistance outside the house.

728 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Special attention is now devoted to the environment, especially environmental modifications that may result because of the impact of insecticides on the environment and its biodiversity.

#### *3.4.1. Environmental Risk Assessment (ERA) and Insect Pest Management IPM*

No pesticide is completely safe. Only through their careful use are we able to gain an under‐ standing of the risks and control them. The environmental impact of biocides is generally studied in the context of scientific investigations conducted beyond the regulatory require‐ ments for approval. This helps to generate better understanding of the biocides and provides opportunity to assess their potential impact and overall effectiveness when used in various control strategies. Although vector control methods are generally confined to urban and suburban areas, these areas may have a significant vegetation cover that provides both refuge and food for wildlife (insects, reptiles, birds, bats etc)...). This shows the need of environmental risk assessments prior to large scale vector control interventions. It also highlights the need for further studies to determine direct, indirect short and long-term potential effects. Risk assessment of control methods must be addressed in an integrated strategy taking into account the relationships between species in regards of the local biodiversity. In fact, environmental risk assessment of these treatments cannot be limited only to consider information on hazards, such as acute toxicity of the biocides used. Every effort needs to be made to minimize the use of chemical pesticides. A great deal of improvement can be made in vector control programs if the existing, methods and materials are more effectively used. The idea of integrated vector control which effectively combines a package of appropriate control methods i.e. insecticidal, environmental, biological and physical, in an orderly and coordinated manner can impact upon insect vectors and diseases with positive results of economic, ecological and sociological consequences [106].

Programs based on Insect Pest Management (IPM) must be designed to reduce vector bites and disease transmission, but also mitigate any potentially negative effects, i.e., such as environmental damage, harm of non-target organisms exposed to insecticides, or increase of insecticide resistant in target organisms [107]. Such programs do already exist notably in the USA (for example in Santa Barbara County) and in Australia [108]. In these programs, process are very well defined step by step: 1) vector surveillance and identification of target vector species to develop species‐specific pest management strategies based on developmental and behavioral considerations for each species; 2) threshold measures to determine when action is necessary; 3) public education, control, prevention; 4) monitoring of efficacy and environmen‐ tal impacts to identify the occurrence of unexpected/unwanted effects of treatments.

#### *3.4.2. Impact of insecticides used for vector control*

The impact of insecticides on the environment depends not only on the active substance, but also the formulation and the method of applying: indoor residual spraying, space spraying or treated nets will have different impacts.

#### *3.4.2.1. Indoor Residual Spraying (IRS)*

Domestic livestock (particularly chickens) and organisms in the environment may be harmed if operations, cleanup, and disposal are not conducted according to best practices.

Table 4 describes the potential ecological effects of each recommended IRS chemical. There is a lack of data concerning toxicity of IRS insecticides on non-target fauna. However, most insecticides are highly toxic for aquatic and terrestrial arthropods like bees (in particular pyrethroid), and some of them can also be toxic for mammals (some pyrethroids and organo‐ phosphates).

#### *3.4.2.2. Space spraying and larviciding*

Space spraying has only occasionally been used in malaria epidemic control program and as a complementary measure against exophilic vectors. Nevertheless, pyrethroids, which have a short remanence, have been the predominant insecticides [123], and then care must be taken to avoid applications near fish-bearing water bodies. It is also recommended that such applications should not be carried out directly over water bodies and that a no-treated barrier of 100 m should be maintained to prevent fish mortality. Home owners should be advised to cover domestic fish tanks and bird cages during the applications [123].

Blom [124] examined the effects of aerial, barrier, and ground based ultra-low volume (ULV) sprays with sumithrin and deltamethrin, in Massachusetts on non-target insects. Malaise traps, targeting the flying insect population, were collected in regular intervals before and after sprays, then the captured insects were sorted by order and counted. The results have shown little effect on non-target insects from the ground based sprays, and a temporary knockdown from the aerial spray. However, Coleoptera were affected in the short term by the ULV sprays and, suffered long term effects from aerial spraying.


Key: 0: non-toxic; +: potentially toxic; ++: highly toxic

#### **Table 4.** Toxicity of chemicals used for IRS on non-target organisms

Davis and Peterson [125] assessed long-term impacts of permethrin on non-target terrestrial arthropods after repeat ULV applications in the context of West Nile Virus Management in the USA. The authors concluded that although small flying insects that were active at the same time as mosquitoes were slightly impacted, effects on non-target arthropods exposed to adulticides applied via ULV sprayer would be small in the ecosystem studied.

Several classes of recommended larvicides are used in vector control management such as: the bio-insecticides (*Bacillus thuringiensis* var. *israelensis (Bti)*, *Bacillus sphaericus*(*Bs*) and spinosad), the organophosphates (chlorpyrifos, fenthion, pirimiphos-methyl, and temephos), and the insect growth regulators (diflubenzuron, methoprene, pyriproxyfen). The results of some studies concerning the environmental risk assessment of these larvicides are summarized in the Table 5.

#### *3.4.2.3. Treated net*

Programs based on Insect Pest Management (IPM) must be designed to reduce vector bites and disease transmission, but also mitigate any potentially negative effects, i.e., such as environmental damage, harm of non-target organisms exposed to insecticides, or increase of insecticide resistant in target organisms [107]. Such programs do already exist notably in the USA (for example in Santa Barbara County) and in Australia [108]. In these programs, process are very well defined step by step: 1) vector surveillance and identification of target vector species to develop species‐specific pest management strategies based on developmental and behavioral considerations for each species; 2) threshold measures to determine when action is necessary; 3) public education, control, prevention; 4) monitoring of efficacy and environmen‐

tal impacts to identify the occurrence of unexpected/unwanted effects of treatments.

The impact of insecticides on the environment depends not only on the active substance, but also the formulation and the method of applying: indoor residual spraying, space spraying or

Domestic livestock (particularly chickens) and organisms in the environment may be harmed

Table 4 describes the potential ecological effects of each recommended IRS chemical. There is a lack of data concerning toxicity of IRS insecticides on non-target fauna. However, most insecticides are highly toxic for aquatic and terrestrial arthropods like bees (in particular pyrethroid), and some of them can also be toxic for mammals (some pyrethroids and organo‐

Space spraying has only occasionally been used in malaria epidemic control program and as a complementary measure against exophilic vectors. Nevertheless, pyrethroids, which have a short remanence, have been the predominant insecticides [123], and then care must be taken to avoid applications near fish-bearing water bodies. It is also recommended that such applications should not be carried out directly over water bodies and that a no-treated barrier of 100 m should be maintained to prevent fish mortality. Home owners should be advised to

Blom [124] examined the effects of aerial, barrier, and ground based ultra-low volume (ULV) sprays with sumithrin and deltamethrin, in Massachusetts on non-target insects. Malaise traps, targeting the flying insect population, were collected in regular intervals before and after sprays, then the captured insects were sorted by order and counted. The results have shown little effect on non-target insects from the ground based sprays, and a temporary knockdown from the aerial spray. However, Coleoptera were affected in the short term by the ULV sprays

cover domestic fish tanks and bird cages during the applications [123].

and, suffered long term effects from aerial spraying.

if operations, cleanup, and disposal are not conducted according to best practices.

*3.4.2. Impact of insecticides used for vector control*

treated nets will have different impacts.

730 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*3.4.2.1. Indoor Residual Spraying (IRS)*

*3.4.2.2. Space spraying and larviciding*

phosphates).

Long-Lasting Insecticide-Treated Nets (LLINs) have many important advantages as there is no need for re-treatment, the insecticide consumption is reduced, and release of insecticide in natural water bodies during washing is also reduced [142]. However, there is considerable misuse of mosquito nets for drying fish and fishing, in particular along Lake Victoria [143]. In their study, Minakawa et al. [143] surveyed 7 fishing villages along the lake and estimated that 239 LLIN were used for fishing and drying fish from the 1040 LLINs distributed by NGO in these villages. This could have an impact on aquatic organisms while the net are immersed into the lake water. On the other hand, LLIN can also moderately impact non-target household


Key: 0: non-toxic; +: potentially toxic; ++: highly toxic

<sup>a</sup> In some cases non-target Nematocera such as Chironomidae can be impacted by *Bti*, depending on the dose and the formulations applied (Boisvert and Lacoursière, 2004)[126].

**Table 5.** Toxicity of larvicides chemicals on non-target organisms

pests such as house fly, American cockroach, head louse, and mosquito bug after 30-min exposure [144].

#### *3.4.3. Environmental management*

Mosquitoes breed in shallow-water habitats, so it is not surprising that most environmental management interventions for malaria control are associated with the manipulation of wetland environments. If applied correctly, these strategies can have very good results by modifying vector-breeding habitats [145]. But these habitats can include freshwater wetlands (swamps, flood plains, riverine forest, and swamp forest), mangroves, and coastal wetlands (lagoons, estuaries, and tidal mudflats) [146]. In some geographical regions, there are also semi-arid grasslands, which maintain areas of temporary flooding. Wetlands provide a wide range of ecological services including soil erosion and flood control, water purification and pollutant and nutrient retention, groundwater discharge and recharge, and provision of habitat and breeding grounds for wildlife. Disturbing wetlands through environmental management may alter the quantity and quality of the services that wetlands provide. Increasing water runoff (or, alternatively, a change in the composition or clearing of wetland vegetation by drainage or clearing vegetation) may also decrease the ability of the wetland to take up pollutants, potentially diminishing the quality of water resources. It may also cause higher peak water flows in streams and rivers during rain events, resulting in flood damage. Vegetation clearance may also decrease spawning ground for aquatic species and decrease breeding habitats for migratory birds and animals [147].

Larvivorous fish (such as *Gambusia*) are often introduced for biological control. However, the introduction of exotic fish species into the natural environment (e.g., wetlands and marshes) could disrupt existing predator–prey relationships and alter ecosystem composition. In some cases, the introduction of *Gambusia* has led to the destruction of native fish [145].

### *3.4.4. Methodological approach for ERA in the context of vector control*

pests such as house fly, American cockroach, head louse, and mosquito bug after 30-min

<sup>a</sup> In some cases non-target Nematocera such as Chironomidae can be impacted by *Bti*, depending on the dose and the

**Larvicides Mammal Bird Fish Aquatic invertebrate Bee References** *Bti* and *Bs* 0 0 0 0a 0 [126] Spinosad 0 0 0 ++ ++ [127-129] Chlorpyrifos + ++ ++ ++ ++ [130] Fenthion ++ ++ ++ ++ ++ [131-132] Pirimiphos-methyl ++ 0 ++ ++ ++ [133-134] Temephos + 0 + + ++ [135-136] Diflubenzuron 0 0 + + + [137-139] Methoprene 0 0 0 ++ + [134] Pyriproxyfen + 0 + ++ + [140-141]

Mosquitoes breed in shallow-water habitats, so it is not surprising that most environmental management interventions for malaria control are associated with the manipulation of wetland environments. If applied correctly, these strategies can have very good results by modifying vector-breeding habitats [145]. But these habitats can include freshwater wetlands (swamps, flood plains, riverine forest, and swamp forest), mangroves, and coastal wetlands (lagoons, estuaries, and tidal mudflats) [146]. In some geographical regions, there are also semi-arid grasslands, which maintain areas of temporary flooding. Wetlands provide a wide range of ecological services including soil erosion and flood control, water purification and pollutant and nutrient retention, groundwater discharge and recharge, and provision of habitat and breeding grounds for wildlife. Disturbing wetlands through environmental management may alter the quantity and quality of the services that wetlands provide. Increasing water runoff (or, alternatively, a change in the composition or clearing of wetland vegetation by drainage or clearing vegetation) may also decrease the ability of the wetland to take up pollutants, potentially diminishing the quality of water resources. It may also cause higher peak water flows in streams and rivers during rain events, resulting in flood damage. Vegetation clearance may also decrease spawning ground for aquatic species and decrease breeding habitats for

Larvivorous fish (such as *Gambusia*) are often introduced for biological control. However, the introduction of exotic fish species into the natural environment (e.g., wetlands and marshes)

exposure [144].

*3.4.3. Environmental management*

Key: 0: non-toxic; +: potentially toxic; ++: highly toxic

732 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

formulations applied (Boisvert and Lacoursière, 2004)[126].

**Table 5.** Toxicity of larvicides chemicals on non-target organisms

migratory birds and animals [147].

Measurements of toxicity based on the impact of a chemical on a species of interest, such as the LC50 (concentration that kills 50% of a population), and the no observable effect concen‐ tration for reproduction, are used extensively in determining ecological risk. But these methods are too simplistic to establish relationship between the results obtained and the response observed [148] and are not always representative of real life settings. As a consequence, new assessment methodologies to predict and anticipate the risks associated with new chemicals, and improve knowledge about existing chemicals are needed. The last decade has seen some development in this area, but there have been very few studies on the effects of large scale vector control published [149]. Recently, indirect effects of *Bti* treatments on birds such as house martins *Delichon urbicum* have been shown via measuring impact on their insect food sources [150]. In this study, the authors have measured foraging rates and chick diet and have shown that clutch size and fledgling survival were significantly lower at treated sites relative to control. Their hypothesis is that intake of Nematocera (Diptera) and their predators (spiders and dragonflies) decreased significantly in the sites treated with *Bti*, hindering the breeding success of the house martins. Another study on *Bti* monitored Chironomidae populations [151] in three wetlands treated with *Bti*-treatment to control mosquitoes, and three untreated wetlands. Results showed no reduced production of chironomids in *Bti*-treated as compared to untreated wetlands. However, the same authors [152] identified possible indirect effects of *Bti*-treatments in a further study that showed a higher specific richness of chironomids in treated wetlands, compared to control wetlands. They hypothesized that this was the result of reduced competition from mosquito larvae.

These studies demonstrate the need for more suitable methodologies and protocols to be developed for long-term monitoring of ecosystems. Several studies in Europe have monitored long term mosquito control effects, including programmes efforts in western France [153-154], and another in Ramsar area of southern France [155] where the Life-Environment European Program has been studying methods for the sustainable management of mosquito control. The French Ministry for Ecology, Sustainable Development and Spatial Planning via the National Programme for Ecotoxicology (PNETOX; APR2003) are studying the harmonisation of mosquito control methods in terms of their impact on non-target invertebrates in Mediterra‐ nean and Atlantic coastal wetlands [156].

**A Life-Environment project**, sustained by the European Commission, called "Control of noxious or vector mosquitoes: implementation of integrated management consistent with sustainable development (IMCM/n° n°LIFE08 ENV/F/000488)" is also under way in France. Its objective is to validate integrated methodologies and techniques allowing (1) a precise and up to date knowledge of target species' presence, biology, colonized habitats, using GIS/GPS tools, (2) the development of control methods fully appropriate to the health and environmental risks faced, (3) an evaluation of nuisance thresholds based on knowledge of social demands through sociological surveys, in order to optimise the communication strat‐ egies, (4) traceability of operations by means of retrospective and prospective analyses, and (5) the adoption of valid procedures and methodologies for the monitoring of the non-inten‐ tional effects on Man and the environment that can result from these control methods. This project will implement these decision-making tools with five public bodies that are involved in mosquito control efforts in Metropolitan France (Entente InterDepartementale pour la Dé‐ moustication du Littoral méditerranéen, EID Méditerranée, Entente InterDépartementale Rhône-Alpes pour la démoustication, EID Rhône-Alpes, General Council of Southern Corsi‐ ca) and overseas (General Councils of Martinique and Guyana). The project prioritises envi‐ ronmental care and uses complementary methods for environmental risk assessment (in aquatic and terrestrial compartments) for mosquito control methods in temperate or tropical zones. All these projects have focused on consideration of the indirect possible effects of mosquito control on the invertebrates' communities in order to preserve the local biodiversi‐ ty and endangered species. These projects have highlighted the importance of using meth‐ odologies adapted to the habitats and specific organisms, with relevant bio-indicators, implemented infield settings that represent the context in which the vector control manage‐ ment is to be undertaken. The studies also underlined the necessity of post-approval moni‐ toring of the insecticides used in vector control management.

## **4. Conclusion — Discussion**

The history of vector control for malaria control can roughly be divided in 3 main periods: before DDT: from general control to "eradication"; the DDT era and the "Malaria Eradication Programme" (MEP); after DDT: insecticide treated nets (ITN-LLIN), Integrated Vector Management (IVM) and new paradigms.

#### **4.1. Before DDT**

Since his discovery of the role of mosquito as vector of malaria parasite, Ross advocated the vector control for malaria control and in 1899, in Sierra-Leone; he "carried out the first project based on his discovery. His principal weapon was "illuminating oil" (kerosene)". It "was a transient success" not sustained due to lack of funds [157]. "In 1907 Ross was invited to Mauritius to organize antimalaria operations there. His recommendations were sound and the results were good if the government had given them more support" (Bruce-Chwatt, loc.cit.). It is interesting to underline some of the main issues observed at that time: the lack of financial and political support and the financial support is still matter of concerns when referring to the recent RBM statement. The greatest and most successful programme was malaria control in the Panama Canal zone by Gorgas [158] who, helped by Joseph Le Prince, successfully planned and implemented "*sanitation measures*" based on the principle to deal with the situation by all available means based on the role of mosquitoes. He could be therefore considered as the actual precursor of IVM.

Still underlined by Bruce-Chwatt (loc. Cit) "among the early projects one carried out by Malcolm Watson in Malaya deserves special mention, because of the ingenious combination of open and subsoil-drainage with naturalistic methods of control of *Anopheles* [159]. These measures were adapted to the behavioral characteristics of malaria in a given area and formed the basis for the concept of "*species sanitation*" [160].

egies, (4) traceability of operations by means of retrospective and prospective analyses, and (5) the adoption of valid procedures and methodologies for the monitoring of the non-inten‐ tional effects on Man and the environment that can result from these control methods. This project will implement these decision-making tools with five public bodies that are involved in mosquito control efforts in Metropolitan France (Entente InterDepartementale pour la Dé‐ moustication du Littoral méditerranéen, EID Méditerranée, Entente InterDépartementale Rhône-Alpes pour la démoustication, EID Rhône-Alpes, General Council of Southern Corsi‐ ca) and overseas (General Councils of Martinique and Guyana). The project prioritises envi‐ ronmental care and uses complementary methods for environmental risk assessment (in aquatic and terrestrial compartments) for mosquito control methods in temperate or tropical zones. All these projects have focused on consideration of the indirect possible effects of mosquito control on the invertebrates' communities in order to preserve the local biodiversi‐ ty and endangered species. These projects have highlighted the importance of using meth‐ odologies adapted to the habitats and specific organisms, with relevant bio-indicators, implemented infield settings that represent the context in which the vector control manage‐ ment is to be undertaken. The studies also underlined the necessity of post-approval moni‐

The history of vector control for malaria control can roughly be divided in 3 main periods: before DDT: from general control to "eradication"; the DDT era and the "Malaria Eradication Programme" (MEP); after DDT: insecticide treated nets (ITN-LLIN), Integrated Vector

Since his discovery of the role of mosquito as vector of malaria parasite, Ross advocated the vector control for malaria control and in 1899, in Sierra-Leone; he "carried out the first project based on his discovery. His principal weapon was "illuminating oil" (kerosene)". It "was a transient success" not sustained due to lack of funds [157]. "In 1907 Ross was invited to Mauritius to organize antimalaria operations there. His recommendations were sound and the results were good if the government had given them more support" (Bruce-Chwatt, loc.cit.). It is interesting to underline some of the main issues observed at that time: the lack of financial and political support and the financial support is still matter of concerns when referring to the recent RBM statement. The greatest and most successful programme was malaria control in the Panama Canal zone by Gorgas [158] who, helped by Joseph Le Prince, successfully planned and implemented "*sanitation measures*" based on the principle to deal with the situation by all available means based on the role of mosquitoes. He could be therefore considered as the actual

Still underlined by Bruce-Chwatt (loc. Cit) "among the early projects one carried out by Malcolm Watson in Malaya deserves special mention, because of the ingenious combination

toring of the insecticides used in vector control management.

**4. Conclusion — Discussion**

734 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Management (IVM) and new paradigms.

**4.1. Before DDT**

precursor of IVM.

After the success of Watson, several other "naturalistic methods" were developed such as altering the salinity of breeding site of *An. ludlowae* control in Indonesia, introduction of natural enemies of mosquitoes, use of *Gambusia* in California, Florida, then in Cyprus, Spain, Italy, Russia, Chile, etc [161].

Some of the best example of environmental modifications based upon drainage for successful malaria control were observed in Italy with reclamation of marshy areas (with resettlement of population in new land) for "*bonifica integrale*" of Pontine Marshes of the Roman Campagna [162-164] or Algeria in the marshy area of Mitidja Plaine [165-166].

Such programs could also be considered as precursor in the field of biological control which currently received great attention with the ecological issues of insecticide and insecticide resistance of main vectors.

In term of chemical control, 2 schools of thought were opposed*:* larva control, based upon Paris Green dust successfully used in Sardinia and Calabria and in several other places such as Brazil to get rid of invaders *An. gambiae* which caused severe epidemics of malaria in 1930s'; and adult control, with the use of the well known oriental daisy *Chrysanthemum cinerariaefolium*, (used for long time as fumigants in China against biting insects) the powder made of it contains powerful insecticide compounds such as pyrethrins and cinerins and as soon as 1932 Park-Ross and De Meillon instituted systematic house to house weekly sprayings of pyrethrum solution in kerosene for the control of adults *Anopheles* in Natal and Zululand and this program is somehow still ongoing with the regular inside resting spraying (with DDT) operations added to case management to control malaria in KwaZulu Natal [167]. Instead of pyrethrins, National Malaria control programme uses now pyrethroid but they are chemically developed from natural pyrethrins used formerly. Somehow history of approaches for malaria control repeats itself.

It is interesting to notice the variety of approaches and techniques involved (species sanitation, sanitation measures, bonifica integrale (reclamation of marshy area and resettlement of populations on the new land), pursued by Italian governments for many years, larval control through different measures from source reduction to Paris Greendust spraying, adult control with spray of pyrethrin, …) based on some knowledge of entomological, ecological and socioeconomical situation for improvement of Public Health, control of outbreak or achievement of large constructions (dams, Panama Canal, etc). In a way these measures paved the way for new approaches developed after the failure of the Global Malaria Eradication Programme and the development of IVM with new paradigms for vector control.

### **4.2. The DDT era 1957 — 1969: Global malaria eradication programme**

"In 1874 a Viennese student of chemistry, Othmar Zeidler, published in the Berichtungen (Proceedings) of the German Chemical Society a paper under the title "Verdindungen von Chlral mit Brom und Chlorbenzol"; the compound described in it was DDT (Bruce-Chwatt, loc cit) but its insecticidal properties remained unknown until 1939 [168].

The first Expert Malaria Committee (Ciuca, Gabaldon, Hamilton, Fairley, Pampana, Russell) met in Geneva in 1947 to deal with "the enormous social and economic damage that malaria was causing to the developing tropical countries", Russell [169] estimating that throughout the world there were some 300 million cases of malaria every year with at least a million deaths, it is interesting to underline that such evaluation of the burden of malaria was regu‐ larly reported during the following decades. And as Bruce-Chwatt [157] rightly underlined: "this was also the time when the new concept of malaria control by imagocidal measures was stimulated by the reports of the extraordinary properties of an obscure compound syn‐ thesized 65 years before the outbreak of the Second World War. They were observed by a Swiss chemist, Müller who was looking for a substance active against clothes moths, and with the biologist Wiesmann they realized in 1939 the insecticidal properties of this product, named Gesarol or Neocid and first used in agriculture [170] then sent to USA and Britain (where it received the acronym DDT). This product presented 3 important operational prop‐ erties: long persistence of residues on sprayed surfaces; high toxicity for insects and low for man; killing insects by simple contact. The advent of DDT revolutionized malaria control as the residual indoor spraying as this product appeared simple, and could be successfully and economically used even in rural areas where malaria was the worse. Actually a lot of suc‐ cessful campaigns were done in Sardinia (Italy) (for eradication of *An. labranchiae*), Cyprus, Greece, Venezuela, British Guiana, Bombay State, etc [171]. In 1955, Pampana and Russell [172] underlined the needs of "plans to eradicate malaria from a territory within a few years, so that eventually the recurring item of malaria control could be struck from the annual budget". And the Eighth World Health Assembly in 1955 decided "that the World Health Organization should take the initiative, provide technical advice, and encourage research and co-ordination of resources in the implementation of a programme having as its ultimate objective the world-wide eradication of malaria".

DDT appeared as a "magic bullet" but the great mistake was that the original policy relied only on the use of residual insecticide, DDT then other organochlorines (BHC, dieldrin,...) along with drug use for reducing human reservoir, with the same strategy to be implement‐ ed everywhere without taking care of biodiversity, epidemiological diversity, social, eco‐ nomical, entomological diversity. The basic concept was one malaria and therefore one strategy to be implemented faster than insecticide resistance spreading, already noticed in the main vectors such as *An. gambiae*. In 1956, the Ninth World Health Assembly recom‐ mended the policy of eradication and stimulation of inter-countries cooperation. The strat‐ egy was defined as "operation aimed at cessation of transmission of malaria and elimination of the reservoir of infected cases in a campaign limited in time and carried to such a degree of perfection that, when it comes to an end, there is no resumption of transmission". It was based upon 3 successive steps: "attack phase" with total coverage with inside residual spraying, then "consolidation phase" to eradicate any remaining foci after the IRS rounds, then the "maintenance phase" where the malaria eradication programme doesn't exist as such and comes under the responsibility of general health services involves in "vigilance" to check any imported cases.

During the following decades malaria was actually eradicated from Europe, part of Russia, Middle East, North America, Australia, Japan, Singapore, Korea, Taiwan, almost all West Indies Islands and about 53% of the population of the originally malarious areas became free of malaria. But "the magnitude of the malaria problem in Tropical Africa has been daunting" (Bruce-Chwatt, loc cit). A re-examination of the global strategy of malaria eradication was carried in the 60' and the results presented at the 22nd World Health Assembly in 1969. One of the conclusion was that "in countries where eradication does not appear to be feasible because of the inadequacy of financial resources, manpower requirements or shortcomings of basic health services, malaria control operations should move to a transitional control programme stage, with the aim of launching of an eradication programme in the future". This is political wording that recognizes the failure of the rigid Global Eradication Programme and the reality that this may translate to "malaria control" involving the use of every available effective method to tackle first malaria mortality and morbidity, rather than malaria transmission specifically, as it was targeted by the MEP.

Chlral mit Brom und Chlorbenzol"; the compound described in it was DDT (Bruce-Chwatt,

The first Expert Malaria Committee (Ciuca, Gabaldon, Hamilton, Fairley, Pampana, Russell) met in Geneva in 1947 to deal with "the enormous social and economic damage that malaria was causing to the developing tropical countries", Russell [169] estimating that throughout the world there were some 300 million cases of malaria every year with at least a million deaths, it is interesting to underline that such evaluation of the burden of malaria was regu‐ larly reported during the following decades. And as Bruce-Chwatt [157] rightly underlined: "this was also the time when the new concept of malaria control by imagocidal measures was stimulated by the reports of the extraordinary properties of an obscure compound syn‐ thesized 65 years before the outbreak of the Second World War. They were observed by a Swiss chemist, Müller who was looking for a substance active against clothes moths, and with the biologist Wiesmann they realized in 1939 the insecticidal properties of this product, named Gesarol or Neocid and first used in agriculture [170] then sent to USA and Britain (where it received the acronym DDT). This product presented 3 important operational prop‐ erties: long persistence of residues on sprayed surfaces; high toxicity for insects and low for man; killing insects by simple contact. The advent of DDT revolutionized malaria control as the residual indoor spraying as this product appeared simple, and could be successfully and economically used even in rural areas where malaria was the worse. Actually a lot of suc‐ cessful campaigns were done in Sardinia (Italy) (for eradication of *An. labranchiae*), Cyprus, Greece, Venezuela, British Guiana, Bombay State, etc [171]. In 1955, Pampana and Russell [172] underlined the needs of "plans to eradicate malaria from a territory within a few years, so that eventually the recurring item of malaria control could be struck from the annual budget". And the Eighth World Health Assembly in 1955 decided "that the World Health Organization should take the initiative, provide technical advice, and encourage research and co-ordination of resources in the implementation of a programme having as its ultimate

DDT appeared as a "magic bullet" but the great mistake was that the original policy relied only on the use of residual insecticide, DDT then other organochlorines (BHC, dieldrin,...) along with drug use for reducing human reservoir, with the same strategy to be implement‐ ed everywhere without taking care of biodiversity, epidemiological diversity, social, eco‐ nomical, entomological diversity. The basic concept was one malaria and therefore one strategy to be implemented faster than insecticide resistance spreading, already noticed in the main vectors such as *An. gambiae*. In 1956, the Ninth World Health Assembly recom‐ mended the policy of eradication and stimulation of inter-countries cooperation. The strat‐ egy was defined as "operation aimed at cessation of transmission of malaria and elimination of the reservoir of infected cases in a campaign limited in time and carried to such a degree of perfection that, when it comes to an end, there is no resumption of transmission". It was based upon 3 successive steps: "attack phase" with total coverage with inside residual spraying, then "consolidation phase" to eradicate any remaining foci after the IRS rounds, then the "maintenance phase" where the malaria eradication programme doesn't exist as such and comes under the responsibility of general health services involves in "vigilance" to

loc cit) but its insecticidal properties remained unknown until 1939 [168].

736 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

objective the world-wide eradication of malaria".

check any imported cases.

After the illusion of the Malaria eradication came the time of pragmatism, and the recognition of the biodiversity concept with IVM which takes into account all biological but also econom‐ ical, socio-cultural components of the vector-borne parasitic disease and tools available (or to be developed) to tailor vector control measures to each epidemiological settings, to reach its full efficacy in the aim of sharply reduce, then eliminating malaria steps by steps. In this concept of biodiversity, a flexible and multifaceted approach is requested and paradigms were developed accordingly. For example, it is generally considered that tools for vector control must have a quick action to kill vectors before they transmit the parasites to any other human being, but slow acting products are now envisaged considering that if life is shortening to become less than the duration of the sporogonic cycle there couldn't be any transmission of the pathogenic agent even if this takes slightly more time than the "killing" product. Another approach is to mix different products for LLIN or IRS to deal with insecticide resistance and even to join IGR usually used against larvae in product targeting adults such as insecticide paints and even LLIN. The main impact should therefore be observed in term of reducing fecundity and fertility which would impact new generations of adults and more generally *Anopheles* populations.

Nevertheless for the time being the only new tools operational for vector control at large is insecticide treated nets (ITN) currently industrialized treated to become Long Lasting nets and which clearly showed their efficacy if well used and maintained. But the field is largely open for new tools mainly dealing with insecticide, and sometimes social resistance.

A great attention is now devoted to the cultural and social aspects of vector control methods implemented from outside, the "non usage" or "mis-usage" of mosquito nets are good example of the misfit between International agencies which gave large number of LLIN free of charge and the local social acceptability or local financial constraints.

A great care is also given to ecological impact and Malaria control programme must take lessons from the large multicountries Onchocerciasis Control Programme for managing insecticide resistance and care of non targeted fauna.

We must keep in mind the sentences of late Prof Bruce-Chwatt [173]: "the present approach to the control of this disease envisages a progressive incorporation of all general and specific antimalarial activities into the primary health care structures. This opens up many possibilities for research on the use of different technical resources together with the involvement of indigenous communities. But this is a different story!".

## **List of abbreviations**


MLT - Median Lethal Time


We must keep in mind the sentences of late Prof Bruce-Chwatt [173]: "the present approach to the control of this disease envisages a progressive incorporation of all general and specific antimalarial activities into the primary health care structures. This opens up many possibilities for research on the use of different technical resources together with the involvement of

indigenous communities. But this is a different story!".

EID - Entente InterDepartementale pour la Démoustication

**List of abbreviations**

ASB - Attractive Sugar Bait

CTN - hand treated ITN DL - Durable wall linings

C - Carbamate

ATSB - Attractive toxic sugar bait

738 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

CS - Microencapsulated formulation

ERA - Environmental Risk Assessment

ITPS - Insecticide-Treated Plastic Sheeting

IVM - Integrated Vector Management

LC100 - absolute Lethal Concentration

LLIN - Long-Lasting Insecticidal Net

LN - Long lasting insecticide treated Net MEP - Malaria Eradication Programme

KDR - KnockDown Resistance

LiST - Lives Saved Tool

KD - KnockDown

IVCC - Innovative Vector Control Consortium

LC50 - median Lethal Concentration of a substance

IGR - Insect Growth Regulator IPM - Insect Pest Management IRS - Indoor Residual Spraying ITN - Insecticide Treated Nets


## **Acknowledgements**

We are grateful to Dr Kate Aultman for her helful suggestions and comments all along the writing of this document.

## **Author details**

Claire Duchet1,2, Richard Allan3 and Pierre Carnevale4

\*Address all correspondence to: pjcarnevale2001@yahoo.fr; cduchet.eid@gmail.com

1 Entente InterDépartementale de Démoustication du Littoral Méditerranéen, Montpellier, France

2 Community Ecology Laboratory, Institute of Evolution and Department of Evolutionary & Environmental Biology, University of Haifa, Israel

3 The MENTOR Initiative, Crawley, UK

4 Portiragnes, France

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## **New Salivary Biomarkers of Human Exposure to Malaria Vector Bites**

Papa M. Drame, Anne Poinsignon, Alexandra Marie, Herbert Noukpo, Souleymane Doucoure, Sylvie Cornelie and Franck Remoue

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55613

## **1. Introduction**

Mosquitoes are the most menacing worldwide arthropod disease vectors. They transmit a broad range of viral, protozoan and metazoan pathogens responsible of the most devastating human and animal diseases [1]. Among the main frequent mosquito-borne diseases, malaria represents the most widespread and serious infection in terms of heavy burden on health and economic development throughout the world. Despite substantial efforts and increasing international funding to eliminate it, malaria is still a major public health problem with nearly a million of deaths per year, especially in children younger than 5 years old (86%) [2]. Ap‐ proximately two thirds of the world's population live in areas at risk for malaria [3, 4]. Understanding mechanisms that govern its transmission remains therefore a major scientific challenge, but also an essential step in the design and the evaluation of effective control programs [5, 6].

Entomological, parasitological and clinical assessments are routinely used to evaluate the exposure of human populations to *Anopheles* vector bites and the risk of malaria transmission. However, these methods are labor intensive and difficult to sustain on large scales, especially when transmission and exposure levels are low (dry season, high altitude, urban settings or after vector control) [7, 8]. In particular, the entomological inoculation rate (EIR), the gold standard measure for mosquito–human transmission intensity of *Plasmodium*, is highly dependent on the density of human-biting *Anopheles* [9]. This latter is estimated by using trapping methods such as human-landing catches (HLC) of adult mosquitoes, the commonly used for sampling host-seeking mosquitoes and then for assessing the human exposure level.

© 2013 Drame et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Drame et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HLC may be limited because of ethical and logistical constraints to relevantly apply it to children [10]. Transmission estimates based on the prevalence or density of human infection are susceptible to micro-heterogeneity caused by climatic factors and the socioeconomic determinants of the host-seeking behavior [8]. Incidence of disease may be the closest logical correlate of the burden of disease on health systems. However, it can be subject to variability between sites and may not be appropriate for the evaluation of early phase studies of vector control or reliable for epidemic prediction [10]. More recently, serological correlates of transmission intensity have been described, yet they represent long-term rather than shortterm exposure data [8]. They are not then suitable in evaluating the short-term impact of vector control programs. Therefore, it is currently emphasized the need to develop new tools assessing reliably human malaria risk and control interventions, and monitoring changes over time at both population and individual levels [5, 6].

Malaria is a parasitic disease caused by protozoan agents of the genus *Plasmodium* (*Aplicom‐ plexa*; *Haemosporida*). Five *Plasmodium* species are pathogen for humans: *P. falciparum*, *P. vivax, P. ovale*, *P. malariae* and *P. knowlesi*. During their complex life cycle in the female *Anopheles* mosquito (*Insecta*; *Diptera*), *Plasmodium* parasites go through several developmental transi‐ tions, traverse the midgut and reach the salivary gland (SG) epithelium. They acquire their maturity within SGs of the vector and can be then transmitted by the bite of the female mosquito. This latter needs, during the first days after emergence, to feed on sugar to meet the energy demands of basic metabolism and flight, but also to feed on vertebrate blood for its eggs' development and maturation [11], and therefore to keep perennial its life cycle and indirectly malaria transmission cycle.

*Anopheles* mouthparts comprise six pieces that form a long stylus allowing to perforate human tissues and to suck the internal liquid. However, it is clear that *Anopheles* mosquito acts not only as syringe injecting parasites during the bite. When taking a blood meal, it also injects into human skin avascular tissue [12] a cocktail of bioactive molecules including enzymes that are injected in human skin by saliva [13, 14]. Some of these salivary compounds are essential to the *Plasmodium* life cycle [15]. They have substantial anti-hemostatic, anti-inflammatory, and immunomodulatory activities that assist the mosquito in the blood-feeding process by inhibiting several defense mechanisms of the human host [16]. Furthermore, many of them are immunogenic and elicit strong immune responses, evidenced by the swelling and itching that accompany a mosquito bite [17]. Specific acquired cellular [18, 19] or/and humoral responses are developed by human individuals when exposed to bites of *Anopheles* mosquitoes [20-23]. These immune responses may play several roles in the pathogen transmission ability and the disease outcomes [24]. In addition, recent studies have demonstrated that the intensity of the antibody response specific to salivary proteins could be a biomarker of the exposure level of human to *Anopheles* bites [22, 25]. Therefore, studying *Anopheles*-human immunological relationships can provide new promising tools for monitoring the real human-*Anopheles* contact and identifying individuals at risk of malaria transmission. It can also allow the development of novel methods for monitoring control and mosquito-release programmes' effectiveness.

However, whole saliva could be inadequate as a biomarker tool, because it is a cocktail of various molecular components with different nature and biological functions. Some of these elements are ubiquitous and may potentially cause cross-reactivities with common salivary epitopes of other haematophagous arthropods [26]. In addition, a lack of reproducibility between collected whole *Anopheles* saliva batches has been observed and difficulties to obtain sufficient quantities needed for large-scale studies were highlighted [26]. Therefore, specific and antigenic proteins have been identified in the secretome of *Anopheles* mosquitoes and a specific biomarker of *Anopheles* bites was developed by coupling bioinformatic and immunoepidemiological approaches. This promising candidate, namely, the gSG6-P1 (*An. gambiae* Salivary Gland Protein-6 peptide 1), has been described to be highly antigenic [26]. It has been then validated as a pertinent biomarker assessing specifically and reliably the exposure level to *Anopheles* bites [27-29] and/or the effectiveness of malaria vector control [30] in all age-classes of human populations (newborns, infants, children and adults) from several malaria epide‐ miological settings (rural, semi-urban and urban areas…) throughout sub-Saharan Africa countries (Senegal, Angola and Benin).

The present chapter contributes therefore to a better understanding of the human-mosquito immunological relationship. It resumes most of the studies highlighting the roles of mosquito saliva on the human physiology and immunology, approaches, techniques, and methods used to develop and validate specific candidate-biomarkers of exposure to *Anopheles* bites and their applications on malaria control in several different epidemiological settings. Effects of various explanatory variables (age, sex, seasonality, differential use of vector control…) on human antibody responses to *Anopheles* salivary antigens are also discussed in the aim to optimize their use in epidemiological and vector-borne disease (VBD) control studies. Finally, different ways of application of such salivary biomarker of exposure of *Anopheles* vector bites in the field of operational research by National Malaria Control Programmes (NMCP) are highlighted.

## **2. Human host-mosquito relationship: Roles of mosquito saliva**

Arthropods represent the vast majority of described metazoan life forms throughout the world, with species' richness estimated between 5 to 10 million [31]. The blood feeding habit has arisen and evolved independently in more than 14,000 species from 400 genera in the arthropod taxonomy [32]. In mosquitoes, only the adult female is hematophagous, whereas both male and female take sugar meals [33]. During the probing and the feeding stages, like all bloodsucking arthropods, female *Anopheles* must circumvent the highly sophisticated barriers represented by human defense systems (Fig. 1): haemostatic and inflammatory reactions, innate and adaptive immune system defenses. Therefore, they express in their saliva potent pharmacological and immunogenic components.

#### **2.1. Pharmacological properties of mosquito saliva**

HLC may be limited because of ethical and logistical constraints to relevantly apply it to children [10]. Transmission estimates based on the prevalence or density of human infection are susceptible to micro-heterogeneity caused by climatic factors and the socioeconomic determinants of the host-seeking behavior [8]. Incidence of disease may be the closest logical correlate of the burden of disease on health systems. However, it can be subject to variability between sites and may not be appropriate for the evaluation of early phase studies of vector control or reliable for epidemic prediction [10]. More recently, serological correlates of transmission intensity have been described, yet they represent long-term rather than shortterm exposure data [8]. They are not then suitable in evaluating the short-term impact of vector control programs. Therefore, it is currently emphasized the need to develop new tools assessing reliably human malaria risk and control interventions, and monitoring changes over

Malaria is a parasitic disease caused by protozoan agents of the genus *Plasmodium* (*Aplicom‐ plexa*; *Haemosporida*). Five *Plasmodium* species are pathogen for humans: *P. falciparum*, *P. vivax, P. ovale*, *P. malariae* and *P. knowlesi*. During their complex life cycle in the female *Anopheles* mosquito (*Insecta*; *Diptera*), *Plasmodium* parasites go through several developmental transi‐ tions, traverse the midgut and reach the salivary gland (SG) epithelium. They acquire their maturity within SGs of the vector and can be then transmitted by the bite of the female mosquito. This latter needs, during the first days after emergence, to feed on sugar to meet the energy demands of basic metabolism and flight, but also to feed on vertebrate blood for its eggs' development and maturation [11], and therefore to keep perennial its life cycle and

*Anopheles* mouthparts comprise six pieces that form a long stylus allowing to perforate human tissues and to suck the internal liquid. However, it is clear that *Anopheles* mosquito acts not only as syringe injecting parasites during the bite. When taking a blood meal, it also injects into human skin avascular tissue [12] a cocktail of bioactive molecules including enzymes that are injected in human skin by saliva [13, 14]. Some of these salivary compounds are essential to the *Plasmodium* life cycle [15]. They have substantial anti-hemostatic, anti-inflammatory, and immunomodulatory activities that assist the mosquito in the blood-feeding process by inhibiting several defense mechanisms of the human host [16]. Furthermore, many of them are immunogenic and elicit strong immune responses, evidenced by the swelling and itching that accompany a mosquito bite [17]. Specific acquired cellular [18, 19] or/and humoral responses are developed by human individuals when exposed to bites of *Anopheles* mosquitoes [20-23]. These immune responses may play several roles in the pathogen transmission ability and the disease outcomes [24]. In addition, recent studies have demonstrated that the intensity of the antibody response specific to salivary proteins could be a biomarker of the exposure level of human to *Anopheles* bites [22, 25]. Therefore, studying *Anopheles*-human immunological relationships can provide new promising tools for monitoring the real human-*Anopheles* contact and identifying individuals at risk of malaria transmission. It can also allow the development of novel methods for monitoring control and mosquito-release programmes'

time at both population and individual levels [5, 6].

756 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

indirectly malaria transmission cycle.

effectiveness.

The first-line of the human host non-specific defense to the insect bite is the haemostatic reaction. It provides an immediate response to the vascular injury caused by the intrusion of

**Figure 1.** Effects of *Anopheles* saliva on hemostatic, inflammatory and immune reactions of the human to the vector bites.

the mosquito mouthparts in host vessels, thus preventing the extensive loss of host blood [32, 34]. The haemostatic reaction consists of three not physiologically distinct mechanisms: i) the blood coagulation that leads to the production of fibrin clots, ii) the thrombus formation and wound healing mediated by platelet aggregation, and iii) the vasoconstriction that leads to restricted influx of blood to the injured site. Each mechanism is activated by several pathways, in response to different exogenous and endogenous stimuli. Platelet aggregation is the first step in the haemostatic cascade and follows the interaction between blood platelets and the exposed extracellular matrix. This latter contains a large number of adhesive macromolecules such as collagen which is abundant underneath endothelial cells (not found in blood). This interaction results to the activation of platelets by mainly collagen and adenosine diphosphate (ADP, released by damaged cells and by activated platelets), the primary agonists of platelet aggregation. Platelets can be also activated by other agonists such as thrombin (produced by the coagulation cascade) and thromboxane A2 (TXA2, produced by activated platelets) [35]. Activated platelets release endogeneous secretions such as serotonin and TXA2, two potent vasoconstrictors. In parallel, the blood coagulation mechanism is getting underway. The main task of the coagulation cascade is to produce fibrin that supports aggregated platelets in a thrombus formation. The coagulation process consists of an enzymatic cascade with two ways of activation, the exogenous and the endogenous, where several amplification points and regulatory mechanisms are known.

However, mosquitoes can successfully engorge on their hosts within a half-minute because antihemostatic components of their saliva facilitate location of blood vessels and the blood sampling [36]. These salivary secretions, named sialogenins (from the Greek *sialo*, saliva; *gen*, origin, source; and *ins* for proteins), are mainly an array of potent anticoagulants, anti-platelets, vasodilators and anti-inflammatory substances [16, 32, 37, 38].

#### *2.1.1. Inhibition of platelet aggregation*

Compared to other blood-sucking arthropods like ticks and sand flies, only a limited number of *Anopheles* mosquito sialogenins involved in the inhibition of platelet aggregation have been characterized. *Apyrase* (Adenosine triphosphate (ATP)-diphosphohydrolase EC 3.6.1.5) is ubiquitous for hematophagous arthropods (mosquitoes, bugs, sand flies, fleas, triatomines, and ticks) and hydrolyses ATP and ADP into adenosine monophosphate (AMP) and inorganic phosphate (*P*<sup>i</sup> ), thus inhibiting platelet aggregation [16]. Three classes of apyrase have been characterized at the molecular level in different blood-sucking arthropods (reviewed by [39]). One named 5′-nucleotidase family is highly expressed in the salivary gland of *Anopheles gambiae* [40]. The *D7 protein family* is one of the most abundantly expressed sialogenins of mosquitoes. Two classes have been described in the saliva of mosquitoes: long (28–30 kDa) and short (15–20 kDa) forms [41-43]. The D7-related proteins may inhibit activation of host plasma. It has been described in *Anopheles* mosquitoes in a short form and may block the platelet activation by scavenging serotonin (agonist-positive feedback loop to increase platelet aggregation), while it principal function is reported to modulate tonus of vessels (vasocon‐ striction) [44]. *Anophelin* from *An. stephensi* saliva is a 30-kDa protein that directly binds to immobilized collagen and specifically inhibits collagen-induced platelet aggregation and the intracellular Ca2+ increase [45]. It can also act by inhibiting the activity of thrombin which plays a role in concentration of platelet aggregation [46].

#### *2.1.2. Inhibition of blood coagulation cascade*

the mosquito mouthparts in host vessels, thus preventing the extensive loss of host blood [32, 34]. The haemostatic reaction consists of three not physiologically distinct mechanisms: i) the blood coagulation that leads to the production of fibrin clots, ii) the thrombus formation and wound healing mediated by platelet aggregation, and iii) the vasoconstriction that leads to restricted influx of blood to the injured site. Each mechanism is activated by several pathways, in response to different exogenous and endogenous stimuli. Platelet aggregation is the first step in the haemostatic cascade and follows the interaction between blood platelets and the exposed extracellular matrix. This latter contains a large number of adhesive macromolecules such as collagen which is abundant underneath endothelial cells (not found in blood). This interaction results to the activation of platelets by mainly collagen and adenosine diphosphate (ADP, released by damaged cells and by activated platelets), the primary agonists of platelet aggregation. Platelets can be also activated by other agonists such as thrombin (produced by the coagulation cascade) and thromboxane A2 (TXA2, produced by activated platelets) [35]. Activated platelets release endogeneous secretions such as serotonin and TXA2, two potent vasoconstrictors. In parallel, the blood coagulation mechanism is getting underway. The main task of the coagulation cascade is to produce fibrin that supports aggregated platelets in a thrombus formation. The coagulation process consists of an enzymatic cascade with two ways of activation, the exogenous and the endogenous, where several amplification points and

**Figure 1.** Effects of *Anopheles* saliva on hemostatic, inflammatory and immune reactions of the human to the vector

regulatory mechanisms are known.

758 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

bites.

Arthropod anticoagulants mostly target factor X-active (fXa), which plays a central role at the nexus of the intrinsic and extrinsic pathways, as well as an ultimate role of thrombin in driving production of fibrin from fibrinogen. However, *Anopheles* mosquitoes produce an antithrombin [38]. In *An. albimanus* for example, *Anophelin* protein has been shown to be a potent anticoagulant that acts as a specific and tight-binding thrombin inhibitor [46], blocking or delaying then the clot formation process until blood meal completion [34]. In addition, a D7 related protein of *An. stephensi* saliva has been characterized as an inhibitor of fXII [47].

#### *2.1.3. Vasodilator effect on host blood vessels*

In human, various types of endogenous vasoconstrictors (serotonin, TXA2, noradrenalin…) are released few seconds after tissue injury in order to stop the blood flow locally at the bite site. Diverse types of vasodilators have been characterized in the saliva of hematophagous arthropods. *Aedes* mosquitoes use sialokinins that mimic the endogenous tachykinin substance P which stimulate the production of nitric oxide (NO), a potent dilator of blood vessels [48, 49]. In contrast, the saliva of the adult female *Anopheles* mosquito has been shown to contain a myeloperoxidase with a vasodilator activity associated with a catechol oxidase/peroxidase activity [50]. This latter drives the H2O2-dependent destruction of noradrenalin and serotonin, two important endogenous vasoconstrictors [50]. In addition, some D7 proteins of *Anopheles* have been described to bind to biogenic amines such as serotonin, histamine, and norepi‐ nephrine [44]. These strategies remove the human host's ability to maintain vascular tone at the bite site, resulting to a weak but persistent local vasodilatation [14].

#### **2.2. Immunological effects of mosquito saliva**

The tissue injury causes an immediate onset of acute inflammation and innate immunity, which promote tissue repair, prevent colonization of the damaged tissues by opportunistic pathogens and initiates adaptive immunity, which is more specific [51]. These responses mobilize multiple elements such as phagocytes and antigen-presenting cells, cytokineproducing cells, T and B lymphocytes (TL and BL) and complement (classical and alternative pathways). It may result to the development of strong cell and humoral immune reactions, thereby altering physiologically the environment at the bite site and leading to the rejection of the blood-sucker [52]. The saliva of *Anopheles* mosquitoes (like blood-feeding arthropods in general) has selected, during evolution, compounds that can counter these host responses by modulating immune cells and cytokines' production [52, 53]. This certainly allows mosquitoes to complete successfully a blood meal in only few seconds. Immunomodulatory effects of *Anopheles* mosquito saliva can therefore affect the transmission of pathogens and the devel‐ opment of associated pathologies [54]. Understanding the mechanisms which govern this immunomodulation could then allow the development of new prevention tools or strategies against malaria transmission [54-56].

#### *2.2.1. Inhibition of host inflammatory reaction*

The host inflammatory reaction following tissue injury consists of the triple response of Lewis: redness, heat and pain, triggering the awareness of the host to the blood sucker action [16]. If redness and heat are ones of the direct consequences of the dilatation of blood vessels, pain is induced by an increased vascular permeability under the effect of ADP, serotonin and histamine released by platelets and mast cells, following activation of the fXII by tissueexposed collagen [16]. The fXIIa converts prekallikrein to kallikrein, which hydrolyzes blood kininogen to produce the vasodilator peptide, bradykinin. This latter induces TNF-α (Tumor Necrosis Factor alpha) release by neutrophils [57], which in turn stimulates the release of IL (interleukin)-1β and IL-6 from various cell types. These cytokines contribute to the phenom‐ enon of hyperalgesia (increased sensitivity to pain) that accompanies inflammation. Host inflammatory reaction to bites has been described as mast cells-dependent in individuals bitten by *Anopheles* mosquitoes [58]. In contrast to ticks which need to be attached to their host for several hours (tick *Argasidæ*) or weeks (tick *Ixodidæ*), mosquitoes take just few seconds for a successful blood meal. This certainly explains the poverty of anti-inflammatory components in their saliva in contrast to the ticks' one. Nevertheless, some salivary components of *Anopheles* mosquitoes can inhibit the human inflammatory reaction. In particular, a 16kDa D7 family proteins of *An. stephensi* (Hamadarin) inhibits the contact system by preventing the mutual activation between the fXIIa and the kallikrein in the presence of Zn2+ [47].

#### *2.2.2. Modulation of host immune response*

a myeloperoxidase with a vasodilator activity associated with a catechol oxidase/peroxidase activity [50]. This latter drives the H2O2-dependent destruction of noradrenalin and serotonin, two important endogenous vasoconstrictors [50]. In addition, some D7 proteins of *Anopheles* have been described to bind to biogenic amines such as serotonin, histamine, and norepi‐ nephrine [44]. These strategies remove the human host's ability to maintain vascular tone at

The tissue injury causes an immediate onset of acute inflammation and innate immunity, which promote tissue repair, prevent colonization of the damaged tissues by opportunistic pathogens and initiates adaptive immunity, which is more specific [51]. These responses mobilize multiple elements such as phagocytes and antigen-presenting cells, cytokineproducing cells, T and B lymphocytes (TL and BL) and complement (classical and alternative pathways). It may result to the development of strong cell and humoral immune reactions, thereby altering physiologically the environment at the bite site and leading to the rejection of the blood-sucker [52]. The saliva of *Anopheles* mosquitoes (like blood-feeding arthropods in general) has selected, during evolution, compounds that can counter these host responses by modulating immune cells and cytokines' production [52, 53]. This certainly allows mosquitoes to complete successfully a blood meal in only few seconds. Immunomodulatory effects of *Anopheles* mosquito saliva can therefore affect the transmission of pathogens and the devel‐ opment of associated pathologies [54]. Understanding the mechanisms which govern this immunomodulation could then allow the development of new prevention tools or strategies

The host inflammatory reaction following tissue injury consists of the triple response of Lewis: redness, heat and pain, triggering the awareness of the host to the blood sucker action [16]. If redness and heat are ones of the direct consequences of the dilatation of blood vessels, pain is induced by an increased vascular permeability under the effect of ADP, serotonin and histamine released by platelets and mast cells, following activation of the fXII by tissueexposed collagen [16]. The fXIIa converts prekallikrein to kallikrein, which hydrolyzes blood kininogen to produce the vasodilator peptide, bradykinin. This latter induces TNF-α (Tumor Necrosis Factor alpha) release by neutrophils [57], which in turn stimulates the release of IL (interleukin)-1β and IL-6 from various cell types. These cytokines contribute to the phenom‐ enon of hyperalgesia (increased sensitivity to pain) that accompanies inflammation. Host inflammatory reaction to bites has been described as mast cells-dependent in individuals bitten by *Anopheles* mosquitoes [58]. In contrast to ticks which need to be attached to their host for several hours (tick *Argasidæ*) or weeks (tick *Ixodidæ*), mosquitoes take just few seconds for a successful blood meal. This certainly explains the poverty of anti-inflammatory components in their saliva in contrast to the ticks' one. Nevertheless, some salivary components of *Anopheles* mosquitoes can inhibit the human inflammatory reaction. In particular, a 16kDa D7 family proteins of *An. stephensi* (Hamadarin) inhibits the contact

the bite site, resulting to a weak but persistent local vasodilatation [14].

**2.2. Immunological effects of mosquito saliva**

760 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

against malaria transmission [54-56].

*2.2.1. Inhibition of host inflammatory reaction*

A role for arthropod saliva in modifying the outcome of transmission and infection is not a novel idea introduced in the context of mosquitoes and malaria parasites. The increased pathogen infectivity in association with ticks, sand flies, and mosquitoes saliva has been described previously [54]. If ticks that take a long time to engorge must additionally necessitate in their saliva anti-inflammatory and immunosuppressive factors, rapidly feeding dipterans, in particular mosquitoes and sand flies, clearly have evolved salivary factors that directly modulate host immune defenses [52]. One possible explanation is that these molecules have evolved because they have long-term beneficial effects for the populations rather than to the individual at the time of feeding [24]. Although the molecular mechanisms by which mosquito saliva induces alteration of the host immune response are unclear [59, 60], data evidently demonstrate that effects depend on the global regulation of the Th1/Th2 cyto‐ kines' balance, as it has been described in sand flies*/Leishmania* model, the most studied striking host-parasite vector system [61]. The Th1 response has been described to lead to a protective immunity and the resistance of the host to intracellular pathogens, while the Th2 response might favor the survivor of pathogens (parasites, virus…) and then the disease transmission and evolution [24]. For mosquitoes, studies have globally shown an enhance‐ ment of transmission and disease when pathogens are introduced in the presence of vector saliva. Mosquito saliva is commonly associated with a downregulation of the expression of Th1 and an upregulation of the Th2-type cytokines. In mouse models, mosquito saliva can potentiate the infection of arboviruses [24, 62, 63]. The co-inoculation of Sindbis virus with *Aedes aegypti* salivary gland extract resulted on a reduced interferon- gamma (IFN-γ) expression, when compared to injection of virus alone [64]. It has been also shown that *Ae. aegypti* saliva contains multiple factors that can affect various components of the host immune response [65]. For example, factor Xa inhibitor may inhibit complement activation and leukocyte migration to the bite site [24] and other factors inhibit TNF-α release from activated mast cells [66]. Chickens subcutaneously infected with *P. gallinaceum* sporozoites in the presence of *Aedes fluviatillis* salivary gland homogenates showed a higher level of parasitae‐ mia when compared to those that received only sporozoites [67]. For *Anopheles*, mice exposed to mosquito feeding in tandem with the inoculation of sporozoites had higher parasitemia and an elevated progression to cerebral malaria. This was associated with, in particular, elevated levels of IL-4 and IL-10, suppression of overall transcription in response to infection, and decreased mobility of dendritic cells and monocytes [19]. It was also described that *Anopheles stephensi* saliva downregulates specific antibody (Ab) immune responses by a mechanism that is mast cell and IL-10 -dependent [60]. IL-10, by inhibiting pro-inflammato‐ ry and Th1 cytokines, stimulates certain T, mast and B cells and has pleiotropic effects in immunoregulation and inflammation, while IL-4 is the prototypical Th2 cytokine (it differentiates CD4+ T-cells and up-regulates MHC class II production). The enhancement of IL-10 expression could account for reduction in secretion of other cytokines because it inhibits antigen presentation, IFN–γ expression, and macrophage activation [68]. However, some data have suggested a paradoxical protective role of mosquito saliva against pathogen transmission and disease infection. *Ae. aegypti* saliva can inhibit infection of dendritic cells by dengue virus, and the pre-sensitization of dendritic cells with saliva prior to infection enhanced this inhibition. Moreover, the proportion of dead cells was also reduced in virusinfected dendritic cell cultures exposed to mosquito saliva, and an enhanced production of IL-12 and TNF-α was detected in these cultures [69]. In addition to these effects on cellular immunity, *Anopheles* saliva can also acts on humoral host immune response. Indeed, specific antibodies (immunoglobulins [Ig] G, M and E) to salivary antigens have been described in several studies [20, 22, 23, 25, 56, 70]. However, the implication of these Ab responses in disease pathogenesis or protection is not yet elucidated.

Therefore, future studies are needed for an overall understanding of mosquito saliva effect, especially *Anopheles* mosquito saliva, in pathogen transmission, disease development and pathogenesis.

#### *2.2.3. Human host-*Anopheles *vector immune relationship and applications*

The study of immunological properties of salivary proteins of *Anopheles* mosquitoes represents a new research thematic which can significantly improve the understanding of *Plasmodium* transmission mechanisms and therefore help for the effective prevention and control of malaria. It can notably lead to major applications in three areas: i) development of vaccines, diagnosis, treatment, ii) prevention of allergies, and iii) development of biomarkers of exposure to bites and malaria disease risk.

The development of parasite transmission-blocking vaccines, by stimulating the immune response against the vector is an attractive alternative way for malaria control. Several studies targeted the effect of Abs specific to the mosquito midgut antigens have shown promising results [71-73]. The study of the immune response induced by vector saliva at the biting site and its potential effect on the transmission and the development of pathogens suggests the possibility to control parasite transmission by vaccinating the host with immunogenic salivary compounds [54, 74]. In a mouse model, it has been shown that two salivary proteins (29 and 100 kDa) of the female *An. gambiae* can induce production of Ab which can block about 75% of the invasion of *An. stephensi* salivary glands by *P. yoelii* sporozoites [75]. In addition, the prior exposition to non infective *An. stephensi* bites induces a Th1 immune response with increased production of IL-12 and IFN-γ. Its effect can subsequently limit future *P. yoelii* infection (reduced rate of liver and blood parasites) and the development of cerebral malaria in mouse [18]. In this context, saliva can be thought as a non-specific "adjuvant" which could be effective at inducing a Th1-biased environment that is known to be protective against malaria infection. However, the development of such vaccines is complex. For example, Ab produced by immunization (with salivary proteins) must be ingested by the mosquito during a bite, cross it midgut and digestive enzymes, migrate to the salivary glands, before they can block the invasion by sporozoites. Nevertheless, the possibility to develop a pan-arthropod vaccine has been recently demonstrated by another mechanism. Indeed, an immune response directed to salivary proteins that adsorb to pathogens can turn the microorganism into an innocent bystander of anti-salivary immunity as it has been recently reported in a salivary protein (Salp15) from the hard tick *Ixodes scapularis* [76] and vaccine candidate for the control of Lyme disease [77]. Unfortunately, any hematophagous arthropod saliva-based vaccine has not yet been tested on humans.

In the field of allergic reactions to salivary proteins of mosquitoes, the first studies were mainly conducted in Canada and Finland. They concerned *Aedes* and *Culex* mosquitoes which express a panel of allergens in their saliva during the blood feeding time [17, 56, 78]. These proteins can thus be used in recombinant form, as diagnostic tool of the level of human exposure to allergens or in immunotherapy injections for desensitization of human [56, 70, 79]. It exists yet no study highlighting the presence and effect of allergens in the *Anopheles* mosquitoes' saliva.

The study of immunological relationship between human-vector by quantifying specific Ab responses to salivary proteins may also allow the identification and characterization of biological markers for epidemiological assessment of the exposure of individuals and popu‐ lations to the *Anopheles* bites and thus to the risk of malaria transmission [22]. The development of such biomarkers or indicators (see next chapter) can be a complementary alternative to current referent entomological and parasitological methods which present several limitations especially in low exposure/transmission contexts.

## **3. Development of biomarkers of human exposure to** *Anopheles* **bites and indicators of malaria vector control effectiveness**

### **3.1. Validation of concept with whole** *Anopheles* **saliva**

data have suggested a paradoxical protective role of mosquito saliva against pathogen transmission and disease infection. *Ae. aegypti* saliva can inhibit infection of dendritic cells by dengue virus, and the pre-sensitization of dendritic cells with saliva prior to infection enhanced this inhibition. Moreover, the proportion of dead cells was also reduced in virusinfected dendritic cell cultures exposed to mosquito saliva, and an enhanced production of IL-12 and TNF-α was detected in these cultures [69]. In addition to these effects on cellular immunity, *Anopheles* saliva can also acts on humoral host immune response. Indeed, specific antibodies (immunoglobulins [Ig] G, M and E) to salivary antigens have been described in several studies [20, 22, 23, 25, 56, 70]. However, the implication of these Ab responses in

Therefore, future studies are needed for an overall understanding of mosquito saliva effect, especially *Anopheles* mosquito saliva, in pathogen transmission, disease development and

The study of immunological properties of salivary proteins of *Anopheles* mosquitoes represents a new research thematic which can significantly improve the understanding of *Plasmodium* transmission mechanisms and therefore help for the effective prevention and control of malaria. It can notably lead to major applications in three areas: i) development of vaccines, diagnosis, treatment, ii) prevention of allergies, and iii) development of biomarkers of

The development of parasite transmission-blocking vaccines, by stimulating the immune response against the vector is an attractive alternative way for malaria control. Several studies targeted the effect of Abs specific to the mosquito midgut antigens have shown promising results [71-73]. The study of the immune response induced by vector saliva at the biting site and its potential effect on the transmission and the development of pathogens suggests the possibility to control parasite transmission by vaccinating the host with immunogenic salivary compounds [54, 74]. In a mouse model, it has been shown that two salivary proteins (29 and 100 kDa) of the female *An. gambiae* can induce production of Ab which can block about 75% of the invasion of *An. stephensi* salivary glands by *P. yoelii* sporozoites [75]. In addition, the prior exposition to non infective *An. stephensi* bites induces a Th1 immune response with increased production of IL-12 and IFN-γ. Its effect can subsequently limit future *P. yoelii* infection (reduced rate of liver and blood parasites) and the development of cerebral malaria in mouse [18]. In this context, saliva can be thought as a non-specific "adjuvant" which could be effective at inducing a Th1-biased environment that is known to be protective against malaria infection. However, the development of such vaccines is complex. For example, Ab produced by immunization (with salivary proteins) must be ingested by the mosquito during a bite, cross it midgut and digestive enzymes, migrate to the salivary glands, before they can block the invasion by sporozoites. Nevertheless, the possibility to develop a pan-arthropod vaccine has been recently demonstrated by another mechanism. Indeed, an immune response directed to salivary proteins that adsorb to pathogens can turn the microorganism into an innocent bystander of anti-salivary immunity as it has been recently reported in a salivary

disease pathogenesis or protection is not yet elucidated.

762 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

exposure to bites and malaria disease risk.

*2.2.3. Human host-*Anopheles *vector immune relationship and applications*

pathogenesis.

To improve the fight against malaria and regarding numerous limitations described with current entomological and parasitological tools, the World Health Organization (WHO) has emphasized the need of new indicators and methods to evaluate, at individual and population levels, the exposure level to *Anopheles* vectors and the effectiveness of vector control strategies. One promising concept is based on the fact that mosquito saliva injected to the human host during the vector bite is antigenic and can induce an adaptive humoral host response (see Figure 1). Therefore, a logical positive correlation between the human exposure level to *Anopheles* bites and human anti-mosquito saliva Ab level can be expected. In this way, antimosquito saliva Ab response can be a pertinent epidemiological biomarker of human exposure to vector bites.

The epidemiological importance of human exposure to the saliva of vectors has been firstly described in Lyme disease [80, 81], leishmaniasis [82] and Chagas disease [83]. During the last decade, studies have provided data on human exposure to anopheline saliva and its interaction with malaria transmission. In particular, Remoue *et al.* [22] have shown that children living in a seasonal malaria transmission region of Senegal developed IgG responses to *An. gambiae* whole saliva (WS). Interestingly, these specific IgG levels were positively associated with an increased rainfall and the *Anopheles* mosquito density, measured by referent entomological methods. Indeed, an increase in the level of IgG was observed according to the *Anopheles* aggressiveness and density in September (Figure 2), the peak of malaria transmission.

**Figure 2.** Anti-saliva IgG according to the intensity of exposure [22]. Individual absorbance (OD) values in September are shown for the three groups with different levels of exposure. Bars indicate the median value for each group. Statis‐ tical significances between each group by non-parametric Mann–Whitney *U*-test are indicated.

Importantly, IgG response to *An. gambiae* WS can predict clinical malaria cases. Indeed, children who developed a malaria attack in December had higher levels of anti-WS IgG in September of the same year, i.e. three months before they develop the disease (Figure 3) [22].

**Figure 3.** Anti-salivary IgG according to malaria morbidity. The results of individual absorbance (OD) values in Septem‐ ber are shown according to subsequent detection of clinical malaria for the age ≥1 year. Bars indicate the median val‐ ue for each group. Statistical significance between groups is indicated by a non-parametric Mann–Whitney *U*-test).

Anti-mosquito saliva Ab appeared transitional. Soldier travelers transiently exposed to *An. gambiae* bites in endemic areas of Africa (especially Ivory Coast and Gabon) developed specific IgG responses to anti-*An. gambiae* WS which strongly decreased several weeks after the end of their trip [21]. In addition, anti-*An. gambiae* saliva IgG levels waned rapidly after 6 weeks of Insecticide-Treated Nets (ITNs) well-use in a semi-urban population in Angola, before a new significant increase two months later following the stop of ITN use [84]. Data on human exposure to anopheline saliva and its interaction with malaria were also provided by studies from other none African areas. In South-eastern Asia, it has been described that anti-*An. dirus* salivary protein Ab occur predominantly in patients with acute *P. falciparum* or *P. vivax* malaria; people from non-endemic areas do not carry such Abs [23]. In the Americas, the presence of anti-*Anopheles* saliva Ab has been also described. In adult volunteers from Brazil, anti-*An. darlingi* WS Ab levels increased with *P. vivax* infections [20]. The presence of anti-*An. albimanus* WS Ab with exposure to mosquito bite has been recently described in Haiti [25]. Specific IgG response to *An. gambiae* WS has also been described as an immunological indicator evaluating the efficacy of malaria vector control strategies. Indeed, Drame *et al*. have recently shown in a semi-urban area (Lobito, Provence Benguela) in Angola that specific IgG levels drastically decreased after the introduction of ITNs and this was associated with a drop in parasite load (Figure 4) [84].

Importantly, IgG response to *An. gambiae* WS can predict clinical malaria cases. Indeed, children who developed a malaria attack in December had higher levels of anti-WS IgG in September of the same year, i.e. three months before they develop the disease (Figure 3) [22].

**Figure 2.** Anti-saliva IgG according to the intensity of exposure [22]. Individual absorbance (OD) values in September are shown for the three groups with different levels of exposure. Bars indicate the median value for each group. Statis‐

low moderate high

tical significances between each group by non-parametric Mann–Whitney *U*-test are indicated.

**Anti-saliva I gG**

**P< 0.01 P< 0.05**

**Exposure level (entomology)**

*n= 132 n= 89*

No Yes

**Figure 3.** Anti-salivary IgG according to malaria morbidity. The results of individual absorbance (OD) values in Septem‐ ber are shown according to subsequent detection of clinical malaria for the age ≥1 year. Bars indicate the median val‐ ue for each group. Statistical significance between groups is indicated by a non-parametric Mann–Whitney *U*-test).

Anti-mosquito saliva Ab appeared transitional. Soldier travelers transiently exposed to *An. gambiae* bites in endemic areas of Africa (especially Ivory Coast and Gabon) developed specific

**P= 0.046**

**Malaria morbidity Sept. to Dec.**

0.0

0.1

0.2

**ΔO.D.**

0.0

0.2

0.4

0.6

0.8

764 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

0.3

**Figure 4.** Evolution of anti-*Anopheles* gambiae saliva IgG and *Plasmodium falciparum* infections before and after ITN implementation, (Ano=*Anopheles*).

Anti-*Anopheles* saliva IgG response has also been recently used to evaluate and compare the effectiveness ofthree malaria vector control strategies in another area (Balombo) ofAngola [85]. Indeed,Brosseau*et al.*[85]have investigatedover aperiodoftwoyears (2008-2009)Ab response to *An. gambiae* WS in children between 2 to 9 years old, before and after the introduction of three different malaria vector control methods: deltamethrin treated long lasting impregnated nets (LLIN) and insecticide treated plastic sheeting (ITPS) - Zero Fly®) (ITPS-ZF), deltamethrin impregnatedDurable(Wall)Lining(ITPS-DL-Zerovector®)alone,andindoorresidualspraying

(IRS) with lambdacyhalothrin alone. They observed considerable decreases in entomological (82.4%), parasitological (54.8%) and immunological criteria analyzed. In particular, the immunologicaldatabasedonthe levelofanti-salivaIgGAbinchildrenofallvillages significant‐ ly dropped from 2008 to 2009, especially with LLIN+ZF and with IRS (Figure 5).

**Figure 5.** Comparison of median values of the IgG antibody response to *Anopheles* saliva obtained before and after implementation of each vector control method [85].

Taken together, these studies indicated that the estimation of human IgG Ab responses specific to *Anopheles* WS could provide a reliable biomarker for evaluating the *Anopheles* exposure level, the risk of malaria transmission, the disease outcomes and the effectiveness of vector control strategies. However, the pertinence and the practical large-scale application of serological tests for epidemiological purposes have been hampered by several limitations. First, WS is a cocktail of various molecular components with different nature and biological functions. Some components are *Anopheles*-specific and other widely distributed within genus, families, orders or classes of bloodsucking *Diptera* or Arthropods [16]. Therefore, the evaluation of *Anopheles* exposure or vector control effectiveness based on the immunogenicity of WS could be skewed and over or underestimated by possible cross-reactivities between common epitopes between mosquito species or other organisms [26]. Second, the collection of saliva or salivary gland extracts is tedious and time-consuming; therefore it will be difficult or impossible to have an adequate production of mosquito saliva needed for large-scale epidemiological studies [26]. Third, saliva composition can be affected by several ecological parameters such as age, feeding status or infectivity of *Anopheles* [86],which in turn may influence the anti-saliva immune response measured and may cause a lack of reproducibility between saliva batches. An alternative for optimizing the specificity of this immunological test would thus be to identify *Anopheles* genus-specific proteins [87].

#### **3.2. Methods for the identification of specific** *Anopheles* **salivary proteins**

(IRS) with lambdacyhalothrin alone. They observed considerable decreases in entomological (82.4%), parasitological (54.8%) and immunological criteria analyzed. In particular, the immunologicaldatabasedonthe levelofanti-salivaIgGAbinchildrenofallvillages significant‐

2008 2009 2008 2009 2008 2009

**P<0.0001 P=0.0001 P<0.0001**

LLIN + ZF DL IRS

**Figure 5.** Comparison of median values of the IgG antibody response to *Anopheles* saliva obtained before and after

Taken together, these studies indicated that the estimation of human IgG Ab responses specific to *Anopheles* WS could provide a reliable biomarker for evaluating the *Anopheles* exposure level, the risk of malaria transmission, the disease outcomes and the effectiveness of vector control strategies. However, the pertinence and the practical large-scale application of serological tests for epidemiological purposes have been hampered by several limitations. First, WS is a cocktail of various molecular components with different nature and biological functions. Some components are *Anopheles*-specific and other widely distributed within genus, families, orders or classes of bloodsucking *Diptera* or Arthropods [16]. Therefore, the evaluation of *Anopheles* exposure or vector control effectiveness based on the immunogenicity of WS could be skewed and over or underestimated by possible cross-reactivities between common epitopes between mosquito species or other organisms [26]. Second, the collection of saliva or salivary gland extracts is tedious and time-consuming; therefore it will be difficult or impossible to have an adequate production of mosquito saliva needed for large-scale epidemiological studies [26].

ly dropped from 2008 to 2009, especially with LLIN+ZF and with IRS (Figure 5).

0.0

implementation of each vector control method [85].

0.5

1.0

1.5

**Anti-saliva IgG responses**

**(O.D.)**

2.0

2.5

3.0

766 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

The isolation of salivary components has been a challenge for many years. Many functional active salivary proteins have been isolated following classical biochemical and molecular biology approaches [88]. Protocols mainly consisted of the isolation of salivary components from hundreds of salivary gland pairs, obtaining amino-terminal or internal peptide sequence of the purified component, screening of a salivary gland library with the information obtained, and isolation of the cDNA or gene of interest (Fig. 6).

**Figure 6.** Classical biochemical and molecular biology protocol used for isolation and characterisation of salivary pro‐ teins and cDNA from vectors of disease [90].

During the last decade, technical advances in molecular biology have allowed the sequencing of the genome, including transcripts of salivary glands [89], of most disease vectors, comprising *Anopheles* mosquitoes [90]. However, protocols do not allow to obtain entire sequences [89]. Nowadays, researchers have switched from testing one salivary molecule at a time to studying the whole complex of genes and secreted proteins in blood-feeding arthropods using tran‐ scriptomic and/or proteomic approaches. The transcriptomic is the complete set of transcripts in an organism for a specific developmental stage or physiological condition. Transcriptomic techniques help to interpret the functional elements of the genome, and to understand the transmission and development of diseases [91]. They aim to catalogue transcript of major *Anopheles* species, including mRNAs, non-coding RNAs and small RNAs; to determine the transcriptional structure of genes and to quantify the changing expression levels of each transcript during development and under different conditions [91]. Proteomic is a large-scale study of the gene expression at the protein level, which ultimately provides direct measure‐ ment of protein expression levels [92]. The proteomic revolution is hitting the vector biology field as well as many other fields. The isolation and sequencing of all the proteins from SGs of disease vectors and, more specifically, secreted salivary proteins, is clarifying the complexity of proteins present in the saliva of various blood-feeding arthropods [93]. During the last years, a comprehensive high-throughput approach has been developed (Figure 7) [88]. It combines massive sequencing protocol of high quality full-length salivary gland cDNA libraries, a proteomic approach to isolate a large set of salivary proteins, and high-throughput computa‐ tional biology and functional assays to analyze and test the biologic activities of these novel molecules. It is a powerful tool which can help easily and rapidly to identify and characterize genes or transcripts encoding for various proteins of SGs (the sialome) of blood-sucking arthropods. This high-throughput approach has then allowed an unprecedented insight into the complexity of salivary gland compounds of mosquito vectors of disease agents, indicating that the diversity of their targets is still larger than previously thought [16].

#### **3.3. Salivary proteins (sialome) of** *Anopheles* **mosquitoes**

The increasing power of large-scale genomic, transcriptomic and proteomic analyses allowed the accumulation of a considerable amount of information on the salivary secretions of bloodsucking arthropods [86]. As far as mosquitoes are concerned, the analysis of salivary tran‐ scriptomes of a number of *Anopheles* have allowed the discovery of a variety of genes that matched the sequence of various protein families, providing some clues on the evolution of blood feeding [15, 41-43, 92, 94-100]. Many of the salivary protein sequences are coded by genes related to intrinsic functions of the cell (housekeeping genes). However, the large number of salivary proteins is secreted during plant or blood feeding. Finally, a little number has no similarities to sequences deposited in databases, representing unknown and novel sequences [41, 94, 101]. This emphasizes how much still need to be learned concerning the biological functions of salivary proteins in blood feeding, pathogen transmission and manipulation of host responses.

The analysis of the adult *Anopheles* sialome has shown that secreted proteins and/or peptides (secretome) can be ubiquitous or specific to arthropod classes, orders, families, genus or species

**Figure 7.** Current high-throughput strategies used for the isolation and characterisation of salivary cDNA and proteins from disease vectors [90].

[44, 101, 102]. In *An. gambiae* salivary gland females over 70 putative secreted salivary proteins have been identified [94].

#### *3.3.1. Ubiquitous salivary proteins*

During the last decade, technical advances in molecular biology have allowed the sequencing of the genome, including transcripts of salivary glands [89], of most disease vectors, comprising *Anopheles* mosquitoes [90]. However, protocols do not allow to obtain entire sequences [89]. Nowadays, researchers have switched from testing one salivary molecule at a time to studying the whole complex of genes and secreted proteins in blood-feeding arthropods using tran‐ scriptomic and/or proteomic approaches. The transcriptomic is the complete set of transcripts in an organism for a specific developmental stage or physiological condition. Transcriptomic techniques help to interpret the functional elements of the genome, and to understand the transmission and development of diseases [91]. They aim to catalogue transcript of major *Anopheles* species, including mRNAs, non-coding RNAs and small RNAs; to determine the transcriptional structure of genes and to quantify the changing expression levels of each transcript during development and under different conditions [91]. Proteomic is a large-scale study of the gene expression at the protein level, which ultimately provides direct measure‐ ment of protein expression levels [92]. The proteomic revolution is hitting the vector biology field as well as many other fields. The isolation and sequencing of all the proteins from SGs of disease vectors and, more specifically, secreted salivary proteins, is clarifying the complexity of proteins present in the saliva of various blood-feeding arthropods [93]. During the last years, a comprehensive high-throughput approach has been developed (Figure 7) [88]. It combines massive sequencing protocol of high quality full-length salivary gland cDNA libraries, a proteomic approach to isolate a large set of salivary proteins, and high-throughput computa‐ tional biology and functional assays to analyze and test the biologic activities of these novel molecules. It is a powerful tool which can help easily and rapidly to identify and characterize genes or transcripts encoding for various proteins of SGs (the sialome) of blood-sucking arthropods. This high-throughput approach has then allowed an unprecedented insight into the complexity of salivary gland compounds of mosquito vectors of disease agents, indicating

that the diversity of their targets is still larger than previously thought [16].

The increasing power of large-scale genomic, transcriptomic and proteomic analyses allowed the accumulation of a considerable amount of information on the salivary secretions of bloodsucking arthropods [86]. As far as mosquitoes are concerned, the analysis of salivary tran‐ scriptomes of a number of *Anopheles* have allowed the discovery of a variety of genes that matched the sequence of various protein families, providing some clues on the evolution of blood feeding [15, 41-43, 92, 94-100]. Many of the salivary protein sequences are coded by genes related to intrinsic functions of the cell (housekeeping genes). However, the large number of salivary proteins is secreted during plant or blood feeding. Finally, a little number has no similarities to sequences deposited in databases, representing unknown and novel sequences [41, 94, 101]. This emphasizes how much still need to be learned concerning the biological functions of salivary proteins in blood feeding, pathogen transmission and manipulation of

The analysis of the adult *Anopheles* sialome has shown that secreted proteins and/or peptides (secretome) can be ubiquitous or specific to arthropod classes, orders, families, genus or species

**3.3. Salivary proteins (sialome) of** *Anopheles* **mosquitoes**

768 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

host responses.

AG5 family proteins are found in the salivary glands of many blood-sucking insects and ticks [102, 103]. In *An. gambiae*, four proteins belonging to this family were identified, but only one (putative gVAG protein precursor) was coding for transcripts enriched in the adult female SGs [94]. A precursor of gVAG protein was also described in *An. funestus* (84% sequence identity) and *An. stephensi* (85% sequence identity) sialome [95, 100]. The function of any AG5 protein in the saliva of any blood-sucking arthropod is still unknown.

Enzymes such as maltase, apyrase, 5′ nucleotidase, and adenosine deaminase, are also secreted during the bite of many blood-sucking arthropods, including *Anopheles* mosquitoes [95]. They generally assist in sugar feeding (maltase) or in degradation of purinergic mediators of platelet aggregation (apyrase, 5′ nucleotidases) and inflammation (adenosine deaminase).

#### *3.3.2. Salivary proteins found exclusively in Diptera*

*D7 family proteins* are specific to SGs of blood-sucking Nematocera, including mosquitoes and sand flies [104, 105]. They are highly represented in the sialome of *Anopheles* mosquitoes in short and long forms [95, 96, 101, 104, 105]. *An. funestus* D7 proteins vary between 64% and 75% identity with their *An. gambiae* closest match [105]. D7 proteins could act as anti-hemostatic factors by trapping agonists of hemostasis [44, 47]. However, further investigations are needed to clearly describe their function.

Other Diptera-specific protein families or peptides have also been described in the sialome of blood-feeding mosquitoes [95]. However their function is still unknown, even if some were known to play a role in antimicrobial property of mosquito saliva.

#### *3.3.3. Protein families found exclusively in mosquitoes*

The 30-kDa antigen family found exclusively in the SGs of adult female mosquitoes has been found in both culicine and anopheline mosquitoes [95, 100, 101, 106-108]. Only one gene enriched in SGs of adult females is known in *An. gambiae*. The *An. funestus* homologue is also abundantly expressed and shares 63% identity with the *An. gambiae* orthologue. The function of this protein family is still unknown [95].

The *gSG (An. gambiae Salivary Gland)-5 family* was first discovered in the SGs of *An. gambiae* and shown to be exclusively expressed in the adult female [94, 109]. This protein shows a high similarity to *Aedes* and *Culex* proteins [101]. Transcripts coding for this family were found in the sialotranscriptome of *An. darlingi* with 46% identical to the *An. gambiae* orthologue and only 26% and 23% identical to the culicine proteins [101]. The function of this mosquito-specific protein remains unknown, but its tissue- and sex-specific expression profile suggests it is possibly related to blood feeding.

The *gSG8 family* is highly divergent with members only found in *An. gambiae* and *Ae. aegypti*. In *An. gambiae*, this protein is specifically expressed in female SGs [109], suggesting a likely role in blood feeding.

Various types of *mucins* have been described in the saliva of adult mosquitoes and may function/act as a lubricant of their mouthparts [15, 41, 94, 102]. Three mucins encoding transcripts have been identified in the *An. gambiae* larval SG [110], suggesting the importance of mucins at multiple developmental stages. Mucins may also play a crucial role in *Anophe‐ les* salivary gland invasion by *P. berghei* sporozoites [111]. Several protein families are also represented in this group, including gSG-3, gSG-10, and 13.5-kDa families [101]. These families were also found abundantly expressed in the sialotranscriptome of *An. gambiae* adult male [112], indicating their function is not related specifically to blood feeding.

#### *3.3.4. Protein families found exclusively in Anophelines*

*Anophelin* was described as a short acidic peptide with strong thrombin inhibitory activity in *An. albimanus* [46]. *An. funestus* anophelin is 59% identical to the *An. gambiae* orthologue [95], and *An. darlingi* anophelin is 86% identical to *An. albimanus* [101].

The 8.2-kDa family is represented in several *Anopheles* species. In *An. funestus*the peptide have 42% identity with the 8.2-kDa salivary peptide of *An. stephensi* and similar proteins from *An. gambiae* and *An. darlingi* [95]. In *An. gambiae*, this peptide was found enriched in adult female SGs, suggesting a role in blood feeding.

The 6.2-kDa family was first described in a sialotranscriptome of *An. gambiae*[94], where it was found enriched in adult female SGs compared to other tissues. The *An. funestus* member of this family is 61% identical to the *An. gambiae* [95], and 53% to an *An. darlingi* [101] homologues.

75% identity with their *An. gambiae* closest match [105]. D7 proteins could act as anti-hemostatic factors by trapping agonists of hemostasis [44, 47]. However, further investigations are needed

Other Diptera-specific protein families or peptides have also been described in the sialome of blood-feeding mosquitoes [95]. However their function is still unknown, even if some were

The 30-kDa antigen family found exclusively in the SGs of adult female mosquitoes has been found in both culicine and anopheline mosquitoes [95, 100, 101, 106-108]. Only one gene enriched in SGs of adult females is known in *An. gambiae*. The *An. funestus* homologue is also abundantly expressed and shares 63% identity with the *An. gambiae* orthologue. The function

The *gSG (An. gambiae Salivary Gland)-5 family* was first discovered in the SGs of *An. gambiae* and shown to be exclusively expressed in the adult female [94, 109]. This protein shows a high similarity to *Aedes* and *Culex* proteins [101]. Transcripts coding for this family were found in the sialotranscriptome of *An. darlingi* with 46% identical to the *An. gambiae* orthologue and only 26% and 23% identical to the culicine proteins [101]. The function of this mosquito-specific protein remains unknown, but its tissue- and sex-specific expression profile suggests it is

The *gSG8 family* is highly divergent with members only found in *An. gambiae* and *Ae. aegypti*. In *An. gambiae*, this protein is specifically expressed in female SGs [109], suggesting a likely

Various types of *mucins* have been described in the saliva of adult mosquitoes and may function/act as a lubricant of their mouthparts [15, 41, 94, 102]. Three mucins encoding transcripts have been identified in the *An. gambiae* larval SG [110], suggesting the importance of mucins at multiple developmental stages. Mucins may also play a crucial role in *Anophe‐ les* salivary gland invasion by *P. berghei* sporozoites [111]. Several protein families are also represented in this group, including gSG-3, gSG-10, and 13.5-kDa families [101]. These families were also found abundantly expressed in the sialotranscriptome of *An. gambiae* adult male

*Anophelin* was described as a short acidic peptide with strong thrombin inhibitory activity in *An. albimanus* [46]. *An. funestus* anophelin is 59% identical to the *An. gambiae* orthologue [95],

The 8.2-kDa family is represented in several *Anopheles* species. In *An. funestus*the peptide have 42% identity with the 8.2-kDa salivary peptide of *An. stephensi* and similar proteins from *An. gambiae* and *An. darlingi* [95]. In *An. gambiae*, this peptide was found enriched in adult female

[112], indicating their function is not related specifically to blood feeding.

and *An. darlingi* anophelin is 86% identical to *An. albimanus* [101].

*3.3.4. Protein families found exclusively in Anophelines*

SGs, suggesting a role in blood feeding.

known to play a role in antimicrobial property of mosquito saliva.

*3.3.3. Protein families found exclusively in mosquitoes*

of this protein family is still unknown [95].

possibly related to blood feeding.

role in blood feeding.

to clearly describe their function.

770 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

The SG-1 family proteins appear to be exclusively expressed in the female SGs of *Anopheles* mosquitoes and not observed in other tissues [94, 101]. However, their function remains to be determined.

The SG-2 family proteins were identified from *An. gambiae* saliva and shown to be expressed in female SGs and adult males but not in other tissues [113]. Related, but very divergent, sequences were obtained from salivary transcriptomes of other anopheline species [95, 101]. Because this protein family is expressed in both male and female *An. gambiae*, and due to its relatively small size, it may display antimicrobial function [101].

The *hyp 8.2* and *hyp 6.2 proteins* are similarly enriched in *An. gambiae* adult female SGs [94]. *An. stephensi* and *An. funestus* also have members of these protein families.

The *SG-7/Anophensin family* is also unique to anophelines. In *An. gambiae*, it is highly enriched in female SGs [94]. More recently, the *An. stephensi* homologue was determined to inhibit kallikrein and production of bradykinin, a pain-producing substance [114]. Four putative alleles representing the homologue(s) of gSG7 in *An. darlingi* were identified. These *An. darlingi* transcripts have no more than 45% identity to the *An. gambiae* gSG7 and *An. stephensi* anophensin [101].

The *SG6 protein* is a small protein first described in *An. gambiae* [109] and a unique sequence codes for a mature peptide/protein of ~10 kDa (116 amino-acids) with ten cysteine residues making probably five disulphide bonds. A homologue was later found in the sialotranscrip‐ tome of *An. stephensi* [100] and *An. funestus* [95]. *An. funestus* SG6/fSG6 (f for funestus) has 81% and 76% identities with *An. stephensi* and *An. gambiae* polypeptides, respectively. It is not found in the transcriptomes of the Culicinae subfamily members analyzed so far, i.e. *C. pipiens quinquefasciatus*, *Ae. aegypti* and *Ae. albopictus* [108, 115, 116]. In *An. gambiae*, the transcript coding for gSG6 (g for *gambiae*) was found to be 16 times more expressed in SGs of adult females than in males [94]. The gSG6 protein plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to *Cellia* and *Anopheles* subgenera [99]. The gSG6 protein, because immunogenic, can be therefore a reliable indicator of human exposure specific to *Anopheles* mosquito bites [99], vectors of malaria.

#### **3.4. Specific salivary biomarker of exposure to** *Anopheles* **bites: The gSG6-P1 peptide candidate**

The SG6 salivary protein has been reported to be immunogenic in travelers exposed for short periods to *Anopheles* bites [21], and in Senegalese children living in a malaria endemic area by animmuno-proteomic, coupling2Dimmunoblotandmass spectrometry[117],andbyanELISA [26]approaches.Recently,its immunogenicityhasbeenconfirmedinindividuals fromamalaria hyperendemicareaofBurkinaFaso[118,119],byusingarecombinantformexpressedaspurified N-terminal His-tagged recombinant protein in the *E. coli* vector pET28b(+) (Novagen) [99, 119].

**Figure 8.** Sequences of the anopheline gSG6 proteins [99]. (A) Clustal alignment of anopheline gSG6 proteins. Signal peptides and conserved Cysteines are boxed. Conserved sites are shaded. (B) Phylogenetic tree (NJ algorithm, boot‐ strapped 10,000 times) constructed from the alignment of the nucleotide sequence encoding the mature gSG6 poly‐ peptides.

In particular, increased anti-gSG6 IgG levels were observed in exposed individuals during the malaria transmission/rainy season [119]. In addition, anti-gSG6 IgG response appeared to be a reliableserologicalindicatorofexposuretobitesofthemainAfricanmalariavectors(*An.gambiae*, *An. arabiensis* and *An. funestus*) in the same area [119]. However, gSG6 recombinant protein has been described to relatively generate a high background in control sera from individuals not exposedto*Anopheles*bites,andconsiderablevariations inspecificAbresponsebetweenchildren supposed to be similarly exposed to *Anopheles* bites [26]. Therefore, with the objective of optimizing *Anopheles* specificity and reproducibility of the immunological assay, a peptide design approach was undertaken using bioinformatic tools [26].

#### *3.4.1. Identification and sequence of gSG6-P1 peptide*

Several algorithms were employed for prediction of potential immunogenic sites of *the* gSG6 protein by using bioinformatics. The prediction of immunogenicity was based on the deter‐ mination of physico-chemical properties of the amino-acid (AA) sequences with BcePred and FIMM databases and on the identification of MHC class 2 binding regions using the ProPred-2 online service. This led to define five gSG6 peptides (gSG6-P1 to gSG6-P5) of 20 to 27 AA residues in length (Fig. 9), overlapping by at least 3 residues and spanning the entire sequence of the mature gSG6 protein. Both predictive methods for putative linear B-cell epitopes (FIMM and BcePred) assigned the highest immunogenicity to gSG6-P1, gSG6-P2, gSG6-P3, and then gSG6-P4.

**Figure 9.** Amino-acid sequence of gSG6 Peptides. Amino-acid sequence of the SG6 protein of *Anopheles gambiae* (gi: 13537666) is presented and sequences of the selected peptides, gSG6-P1 to gSG6-P5, are underlined. Signal peptide (SP) sequence is indicating by dotted underline [26].

Similarities were also searched using the Blast family programs, including both the genome/EST libraries of other vector arthropods available in Vectorbase and of pathogens/ organisms in non-redundant GenBank CDS databases. No relevant identity was found with proteins of other blood-sucking arthropods. Indeed, the longest perfect match was 6 AAs between a putative protein from *Pediculus humanus* and gSG6-P2 and gSG6-P3 peptides. In the case of gSG6-P1, the best match was 4 AAs in length with *Culex pipiens quinquefasciatus* salivary adenosine deaminase. Moreover, no relevant similarity was found with sequences from pathogens or other organisms. The highest hits of gSG6-P1 were with the cyanobacterium *Microcystis aeruginosa* (3 AAs) and with *Ostreococcus* OsV5 virus (4 AAs). Altogether, this analysis confirmed the *bona fide* high specificity of the five selected gSG6 peptides for the *Anopheles* species. Peptides were then synthesized.

### *3.4.2. Antigenicity of gSG6 peptides*

In particular, increased anti-gSG6 IgG levels were observed in exposed individuals during the malaria transmission/rainy season [119]. In addition, anti-gSG6 IgG response appeared to be a reliableserologicalindicatorofexposuretobitesofthemainAfricanmalariavectors(*An.gambiae*, *An. arabiensis* and *An. funestus*) in the same area [119]. However, gSG6 recombinant protein has been described to relatively generate a high background in control sera from individuals not exposedto*Anopheles*bites,andconsiderablevariations inspecificAbresponsebetweenchildren supposed to be similarly exposed to *Anopheles* bites [26]. Therefore, with the objective of optimizing *Anopheles* specificity and reproducibility of the immunological assay, a peptide

**Figure 8.** Sequences of the anopheline gSG6 proteins [99]. (A) Clustal alignment of anopheline gSG6 proteins. Signal peptides and conserved Cysteines are boxed. Conserved sites are shaded. (B) Phylogenetic tree (NJ algorithm, boot‐ strapped 10,000 times) constructed from the alignment of the nucleotide sequence encoding the mature gSG6 poly‐

Several algorithms were employed for prediction of potential immunogenic sites of *the* gSG6 protein by using bioinformatics. The prediction of immunogenicity was based on the deter‐ mination of physico-chemical properties of the amino-acid (AA) sequences with BcePred and FIMM databases and on the identification of MHC class 2 binding regions using the ProPred-2 online service. This led to define five gSG6 peptides (gSG6-P1 to gSG6-P5) of 20 to 27 AA

design approach was undertaken using bioinformatic tools [26].

*3.4.1. Identification and sequence of gSG6-P1 peptide*

772 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

peptides.

IgG Ab responses to the five gSG6 peptides were evaluated by ELISA in a randomly selected subsample of children (n<30) living in a rural area of Senegal. All peptides were immunogenic, but the intensity of the IgG level was clearly peptide-dependent; weak immunogenicity was observed for gSG6-P3, gSG6-P4 and gSG6-P5, whereas gSG6-P1 and gSG6-P2 appeared highly immunogenic (Fig. 10).

**Figure 10.** IgG antibody response according to gSG6 peptides [26]. For each peptide, the IgG Ab level was evaluated in a subsample of exposed children. Results at the peak of the season of *Anopheles* exposure are reported according to gSG6 peptides. Results are presented by box plot graph where lines of the boxes represent the 75th percentile, median and 25th percentile of individual average ΔOD values; whiskers represent the lower and upper adjacent values.

#### *3.4.3. Validation as a biomarker of exposure in several epidemiological settings*

The specific IgG level to the two most antigenic gSG6 peptides (gSG6-P1 et gSG6-P2) was then evaluated according to the level of exposure (estimated by entomological data) in a larger sample (n=241) of children living in a malaria seasonal area [26]. A positive trend was found for both peptides, but only significant for gSG6-P1 (Figure 11). Altogether, these results indicated that only the IgG response to gSG6-P1 is suitable to be a pertinent biomarker of exposure to *Anopheles* bites and thus to risk of malaria.

**Figure 11.** IgG response to gSG6-P1 and gSG6-P2 according to intensity of exposure to *Anopheles* gambiae bites [26]. Individual ΔOD (Optical Density) values in September (peak of the season of *Anopheles* exposure) are shown for the three different exposure groups. Results are presented for the same children (n=241) for gSG6-P1 (A) and gSG6-P2 (B). Exposure groups were defined by entomological data. Bars indicate median value for each exposure group. Statistical significance between the 3 groups is indicated (non-parametric Mann-Whitney U-test).

Therefore, the gSG6-P1 was selected as the most pertinent candidate as marker of exposure. Indeed, this peptide appeared to satisfy several requirements that an exposure biomarker should fulfill. First, it thus far appears to be specific to *Anopheles* genus and therefore, no rel‐ evant cross-reactivity phenomena with epitopes from other proteins of arthropods or patho‐ gens would be expected. Second, because it is of a synthetic nature, it guarantees high reproducibility of the immunological assay. Third, it elicits a specific Ab response which correlates well with the level of exposure to *An. gambiae* bites.

#### *3.4.3.1. Biomarker of* Anopheles *vector bites*

**IgG response to gSG6 peptides**

**Figure 10.** IgG antibody response according to gSG6 peptides [26]. For each peptide, the IgG Ab level was evaluated in a subsample of exposed children. Results at the peak of the season of *Anopheles* exposure are reported according to gSG6 peptides. Results are presented by box plot graph where lines of the boxes represent the 75th percentile, median and 25th

The specific IgG level to the two most antigenic gSG6 peptides (gSG6-P1 et gSG6-P2) was then evaluated according to the level of exposure (estimated by entomological data) in a larger sample (n=241) of children living in a malaria seasonal area [26]. A positive trend was found for both peptides, but only significant for gSG6-P1 (Figure 11). Altogether, these results indicated that only the IgG response to gSG6-P1 is suitable to be a pertinent biomarker of

**gSG6-P2**

**Medium**

**High 62.2** 

percentile of individual average ΔOD values; whiskers represent the lower and upper adjacent values.

**High 62.2**

significance between the 3 groups is indicated (non-parametric Mann-Whitney U-test).

**0.0**

mean/trap/village (In September)

**Figure 11.** IgG response to gSG6-P1 and gSG6-P2 according to intensity of exposure to *Anopheles* gambiae bites [26]. Individual ΔOD (Optical Density) values in September (peak of the season of *Anopheles* exposure) are shown for the three different exposure groups. Results are presented for the same children (n=241) for gSG6-P1 (A) and gSG6-P2 (B). Exposure groups were defined by entomological data. Bars indicate median value for each exposure group. Statistical

**0.5**

**1.0**

**DO**

**1.5**

P<0.05

**Low 1.75**

**33.5** *An. gambiae*

*3.4.3. Validation as a biomarker of exposure in several epidemiological settings*

exposure to *Anopheles* bites and thus to risk of malaria.

774 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**gSG6-P1**

**Medium 33.5** 

**DO**

**0.0**

**0.5**

**1.0**

**1.5**

P<0.0001

**Low 1.75** As previously suggested, anti-gSG6-P1 IgG response was described as a biomarker of *An. gambiae* bites in children living in Senegalese villages where malaria transmission seasonally and moderately occurred [26]. In the same area, a specific IgG response to the peptide has been detected in 36% of children living in villages where very few *An. gambiae,* or none, were collected by classical entomological methods [28]. This deals with a high sensitivity and specificity of the gSG6-P1 epitope(s) after a low immunological boost induced by weak bites exposure. This result points to the potential use of such serological tool as an epidemiological biomarker of *An. gambiae* bites in very low exposure areas, where the sensitivity of current entomological methods of malaria risk assessment is weak.

One study aimed to evaluate the risk of malaria transmission in children and adults living in urban area of Senegal (Dakar region) by using the gSG6-P1 peptide biomarker. Results showed considerable individual variations in anti-gSG6-P1 IgG levels between and within districts, in spite of a context of a global low *Anopheles* exposure level and malaria transmission [27]. Despite this individual heterogeneity, the median level of specific IgG and the percentage of immune responders differed significantly between districts. In addition, a positive association was observed between the exposure levels to *An. gambiae* bites, estimated by classical ento‐ mological methods, and the median IgG levels or the percentage of immune responders reflecting the real contact between human populations and *Anopheles* mosquitoes [27]. Differences in exposure levels to *An. gambiae* bites could then partly explain district and/or group-variations in anti-gSG6-P1 IgG Ab response as previously described in a low-exposure rural area of Senegal [28]. Interestingly, in urban Dakar area, immunological parameters seemed to better discriminate the *Anopheles* exposure level between different groups compared to referent entomological data. Moreover, in this study, some discrepancies were observed in the correlation between immunological parameters and the exposure level to *An. gambiae* bites assessed by entomological data in districts. This suggests the main role of the human behavior influencing the contact with vectors. A differential use of Vector Control Measures (ITNs, sprays, curtains) can for example drastically reduce human-vector contact. Many household characteristics (height, type, use of air conditioning, well-closed windows), which can differ between districts, could also be crucial factors. Importantly, the effect of these factors may be not taken into account by assessing the mosquito exposure level and malaria risk with classical entomological tools. This strengthens the usefulness of such biomarker as an alternative tool in the evaluation of exposure levels to *Anopheles* bites, especially in low/very low exposure, where current entomological methods can give inaccurate estimations of the human-mosquito contact [27].

In a population from a malaria hyperendemic area of Burkina Faso, the use of gSG6 recombi‐ nant protein as reliable indicator of exposure to the 3 main African malaria vectors (*An. gambiae s.s.*, *An. arabiensis* and *An. funestus*) has been suggested [119]. This probably could be relied to a wide cross-reactivity between SG6 sequences of principal *Anopheles* vectors, which highly share identical epitopes between species. Moreover, the gSG6-P1 peptide has been used to accurately evaluate the exposure level to *An. funestus* bites in a rural area in Senegal [29]. Indeed, two-thirds of 2-9 years old children from this area developed an IgG response to gSG6- P1, in an area where *An. funestus* only was reported. In addition, IgG response increased during the *An. funestus* exposure season, and a positive association was observed with the level of exposure to *An. funestus* bites [29]. This result deals with the cross-reactivity between *An. gambiae* gSG6-P1 and *An. funestus* fSG6-P1 sequences which share a high level of identity. Indeed, these sequences differ only by the substitution of two AAs: asparagine by glutamine (position 9) and leucine by isoleucine (position 15) (Fig. 12).


**Figure 12.** Sequences of the SG6-P1 salivary peptide [29]. Sequences are shown for *An. funestus* (fSG6-P1), for *An. gambiae* (gSG6-P1). Identities are marked with '\*' and strong AA conservations with ':'.

AAs from fSG6-P1 are close in terms of polarity and charge to those from *An. gambiae* gSG6- P1. The main consequence is that individuals exposed to *An. funestus* bites can sufficiently develop a specific Ab response against gSG6-P1 *An. gambiae* antigen. This observation, in conjunction with present results, suggests that these substitutions do not alter the synthesis and the recognition of specific Ab because epitope appears to be conserved.

All mentioned studies were conducted on subjects older than 1 year. However, to be more relevant in epidemiological surveys and studies on malaria, such biomarker tool must pertinently be applicable to all human age-classes, including newborns and young infants (<1 year old) who can be also bitten by *Anopheles* and at high risk of malaria transmission [120]. In this way, a recent study has indicated that human Ab responses to gSG6-P1 biomarker help to assess *Anopheles* exposure level and the risk of malaria in younger than 1 year old infants living in moderate to high transmission area of Benin (Drame *et al.*, submitted).

Indeed, the presence of anti-gSG6-P1 IgG and IgM in the blood of respectively 93.28 and 41.79% of 3-months old infants (the majority of infants) and their gradual increasing levels until 12 months (Fig. 13), whatever the *Anopheles* exposure level or the season. These observations are consistent with the development and maturation patterns of the newborn immune system during the first months of life. Indeed, the immature human immune system completes its maturation during infancy following exposition to antigens. Therefore, newborns are naive and increasingly susceptible to infectious agents; their immune system is not or insufficiently

**Figure 13.** IgG and IgM responses to *Anopheles* gSG6-P1 salivary peptide in the first year-life. Individual IgG (A) and IgM (B) responses to the *Anopheles* gSG6-P1 are represented for infants in months 3 (white), 6 (light-gray), 9 (darkgray) and 12 (black box) after their birth. Horizontal lines in the boxes indicate medians of the individual data. Hori‐ zontal black dotted lines represent the cut-off of IgG (0.204) and IgM (0.288) responder. Statistical significant differences between all age groups (multivariate linear mixed model analysis) are indicated.

stimulated by antigens. In endemic malaria transmission area, they are progressively exposed to salivary antigens of *Anopheles* [121], probably explaining the progressive increase of antigSG6-P1 IgG and IgM from 3 to 12 months-old. Individual or population factors and behaviors enhancing the level of the human-*Anopheles* contact with age can play a crucial role on accelerating this gradual acquisition [122, 123].

#### *3.4.3.2. Factors of variation of antibody response to gSG6-P1 and their consequences*

Specific gSG6-P1 Ab responses can be influenced by several determinant factors in their variations between individuals, districts, villages, regions... Therefore, identifying effects of human intrinsic (gender, age…) and extrinsic (period of sampling, use of vector control measure…) factors will be useful to the application of the gSG6-P1 biomarker in epidemio‐ logical studies or monitoring, evaluation and surveillance of risk of malaria programmes.

#### **Effect of age**

In a population from a malaria hyperendemic area of Burkina Faso, the use of gSG6 recombi‐ nant protein as reliable indicator of exposure to the 3 main African malaria vectors (*An. gambiae s.s.*, *An. arabiensis* and *An. funestus*) has been suggested [119]. This probably could be relied to a wide cross-reactivity between SG6 sequences of principal *Anopheles* vectors, which highly share identical epitopes between species. Moreover, the gSG6-P1 peptide has been used to accurately evaluate the exposure level to *An. funestus* bites in a rural area in Senegal [29]. Indeed, two-thirds of 2-9 years old children from this area developed an IgG response to gSG6- P1, in an area where *An. funestus* only was reported. In addition, IgG response increased during the *An. funestus* exposure season, and a positive association was observed with the level of exposure to *An. funestus* bites [29]. This result deals with the cross-reactivity between *An. gambiae* gSG6-P1 and *An. funestus* fSG6-P1 sequences which share a high level of identity. Indeed, these sequences differ only by the substitution of two AAs: asparagine by glutamine

**Figure 12.** Sequences of the SG6-P1 salivary peptide [29]. Sequences are shown for *An. funestus* (fSG6-P1), for *An.*

AAs from fSG6-P1 are close in terms of polarity and charge to those from *An. gambiae* gSG6- P1. The main consequence is that individuals exposed to *An. funestus* bites can sufficiently develop a specific Ab response against gSG6-P1 *An. gambiae* antigen. This observation, in conjunction with present results, suggests that these substitutions do not alter the synthesis

All mentioned studies were conducted on subjects older than 1 year. However, to be more relevant in epidemiological surveys and studies on malaria, such biomarker tool must pertinently be applicable to all human age-classes, including newborns and young infants (<1 year old) who can be also bitten by *Anopheles* and at high risk of malaria transmission [120]. In this way, a recent study has indicated that human Ab responses to gSG6-P1 biomarker help to assess *Anopheles* exposure level and the risk of malaria in younger than 1 year old infants

Indeed, the presence of anti-gSG6-P1 IgG and IgM in the blood of respectively 93.28 and 41.79% of 3-months old infants (the majority of infants) and their gradual increasing levels until 12 months (Fig. 13), whatever the *Anopheles* exposure level or the season. These observations are consistent with the development and maturation patterns of the newborn immune system during the first months of life. Indeed, the immature human immune system completes its maturation during infancy following exposition to antigens. Therefore, newborns are naive and increasingly susceptible to infectious agents; their immune system is not or insufficiently

(position 9) and leucine by isoleucine (position 15) (Fig. 12).

776 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*gambiae* (gSG6-P1). Identities are marked with '\*' and strong AA conservations with ':'.

and the recognition of specific Ab because epitope appears to be conserved.

living in moderate to high transmission area of Benin (Drame *et al.*, submitted).

Studies have globally reported an increasing anti-gSG6-P1 Ab level according to individual age. In a moderate transmission semi-urban area in Angola, the lowest and highest specific IgG levels have been described in young children (0-7 years old) and in teenagers/ adults (>14 years old) respectively [30]. In a low malaria transmission urban area (Dakar region) in Senegal, specific IgG levels were significantly higher in adults (>18 years old) compared to 6-10 years old children and in this latter group compared to those aged from 2 to 5 years [27] [124]. In Tori Bossito, moderate-high rural transmission area of Benin, both anti-gSG6-P1 IgG and IgM levels were low at 3 months of age and gradually increased until 12 months after birth (Drame *et al.*, *submitted*). The increase of specific IgG response with age is consistent with the gradual acquired immunity against *Anopheles* mosquito saliva [30] following the development of individual factors and behaviors enhancing the probability of human-vector contact [122, 123]. However, few data have reported a decrease of IgG levels to gSG6-P1 peptide [28] or to SG6 protein [118] with age. In particular, in Senegalese children (0 to 60 months old), the highest specific IgG levels were reported in the youngest children in spite of a probable very weak exposure to *An. gambiae* [30]. It can be explained by a passive IgG transfer from mother to child during pregnancy or breastfeeding as recently reported in young infants from Benin (Drame *et al.*, *submitted*). This represents a way of overestimation of the assessment of human-*Anopheles* contact level and the risk of malaria in young infants by using anti-gSG6-P1 IgG Ab. Therefore, the evaluation of specific IgM Ab levels could be a relevant solution to bias in IgG measurements. Indeed, IgM Ab, in a form of polymers (usually pentamers) in the human organism, could not cross the maternal-foetal barrier [125] and are the first Ab to appear in response to initial or primary exposure to antigen [126]. Interestingly, in Tori Bossito, specific IgM levels seemed to be a serological marker only during the first 6-months of exposure. In infants older to 6 months, the assessment of gSG6-P1-specific IgG showed a more pertinent evaluation of exposure level.

#### **Effect of sex**

Some studies have reported higher levels of anti-gSG6-P1 in female individuals (children and women) compared to males (children and men) [27, 30] ([124]; Drame *et al.*, *submitted*). However, this difference was not significant, suggesting that it might be only physiological.

#### **The season of** *Anopheles* **exposure**

TheseasonofindividualsamplingmaybealsoafactorofconfusionintheusegSG6-P1biomarker in epidemiological studies on malaria risk assessment or control. Indeed, significant seasonal‐ ly variations in anti-gSG6-P1 IgG or/and IgM levels have been reported in studies conducted in newborns, children or/and adults from endemic malaria areas in Senegal [27-29, 124], Angola [30] and Benin (Drame *et al.*, submitted). In Senegal, in particular, specific gSG6-P1 in urban children and adults steadily waned from the beginning (October) to the end (December) of the study, due to an important drop in human exposure level to *An. gambiae s. l.* bites from the end of rainfalls (October) to the beginning of the dry season (December) [127, 128].

One direct application of a salivary biomarker of exposure could serve in the elaboration of maps representing the risk of exposure to *Anopheles* bites. Such immuno-epidemiological marker might represent a quantitative tool applied to field conditions and a complementary tool to those currently available, such as entomological, ecological and environmental data [59, 129]. It could represent a geographic indicator of the risks of malaria transmission and thus a useful tool for predicting malaria morbidity risk as previously described [22]. Furthermore, it may represent a powerful tool for evaluation of vector control strategies (impregnated bednet, intradomiciliary aspersion, etc.) and could here constitute a direct criterion for effective‐ ness and appropriate use (malaria control program) [84].

#### *3.4.3.3. Indicator of malaria vector control effectiveness*

IgG levels have been described in young children (0-7 years old) and in teenagers/ adults (>14 years old) respectively [30]. In a low malaria transmission urban area (Dakar region) in Senegal, specific IgG levels were significantly higher in adults (>18 years old) compared to 6-10 years old children and in this latter group compared to those aged from 2 to 5 years [27] [124]. In Tori Bossito, moderate-high rural transmission area of Benin, both anti-gSG6-P1 IgG and IgM levels were low at 3 months of age and gradually increased until 12 months after birth (Drame *et al.*, *submitted*). The increase of specific IgG response with age is consistent with the gradual acquired immunity against *Anopheles* mosquito saliva [30] following the development of individual factors and behaviors enhancing the probability of human-vector contact [122, 123]. However, few data have reported a decrease of IgG levels to gSG6-P1 peptide [28] or to SG6 protein [118] with age. In particular, in Senegalese children (0 to 60 months old), the highest specific IgG levels were reported in the youngest children in spite of a probable very weak exposure to *An. gambiae* [30]. It can be explained by a passive IgG transfer from mother to child during pregnancy or breastfeeding as recently reported in young infants from Benin (Drame *et al.*, *submitted*). This represents a way of overestimation of the assessment of human-*Anopheles* contact level and the risk of malaria in young infants by using anti-gSG6-P1 IgG Ab. Therefore, the evaluation of specific IgM Ab levels could be a relevant solution to bias in IgG measurements. Indeed, IgM Ab, in a form of polymers (usually pentamers) in the human organism, could not cross the maternal-foetal barrier [125] and are the first Ab to appear in response to initial or primary exposure to antigen [126]. Interestingly, in Tori Bossito, specific IgM levels seemed to be a serological marker only during the first 6-months of exposure. In infants older to 6 months, the assessment of gSG6-P1-specific IgG showed a more pertinent

Some studies have reported higher levels of anti-gSG6-P1 in female individuals (children and women) compared to males (children and men) [27, 30] ([124]; Drame *et al.*, *submitted*). However, this difference was not significant, suggesting that it might be only physiological.

TheseasonofindividualsamplingmaybealsoafactorofconfusionintheusegSG6-P1biomarker in epidemiological studies on malaria risk assessment or control. Indeed, significant seasonal‐ ly variations in anti-gSG6-P1 IgG or/and IgM levels have been reported in studies conducted in newborns, children or/and adults from endemic malaria areas in Senegal [27-29, 124], Angola [30] and Benin (Drame *et al.*, submitted). In Senegal, in particular, specific gSG6-P1 in urban children and adults steadily waned from the beginning (October) to the end (December) of the study, due to an important drop in human exposure level to *An. gambiae s. l.* bites from the end

One direct application of a salivary biomarker of exposure could serve in the elaboration of maps representing the risk of exposure to *Anopheles* bites. Such immuno-epidemiological marker might represent a quantitative tool applied to field conditions and a complementary tool to those currently available, such as entomological, ecological and environmental data [59, 129]. It could represent a geographic indicator of the risks of malaria transmission and thus a

of rainfalls (October) to the beginning of the dry season (December) [127, 128].

evaluation of exposure level.

778 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**The season of** *Anopheles* **exposure**

**Effect of sex**

#### **Long and short-term evaluation of ITN efficacy**

A longitudinal study associating parasitological, entomological and immunological assess‐ ments of the efficacy of ITN-based strategies using the gSG6-P1 biomarker has been conducted in a malaria-endemic area in Angola. Human IgG responses to gSG6-P1 peptide were evalu‐ ated in 105 individuals (adults and children) before and after the introduction of ITNs and compared to entomo-parasitological data. A significant decrease of anti-gSG6-P1 IgG response was observed just after the effective use of ITNs (Fig. 14). The drop in gSG6-P1 IgG levels was associated with a considerable decrease of *P. falciparum* parasitaemia, the current WHO criterion for vector control efficacy [130]. It was particularly marked in April-August 2006, corresponding to the season peak of *An. gambiae* exposure. Interestingly, the entomological data indicated that this season-dependent peak was of similar intensity before (2005) and after (2006) ITN use, suggesting ITN installation had no impact on *An. gambiae* density, probably because of the low percentage of the overall human population covered in the studied area [131]. This study indicated also that the drop of anti-gSG6-P1 IgG response was associated with correct ITN use and not due to low *Anopheles* density. In addition, this was observed in all age groups studied (<7 years, 7–14 years, and >14 years), suggesting that this biomarker is relevant for ITN evaluation in all age groups. This rapid decrease after correct ITN usage appears to be a special property of anti-gSG6-P1 IgG which is short-lived (4-6 weeks) in the absence of ongoing antigenic stimulation, at/for all age classes.

The response does not seem to build up but wanes rapidly, when exposure failed. This property represents a major strength when using such salivary biomarker of exposure for evaluating the efficacy of vector control. In addition, using a response threshold (ΔOD=0.204) combined with ΔODITNs - the difference between April (after ITNs) and January 2006 (before) - makes possible the use of this operational biomarker at individual level (Fig. 15). The threshold response (TR) represents the non-specific background IgG response (the cut-off of immune response) and was calculated in non-*Anopheles* exposed individuals (n= 14- neg; North of France) by using this formula: TR= mean (∆DOneg) + 3SD = 0.204. An exposed individual was then classified as an immune responder if its ΔOD> 0.204. If the ΔODITNs value is comprised between -0.204 and +0.204, no clear difference in exposure level to *Anopheles* bites can be defined.

In contrast, if the individual ΔODITNs value <−0.204, it could be concluded with a high level of confidence that this individual is benefiting from ITN installation. The ΔODITNs parameter could therefore provide a measure of ITN efficacy at the individual level. An individual bio‐ marker would also be relevant at the large-scale operational studies or surveillance in the field, e.g. in National Malaria Control Programs (NMCP). In addition, the high sensitivity and specificity of the gSG6-P1 Ab response make it ideal for the evaluation of low-level ex‐

**Figure 14.** IgG Ab responses to gSG6-P1 before and after ITN use [30]. The percentage (%) of anti-gSG6-P1 IgG im‐ mune responders (thick-dotted line) in the "immunological" sub-population (n=105), before (2005) and after (2006 and January 2007) the installation of ITNs (A). These results are presented together with the intensity of *P. falciparum* infection (mean parasitaemia – fine-dotted line) measured in the same population and the mean of number of *An. gambiae* (solid line) in the studied area (A). Entomological data were not available in December 2006 and January 2007 (the last two months of the study). Arrows indicate the installation of Insecticide Treated Nets (ITNs) in February 2006. Individual anti-gSG6-P1 IgG levels (ΔOD) are presented before (2005) and after (2006) the installation of ITNs (B). Bars indicate the median value for each studied month. Statistically significant differences between months are indicated.

posure to *Anopheles bites* [27, 28], even when exposure or transmission is curtailed by NMCP efforts. Taken together, the estimation of human IgG responses to *Anopheles* gSG6-P1 could provide a reliable indicator for evaluating the efficacy of ITN-based strategies against malar‐ ia vectors, at individual and population levels, even after vector control generating particu‐ lar low exposure/transmission contexts. This salivary biomarker is a relevant tool for the evaluation of short-term efficacy as well as longer-term monitoring of malaria VCMs.

#### **Evaluation of effectiveness of diverse vector control measures**

A recent cross-sectional study conducted from October to December 2008 on 2,774 residents (children and adults) of 45 districts of urban Dakar (Senegal) has validated IgG responses to gSG6-P1 as an epidemiological indicator evaluating the effectiveness of a range of VCMs. Indeed, in this area, IgG levels to gSG6-P1 as well as the use of diverse malaria VCMs (ITNs, mosquito coils, spray bombs, ventilation and/or incense) highly varied between districts [124]. This difference of use suggests some socio-economical and cultural discrepancies between householders as described in large cities of Ivory Coast [132] and Tanzania [123]. At the district level, specific IgG levels significantly decreased with VCM use in children as well as in adults.

**Figure 15.** IgG response to gSG6-P1 as biomarker for short-term ITN efficacy. Changes in individual IgG levels (ΔOD) are presented between "just before" (January 2006) and "just after" (April 2006) ITN introduction (n=105; children and adults) (A). The arrow indicates the installation of Insecticide Treated Nets (ITNs) in February 2006. Individual IgG level changes from January (before) to April are presented (B) by individual ΔODITNs, value (ΔODITNs=ΔODApril06, - ΔODJanuary06). The threshold of specific IgG responders (TR=0.204) is indicated (dotted line). Significant positive (ΔOD>0.204) or negative (ΔOD<−0.204) changes are therefore individually presented.

Among used VCM, ITNs, the 1st chosen preventive method (43.35% rate of use), by reducing drastically the human-*Anopheles* contact level and specific IgG levels in children as well as in adults, were by far the most efficient whatever age, period of sampling or the exposure level to mosquito bites. Spray bombs were secondarily associated to a decrease of specific IgG level, due certainly to their power and fast knock-down action. But, their effects can be limited by the non-persistence of used products and some socio-economic considerations [133]. In addition, they only have been recently adopted and are more expensive in the majority of sub-Saharan Africa cities [133], explaining their less frequent use (9.57% rate of use) in the Dakar area. The non-effect of mosquito coil use is surprising, regardless to their well-adoption by residents (36.68% of rate of use), but it can be explained by their power deterrent effect which tends to push *Anopheles* vectors outside where they can remain active [133]. However, the protection ensured by ITN use seemed to be insufficient because anti-gSG6-P1 IgG levels in ITN users were specifically high in some periods of fairly high exposure to *Anopheles* bites. Changes in *An. arabiensis* behaviour, the major malaria vector in the area, can also explain this lack of protection. It can bite outside the rooms/ habitations with a maximal activity around 10.00 pm, when people are not in bed and ITNs not hanged [123]. Therefore, ITNs must be associated to a complementary VCM for an effective protection against *Anopheles* bites.

posure to *Anopheles bites* [27, 28], even when exposure or transmission is curtailed by NMCP efforts. Taken together, the estimation of human IgG responses to *Anopheles* gSG6-P1 could provide a reliable indicator for evaluating the efficacy of ITN-based strategies against malar‐ ia vectors, at individual and population levels, even after vector control generating particu‐ lar low exposure/transmission contexts. This salivary biomarker is a relevant tool for the

**Figure 14.** IgG Ab responses to gSG6-P1 before and after ITN use [30]. The percentage (%) of anti-gSG6-P1 IgG im‐ mune responders (thick-dotted line) in the "immunological" sub-population (n=105), before (2005) and after (2006 and January 2007) the installation of ITNs (A). These results are presented together with the intensity of *P. falciparum* infection (mean parasitaemia – fine-dotted line) measured in the same population and the mean of number of *An. gambiae* (solid line) in the studied area (A). Entomological data were not available in December 2006 and January 2007 (the last two months of the study). Arrows indicate the installation of Insecticide Treated Nets (ITNs) in February 2006. Individual anti-gSG6-P1 IgG levels (ΔOD) are presented before (2005) and after (2006) the installation of ITNs (B). Bars indicate the median value for each studied month. Statistically significant differences between months are

A recent cross-sectional study conducted from October to December 2008 on 2,774 residents (children and adults) of 45 districts of urban Dakar (Senegal) has validated IgG responses to gSG6-P1 as an epidemiological indicator evaluating the effectiveness of a range of VCMs. Indeed, in this area, IgG levels to gSG6-P1 as well as the use of diverse malaria VCMs (ITNs, mosquito coils, spray bombs, ventilation and/or incense) highly varied between districts [124]. This difference of use suggests some socio-economical and cultural discrepancies between householders as described in large cities of Ivory Coast [132] and Tanzania [123]. At the district level, specific IgG levels significantly decreased with VCM use in children as well as in adults.

evaluation of short-term efficacy as well as longer-term monitoring of malaria VCMs.

**Evaluation of effectiveness of diverse vector control measures**

indicated.

780 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Taken together, these results suggest that the assessment of human IgG responses to *Anophe‐ les* gSG6-P1 salivary peptide can provide a reliable evaluation of the effectiveness of malaria vector control in urban settings of Dakar whatever the age, sex, level of exposure to bites or period of malaria transmission. Therefore, this salivary biomarker can be used to compare the effectiveness of different anti-malaria vector strategies in order to identify the most suitable for a given area.

#### **Comparing effectiveness of combined or not vector control measures**

In parallel to an entomological and parasitological evaluation, IgG responses to gSG6-P1 were also used to assess, in a randomized controlled trial in 28 villages in southern Benin, four malaria vector control interventions: Long-Lasting Insecticide-treated Net (LLIN) targeted coverage to pregnant women and children younger than 6 years (TLLIN, reference group), LLIN universal coverage of all sleeping units (ULLIN), TLLIN plus full coverage of carbamateindoor residual spraying (IRS) applied every 8 months (TLLIN+IRS), and ULLIN plus full coverage of carbamate-treated plastic sheeting (CTPS) lined up to the upper part of the household walls (ULLIN+CTPS). Results from this study have shown that specific IgG levels were similar in the 4 groups before intervention and only significantly lower in the ULLIN group compared to the others after intervention. In contrast to immunological data, clinical incidence density of malaria, the prevalence and parasite density of asymptomatic infections, and the density and aggressiveness of *Anopheles* mosquitoes, were not significantly different between the four groups before as well as after interventions [134]. These findings mean that LLIN used along by all the population of a given area may be more suitable in reducing the contact between human populations and the *Anopheles* vectors, even if any effect on malaria morbidity, infection, and transmission was not observed. Therefore, combining anti-vector tools do not undeniably reduce individual exposure to malaria vectors, even if significant effect on reducing more rapidly malaria transmission and burden has been reported [135]. These findings confirm that anti-vector saliva Ab response as a biomarker of exposure is also important for NMCPs and should help the design of more cost-effective strategies for malaria control and elimination.

#### *3.4.4. Importance to develop a specific biomarker of infecting* Anopheles *bites*

Recent data have shown that the use of the gSG6-P1 biomarker for the assessment of the differential risk of the disease transmission may have some limitations in high exposure areas (Drame *et al.*, submitted). Indeed, the gSG6-P1 assesses the exposure level to both infective and not infective *Anopheles* bites. In malaria hyperendemic areas, resident people are highly exposed to mainly not infective bites and present almost all Ab specific to gSG6-P1 levels relatively high. Therefore it should be relevant to develop a biomarker of exposure specific to infective bites in order to assess the human risk of malaria transmission in such contexts. Such epidemiological parameter would be important to define in the context of malaria control. The transmission depends on the density of competent *Anopheles*, of their *Plasmodium* infective rate and of the intensity of human-vector contact. In addition, current methods to measure the intensity of malaria transmission show several limitations, especially in low transmission areas. The EIR (entomological inoculation rate) is a commonly used metric rate that estimates the number of bites by infectious mosquitoes per person per unit time. It is the product of the "human biting rate" – the number of bites per person per day by vector mosquitoes – and the fraction of vector mosquitoes that are infectious (the "sporozoite rate"). The classical method to estimate the density of sporozoites in mosquitoes is the dissection of salivary glands and the sporozoites counting under microscope. But in area of low exposure and because few mosquitoes are infected, many mosquitoes must be caught and dissected. The salivary glands dissection is a tedious technique which required well trained and studious personnel. More‐ over this technique cannot differentiate *Plasmodium* species. Another technique named CSP-ELISA detects the CSP (Circumsporozoite protein) parasite surface protein and is generally done on head/thorax of mosquitoes. However the CSP protein is expressed at the oocyst stage, consequently the CSP can be detected in the mosquito before the sporozoites have reached the salivary glands (until 2-3 days) [136, 137]. Therefore, this method induced a bias with an overestimation of sporozoites index [138, 139]. Other traditional epidemiological estimates mainly based on parasitological tests are very sensitive and specific allowing the determination of parasite species, but the examination of finger prick and thick blood smear is also labour intensive and time-consuming requiring well trained staff for a reliable examination [140]. To improve the measure of transmission, antibody responses against parasite proteins (CSP, AMA1, MSP1, MSP3, etc…) could be used but several studies have highlighted limits of this approach. Actually, people exposed to malaria can be seropositive during several months [141, 142], even after transmission has stopped [141] or in the context of low transmission [143]. So by using this method we are not able to distinguish old and new infection which is particularly important in the context of evaluation of the effectiveness of vector control program. Consid‐ ering these limits, these serological parameters seem inappropriate to assess the malaria exposure at the individual level. Some proteomic and transcriptomic studies highlighted that the composition of *Anopheles* salivary glands could be modified with the presence of *Plasmo‐ dium* parasite [15, 144, 145]. Therefore, the development of a biomarker specific of infective bites based on the analysis of antibody response against salivary proteins should represent an alternative method to assess the parasite transmission to the human.

The principle of biomarker of infective bites is based on the use of immunogenic salivary protein like marker of transmission. The expression of some salivary proteins could be induced or regulated when the salivary glands are infected. Therefore, if one of such protein presents also immunogenic properties, we can probably use the specific immune response to this protein like a marker of transmission in human. Such a biomarker will be also particularly relevant in the context of re-emergence after malaria transmission reduction or in area of low exposure. This tool will allow focusing the intervention (vector control strategies and drugs distribution) on the most exposed and the most susceptible population.

## **4. Conclusions**

**Comparing effectiveness of combined or not vector control measures**

782 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*3.4.4. Importance to develop a specific biomarker of infecting* Anopheles *bites*

Recent data have shown that the use of the gSG6-P1 biomarker for the assessment of the differential risk of the disease transmission may have some limitations in high exposure areas (Drame *et al.*, submitted). Indeed, the gSG6-P1 assesses the exposure level to both infective and not infective *Anopheles* bites. In malaria hyperendemic areas, resident people are highly exposed to mainly not infective bites and present almost all Ab specific to gSG6-P1 levels relatively high. Therefore it should be relevant to develop a biomarker of exposure specific to infective bites in order to assess the human risk of malaria transmission in such contexts. Such epidemiological parameter would be important to define in the context of malaria control. The transmission depends on the density of competent *Anopheles*, of their *Plasmodium* infective rate and of the intensity of human-vector contact. In addition, current methods to measure the intensity of malaria transmission show several limitations, especially in low transmission areas. The EIR (entomological inoculation rate) is a commonly used metric rate that estimates the number of bites by infectious mosquitoes per person per unit time. It is the product of the "human biting rate" – the number of bites per person per day by vector mosquitoes – and the fraction of vector mosquitoes that are infectious (the "sporozoite rate"). The classical method to estimate the density of sporozoites in mosquitoes is the dissection of salivary glands and the sporozoites counting under microscope. But in area of low exposure and because few mosquitoes are infected, many mosquitoes must be caught and dissected. The salivary glands

control and elimination.

In parallel to an entomological and parasitological evaluation, IgG responses to gSG6-P1 were also used to assess, in a randomized controlled trial in 28 villages in southern Benin, four malaria vector control interventions: Long-Lasting Insecticide-treated Net (LLIN) targeted coverage to pregnant women and children younger than 6 years (TLLIN, reference group), LLIN universal coverage of all sleeping units (ULLIN), TLLIN plus full coverage of carbamateindoor residual spraying (IRS) applied every 8 months (TLLIN+IRS), and ULLIN plus full coverage of carbamate-treated plastic sheeting (CTPS) lined up to the upper part of the household walls (ULLIN+CTPS). Results from this study have shown that specific IgG levels were similar in the 4 groups before intervention and only significantly lower in the ULLIN group compared to the others after intervention. In contrast to immunological data, clinical incidence density of malaria, the prevalence and parasite density of asymptomatic infections, and the density and aggressiveness of *Anopheles* mosquitoes, were not significantly different between the four groups before as well as after interventions [134]. These findings mean that LLIN used along by all the population of a given area may be more suitable in reducing the contact between human populations and the *Anopheles* vectors, even if any effect on malaria morbidity, infection, and transmission was not observed. Therefore, combining anti-vector tools do not undeniably reduce individual exposure to malaria vectors, even if significant effect on reducing more rapidly malaria transmission and burden has been reported [135]. These findings confirm that anti-vector saliva Ab response as a biomarker of exposure is also important for NMCPs and should help the design of more cost-effective strategies for malaria

> Inthepresentchapter,wehavedescribedthedevelopmentofabiomarker(the*An.gambiae*gSG6- P1 peptide) of *Anopheles* mosquito bites by using an original approach coupling bioinformatic tools and immuno-epidemiological assays. Then, measurements of IgG level specific to gSG6- P1 at individual as well as population level, represent a tool/biomarker for accurately evaluate the level of human exposure to *Anopheles* bites and the risk of malaria in all age-classes of populations (newborns, infants, children, adults) living in various settings (very-low, low, moderate, and high malaria transmission areas) of rural, semi-urban and urban regions of Senegal, Angola and Benin. In the majority of these areas, this biomarker appeared to be promisingandcomplementarytoclassical entomologicalmethods,because it cangive a reliable

evaluation of the individual contact with anthropophilic *Anopheles* even if exposure to bites is low/very low (urban area). Therefore, such biomarker would be particularly relevant in places where malaria transmission is low, e.g. in foci of urban, high-altitude or seasonal malaria, and intravelersinendemicareas.Thischapterhasalsoshownthattheavailabilityofsuchabiomarker could allow the evaluation of the exposure to the main *P. falciparum* vectors (*An. gambiae s.s.*, *An. arabiensis*, *An. funestus*, *An. melas*) in Africa where different species of malaria vector co-inhab‐ it. One direct application of such a gSG6 peptide marker of exposure could be in the elabora‐ tion of maps representing the risk of exposure to *Anopheles* bites.It couldrepresent a geographic indicator of the risks of malaria transmission and thus a useful tool for predicting malaria morbidity risk as previously described. Furthermore, it represents a powerful and reliable tool for the evaluation of the effectiveness of vector control strategies. Such an indicator could also represent an alternative to classical entomological-parasitological monitoring methods for measuring and following the effectiveness of vector control strategies used by the National Malaria Control Programmes in various settings across Africa. Finally, this biomarker ap‐ proachcouldbe similarlyappliedtovector-control strategies forothermosquito-bornediseases such as emergent or re-emergent arbovirus diseases and trypanosomiasis.

## **Author details**

Papa M. Drame1\*, Anne Poinsignon1 , Alexandra Marie1 , Herbert Noukpo2 , Souleymane Doucoure1 , Sylvie Cornelie1,2 and Franck Remoue1,2

\*Address all correspondence to: drpapamak@gmail.com

1 Universités Montpellier 1 et 2, Institut de Recherche pour le Développement (IRD), Mont‐ pellier, France

2 Universités Montpellier 1 et 2, Centre de Recherche Entomologique de Cotonou (CREC), Cotonou, Bénin

### **References**


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evaluation of the individual contact with anthropophilic *Anopheles* even if exposure to bites is low/very low (urban area). Therefore, such biomarker would be particularly relevant in places where malaria transmission is low, e.g. in foci of urban, high-altitude or seasonal malaria, and intravelersinendemicareas.Thischapterhasalsoshownthattheavailabilityofsuchabiomarker could allow the evaluation of the exposure to the main *P. falciparum* vectors (*An. gambiae s.s.*, *An. arabiensis*, *An. funestus*, *An. melas*) in Africa where different species of malaria vector co-inhab‐ it. One direct application of such a gSG6 peptide marker of exposure could be in the elabora‐ tion of maps representing the risk of exposure to *Anopheles* bites.It couldrepresent a geographic indicator of the risks of malaria transmission and thus a useful tool for predicting malaria morbidity risk as previously described. Furthermore, it represents a powerful and reliable tool for the evaluation of the effectiveness of vector control strategies. Such an indicator could also represent an alternative to classical entomological-parasitological monitoring methods for measuring and following the effectiveness of vector control strategies used by the National Malaria Control Programmes in various settings across Africa. Finally, this biomarker ap‐ proachcouldbe similarlyappliedtovector-control strategies forothermosquito-bornediseases

such as emergent or re-emergent arbovirus diseases and trypanosomiasis.

, Alexandra Marie1

1 Universités Montpellier 1 et 2, Institut de Recherche pour le Développement (IRD), Mont‐

2 Universités Montpellier 1 et 2, Centre de Recherche Entomologique de Cotonou (CREC),

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, Sylvie Cornelie1,2 and Franck Remoue1,2

, Herbert Noukpo2

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**Author details**

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