3. Insecticide resistance mechanisms

A proportion of insect populations can tolerate doses of insecticides which have been proved lethal to the majority of the individuals in a normal population of the same species through various mechanisms such as: (i) insecticide can be broken down or detoxified much faster in the resistant mosquitoes than in the susceptible ones, hence quickly eliminated from their body (metabolic resistance); (ii) the target of the insecticide can be genetically altered to prevent the insecticide from binding thereby reducing the insecticide effect (target-site resistance); or (iii) resistant mosquitoes may absorb the toxin slower than susceptible insects (penetration resistance). An illustration of these mechanisms is represented in Figure 4.

Figure 4. Illustration of the physiological insecticide resistance mechanisms in mosquitoes. (1) Reduced penetration: physiological changes to the cuticle of the mosquitoes prevent the absorption or penetration of insecticide. (2) Target-site resistance: insecticides have a target site within the mosquito. This site can become modified so that the insecticide no longer binds to it. (3) Metabolic resistance: enhanced enzyme systems break down insecticides before they can have a toxic effect on the mosquito.

#### 3.1. Methods used to study resistance mechanisms

Insecticide resistance monitoring is essential to understand the actual threat and how resistance is spreading among malaria vectors [7]. Once resistance has reached very high levels (fixed in the population), most insecticide resistance management strategies, which are based to restore susceptibility, would not work. Thus, regular monitoring is crucial. Three detection methods (Table 1) can be used to monitor insecticide resistance, each method providing different information. Bioassays are the most popular way to monitor resistance where mosquitoes are exposed fixed doses of insecticides for a fixed time and the percentage mortality is recorded 24 h post-exposure [73]. Even though they are simple to perform, bioassays have several disadvantages such as requiring a large number of mosquitoes, affected by variations in humidity, temperature and time of the day [74]. Some authors argue that bioassays should be supplemented with DNA markers or even partially replaced by these DNA markers [75]. It should be noted that DNA markers are usually specific to certain mechanisms hence the need to perform them is to avoid unknown mechanisms going undetected. Until now, no assay has been developed that is suitable to monitor cuticular or behavioural resistance.

#### 3.2. Target-site resistance

pseudopunctipennis, have shown susceptibility to PY across most range of their distribution with only two exceptions: one An. benarrochi population at the border between Peru and Brazil resistant to permethrin, and one An. pseudopunctipennis population in the northwest Peru resistant to permethrin, cypermethrin, deltamethrin and lambda-cyhalothrin [36], the latter

In conclusion, resistance to insecticide is steadily spreading worldwide in most vectors as shown by the comparison of resistance profile between 1985 and 2000 (Figure 3A) and 1985 to 2017 (Figure 3B) from IR mapper (http://www.irmapper.com/). This represents a serious

A proportion of insect populations can tolerate doses of insecticides which have been proved lethal to the majority of the individuals in a normal population of the same species through various mechanisms such as: (i) insecticide can be broken down or detoxified much faster in the resistant mosquitoes than in the susceptible ones, hence quickly eliminated from their body (metabolic resistance); (ii) the target of the insecticide can be genetically altered to prevent the insecticide from binding thereby reducing the insecticide effect (target-site resistance); or (iii) resistant mosquitoes may absorb the toxin slower than susceptible insects (penetration

Figure 4. Illustration of the physiological insecticide resistance mechanisms in mosquitoes. (1) Reduced penetration: physiological changes to the cuticle of the mosquitoes prevent the absorption or penetration of insecticide. (2) Target-site resistance: insecticides have a target site within the mosquito. This site can become modified so that the insecticide no longer binds to it. (3) Metabolic resistance: enhanced enzyme systems break down insecticides before they can have a

challenge to malaria control, which relies heavily on insecticide-based tools.

resistance). An illustration of these mechanisms is represented in Figure 4.

population also showed cross-resistance to OP (malathion).

3. Insecticide resistance mechanisms

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toxic effect on the mosquito.

One of the mechanisms mosquito becomes resistant is by altering the target site of the insecticide thereby preventing it from binding effectively hence the insecticide has little or no effect on the insect. Most insecticide targets are found within the nervous system and mutations in these target sites (mainly receptors) lead to reduced sensitivity. For example, PYs and DDT act on the voltage-gated sodium channels (VGSCs) and mutation in the amino sequence of this gene results in reduced sensitivity of the channels preventing PYs and DDT from binding [76]. Insects with this mutation can withstand prolong exposure to insecticide without being knocked down, hence the name "knockdown resistance" (kdr) [77]. The replacement of the leucine residue for a phenylalanine or a serine at position 1014 in the VGSC is one of the most common amino acid substitution associated with PY resistance in malaria vector [77]. Also an alanine to serine substitution at position 302 (or 296) of the γ-amino-butyric acid (GABA) receptor is found in the dieldrin-resistant (rdl) insect species including An. gambiae [78] and An. funestus [79]. Similarly, mutations in the gene coding for the neurotransmitter acetylcholinesterase (ace-1), the target site of OPs and CAs, have been found [80], which reduces the inhibition effect of the insecticide on the enzyme [81, 82]. Substitution of glycine to serine at position 119 has been reported in An. albimanus and An. gambiae, and this mutation confers resistance to OPs and CAs [83]. Duplication of the ace-1 gene has been reported in the An. gambiae and An. coluzzii [84]. However, in species such as An. funestus, other mutations were detected in ace-1 including the N485I shown to be associated with bendiocarb resistance in southern African populations [85].

#### 3.3. Metabolic resistance

Metabolic resistance is the most common and challenging of all insecticide resistance mechanisms. Mosquitoes have enzyme systems that protect them from xenobiotic compounds and

#### Susceptibility bioassay tests Biochemical assays Molecular assays

Vectors are exposed to fixed insecticide concentrations, and the level of vector mortality is subsequently recorded. The results are expressed as the percentage of vectors knocked down, alive or dead. Susceptibility testing requires samples of at least 100 live mosquitoes per testing site. These susceptibility tests are generally used for routine monitoring, as they can be applied in the field. They provide standardised data that are relatively easily interpreted. Either WHO paper bioassays or CDC bottle bioassays can be used. The results obtained with the two methods are not comparable. In order to observe longitudinal or temporal patterns in resistance, countries and academic institutions in all regions must therefore use the same method consistently over time.

Susceptibility tests identify the existence of resistance once it is at a detectable level but do not establish the resistance mechanism involved. They may also not identify resistance if the frequency is too low. Several countries have reported shortages in the supply of testing materials and have switched between the WHO and CDC tests, making results difficult to compare. In some cases, they have limited their testing.

Description Description Description

Biochemical assays detect the presence of a particular resistance mechanism or an increase in enzyme activity. They require fresh mosquitoes, but much fewer than for bioassays. Unlike bioassays, biochemical assays can identify some specific resistance mechanisms and indicate an increase in metabolic enzyme activity. Biochemical assays are normally used in conjunction with synergist and molecular assays.

#### Limitations Limitations Limitations

The method is more difficult to use in the field as it requires sophisticated equipment, and interpretation of the results requires strong technical skills. Further, the correlation between chemical reactions in these tests and increased ability to metabolise insecticides is not yet well defined.

Molecular tests are used on the actual gene, allowing detailed and direct analysis of resistance genes. Testing can be done with straightforward polymerase chain reaction techniques (30) with DNA or in more elaborate microarray tests with RNA. More advanced molecular methods can provide complex genetic information including whether the mutation is unique or has spread. These are the most accurate tests for measuring resistance frequency in vector populations. Molecular tests must, however, be correlated with susceptibility testing.

glutathione S-transferases (GSTs) and esterases are the three major enzyme families that are

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Of the six families of P450s, genes belonging to the CYP4, CYP6 and CYP9 have been observed in resistant mosquitoes with increased transcriptional level [89], with the majority of those implicated in resistance belonging to the CYP6 family. For a P450 to be involved in resistance, it does not only have to be overexpressed but also must be able to metabolise/sequester the insecticide to which the insect is resistant and also be better metaboliser than those for the susceptible strain [90]. In An. gambiae, CYP6P3 and CYP6M2 have been shown to metabolise type I and type II PYs [91], and CYP6M2 can metabolise DDT [92]. In An. funestus, the duplicated P450 CYP6P9a and CYP6P9b and CYP6M7 have been shown to metabolise PYs [93, 94], whereas CYP6Z1 confers cross-resistance to both pyrethroids and carbamates [85]. Furthermore, allelic variation of P450 genes, such as CYP6P9a/b, has been shown to drive pyrethroid resistance in field populations of An. funestus [95] with signature of selective sweep

The GSTs are involved in the phase two of the detoxification of xenobiotic compounds where they conjugate the substrate with glutathione enhancing solubility thus facilitating the excretion. In insect, six classes of GSTs, i.e., delta, sigma, epsilon, omega, theta and zeta have been identified [97]. Insects resistant to major classes of insecticide show elevated levels of GSTs activities. For example, GSTs confer resistance to DDT in mosquitoes including An. gambiae [98], An. dirus [99], Aedes aegypti [100] and An. funestus [26]. A single amino acid change in GSTe2 (L119F) has been shown to confer a cross-resistance to DDT and PYs in An. funestus [26],

CAs and OPs are the main insecticides that are metabolised or sequestered by esterasemediated insecticide resistance. Esterase levels in the resistant mosquitoes can either be elevated like in Culex or non-elevated like in Anopheles species (An. arabiensis, An. stephensi and An. culicifacies). Esterase-mediated insecticide resistance in Anopheles spp. was associated with allelic variants that can metabolise the insecticide at a faster rate than those of the susceptible and shown to confer resistance to malathion [102, 103]. The role of esterases in PY resistance

Cuticular resistance occurs when mosquitoes reduce the absorption of insecticide into their bodies by altering the structure or composition of the cuticle. A wide range of insecticides are threatened by this mechanism as for their lethal effect to occur, most insecticides must cross the cuticle in order to reach their site of action. Cuticular resistance enhances the resistance

whereas a similar change is also reported in An. gambiae (I114T) [101].

involved in breaking down of insecticides.

3.3.1. Cytochrome P450 monooxygenases

associated with scale-up of bed nets [96].

3.3.2. Glutathione S-transferases

still needs to be investigated.

3.4. Cuticular or reduced penetration resistance

3.3.3. Esterases

The method requires sophisticated equipment and entomological capacity. It can be used to detect target site resistance and a few identified metabolic mechanisms. Therefore, susceptibility tests should be used to complement molecular results, as the absence of identifiable genotypic resistance does not necessarily mean that resistance does not exist.

Table 1. Different methods for monitoring insecticide resistance in mosquito vectors and their limitations.

some of these enzyme systems can break down insecticide before it can reach its site of action. In metabolic resistance, enzymes that detoxify the insecticide can be overexpressed or alter the affinity of the enzyme for the insecticide through amino acid substitutions [86]. Overexpression of insecticide resistance genes is the most frequent mechanism in resistant mosquitoes. This increased expression of insecticide resistance genes can be due to cis- or trans-acting elements in the promoter or gene amplification [87, 88]. This overexpression results in the high level of enzyme production in the resistant mosquitoes that enables them to break down the insecticide at a faster rate before it reaches the target site. Cytochrome P450 monooxygenases, glutathione S-transferases (GSTs) and esterases are the three major enzyme families that are involved in breaking down of insecticides.

#### 3.3.1. Cytochrome P450 monooxygenases

Of the six families of P450s, genes belonging to the CYP4, CYP6 and CYP9 have been observed in resistant mosquitoes with increased transcriptional level [89], with the majority of those implicated in resistance belonging to the CYP6 family. For a P450 to be involved in resistance, it does not only have to be overexpressed but also must be able to metabolise/sequester the insecticide to which the insect is resistant and also be better metaboliser than those for the susceptible strain [90]. In An. gambiae, CYP6P3 and CYP6M2 have been shown to metabolise type I and type II PYs [91], and CYP6M2 can metabolise DDT [92]. In An. funestus, the duplicated P450 CYP6P9a and CYP6P9b and CYP6M7 have been shown to metabolise PYs [93, 94], whereas CYP6Z1 confers cross-resistance to both pyrethroids and carbamates [85]. Furthermore, allelic variation of P450 genes, such as CYP6P9a/b, has been shown to drive pyrethroid resistance in field populations of An. funestus [95] with signature of selective sweep associated with scale-up of bed nets [96].

#### 3.3.2. Glutathione S-transferases

The GSTs are involved in the phase two of the detoxification of xenobiotic compounds where they conjugate the substrate with glutathione enhancing solubility thus facilitating the excretion. In insect, six classes of GSTs, i.e., delta, sigma, epsilon, omega, theta and zeta have been identified [97]. Insects resistant to major classes of insecticide show elevated levels of GSTs activities. For example, GSTs confer resistance to DDT in mosquitoes including An. gambiae [98], An. dirus [99], Aedes aegypti [100] and An. funestus [26]. A single amino acid change in GSTe2 (L119F) has been shown to confer a cross-resistance to DDT and PYs in An. funestus [26], whereas a similar change is also reported in An. gambiae (I114T) [101].

#### 3.3.3. Esterases

some of these enzyme systems can break down insecticide before it can reach its site of action. In metabolic resistance, enzymes that detoxify the insecticide can be overexpressed or alter the affinity of the enzyme for the insecticide through amino acid substitutions [86]. Overexpression of insecticide resistance genes is the most frequent mechanism in resistant mosquitoes. This increased expression of insecticide resistance genes can be due to cis- or trans-acting elements in the promoter or gene amplification [87, 88]. This overexpression results in the high level of enzyme production in the resistant mosquitoes that enables them to break down the insecticide at a faster rate before it reaches the target site. Cytochrome P450 monooxygenases,

Table 1. Different methods for monitoring insecticide resistance in mosquito vectors and their limitations.

Susceptibility bioassay tests Biochemical assays Molecular assays Description Description Description

Limitations Limitations Limitations

The method is more difficult to use in the field as it requires sophisticated equipment, and interpretation of the results requires strong technical skills. Further, the correlation between chemical reactions in these tests and increased ability to metabolise insecticides is not yet well defined.

Biochemical assays detect the presence of a particular resistance mechanism or an increase in enzyme activity. They require fresh mosquitoes, but much fewer than for bioassays. Unlike bioassays, biochemical assays can identify some specific resistance mechanisms and indicate an increase in metabolic enzyme activity. Biochemical assays are normally used in conjunction with synergist and molecular assays.

Molecular tests are used on the actual gene, allowing detailed and direct analysis of resistance genes. Testing can be done with straightforward polymerase chain reaction techniques (30) with DNA or in more elaborate microarray tests with RNA. More advanced molecular methods can provide complex genetic information including whether the mutation is unique or has spread. These are the most accurate tests for measuring resistance frequency in vector populations. Molecular tests must, however, be correlated with susceptibility testing.

The method requires sophisticated equipment and entomological capacity. It can be used to detect target site resistance and a few identified metabolic mechanisms. Therefore, susceptibility tests should be used to complement molecular results, as the absence of identifiable genotypic resistance does not necessarily mean that resistance does not exist.

Vectors are exposed to fixed insecticide concentrations, and the level of vector mortality is subsequently recorded. The results are expressed as the percentage of vectors knocked down, alive or dead. Susceptibility testing requires samples of at least 100 live mosquitoes per testing site. These susceptibility tests are generally used for routine monitoring, as they can be applied in the field. They provide standardised data that are relatively easily interpreted. Either WHO paper bioassays or CDC bottle bioassays can be used. The results obtained with the two methods are not comparable. In order to observe longitudinal or temporal patterns in resistance, countries and academic institutions in all regions must therefore use the same method consistently over

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time.

Susceptibility tests identify the existence of resistance once it is at a detectable level but do not establish the resistance mechanism involved. They may also not identify resistance if the frequency is too low. Several countries have reported shortages in the supply of testing materials and have switched between the WHO and CDC tests, making results difficult to compare. In some cases, they have limited their testing.

CAs and OPs are the main insecticides that are metabolised or sequestered by esterasemediated insecticide resistance. Esterase levels in the resistant mosquitoes can either be elevated like in Culex or non-elevated like in Anopheles species (An. arabiensis, An. stephensi and An. culicifacies). Esterase-mediated insecticide resistance in Anopheles spp. was associated with allelic variants that can metabolise the insecticide at a faster rate than those of the susceptible and shown to confer resistance to malathion [102, 103]. The role of esterases in PY resistance still needs to be investigated.

#### 3.4. Cuticular or reduced penetration resistance

Cuticular resistance occurs when mosquitoes reduce the absorption of insecticide into their bodies by altering the structure or composition of the cuticle. A wide range of insecticides are threatened by this mechanism as for their lethal effect to occur, most insecticides must cross the cuticle in order to reach their site of action. Cuticular resistance enhances the resistance conferred by other mechanisms. This mechanism has not been extensively studied as compared to the other mechanisms because there are very few examples. Recently, Yahouédo et al. [104] studied the role of the cuticular resistance in PY-resistant strain of An. gambiae called MRS, free of kdr mutations. They succeeded to show that lower amount of insecticide was absorbed in the MRS strain than in the susceptible strain and also that the MRS strain had a significantly thicker cuticle layers than those of the susceptible strain. CPLCG3 gene, which codes for a structural protein contributing to the cuticle thickness, was found to be constitutively upregulated. Similar evidences of cuticular resistance were shown for An. funestus with proofs of cuticle thickening in PY-resistant mosquitoes [105].

vectors, thereby increasing infectiousness of parasites and threatening vector control. However, the development of resistance in a mosquito often comes with a price subsequently affecting the fitness of the vector [115]. As a consequence of the fitness cost of insecticide resistance on the life traits (mentioned above) of the vectors, reversion to susceptibility is expected. A good example of reversal to susceptibility occurred in An. arabiensis in Sudan. In this country, after antimalarial house spraying in the early 1980s, resistance to malathion was noticed. This prompted a switch of insecticide treatment to fenitrothion (OP insecticide), and susceptibility to malathion was restored in the following years [10]. However, reversal rates are variable and may be very slow, particularly when an insecticide has been used for many years. For example, the same rdl gene has been reported to be maintained in field populations of Sri Lanka despite the withdrawal of cyclodiene insecticides for mosquito control for more than 30 years [116]. Before implementing any resistance management strategy in the field,

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4.3. Epidemiological consequences of the insecticide resistance on malaria incidence

There are large number of confounding factors threatening the assessment of epidemiological consequences of the insecticide resistance on malaria incidence and data interpretation [117]. For this reason, only few studies have assessed the epidemiological impact of insecticide resistance. Impact of PY resistance on control failure was reported from the borders of Mozambique and South Africa. In 1996, the malaria control programme in KwaZulu-Natal (South Africa) switched from using DDT to deltamethrin for indoor spraying [118]. After four years of deltamethrin spraying, reported malaria cases increased approximately fourfold. An. funestus, previously eradicated, had reappeared and was observed emerging alive from PY-sprayed houses. Bioassays showed that this species was resistant to PYs but susceptible to DDT [119]. The decision to revert to IRS with DDT was accompanied by a decline in malaria cases by 91% [120]. On the Bioko Island on the West African coast, increased density of PY-resistant An. gambiae was also reported after IRS campaign with lambda-cyhalothrin, although a significant reduction in transmission index and malaria reported cases was observed [121, 122]. High frequencies of the L1014F kdr allele were observed in the local An. gambiae population. When PYs were replaced by CAs (bendiocarb), mosquito population declined [122]. Nevertheless, in an operational scale programme such as this, the possible contribution of other factors to the failure of PY IRS to control mosquito population density cannot be overlooked; thus, the direct consequence of the high kdr frequency is uncertain. After initiation of interventions combining IRS with PYs and ITNs in the highland provinces of Burundi in 2002, significant reduction was recorded in Anopheles density by 82% [123]. Consequently, transmission intensity was reduced by 90% and occurrence of malaria cases by 43% in children, despite high frequencies of the L1014S kdr allele in the main vector An. gambiae s.s. [123]. Many interventions took place in Africa in order to investigate the efficacy of ITNs for malaria prevention [124]. However, the extent to which PY resistance might affect the effectiveness of such interventions is not well elucidated. In Korhogo area, north of Côte d'Ivoire where the 1014F kdr allele frequency in An. gambiae is up to 80% [125], and malaria is endemic, lambda-cyhalothrin-treated nets had a significant impact on the entomological inoculation rate with around 55% reduction. Malaria

knowledge of the reversal rate is crucial.

4.3.1. Past and current evidences
