**Global Distribution of the Dominant Vector Species of Malaria**

Marianne E. Sinka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54163

## **1. Introduction**

Human malarial protozoa are transmitted by mosquitoes of the genus *Anopheles*. There are 465 formally recognised species and more than 50 unnamed members of species complexes [1]. Approximately 70 of these species have the capacity to transmit human malaria parasites [2] and 41 are considered here to be dominant vector species/species complexes (DVS), capable of transmitting malaria at a level of major concern to public health [3, 4] (Tables 1-3).

The aim of this chapter is to document the distribution of these DVS using global and regional maps. In addition, behavioural summaries are provided for the most important species, i.e. those on each continent that are considered the most dangerous and responsible for most ma‐ laria transmission, and hence have the greatest impact on human health. Only the primary vec‐ tors in those regions with current and problematic malaria transmission are discussed further here (i.e. the vectors of Europe and the Middle-East are not included – but more details can be found in Sinka *et al.* [5]) The regions covered include the Americas, Africa and the Asian-Pacific.

The maps presented (e.g. Figure 1) provide species location information and highlight the existence of a greater number of vector species than is often considered, many in sympatry, across the malarial zones. Amongst these DVS, there are often important behavioural charac‐ teristics that must be considered if successful vector control is to be applied. For example, some species do not always enter houses to bite, are most active in the early evening, and prefer to rest outdoors after feeding, such as many of the species common in South America (e.g. *An. albopictus*, *An. marajoara, An. nuneztovari*), *An. dirus* in South-East Asia and *An. farauti* in the Australian-Pacific region. Others are highly opportunistic in their feeding habits (including *An. darlingi*, the most 'dominant' south American species amongst the South American DVS); biting readily indoors or out. As such, a large investment in insecticide treated bednets (ITNs) or insecticide residual spraying (IRS) will not reduce malaria transmission where such species

© 2013 Sinka; 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 Sinka; 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.

occur. Thus, to appreciate where different malaria control methods are best applied, one must know what *Anopheles* species exists in an area *and* understand their behaviour. A map clearly and simply addresses the first of these needs, and if accompanied by a behavioural summary, than informed decisions about how to combat malaria transmission can be made.

Maps clearly illustrate the spatial extent of a species' distribution. Often, even within a single *Anopheles* species range, behaviour can vary depending on location. The best known example is the *An. gambiae* complex. The *An. gambiae* complex was initially considered as a single species. Clear differences in behaviour reported across its distribution caused it to be examined more closely and now this complex is considered to include eight species [1, 6] including the DVS: *An. arabiensis, An. gambiae, An melas* and *An. merus. Anopheles arabiensis,* is considered mostly zoo‐ philic, when compared to the highly anthropophilic *An. gambiae,* but still plays a very impor‐ tant role in malaria transmission – indeed, its presence and propensity to rest outdoors is attributed (amongst other factors) to the 'failure' of the mass indoor residual spraying program intended to control malaria in Nigeria during the Garki project [7]. Variability in behaviour within the *An. gambiae* species (rather than the complex) is also commonly reported [5]. Such spatially dependant variability amongst the DVS will be discussed further within this chapter.

The maps presented in this chapter are not a comprehensive analysis of all anophelines. They show only those species designated as DVS; a categorisation initially based on information taken from a number of authoritative reviews [8-12] ([10] translated and updated:[13]) and with additional guidance from a technical advisory group of vector experts [3, 5, 14, 15]. This chapter will also briefly touch upon the methodology behind creating the distribution maps for these DVS including what information is needed to ensure increasingly accurate maps can be produced in future.

## **2. Global**

The global DVS map (Figure 1) gives a clear overview of the variability in vector complexity across the world. Africa appears to show a relatively simple picture of a small number of highly dominant species covering large areas of the continent and although the 'secondary DVS' are not shown (see Figure 4), even with their influence, the comparative complexity between African and Asia is very different. The Asian-Pacific region has 19 DVS [14] (16 of which are shown on the multi-species maps presented here (Figures 1 & 5) – see below) whereas Africa has only seven DVS [5], with the three 'primary' DVS shown on the global map (Figure 1 - see below). Of the 19 species in the Asian-Pacific, nine are now considered species complexes, whereas of the seven African DVS, only *An. nili* is a confirmed species complex (the *An. gambiae* complex is not included here, as specific individual members of the complex are categorised within these seven African DVS) [1, 6, 16]. It is unclear what is the cause of the high diversity of vectors found in the Asian-Pacific region, but it may be simply a factor of the large number of islands, and hence a consequence of limitations in dispersal and specialisation within a restricted environment. Whatever the reason, the Asian-Pacific region maintains a high number of vectors and species complexes and even within individual species, behaviours can vary hugely depending on location (e.g. *An. annularis* – see below).

North America (excluding Mexico) shows a simple vector profile (Figure 2). There are only two species considered here as DVS: *An. freeborni* found in northwestern USA and the *An. quadrimaculatus* complex, found in the southern regions of the country. In Latin America, however, the situation is a little more complex. Despite a number of sympatric species on the continent, *An. darlingi* is considered the most important vector in the neotropical region [13] and hence is shown dominating all localities where it occurs. In Central America this species does not have such a great influence and both *An. albimanus* and *An. pseudopunctipennis* are considered of greater importance.

The individual regions (Americas, Africa, and Asia-Pacific) are discussed in more detail in the following sections.

**Figure 1.** The global distribution of 34 DVS. (Map reproduced from Sinka *et al.* [4]); s.l.: *sensu lato*, meaning 'in the broad sense' referring to species complex

## **3. The Americas**

occur. Thus, to appreciate where different malaria control methods are best applied, one must know what *Anopheles* species exists in an area *and* understand their behaviour. A map clearly and simply addresses the first of these needs, and if accompanied by a behavioural summary,

Maps clearly illustrate the spatial extent of a species' distribution. Often, even within a single *Anopheles* species range, behaviour can vary depending on location. The best known example is the *An. gambiae* complex. The *An. gambiae* complex was initially considered as a single species. Clear differences in behaviour reported across its distribution caused it to be examined more closely and now this complex is considered to include eight species [1, 6] including the DVS: *An. arabiensis, An. gambiae, An melas* and *An. merus. Anopheles arabiensis,* is considered mostly zoo‐ philic, when compared to the highly anthropophilic *An. gambiae,* but still plays a very impor‐ tant role in malaria transmission – indeed, its presence and propensity to rest outdoors is attributed (amongst other factors) to the 'failure' of the mass indoor residual spraying program intended to control malaria in Nigeria during the Garki project [7]. Variability in behaviour within the *An. gambiae* species (rather than the complex) is also commonly reported [5]. Such spatially dependant variability amongst the DVS will be discussed further within this chapter. The maps presented in this chapter are not a comprehensive analysis of all anophelines. They show only those species designated as DVS; a categorisation initially based on information taken from a number of authoritative reviews [8-12] ([10] translated and updated:[13]) and with additional guidance from a technical advisory group of vector experts [3, 5, 14, 15]. This chapter will also briefly touch upon the methodology behind creating the distribution maps for these DVS including what information is needed to ensure increasingly accurate maps can

The global DVS map (Figure 1) gives a clear overview of the variability in vector complexity across the world. Africa appears to show a relatively simple picture of a small number of highly dominant species covering large areas of the continent and although the 'secondary DVS' are not shown (see Figure 4), even with their influence, the comparative complexity between African and Asia is very different. The Asian-Pacific region has 19 DVS [14] (16 of which are shown on the multi-species maps presented here (Figures 1 & 5) – see below) whereas Africa has only seven DVS [5], with the three 'primary' DVS shown on the global map (Figure 1 - see below). Of the 19 species in the Asian-Pacific, nine are now considered species complexes, whereas of the seven African DVS, only *An. nili* is a confirmed species complex (the *An. gambiae* complex is not included here, as specific individual members of the complex are categorised within these seven African DVS) [1, 6, 16]. It is unclear what is the cause of the high diversity of vectors found in the Asian-Pacific region, but it may be simply a factor of the large number of islands, and hence a consequence of limitations in dispersal and specialisation within a restricted environment. Whatever the reason, the Asian-Pacific region maintains a high number of vectors and species complexes and even within individual species, behaviours

can vary hugely depending on location (e.g. *An. annularis* – see below).

than informed decisions about how to combat malaria transmission can be made.

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

be produced in future.

**2. Global**

On a global scale, the nations of the Americas benefit from having the lowest *P. falciparum* morbidity, with stable risk areas typically having low levels of endemicity (*Pf*PR2-10≤ 5%) [17]. Such reduced levels of malaria transmission coupled with continuing reports of decreasing mortality and morbidity for all major *Plasmodium* species across the region (e.g. between 2000 and 2007) [18] have been credited to an increasing use of integrated vector control [19]. Integrated vector control/management relies on a number of factors, but foremost (as given in the World Health Organisation (WHO), strategic framework for integrated vector manage‐ ment [20]) is the 'selection of proven vector control methods based on knowledge of local vector biology and ecology, disease transmission and morbidity'; essentially, knowing which vector species is present and understanding how it behaves.

There are nine DVS in the Americas (Figure 2, Table 1) [15], with two species having their distributions contained entirely within North America (*An. freeborni* and *An. quadrimaculatus*), and the remaining six species encompassing areas from southern North America, through Central America and into South America, incorporating the northern reaches of Argentina. As stated above, in South America, *An. darlingi* is considered to be the most important of the DVS where it is found [13]. However there is increasing evidence of the importance of other species, including members of the *An. albitarsis* complex (e.g. *An. marajoara*), that may have a higher influence in malaria transmission than previously thought [21]. As such, and due to the dominance across the continent indicated by the *An. darlingi* distribution in Figure 2, single species maps are also shown for *An. albitarsis, An. marajoara, An. nuneztovari* and *An. pseudo‐ punctipennis* (Figure 3)*.*

Many of the American species show great variability in their adult behaviour, with most showing little preference for biting either humans or animals [15] (Table 1), tending to feed on whichever host they first encounter. This variability is also reflected in their propensity to bite both indoors and out. Overall, the majority of DVS in the Americas will rest outside after biting (Table 1, [15]).

Despite similar adult behaviour amongst many of the South American DVS, there are a number of behavioural characteristics found in the larval stages that do differentiate the species (Table 1). For example *An. aquasalis*, whose name means salt (salis) water (aqua), is a vector found in coastal environments. Its larvae prefer clear, non-polluted water bodies such as mangrove swamps, lagoons and ditches [22, 23]. They can develop in fresh water sites, but it is considered a poor competitor in such habitats, especially against *An. albimanus* [15], which may be causal in tending to restrict the range of *An. aquasalis* to brackish locations.

*Anopheles darlingi* larvae are characteristically associated with patches of floating debris found along river margins in rural and lowland forested areas [24]. *Anopheles marajoara* is also found in lowland areas but is more common in secondary forests and is able to adapt to environments that have undergone some human intervention [21, 25] which may be a causal factor in its increasing dominance over *An. darlingi* in some localities. Forest clearance and pollution will decrease sites suitable for *An. darlingi* but increase the availability of sunlit marshy areas and ponds more suitable for *An. marajoara* [21, 26].

The *An. pseudopunctipennis* complex is known to be able to survive and transmit malaria at altitudes higher than many other DVS, up to 3000 m [27, 28]. Its larvae also have a defining characteristic; an apparent obligate association with filamentous *Spirogyra*-type green algae [23, 27, 29-31] (Table 1). Indeed, the removal of such algae has been shown to be a viable method of control for this species [31].


Table 1: Summary of bionomics of the DVS of the Americas (created by cross referencing TAG and literature searches)

Integrated vector control/management relies on a number of factors, but foremost (as given in the World Health Organisation (WHO), strategic framework for integrated vector manage‐ ment [20]) is the 'selection of proven vector control methods based on knowledge of local vector biology and ecology, disease transmission and morbidity'; essentially, knowing which vector

There are nine DVS in the Americas (Figure 2, Table 1) [15], with two species having their distributions contained entirely within North America (*An. freeborni* and *An. quadrimaculatus*), and the remaining six species encompassing areas from southern North America, through Central America and into South America, incorporating the northern reaches of Argentina. As stated above, in South America, *An. darlingi* is considered to be the most important of the DVS where it is found [13]. However there is increasing evidence of the importance of other species, including members of the *An. albitarsis* complex (e.g. *An. marajoara*), that may have a higher influence in malaria transmission than previously thought [21]. As such, and due to the dominance across the continent indicated by the *An. darlingi* distribution in Figure 2, single species maps are also shown for *An. albitarsis, An. marajoara, An. nuneztovari* and *An. pseudo‐*

Many of the American species show great variability in their adult behaviour, with most showing little preference for biting either humans or animals [15] (Table 1), tending to feed on whichever host they first encounter. This variability is also reflected in their propensity to bite both indoors and out. Overall, the majority of DVS in the Americas will rest outside after biting

Despite similar adult behaviour amongst many of the South American DVS, there are a number of behavioural characteristics found in the larval stages that do differentiate the species (Table 1). For example *An. aquasalis*, whose name means salt (salis) water (aqua), is a vector found in coastal environments. Its larvae prefer clear, non-polluted water bodies such as mangrove swamps, lagoons and ditches [22, 23]. They can develop in fresh water sites, but it is considered a poor competitor in such habitats, especially against *An. albimanus* [15], which may be causal

*Anopheles darlingi* larvae are characteristically associated with patches of floating debris found along river margins in rural and lowland forested areas [24]. *Anopheles marajoara* is also found in lowland areas but is more common in secondary forests and is able to adapt to environments that have undergone some human intervention [21, 25] which may be a causal factor in its increasing dominance over *An. darlingi* in some localities. Forest clearance and pollution will decrease sites suitable for *An. darlingi* but increase the availability of sunlit marshy areas and

The *An. pseudopunctipennis* complex is known to be able to survive and transmit malaria at altitudes higher than many other DVS, up to 3000 m [27, 28]. Its larvae also have a defining characteristic; an apparent obligate association with filamentous *Spirogyra*-type green algae [23, 27, 29-31] (Table 1). Indeed, the removal of such algae has been shown to be a viable method

in tending to restrict the range of *An. aquasalis* to brackish locations.

ponds more suitable for *An. marajoara* [21, 26].

of control for this species [31].

species is present and understanding how it behaves.

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

*punctipennis* (Figure 3)*.*

(Table 1, [15]).

**Table 1.** Summary of bionomics of the DVS of the Americas (created by cross referencing TAG and literature searches). Filled dot (∙) indicates typical behaviour, open dot (◦) indicates non-typical behaviour but examples exist, and dashes (-) indicate no data.

**Figure 2.** Multi-species map of the nine DVS of the Americas (map reproduced from Sinka *et al*. [4])

## **4. Africa**

Across the huge and variable landscape of the African continent, there is a corresponding variability in the intensity of malaria transmission [32, 33]. Sub-saharan Africa is, however, home to localities suffering from the highest global malaria transmission levels, and hence, morbidity and mortality of malaria [17, 32, 34-36]; a consequence of the wide spread presence of the most effective and efficient vector currently known, *An. gambiae* [37, 38]. *Anopheles gambiae* is a member of the *An. gambiae* complex, which also contains other DVS including *An. arabiensis, An. merus* and *An. melas* [6, 39-42]*.* Also found in Africa is the widespread *An.*

**Figure 3.** Predicted distribution maps for a) *An. albitarsis* s.l. (n = 138); b) *An. marajoara* (n = 56); c) *An. nuneztovari* (n = 171); d) *An. pseudopunctipennis* (n = 156). The insert map in each shows the expert opinion distribution for that spe‐ cies (Maps reproduced from Sinka et al, [15]).

**Figure 2.** Multi-species map of the nine DVS of the Americas (map reproduced from Sinka *et al*. [4])

Across the huge and variable landscape of the African continent, there is a corresponding variability in the intensity of malaria transmission [32, 33]. Sub-saharan Africa is, however, home to localities suffering from the highest global malaria transmission levels, and hence, morbidity and mortality of malaria [17, 32, 34-36]; a consequence of the wide spread presence of the most effective and efficient vector currently known, *An. gambiae* [37, 38]. *Anopheles gambiae* is a member of the *An. gambiae* complex, which also contains other DVS including *An. arabiensis, An. merus* and *An. melas* [6, 39-42]*.* Also found in Africa is the widespread *An.*

**4. Africa**

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

*funestus* subgroup of which *An. funestus* is another highly effective vector, and possibly the first species to adapt to make use of humans as a food/blood source [43]. The more restricted, but still highly anthropophilic *An. moucheti* and the more widespread *An. nili* complex add to a suite of vectors within Africa that have proved highly efficient in malaria transmission and equally difficult to control [5].

Figure 1 shows those vector species that can be considered the 'primary' DVS of Africa: *An. gambiae, An. arabiensis* and *An. funestus*. Figure 4 indicates the more 'secondary' DVS, including *An. moucheti, An. nili, An. melas* and *An. merus*. Examining only the 'primary' species (Figure 1), the vector situation in Africa appears relatively simple. However for each of these species to have an extensive spread across such a large geographical area suggests a high level of adaptability and plasticity in behaviours and tolerances within all of these DVS. This plasticity is becoming more apparent as the taxonomy of the species complexes are untangled. The *An. gambiae* complex is a case in point. Originally considered as one species, the discovery of saline tolerant larval '*An. gambiae*' coastal specimens which, in cross mating experiments, produced sterile male progeny, confirmed that the salt-water tolerant and fresh-water '*An. gambiae*' were reproductively incompatible, and identified *An. melas* on the west coast and *An. merus* on the east [44-47]. The Gambiae complex is now known to consist of at least eight species [1, 6] yet this taxonomic categorisation is still a relatively recent occurrence, with the provisional inclusion of *An. quadriannulatus* B only reported in 1998 [6, 48]. Moreover, behavioural and ecological plasticity within the *An. gambiae* species itself have highlighted further potential speciation; there are now five recognised chromosomal forms (Savanna, Mopti, Forest, Bamako and Bissau) and two molecular forms (M and S) [49-51]. The M and S forms have distinctive and separate behaviours, specifically in terms of preferred larval habitats, with the S form utilising larval sites considered typical for *An. gambiae* (i.e. temporary pools or puddles that only occur after rain) whereas larvae from the M form are found in more permanent sites such as rice fields or flooded areas [52-57]. Overall, *An. gambiae* is considered highly anthropophilic (Table 2), a characteristic that is held as greatly influential in the designation of this species as the most effective malaria vector. It also tends to be reported as biting indoors and during the night when people are asleep and therefore more vulnerable [5]. Although these traits do tend to hold true in a general sense, the variability of *An. gambiae* does extend to adult behaviour and there are a number of localities where this species does not follow these commonly reported behaviours [5].

The extensive distribution of *An. arabiensis* (Figure 1) also indicates a vector with a wide range of behaviours [40, 58, 59] and although it is classified as zoophilic and exophagic (Table 2) this is often only reported in comparison with the generally highly anthropophilic and endophagic *An. gambiae* or *An. funestus* [5, 60]. *Anopheles arabiensis* is more tolerant of drier environments than the other DVS, as can be seen in Figure 1 where its range extends north (the Sahel) and south (desert and steppe of Namibia and Botswana) beyond those of either *An. gambiae* or *An. funestus.* It is noticeably absent from the humid, forested areas of western Africa (Figure 1).

Despite the zoophilic label, the feeding behaviour of *An. arabiensis* varies greatly depending upon location, host availability and the local genotype [40, 58, 59, 61] and there is some suggestion that *An. arabiensis* populations are more anthropophilic, endophilic and endopha‐ gic in western Africa whereas those in the east are more zoophilic and exophilic [62].

Beside the apparent inability to exist in the forested west of Africa, *An. arabiensis* appears to tolerate a much greater range of larval sites than *An. gambiae*. Similar to its sibling, it makes use of sunlit, temporary, shallow fresh-water habitats and the larger more permanent sites as characterised by *An. gambiae* M form, but it is also able to survive in flowing water, turbid or polluted sites and even, on occasion, brackish habitats [5, 63-66]. It readily makes use of rice fields, although its propensity for sunlit water means it is primarily found when the rice plants are small and larval numbers reduce substantially as the plants mature [67-70]. The adapta‐


Table 2: Summary of bionomics of the DVS of Africa (created by cross referencing TAG and literature searches)

to have an extensive spread across such a large geographical area suggests a high level of adaptability and plasticity in behaviours and tolerances within all of these DVS. This plasticity is becoming more apparent as the taxonomy of the species complexes are untangled. The *An. gambiae* complex is a case in point. Originally considered as one species, the discovery of saline tolerant larval '*An. gambiae*' coastal specimens which, in cross mating experiments, produced sterile male progeny, confirmed that the salt-water tolerant and fresh-water '*An. gambiae*' were reproductively incompatible, and identified *An. melas* on the west coast and *An. merus* on the east [44-47]. The Gambiae complex is now known to consist of at least eight species [1, 6] yet this taxonomic categorisation is still a relatively recent occurrence, with the provisional inclusion of *An. quadriannulatus* B only reported in 1998 [6, 48]. Moreover, behavioural and ecological plasticity within the *An. gambiae* species itself have highlighted further potential speciation; there are now five recognised chromosomal forms (Savanna, Mopti, Forest, Bamako and Bissau) and two molecular forms (M and S) [49-51]. The M and S forms have distinctive and separate behaviours, specifically in terms of preferred larval habitats, with the S form utilising larval sites considered typical for *An. gambiae* (i.e. temporary pools or puddles that only occur after rain) whereas larvae from the M form are found in more permanent sites such as rice fields or flooded areas [52-57]. Overall, *An. gambiae* is considered highly anthropophilic (Table 2), a characteristic that is held as greatly influential in the designation of this species as the most effective malaria vector. It also tends to be reported as biting indoors and during the night when people are asleep and therefore more vulnerable [5]. Although these traits do tend to hold true in a general sense, the variability of *An. gambiae* does extend to adult behaviour and there are a number of localities where this species does not follow these commonly

The extensive distribution of *An. arabiensis* (Figure 1) also indicates a vector with a wide range of behaviours [40, 58, 59] and although it is classified as zoophilic and exophagic (Table 2) this is often only reported in comparison with the generally highly anthropophilic and endophagic *An. gambiae* or *An. funestus* [5, 60]. *Anopheles arabiensis* is more tolerant of drier environments than the other DVS, as can be seen in Figure 1 where its range extends north (the Sahel) and south (desert and steppe of Namibia and Botswana) beyond those of either *An. gambiae* or *An. funestus.* It is noticeably absent from the humid, forested areas of western Africa (Figure 1).

Despite the zoophilic label, the feeding behaviour of *An. arabiensis* varies greatly depending upon location, host availability and the local genotype [40, 58, 59, 61] and there is some suggestion that *An. arabiensis* populations are more anthropophilic, endophilic and endopha‐

Beside the apparent inability to exist in the forested west of Africa, *An. arabiensis* appears to tolerate a much greater range of larval sites than *An. gambiae*. Similar to its sibling, it makes use of sunlit, temporary, shallow fresh-water habitats and the larger more permanent sites as characterised by *An. gambiae* M form, but it is also able to survive in flowing water, turbid or polluted sites and even, on occasion, brackish habitats [5, 63-66]. It readily makes use of rice fields, although its propensity for sunlit water means it is primarily found when the rice plants are small and larval numbers reduce substantially as the plants mature [67-70]. The adapta‐

gic in western Africa whereas those in the east are more zoophilic and exophilic [62].

reported behaviours [5].

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

**Table 2.** Summary of bionomics of the DVS of Africa (created by cross referencing TAG and literature searches) Filled dot (∙) indicates typical behaviour, open dot (◦) indicates non-typical behaviour but examples exist, and dashes (-) indicate no data.

bility, plasticity and general tendencies for *An. arabiensis* to feed outdoors on animals (Table 2) means that this species does not readily succumb to traditional methods of control such as IRS or ITNs [59, 71, 72].

*Anopheles funestus,* is a highly adaptable species with a large distribution across sub-saharan Africa (Figure 1). It is also a highly effective vector, and in some cases, due to a relatively high longevity plus a preference for human blood and late night biting (Table 2), is even more efficient at transmitting malaria than *An. gambiae* [38, 40, 73]. *Anopheles funestus* is the only member within the Funestus Subgroup regarded as an important vector [73], and can only be morphologically distinguished from other members at certain stages in their development, again highlighting the importance of correct species identification [38, 40, 73, 74]. Indeed, for this subgroup, such identification is rarely reported, and hence the distributions illustrated here (Figure 1) cannot distinguish the true range of this specific vector. For example in Ethiopia, only one known study has performed PCR identifications of the Funestus Group [75], indicating that only *An. parensis* (a non-vector member of the Funestus Subgroup) is present.

*Anopheles funestus* is a highly anthropophilic mosquito [5, 38, 76, 77] and its endophilic behaviour adds to a suite of behaviours that enhance its ability to effectively transmit malaria [5]. It is comparably consistent in its behaviour and has been subject to successful control via both IRS and ITNs, but some populations have shown a rapid development of insecticide resistance to pyrethroids which was considered the primary cause of epidemic malaria reported in South Africa in the late 1990s [73, 78].

The larvae of *An. funestus* are found in large permanent or semi-permanent bodies of fresh‐ water such as swamps, large ponds or lake edges [5]. They are also associated with rice cultivation in some localities, favouring older fields with mature rice plants [79-81].

## **5. Asia**

The region of Central, South and East Asia is home to 46% of the global populations at risk (PAR) of stable falciparum malaria [82] and suffers a particularly high impact of vivax malaria, with an estimated 82% of the world's PAR of *P. vivax* transmission [83]. Indeed within the 'top 10' of countries with the highest global *P. vivax* PAR estimates, seven are from Asia (China, Indonesia, Pakistan, Vietnam, the Philippines, Myanmar and Thailand) [84]. The complexity of the vector situation in the Asian-Pacific region increases the problems associated with understanding the vector/transmission environment. This region has a greater number of DVS than any other and amongst these, there are a greater number of species complexes and taxonomic complexities than anywhere else [1, 6, 16].

With at least nine out of 19 DVS found in the Asian-Pacific now considered as a species complex [1, 6], the impetus to correctly identify both the vectors and their behaviours at a specific loca‐ tion is even greater in this region than elsewhere. Indeed, even within those species not current‐ ly considered as part of a complex, behavioural variability is common, depending upon location, and in some cases to such an extent that a species considered a vector in one location

bility, plasticity and general tendencies for *An. arabiensis* to feed outdoors on animals (Table 2) means that this species does not readily succumb to traditional methods of control such as

*Anopheles funestus,* is a highly adaptable species with a large distribution across sub-saharan Africa (Figure 1). It is also a highly effective vector, and in some cases, due to a relatively high longevity plus a preference for human blood and late night biting (Table 2), is even more efficient at transmitting malaria than *An. gambiae* [38, 40, 73]. *Anopheles funestus* is the only member within the Funestus Subgroup regarded as an important vector [73], and can only be morphologically distinguished from other members at certain stages in their development, again highlighting the importance of correct species identification [38, 40, 73, 74]. Indeed, for this subgroup, such identification is rarely reported, and hence the distributions illustrated here (Figure 1) cannot distinguish the true range of this specific vector. For example in Ethiopia, only one known study has performed PCR identifications of the Funestus Group [75], indicating that only *An. parensis* (a non-vector member of the Funestus Subgroup) is present.

*Anopheles funestus* is a highly anthropophilic mosquito [5, 38, 76, 77] and its endophilic behaviour adds to a suite of behaviours that enhance its ability to effectively transmit malaria [5]. It is comparably consistent in its behaviour and has been subject to successful control via both IRS and ITNs, but some populations have shown a rapid development of insecticide resistance to pyrethroids which was considered the primary cause of epidemic malaria

The larvae of *An. funestus* are found in large permanent or semi-permanent bodies of fresh‐ water such as swamps, large ponds or lake edges [5]. They are also associated with rice

The region of Central, South and East Asia is home to 46% of the global populations at risk (PAR) of stable falciparum malaria [82] and suffers a particularly high impact of vivax malaria, with an estimated 82% of the world's PAR of *P. vivax* transmission [83]. Indeed within the 'top 10' of countries with the highest global *P. vivax* PAR estimates, seven are from Asia (China, Indonesia, Pakistan, Vietnam, the Philippines, Myanmar and Thailand) [84]. The complexity of the vector situation in the Asian-Pacific region increases the problems associated with understanding the vector/transmission environment. This region has a greater number of DVS than any other and amongst these, there are a greater number of species complexes and

With at least nine out of 19 DVS found in the Asian-Pacific now considered as a species complex [1, 6], the impetus to correctly identify both the vectors and their behaviours at a specific loca‐ tion is even greater in this region than elsewhere. Indeed, even within those species not current‐ ly considered as part of a complex, behavioural variability is common, depending upon location, and in some cases to such an extent that a species considered a vector in one location

cultivation in some localities, favouring older fields with mature rice plants [79-81].

IRS or ITNs [59, 71, 72].

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

**5. Asia**

reported in South Africa in the late 1990s [73, 78].

taxonomic complexities than anywhere else [1, 6, 16].

**Figure 4.** Multi-species map of Africa indicating the distributions of the four 'secondary' but still important, DVS. (map reproduced from Sinka et al. [4]).

may be only of secondary importance, or even a non vector in another [14]. For example, *An. an‐ nularis* has a range extending across India, down through South-East Asia, across many of the Indonesian islands down to and including Timor Island [14]. However, it only has a focal role in malaria transmission in selected areas of India. Elsewhere it is considered of little importance [85-91] (hence, *An. annularis,* along with *An. aconitus* and *An. subpictus*; all listed in Table 3 as DVS, are not included in the multi-species maps shown here in Figure 1 and Figure 5, as overall, they do not have as great an impact in malaria transmission as other species in the region).

Table 3a: Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches)


**Table 3a.** Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches) Filled dot (∙) indicates typical behaviour, open dot (◦) indicates non-typical behaviour but examples exist, and dashes (-) indicate no data.


Table 3b: Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches)

Host Biting Resting

Table 3a: Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches)

Host Biting Resting

Other Anthropo-

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

philic Zoophilic Endophagic Exophagic Endophilic Exophilic

Species Larval site characteristics

and dashes (-) indicate no data.

*An. aconitus* 

*An. annularis*

Sunlit, fresh, clear (occasionally turbid), still or

flowing water with higher plants and algae

Particularly favours both coast plain and upland rice fields as larval sites

Vector role depends on location. Possible

complex of two (species A and B) siblings,

but these do not appear to be linked to

variable vector capacity

Primarily found in forested environments

The siblings within the complex are yet to

be fully resolved and their distributions are

unclear.

Bionomics dependent on sibling: Sp E = Anthropophilic; Sp A, B, C , D = Zoophilic)

Bionomics dependent on sibling but the

two main vectors are *An. dirus* and *An.* 

*baimaii. Anopheles scanloni* is also

anthropophilic but plays more focal role in

transmission in Thailand

*Anopheles farauti*, *An. hinesorum* and *An.* 

*farauti* No. 4 are the only siblings considered

to be important malaria vectors

Historically confused/misidentified as *An.* 

*minimus*. All records of *An. minimus* from

the Philippines, Sabah (Malaysia) and

Indonesia are now considered to be *An.* 

*flavirostris* 

Bionomics dependent on sibling. Species S is

the most anthropophilic and endophilic and

is the main vector of the complex. Species T

and U are primarily zoophilic, exophagic

and exophilic and non or poor vectors in

India.

(occasionally without vegetation)

Sunlit, fresh, clear (occasionally turbid), still or

flowing water with higher plants and algae

(occasionally without vegetation)

*An. balabacensis* Shaded (occasionally sunlit), fresh, still water with or without higher plants or algae

Sunlit or shaded, clear or turbid, still or flowing

water with higher plants or algae (occasionally

without vegetation)

Sunlit, fresh (occasionally brackish) clear

(occasionally turbid), still or flowing water with or

without higher plants or algae

Shaded, fresh, clear or turbid, still water without vegetation

Sunlit or shaded, fresh or brackish, clear or turbid,

stagnant (occasionally flowing) water with higher

plants or algae (occasionally without vegetation)

Shaded, fresh, clear, flowing (occasionally still)

water with higher plants or algae (occasionally

without vegetation)

Sunlit, fresh, flowing (occasionally still), water with

higher plants or algae (occasionally without

vegetation)


*An. barbirostris* 

complex

*An. culicifacies* 

**Table 3a.** Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches) Filled dot (∙) indicates typical behaviour, open dot (◦) indicates non-typical behaviour but examples exist,

complex

*An. dirus* complex *An. farauti* complex

*An. flavirostris* 

*An. fluviatilis* 

complex


**Table 3b.** Summary of bionomics of the DVS of the Asian-Pacific (created by cross referencing TAG and literature searches) Filled dot (∙) indicates typical behaviour, open dot (◦) indicates non-typical behaviour but examples exist, and dashes (-) indicate no data.

Unfortunately the high number of vectors in this region, and their complexity, have not equated to a higher level of knowledge, despite considerable effort from local scientists as well as from US military entomologists during WWII and the Vietnam War. Indeed, amongst all 41 DVS mapped [5, 14, 15], the two species with the lowest number of occurrence points, were both from the Asian-Pacific region (*An. leucosphyrus/An. latens* (12 points) and *An. balabacen‐ sis* (14 points)). There are also limitations when attempting to categorise vector behaviour as again, some species are very poorly studied, or those data that do exist are compromised by unreliable identifications due to the lack of robust techniques that are now available. Hence the summaries given here should be considered as potentially transient and may be updated as more data is collected and systematic PCR-based assays for species identification are applied [92-94]. Accepting these caveats, it must also be noted that the behavioural information presented is the culmination of a comprehensive review of the published literature combined with the 'on the ground' knowledge of highly competent and experienced experts and as such, do represent the best currently available species distribution maps and bionomics knowledge. Here focus is on the species and species complexes designated as the most influential across the region, including *An. culicifacies, An. fluviatilis* and *An. stephensi* across the Indian subcon‐ tinent; *An. dirus* and *An. minimus* within south-east Asia and the DVS members of the Punc‐ tulatus Group in the Pacific region. More detailed bionomics information and single species distribution maps for all 19 Asian-Pacific DVS are given in Sinka *et al.* [14].

#### **5.1. Indian subcontinent (Bangladesh, Bhutan, India, Nepal, Pakistan, Sri Lanka)**

The Indian subcontinent is densely populated giving rise to very high figures for the popula‐ tion at risk from malaria, however, the levels of risk are typically lower than those found in sub-saharan Africa. The majority of people at risk are living in areas of low endemicity (<5% prevalence) or areas of unstable malaria transmission where the disease is not endemic. This is true for both falciparum and vivax malaria. A smaller number of people living in India itself are at risk of much higher levels of falciparum malaria (>40% prevalence), possibly equalling the levels of risk found in sub-saharan Africa although there is a need for more data to support these figures [82].

The range of the *An. culicifacies* complex extends far beyond the Indian subcontinent; it also encompasses large areas of Southeast Asia including Vietnam, Cambodia, Lao PDR and southern China and reaches as far as Yemen in the Middle East with a small distribution in Eritrea as well as Nepal, India, Pakistan and Sri Lanka [14] (Figure 1). Despite this extensive distribution, the complex has only been studied in any detail in India and Sri Lanka [95-100]. Of the five species (A, B, C, D, E) of the complex, four are considered vectors in India (A, C, D and E) [101]. Of these four, species E is a particularly efficient vector due to its highly anthro‐ pophilic and endophilic behaviour and is considered the most important vector of both *P. falciparum* and *P. vivax* in southern India and Sri Lanka [102, 103]. The remaining three species (A, C and D) are primarily zoophilic and tend to be considered as playing more minor roles in malaria transmission [97]. Indeed, the highly zoophilic behaviour of Species B means it is often considered a non-vector [99, 104].

Members of the complex are found at a wide range of altitudes, from plains to hilly and mountainous areas [100]. The habitats they utilise are also varied and include forested and deforested ecotypes and irrigated areas. Consequentially, the larval sites they inhabit are also wide-ranging and include man-made habitats such as irrigation canals, borrow pits, domestic wells, tanks and gutters as well as natural sites such as stream margins and rock pools [96, 100, 101, 104-107]. A tolerance to brackish water has also been reported [96, 108], although fresh‐ water sites appear to be preferred. With many aspects of behaviour dependent on sibling, further investigations, coupled with confirmed identifications of each species, are needed before targeted vector control can be applied.

Unfortunately the high number of vectors in this region, and their complexity, have not equated to a higher level of knowledge, despite considerable effort from local scientists as well as from US military entomologists during WWII and the Vietnam War. Indeed, amongst all 41 DVS mapped [5, 14, 15], the two species with the lowest number of occurrence points, were both from the Asian-Pacific region (*An. leucosphyrus/An. latens* (12 points) and *An. balabacen‐ sis* (14 points)). There are also limitations when attempting to categorise vector behaviour as again, some species are very poorly studied, or those data that do exist are compromised by unreliable identifications due to the lack of robust techniques that are now available. Hence the summaries given here should be considered as potentially transient and may be updated as more data is collected and systematic PCR-based assays for species identification are applied [92-94]. Accepting these caveats, it must also be noted that the behavioural information presented is the culmination of a comprehensive review of the published literature combined with the 'on the ground' knowledge of highly competent and experienced experts and as such, do represent the best currently available species distribution maps and bionomics knowledge. Here focus is on the species and species complexes designated as the most influential across the region, including *An. culicifacies, An. fluviatilis* and *An. stephensi* across the Indian subcon‐ tinent; *An. dirus* and *An. minimus* within south-east Asia and the DVS members of the Punc‐ tulatus Group in the Pacific region. More detailed bionomics information and single species

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

distribution maps for all 19 Asian-Pacific DVS are given in Sinka *et al.* [14].

these figures [82].

often considered a non-vector [99, 104].

**5.1. Indian subcontinent (Bangladesh, Bhutan, India, Nepal, Pakistan, Sri Lanka)**

The Indian subcontinent is densely populated giving rise to very high figures for the popula‐ tion at risk from malaria, however, the levels of risk are typically lower than those found in sub-saharan Africa. The majority of people at risk are living in areas of low endemicity (<5% prevalence) or areas of unstable malaria transmission where the disease is not endemic. This is true for both falciparum and vivax malaria. A smaller number of people living in India itself are at risk of much higher levels of falciparum malaria (>40% prevalence), possibly equalling the levels of risk found in sub-saharan Africa although there is a need for more data to support

The range of the *An. culicifacies* complex extends far beyond the Indian subcontinent; it also encompasses large areas of Southeast Asia including Vietnam, Cambodia, Lao PDR and southern China and reaches as far as Yemen in the Middle East with a small distribution in Eritrea as well as Nepal, India, Pakistan and Sri Lanka [14] (Figure 1). Despite this extensive distribution, the complex has only been studied in any detail in India and Sri Lanka [95-100]. Of the five species (A, B, C, D, E) of the complex, four are considered vectors in India (A, C, D and E) [101]. Of these four, species E is a particularly efficient vector due to its highly anthro‐ pophilic and endophilic behaviour and is considered the most important vector of both *P. falciparum* and *P. vivax* in southern India and Sri Lanka [102, 103]. The remaining three species (A, C and D) are primarily zoophilic and tend to be considered as playing more minor roles in malaria transmission [97]. Indeed, the highly zoophilic behaviour of Species B means it is

Again, despite a large distribution (Figure 1) [14], the behaviour and ecology of the *An. fluviatilis* complex has only been studied in any detail in two countries: India and Iran. The complex consists of three species, currently and informally designated species S, T and U [109] and an as yet unconfirmed form V [110]. The complex is distributed widely across the forested hills and mountains of southwestern Asia (Iran, Pakistan, Afghanistan, India, Nepal, Bangla‐ desh and Myanmar) [104, 111-114]. Members of the complex also exhibit behavioural differ‐ ences, with the anthropophilic and endophilic *An. fluviatilis* S categorised as a highly efficient vector in India [112], whereas both the zoophilic Species T and U, which also tend to feed and rest outdoors, are considered poor or non-vectors [115, 116]. However, species T is considered an important vector in Pakistan, Nepal and Iran [117, 118]. The larvae of this complex are associated with slow-flowing water in streams or river margins [119-124] (Table 3a).

The ability of the larval stages of *An. stephensi* (Table 3b) to develop in urban areas, making use of artificial containers such as domestic wells, overhead water tanks, room coolers, cisterns and roof gutters and in water bodies in construction sites and other industrial localities, brings malaria transmission into densely populated areas including the major cities of India such as Delhi [125, 126]. In general, malaria is considered to be a disease confined to rural environ‐ ments, as a simple consequence of the tendency of anophelines to search for clean and unpolluted larval habitats and thus the existence of *An. stephensi* in such areas is a defining characteristic of the species.

*Anopheles stephensi* is found across the Indian subcontinent [14], extending from the Arabian Peninsula, through Iran and Iraq, across to Bangladesh, southern China, Myanmar and Thailand (Figure 1) [127-129]. It is typically described as an endophilic and endophagic species despite a tendency to bite outdoors during warmer months when people are more active outdoors [130, 131]. Host availability seems to be a driver to a variable anthropophily for this species, and therefore in urban areas, there appears to be a greater tendency for biting humans [132, 133], and therefore an increased risk of malaria transmission.

#### **5.2. Southeast Asia (Cambodia, Laos, Myanmar, Thailand, Vietnam)**

Human populations in Southeast Asia, with the exception of Myanmar, are typically exposed to low levels of falciparum and vivax malaria endemicity, unstable malaria transmission or are living in malaria-free areas. The majority of the population in Myanmar live in areas with low malaria endemicity but significant numbers live in areas of moderate (5-40% prevalence) and high (>40% falciparum prevalence or >7% vivax prevalence) risk. There is increasing evidence that knowlesi malaria is transmitted from monkeys to humans in this region, particularly in the South, but the level of risk is currently unmeasured [82, 83, 134, 135].

The Dirus and Minimus complexes both contain species considered particularly efficient in transmitting malaria. Indeed, the *An. dirus* complex, due to its longevity and the highly anthropophilic behaviour of its members (Table 3a), is considered to be the dominant vector group in any area where its species exist [136]. However, due to its close relationship with members of the Leucosphyrus Complex, there has been considerable confusion in its identity in the published literature [137]. Species of the *An. dirus* complex are forest dwellers, existing in mountains and foothills, cultivated forests and forest fringes. There are eight members, *An*. *dirus* (formerly *An. dirus* species A), *An*. *cracens* (formerly sp. B), *An*. *scanloni* (formerly sp. C), *An*. *baimaii* (formerly sp. D), *An*. *elegans* (formerly sp. E), *An*. *nemophilous* (formerly sp. F), *An. takasagoensis* and the recently added species informally named *An.* Aff. *Takasagoensis*[138-142]. Of these species, *An. dirus* and *An. baimaii* are vectors of particular note [143-146]. As mentioned above, they are both highly anthropophilic, but their efficiency in transmitting both vivax and falciparum malaria is enhanced through biting humans both in and outdoors and of avoiding most conventional control methods by resting mainly outdoors (Table 3a) [111, 143, 145, 147-150].

Larvae are typically found in small, temporary, shallow and shaded pools of fresh water within the forest environment, such as puddles, pits, animal footprints, wheel ruts, hollow logs and slow flowing streams (Table 3a) [151-154].

Species of the *An. minimus* complex are also found in the hilly forested regions, but unlike the Dirus Complex, are restricted to mainland Southeast Asia (Figure 1) [14]. The complex contains three sibling species, *An. minimus* (formerly species A), *An. harrisoni* (formerly sp. C) and *An. yaeyamaensis* (formerly sp. E) [155-157]. This latter species has a very restricted distribution, only being found in the Ryukyu Archipelago in southern Japan, where it was considered a major malaria vector before the successful eradication of the disease in 1962 [158, 159]. Both *An. minimus* and *An. harrisoni* are, however, still considered primary vectors across their range, which encompasses much of Southeast Asia [14], although all historical records of *An. minimus* in Indonesia are now considered to actually be *An. flavirostris*. *Anopheles minimus* has a more adaptable nature than *An. harrisoni* allowing it to occupy a large variety of habitats, including dense canopy forests to open rice fields [14] and therefore has a greater distribution. *Anopheles harrisoni* tends to be restricted to deforested agricultural sites [160, 161]. *Anopheles minimus* is also highly variable in its behaviour (Table 3b), being an opportunistic mosquito, although in some reports this may be a consequence of the species complex not being fully identified [160]. Larvae of the *An. minimus* complex are found in small or moderate streams with slow-running, clear water. Females lay their eggs in the partially shaded grassy margins (Table 3b)[162-165]. Larvae have also been found in water containers in Hanoi [166].

*Anopheles minimus* is considered primarily anthropophilic, but its choice of blood meal can also be influenced by the availability of alternative animal hosts such as domestic cattle [148, 167, 168]. Biting habit is also variable (Table 3b), and dependent on location, with reports of endophagic behaviour in India, Thailand and central Vietnam, but exophagic behaviour in Cambodia and northern Vietnam [148, 165, 169]. The same is found for resting behaviour, although there appears to be a large influence of the use of IRS on resting location and population densities for this species [170, 171]. Overall, *An. harrisoni* appears more consistent in behaviour, generally reported as exophagic, exophilic and zoophilic and thus potentially the less dominant vector [172, 173].

evidence that knowlesi malaria is transmitted from monkeys to humans in this region, particularly in the South, but the level of risk is currently unmeasured [82, 83, 134, 135].

The Dirus and Minimus complexes both contain species considered particularly efficient in transmitting malaria. Indeed, the *An. dirus* complex, due to its longevity and the highly anthropophilic behaviour of its members (Table 3a), is considered to be the dominant vector group in any area where its species exist [136]. However, due to its close relationship with members of the Leucosphyrus Complex, there has been considerable confusion in its identity in the published literature [137]. Species of the *An. dirus* complex are forest dwellers, existing in mountains and foothills, cultivated forests and forest fringes. There are eight members, *An*. *dirus* (formerly *An. dirus* species A), *An*. *cracens* (formerly sp. B), *An*. *scanloni* (formerly sp. C), *An*. *baimaii* (formerly sp. D), *An*. *elegans* (formerly sp. E), *An*. *nemophilous* (formerly sp. F), *An. takasagoensis* and the recently added species informally named *An.* Aff. *Takasagoensis*[138-142]. Of these species, *An. dirus* and *An. baimaii* are vectors of particular note [143-146]. As mentioned above, they are both highly anthropophilic, but their efficiency in transmitting both vivax and falciparum malaria is enhanced through biting humans both in and outdoors and of avoiding most conventional control methods by resting mainly outdoors (Table 3a) [111, 143, 145,

Larvae are typically found in small, temporary, shallow and shaded pools of fresh water within the forest environment, such as puddles, pits, animal footprints, wheel ruts, hollow logs and

Species of the *An. minimus* complex are also found in the hilly forested regions, but unlike the Dirus Complex, are restricted to mainland Southeast Asia (Figure 1) [14]. The complex contains three sibling species, *An. minimus* (formerly species A), *An. harrisoni* (formerly sp. C) and *An. yaeyamaensis* (formerly sp. E) [155-157]. This latter species has a very restricted distribution, only being found in the Ryukyu Archipelago in southern Japan, where it was considered a major malaria vector before the successful eradication of the disease in 1962 [158, 159]. Both *An. minimus* and *An. harrisoni* are, however, still considered primary vectors across their range, which encompasses much of Southeast Asia [14], although all historical records of *An. minimus* in Indonesia are now considered to actually be *An. flavirostris*. *Anopheles minimus* has a more adaptable nature than *An. harrisoni* allowing it to occupy a large variety of habitats, including dense canopy forests to open rice fields [14] and therefore has a greater distribution. *Anopheles harrisoni* tends to be restricted to deforested agricultural sites [160, 161]. *Anopheles minimus* is also highly variable in its behaviour (Table 3b), being an opportunistic mosquito, although in some reports this may be a consequence of the species complex not being fully identified [160]. Larvae of the *An. minimus* complex are found in small or moderate streams with slow-running, clear water. Females lay their eggs in the partially shaded grassy margins

(Table 3b)[162-165]. Larvae have also been found in water containers in Hanoi [166].

*Anopheles minimus* is considered primarily anthropophilic, but its choice of blood meal can also be influenced by the availability of alternative animal hosts such as domestic cattle [148, 167, 168]. Biting habit is also variable (Table 3b), and dependent on location, with reports of endophagic behaviour in India, Thailand and central Vietnam, but exophagic behaviour in Cambodia and northern Vietnam [148, 165, 169]. The same is found for resting behaviour,

147-150].

slow flowing streams (Table 3a) [151-154].

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

#### **5.3. Asia-Pacific (Indonesia, Papua New Guinea, Philippines, Solomon Islands, Vanuatu, Timor Leste)**

Human populations in the Asia-Pacific, with the exception of Papua New Guinea and Indonesian Papua, typically live in areas with low levels of falciparum and vivax malaria endemicity (<5% prevalence), or unstable malaria transmission or that are malaria-free. The majority of the population in Papua New Guinea live in areas with low malaria endemicity (<5% prevalence) but significant numbers live in areas of moderate (5-40% prevalence) risk. [82, 83].

The DVS in the Asia-Pacific region (as categorised here) are dominated by three of the 12 members of the Punctulatus Group, namely *An. farauti* complex, *An. koliensis* and *An. punctu‐ latus* complex (Figure 5). *Anopheles farauti* complex has the widest distribution of these vectors (and of the Punctulatus Group as a whole), extending from the Maluku island group (Indo‐ nesia) in the west to Vanuatu in the east, including northern Australia in between. Of the eight species within the *An. farauti* complex, only three are considered to be main vectors, *An. farauti* s.s., *An. hinesorum* (formerly *An. farauti* No. 2) and *An. farauti* No. 4, although there is some, albeit limited and circumstantial, evidence of *An. farauti* No. 6 as a primary vector in the highlands, river valleys and intramontane plains of New Guinea [174, 175].

Despite being the most studied member of the Punctulatus Group, there are still many unknowns regarding the ecology and behaviour of the species of the *An. farauti* complex, with added uncertainty due to apparent variability in behaviour depending on location (based on reports of undifferentiated members of the complex). However, there are some trends that appear relatively consistent, for example, members seem to be mainly anthropophilic, although they will feed on domestic livestock, birds and other animals where available (Table 3a) [14]. Both endo- and exophagic feeding on humans has been reported, and some, albeit limited, endophilic behaviour. On the whole, females tend to be early biters (18.00 – 20.00), biting and resting outdoors [14, 176].

The larvae of *An. farauti* complex are able to make use of a large variety of water sources, both sunlit and shaded, but tend to be found in natural, rain-fed temporary pools through to semipermanent/permanent bodies of ground water, often with floating or emergent vegetation. Within the complex, a defining trait of *An. farauti* s.s. is its ability to tolerate brackish larval sites, and hence this species is found mainly on the coast in pools within mangroves containing high organic debris and subject to tidal fluctuations. They are also found in natural swamps, oxbows, fish ponds, ditches, borrow pits and pools along stream or river margins [14]. Other members of the complex may have greater or lesser salinity tolerance, but this is one uncer‐ tainty that still needs to be confirmed, along with many other aspects of behaviour within the complex.

*Anopheles koliensis* plays an important role in transmitting malaria wherever it is found, possibly due to its strong anthropophily (Table 3b). Its range is essentially limited to New Guinea Island (Figure 5) with only a patchy distribution in the Solomon Islands, where it is absent in some areas despite a presence of apparently suitable environments [177-179]. Where it is found, it will bite in or outdoors, but is rarely found resting indoors [180]. The larvae of this species are found in more permanent habitats than those of either the *An. farauti* or *An. punctulatus* complexes [180, 181]. Typical larval sites include sunlit irrigation ditches, and ponds containing floating and/or emergent vegetation, often in close association with humans. They are never found in brackish water [14].

**Figure 5.** Multi-species map of South-East Asia and the Asia-Pacific region indicating the distribution of 13 DVS of par‐ ticular importance. (map reproduced from Sinka et al. [4]).

The last DVS in this region is a member of the *An. punctulatus* complex. The complex contains two species, *An. punctulatus* and *An.* sp. near *punctulatus* [182] of which the latter is relatively uncommon and restricted to a few remote highland localities on New Guinea Island where little is known of its ability to transmit malaria. On the other hand, *Anopheles punctulatus* is a highly efficient malaria vector across much of its range, which extends within lowland valleys and plains, and up to altitudes of 2000m across New Guinea Island and the Solomon Islands [174], although its impact and importance as a vector appears reduced in this eastward end of its range [14, 178, 179]. As with other vectors within the Punctulatus Group, *An. punctulatus* feeds readily on humans both in and outdoors and is mainly found resting outdoors (Table 3b)[181, 183, 184]. This species is particularly adept at exploiting disturbed environments, such as those caused by land clearance or areas subject to drought conditions, where receding rivers result in small temporary pools rapidly colonised by larvae. Such colonisations can result in explosive adult populations and subsequent severe and unpredictable outbreaks of malaria [185, 186]. The typical larval sites utilised by this species reflect the conditions found in such disturbed ecologies, i.e. scattered temporary pools of fresh water, generally sunlit and shallow, containing either clear or turbid water with little or no vegetation [14]. Eggs can cope with some level of desiccation and larvae can survive in damp mud for several days during drought conditions [183]. They are also able to withstand high water temperatures (over 40o C) where they grow rapidly with particularly short development time (5-9 days to adults), occasionally resorting to cannibalism to survive, (Bangs, pers com; [14]).

## **6. Map methodology**

*Anopheles koliensis* plays an important role in transmitting malaria wherever it is found, possibly due to its strong anthropophily (Table 3b). Its range is essentially limited to New Guinea Island (Figure 5) with only a patchy distribution in the Solomon Islands, where it is absent in some areas despite a presence of apparently suitable environments [177-179]. Where it is found, it will bite in or outdoors, but is rarely found resting indoors [180]. The larvae of this species are found in more permanent habitats than those of either the *An. farauti* or *An. punctulatus* complexes [180, 181]. Typical larval sites include sunlit irrigation ditches, and ponds containing floating and/or emergent vegetation, often in close association with humans.

**Figure 5.** Multi-species map of South-East Asia and the Asia-Pacific region indicating the distribution of 13 DVS of par‐

The last DVS in this region is a member of the *An. punctulatus* complex. The complex contains two species, *An. punctulatus* and *An.* sp. near *punctulatus* [182] of which the latter is relatively uncommon and restricted to a few remote highland localities on New Guinea Island where little is known of its ability to transmit malaria. On the other hand, *Anopheles punctulatus* is a highly efficient malaria vector across much of its range, which extends within lowland valleys and plains, and up to altitudes of 2000m across New Guinea Island and the Solomon Islands [174], although its impact and importance as a vector appears reduced in this eastward end of its range [14, 178, 179]. As with other vectors within the Punctulatus Group, *An. punctulatus*

They are never found in brackish water [14].

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

ticular importance. (map reproduced from Sinka et al. [4]).

A full description of the methodology used to create the individual and multi-species maps is given in Sinka *et al* [15] and Sinka *et al* [4].

The maps presented here were created using the Boosted Regression Tree (BRT) environmental niche modelling method [187, 188]. This method uses spatially defined presence data and environmental and climatic variables to identify the conditions that typify a species' habitat. The model then identifies all locations where such conditions exist and therefore other localities where the species could potentially occur (i.e. its fundamental niche). It also provides an estimate of the probability of occurrence, i.e. applying a numerical value to indicate the conditions within the acceptable range of a species. The multi-species maps show only presence pixels with a probability value greater than 0.5 for each species.

To create the multi-species maps, the individual species distributions were overlaid ensuring the most dominant species (established through consultation with a technical advisory group of vector experts) was uppermost. Where more than one species was considered dominant in an area the species distributions were merged.

## **7. Conclusions**

The maps given in this chapter are presented with the caveat that they represent only the beginning of a process to establish the distribution of these vectors. As with all species distribution modelling, the accuracy of the output is limited by the amount and quality of the data that is available to the model. The data must be accurately geo-referenced and reflect the true and full identity of the species to be modelled. Our maps were created using the most comprehensive database of species occurrence currently available, yet still, for many of the DVS, the quality of the data is ambiguous and the quantity is poor. However, as more reliable and repeatable methods of species identification are developed, species occurrence data and the corresponding bionomics will be better understood as the taxonomy of many of these species are resolved. Moreover, a greater commitment for data sharing between research groups, public health officials, modellers and map makers is beginning to increase the quantity and quality of data available and subsequently, increasingly accurate maps and a greater understanding of transmission dynamics, combined with the benefits of targeted vector control, is making the prospect of the global elimination of malaria a much more realistic goal.

## **Acknowledgements**

The information detailed in this chapter is based on a study originally conceived by Simon Hay and completed in collaboration with an esteemed group of vector experts, who were generous with both their time and expertise, and without whom, the bionomics sections would be a great deal shorter and the maps a great deal poorer. I would therefore like to thank Michael J. Bangs, Theeraphap Chareonviriyaphap, Maureen Coetzee, Ralph E. Harbach, Janet Hemi‐ ngway, Sylvie Manguin, Charles M. Mbogo and Yasmin Rubio-Palis. Thanks also to Catherine Moyes for providing malaria parasite backgound information and for proof reading this work.

## **Author details**

Marianne E. Sinka\*

Address all correspondence to: marianne.sinka@zoo.ox.ac.uk

Spatial Ecology and Epidemiology Group, Tinbergen Building, Department of Zoology, University of Oxford, South Parks Road, Oxford, UK

## **References**


[4] Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, Mbogo CM, Hemingway J, Patil AP, Temperley WH, Gething PW, Kabaria CW, Burkot TR, Harbach RE, Hay SI. A global map of dominant malaria vectors. Parasit Vectors. 2012;5(69).

the corresponding bionomics will be better understood as the taxonomy of many of these species are resolved. Moreover, a greater commitment for data sharing between research groups, public health officials, modellers and map makers is beginning to increase the quantity and quality of data available and subsequently, increasingly accurate maps and a greater understanding of transmission dynamics, combined with the benefits of targeted vector control, is making the prospect of the global elimination of malaria a much more realistic goal.

The information detailed in this chapter is based on a study originally conceived by Simon Hay and completed in collaboration with an esteemed group of vector experts, who were generous with both their time and expertise, and without whom, the bionomics sections would be a great deal shorter and the maps a great deal poorer. I would therefore like to thank Michael J. Bangs, Theeraphap Chareonviriyaphap, Maureen Coetzee, Ralph E. Harbach, Janet Hemi‐ ngway, Sylvie Manguin, Charles M. Mbogo and Yasmin Rubio-Palis. Thanks also to Catherine Moyes for providing malaria parasite backgound information and for proof reading this work.

Spatial Ecology and Epidemiology Group, Tinbergen Building, Department of Zoology,

[1] Genus *Anopheles* Meigen, 1818. *Mosquito Taxonomic Inventory* [database on the Inter‐ net]2011 [cited 2 December 2011]. Available from: http://mosquito-taxonomic-inven‐

[2] Service MW, Townson H. The *Anopheles* vector. In: Gilles HM, Warrell DA, editors.

[3] Hay SI, Sinka ME, Okara RM, Kabaria CW, Mbithi PM, Tago CC, Benz D, Gething PW, Howes RE, Patil AP, Temperley WH, Bangs MJ, Chareonviriyaphap T, Elyazar IR, Harbach RE, Hemingway J, Manguin S, Mbogo CM, Rubio-Palis Y, Godfray HC. Developing global maps of the dominant *Anopheles* vectors of human malaria. PLoS

Essential Malariology. Fourth edition ed. London: Arnold; 2002. p. 59-84.

**Acknowledgements**

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

**Author details**

Marianne E. Sinka\*

**References**

tory.info.

Med. 2010;7(2):e1000209.

Address all correspondence to: marianne.sinka@zoo.ox.ac.uk

University of Oxford, South Parks Road, Oxford, UK


[29] Bond JG, Novelo-Gutierrez R, Ulloa A, Rojas JC, Quiroz-Martinez H, Williams T. Diversity, abundance, and disturbance response of Odonata associated with breeding sites of *Anopheles pseudopunctipennis* (Diptera: Culicidae) in southern Mexico. Environ Entomol. 2006 Dec;35(6):1561-8.

[16] Manguin S, Boëte C. Global impact of mosquito biodiversity, human vector-borne diseases and environmental change. In: Lopez-Pujol J, (ed.). The importance of biological interactions in the study of biodiversity. Rijeka, Croatia: InTech open access

[17] Hay SI, Guerra CA, Gething PW, Patil AP, Tatem AJ, Noor AM, Kabaria CW, Manh BH, Elyazar IR, Brooker S, Smith DL, Moyeed RA, Snow RW. A world malaria map: *Plasmodium falciparum* endemicity in 2007. PLoS Med. 2009 Mar 24;6(3):e1000048.

[18] WHO/PAHO (World Health Organization/Pan American Health Organization). Malaria in the Americas: time series epidemiological data from 2000 to 2007. Wash‐ ington DC: Pan American Health Organization, Regional Office for the Americas 2008.

[19] WHO/PAHO (World Health Organization/Pan American Health Organization). Regional strategic plan for malaria in the Americas 2006-2010. Washington, DC: Pan

[20] WHO (World Health Organization). Global strategic framework for integrated vector

[21] Conn JE, Wilkerson RC, Segura MN, de Souza RT, Schlichting CD, Wirtz RA, Povoa MM. Emergence of a new neotropical malaria vector facilitated by human migration

[22] Grillet ME, Montañez H, Berti J. Estudio biosistemático y ecológico de *Anopheles aquasalis* y sus implicaciones para el control de la malaria en el estado Sucre: II. Ecología

[23] Manguin S, Peyton EL, James AC, Roberts DR. Apparent changes in the abundance and distribution of *Anopheles* species on Grenada Island. J Am Mosq Control Assoc.

[24] Brochero HL, Rey G, Buitrago LS, Olano VA. Biting activity and breeding sites of *Anopheles* species in the municipality Villavicencio, Meta, Colombia. J Am Mosq

[25] Moreno JE, Rubio-Palis Y, Acevedo P. Identificación de criaderos de anofelinos en un área endémica del estado Bolívar, Venezuela. Bol Malariol San Amb 2000;40:21-30.

[26] Moreno JE, Rubio-Palis Y, Paez E, Perez E, Sanchez V. Abundance, biting behaviour and parous rate of anopheline mosquito species in relation to malaria incidence in gold-

mining areas of southern Venezuela. Med Vet Entomol. 2007 Dec;21(4):339-49.

[27] Manguin S, Roberts DR, Peyton EL, Rejmankova E, Pecor J. Characterization of *Anopheles pseudopunctipennis* larval habitats. J Am Mosq Control Assoc. 1996;12(4):

[28] Rubio-Palis Y, Zimmerman RH. Ecoregional classification of malaria vectors in the

neotropics. J Med Entomol. 1997 Sep;34(5):499-510.

American Health Organization, Regional Office for the Americas 2006.

management. Geneva: World Health Organization 2004.

and changes in land use. Am J Trop Med Hyg. 2002;66(1):18-22.

de sus criaderos. Bol Dir Malariol San Amb 1998;38(1):38-46.

Publisher; 2011. p. 390.

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

1993;9(4):403-7.

619-26.

Control Assoc. 2005;21(2):182-6.


[56] Diabate A, Dabire RK, Heidenberger K, Crawford J, Lamp WO, Culler LE, Lehmann T. Evidence for divergent selection between the molecular forms of *Anopheles gambiae*: role of predation. BMC Evol Biol. 2008 Jan 11;8(1):5.

[42] Bryan JH. *Anopheles gambiae* and *A. melas* at Brefet, The Gambia, and their role in malaria

[43] Charlwood JD, Smith T, Kihonda J, Heiz B, Billingsley PF, Takken W. Densityindependent feeding success of malaria vectors (Diptera: Culicidae) in Tanzania. Bull

[44] Gelfand HM. *Anopheles gambiae* Giles and *Anopheles melas* Theobald in a coastal area of

[45] Muirhead-Thomson RC. Studies on salt-water and fresh-water *Anopheles gambiae* on

[46] Muirhead-Thomson RC. Studies on *Anopheles gambiae* and *A. melas* in and around

[47] Muirhead-Thomson RC. Studies on the breeding places and control of *Anopheles gambiae* and *A. gambiae* var. *melas* in coastal districts of Sierra Leone. Bull Entomol Res.

[48] Hunt RH, Coetzee M, Fettene M. The *Anopheles gambiae* complex: a new species from

[49] Coluzzi M, Petrarca V, Dideco MA. Chromosomal inversion intergradation and

[50] Della Torre A, Fanello C, Akogbeto M, Dossou-yovo J, Favia G, Petrarca V, Coluzzi M. Molecular evidence of incipient speciation within *Anopheles gambiae* s.s. in West Africa.

[51] Fanello C, Petrarca V, della Torre A, Santolamazza F, Dolo G, Coulibaly M, Alloueche A, Curtis CF, Toure YT, Coluzzi M. The pyrethroid knock-down resistance gene in the *Anopheles gambiae* complex in Mali and further indication of incipient speciation within

[52] Caputo B, Nwakanma D, Jawara M, Adiamoh M, Dia I, Konate L, Petrarca V, Conway DJ, Della Torre A. *Anopheles gambiae* complex along The Gambia river, with particular reference to the molecular forms of *An. gambiae* s.s. Malar J. 2008 Sep 22;7(1):182.

[53] Costantini C, Ayala D, Guelbeogo WM, Pombi M, Some CY, Bassole IH, Ose K, Fotsing JM, Sagnon N, Fontenille D, Besansky NJ, Simard F. Living at the edge: biogeographic patterns of habitat segregation conform to speciation by niche expansion in *Anopheles*

[54] Della Torre A, Costantini C, Besansky NJ, Caccone A, Petrarca V, Powell JR, Coluzzi M. Speciation within *Anopheles gambiae*-the glass is half full. Science. 2002 Oct

[55] Della Torre A, Tu ZJ, Petrarca V. On the distribution and genetic differentiation of *Anopheles gambiae* s.s. molecular forms. Insect Biochem Molec. 2005 Jul;35(7):755-69.

Liberia, West Africa. Trans R Soc Trop Med Hyg. 1955 Nov;49(6):508-27.

transmission. Ann Trop Med Parasitol. 1983 Feb;77(1):1-12.

the East African coast. Bull Entomol Res. 1951;41:487-502.

Ethiopia. Trans R Soc Trop Med Hyg. 1998 Mar-Apr;92(2):231-5.

incipient speciation in *Anopheles gambiae*. B Zool. 1985;52(1-2):45-63.

Entomol Res. 1995 Mar;85(1):29-35.

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

Lagos. Bull Entomol Res. 1948;38:527-58.

Insect Mol Biol. 2001;10(1):9-18.

*An. gambiae* s.s. Insect Mol Biol. 2003;12(3):241-5.

*gambiae*. BMC Ecol. 2009 May 21;9(1):16.

4;298(5591):115-7.

1946;36:185-252.


[81] Sogoba N, Doumbia S, Vounatsou P, Bagayoko MM, Dolo G, Traore SF, Maiga HM, Toure YT, Smith T. Malaria transmission dynamics in Niono, Mali: the effect of the irrigation systems. Acta Trop. 2007 Mar;101(3):232-40.

[69] Mwangangi J, Shililu J, Muturi E, Gu WD, Mbogo C, Kabiru E, Jacob B, Githure J, Novak R. Dynamics of immature stages of *Anopheles arabiensis* and other mosquito species (Diptera: Culicidae) in relation to rice cropping in a rice agro-ecosystem in Kenya. J

[70] Mutero CM, Blank H, Konradsen F, van der Hoek W. Water management for control‐ ling the breeding of *Anopheles* mosquitoes in rice irrigation schemes in Kenya. Acta

[71] Ameneshewa B, Service MW. Resting habits of *Anopheles arabiensis*in the Awash River

[72] Coluzzi M, Sabatini A, Petrarca V, Di Deco MA. Chromosomal differentiation and adaptation to human environments in the *Anopheles gambiae* complex. Trans R Soc Trop

[73] Coetzee M, Fontenille D. Advances in the study of *Anopheles funestus*, a major vector of

[74] Cohuet A, Simard F, Toto JC, Kengne P, Coetzee M, Fontenille D. Species identification within the *Anopheles funestus* group of malaria vectors in Cameroon and evidence for

[75] Weeto MM, Koekemoer LL, Kamau L, Hunt RH, Coetzee M. Evaluation of a speciesspecific PCR assay for the *Anopheles funestus* group from eleven African countries and

[76] Antonio-Nkondjio C, Kerah CH, Simard F, Awono-Ambene P, Chouaibou M, Tchuin‐ kam T, Fontenille D. Complexity of the malaria vectorial system in Cameroon: contri‐ bution of secondary vectors to malaria transmission. J Med Entomol. 2006;43(6):

[77] Awolola TS, Oyewole IO, Koekemoer LL, Coetzee M. Identification of three members of the *Anopheles funestus* (Diptera: Culicidae) group and their role in malaria transmis‐ sion in two ecological zones in Nigeria. Trans R Soc Trop Med Hyg. 2005;99(7):525-31.

[78] Hargreaves K, Koekemoer LL, Brooke BD, Hunt RH, Mthembu J, Coetzee M. *Anopheles funestus* resistant to pyrethroid insecticides in South Africa. Med Vet Entomol. [Article].

[79] Carnevale P, Guillet P, Robert V, Fontenille D, Doannio J, Coosemans M, Mouchet J. Diversity of malaria in rice growing areas of the Afrotropical region. Parassitologia.

[80] Chandler JA, Highton RB, Hill MN. Mosquitoes of the Kano Plain, Kenya. I. Results of indoor collections in irrigated and nonirrigated areas using human bait and light traps.

valley of Ethiopia. Ann Trop Med Parasitol. 1996;90(5):515-21.

malaria in Africa. Insect Biochem Mol Biol. 2004 Jul;34(7):599-605.

Madagascar. Trans R Soc Trop Med Hyg. 2004 Mar;98(3):142-7.

a new species. Am J Trop Med Hyg. 2003;69(2):200-5.

Vector Ecol. 2006 Dec;31(2):245-51.

Trop. 2000;76(3):253-63.

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

Med Hyg. 1979;73(5):483-97.

1215-21.

2000 Jun;14(2):181-9.

1999;41(1-3):273-6.

J Med Entomol. 1975 Dec 30;12(5):504-10.


Genetic diversity and molecular identification of mosquito species in the *Anopheles maculatus* group using the ITS2 region of rDNA. Infect Genet Evol. 2007 Jan;7(1):93-102.


[107] Amerasinghe FP, Ariyasena TG. Larval survey of surface water-breeding mosquitoes during irrigation development in the Mahaweli Project, Sri Lanka. J Med Entomol. 1990;27(5):789-802.

Genetic diversity and molecular identification of mosquito species in the *Anopheles maculatus* group using the ITS2 region of rDNA. Infect Genet Evol. 2007 Jan;7(1):93-102.

[94] Walton C, Handley JM, Kuvangkadilok C, Collins FH, Harbach RE, Baimai V, Butlin RK. Identification of five species of the *Anopheles dirus* complex from Thailand, using allele-specific polymerase chain reaction. Med Vet Entomol. 1999 Feb;13(1):24-32.

[95] Surendran SN, Abhayawardana TA, De Silva BG, Ramasamy R, Ramasamy MS. *Anopheles culicifacies* Y-chromosome dimorphism indicates sibling species (B and E) with different malaria vector potential in Sri Lanka. Med Vet Entomol. 2000;14(4):

[96] Jude PJ, Dharshini S, Vinobaba M, Surendran SN, Ramasamy R. *Anopheles culicifacies* breeding in brackish waters in Sri Lanka and implications for malaria control. Malar J.

[97] Subbarao SK, Sharma VP. Anopheline species complexes and malaria control. Indian

[98] Amerasinghe PH, Amerasinghe FP, Konradsen F, Fonseka KT, Wirtz RA. Malaria vectors in a traditional dry zone village in Sri Lanka. Am J Trop Med Hyg. 1999;60(3):

[99] Subbarao SK. The *Anopheles culicifacies* complex and control of malaria. Parasitol Today.

[100] Barik TK, Sahu B, Swain V. A review on *Anopheles culicifacies*: from bionomics to control with special reference to Indian subcontinent. Acta Trop. 2009 Feb;109(2):87-97.

[102] Surendran SN, De Silva BG, Srikrishnaraj KA, Ramasamy MS, Ramasamy R. Estab‐ lishment of species E, not B as the major vector of malaria in the *Anopheles culicifacies*

[103] Subbarao SK, Vasantha K, Raghavendra K, Sharma VP, Sharma GK. *Anopheles culicif‐ acies*: siblings species composition and its relationship to malaria incidence. J Am Mosq

[104] Vatandoost H, Shahi H, Abai MR, Hanafi-Bojd AA, Oshaghi MA, Zamani G. Larval habitats of main malaria vectors in Hormozgan Province and their susceptibility to different larvicides. Southeast Asian J Trop Med Public Health. 2004;35 Suppl 2:22-5.

[105] Surendran SN, Ramasamy R. Some characteristics of the larval breeding sites of *Anopheles culicifacies* species B and E in Sri Lanka. J Vector Borne Dis. 2005;42(2):39-44.

[106] Sabesan S, Krishnamoorthy K, Jambulingam P, Rajendran G, Kumar NP, Rajagopalan PK. Breeding habitats of *Anopheles culicifacies* in Rameswaram Island. Indian J Med Res.

complex in the country. Proc Sri Lanka Assoc Advmt Sci. 2003;59:18.

[101] Sharma VP. Fighting malaria in India. Curr Sci. 1998;75:1127-40.

437-40.

421-9.

1988;4(3):72-5.

1986;84:44-52.

2010 Apr 21;9(1):106.

J Med Res. 1997;106:164-73.

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

Control Assoc. 1988;4(1):29-33.


[136] Rosenberg R, Andre RG, Somchit L. Highly efficient dry season transmission of malaria in Thailand. Trans R Soc Trop Med Hyg. 1990;84(1):22-8.

[121] Reisen WK, Pradhan SP, Shrestha JP, Shrestha SL, Vaidya RG, Shrestha JD. Anopheline mosquito (Diptera: Culicidae) ecology in relation to malaria transmission in the inner

[122] Hanafi-Bojd AA, Vatandoost H, Jafari R. Susceptibility status of *Anopheles dthali* and *An. fluviatilis* to commonly used larvicides in an endemic focus of malaria, southern

[123] Gunasekaran K. Age composition, natural survival and population growth of *Anopheles fluviatilis* James, 1902, the major malaria vector in the endemic belt of Koraput District,

[124] Bhatt RM, Srivastava HC, Pujara PK. Biology of malaria vectors in central Gujarat.

[125] Batra CP, Reuben R. Breeding of *Anopheles stephensi* (Liston) in wells and cisterns in

[126] Batra CP, Mittal PK, Adak T, Subbarao SK. Efficacy of Agnique MMF monomolecular surface film against *Anopheles stephensi* breeding in urban habitats in India. J Am Mosq

[127] Vatandoost H, Oshaghi MA, Abaie MR, Shahi M, Yaaghoobi F, Baghaii M, Hanafi-Bojd AA, Zamani G, Townson H. Bionomics of *Anopheles stephensi* Liston in the malarious area of Hormozgan Province, southern Iran, 2002. Acta Trop. 2006;97(2):196-203.

[128] Manouchehri AV, Javadian E, Eshighy N, Motabar M. Ecology of *Anopheles stephensi*

[129] Krishnan KS. *A. stephensi* Liston, 1901. Vectors of Malaria in India. 2nd ed. Delhi: National Society of India for Malaria and Other Mosquito-born Disease; 1961. p. 39-58.

[130] Zaim M, Ershadi MR, Manouchehri AV, Hamdi MR. The use of CDC light traps and other procedures for sampling malaria vectors in southern Iran. J Am Mosq Control

[131] Reisen WK. Population dynamics of some Pakistan mosquitoes: the impact of residual organophosphate insecticide spray on anopheline relative abundance. Ann Trop Med

[132] Sweet WC, Rao B. Races of *Anopheles stephensi* Liston, 1901. Ind Med Gaz.

[134] Cox-Singh J, Singh B. Knowlesi malaria: newly emergent and of public health impor‐

[135] Vythilingam I. *Plasmodium knowlesi* and *Wuchereria bancrofti*: their vectors and chal‐

[133] Singh OP. Bionomics of malaria vectors in India: Malaria Research Centre 2002.

Salem, Tamil Nadu. Indian J Med Res. 1979 Dec;70 Suppl:114-22.

Liston in southern Iran. Trop Geogr Med. 1976 Sep;28(3):228-32.

Orissa, India. Southeast Asian J Trop Med Public Health. 1994;25(1):196-200.

and outer terai of Nepal, 1987-1989. J Med Entomol. 1993;30(4):664-82.

Iran. J Vector Borne Dis. 2006;43(1):34-8.

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

Indian J Malariol. 1994;31(2):65-76.

Control Assoc. 2006;22(3):426-32.

Assoc. 1986;2(4):511-5.

Parasitol. 1986;80(1):69-75.

tance? Trends Parasitol. 2008 Sep;24(9):406-10.

lenges for the future. Front Syst Biol. 2012;3(115).

1937;72:665-74.


*Anopheles minimus* A and C breeding habitats in western Thailand. Southeast Asian J Trop Med Public Health. 2005 Sep;36(5):1145-52.

[162] Rattanarithikul R, Green CA, Panyim S, Noigamol C, Chanaimongkol S, Mahapibul P. Larval habitats of malaria vectors and other *Anopheles* mosquitoes around a transmis‐ sion focus in northwestern Thailand. J Am Mosq Control Assoc. 1995;11(4):428-33.

[148] Trung HD, Bortel WV, Sochantha T, Keokenchanh K, Briet OJ, Coosemans M. Behav‐ ioural heterogeneity of *Anopheles* species in ecologically different localities in Southeast

[149] Misra SP, Nandi J, Narasimham MV, Rajagopal R. Malaria transmission in Nagaland, India. Part I. Anophelines and their seasonality. J Commun Dis. 1993;25(2):62-6.

[150] Baimai V. Population cytogenetics of the malaria vector *Anopheles leucosphyrus* group.

[151] Prakash A, Bhattacharyya DR, Mohapatra PK, Mahanta J. Physico-chemical character‐ istics of breeding habitats of *Anopheles dirus* (Diptera: Culicidae) in Assam, India. J

[152] Prakash A, Bhattacharyya DR, Mohapatra PK, Mahanta J. Breeding and day resting habitats of *Anopheles dirus* in Assam, India. Southeast Asian J Trop Med Public Health.

[153] Oo TT, Storch V, Becker N. Studies on the bionomics of *Anopheles dirus* (Culicidae: Diptera) in Mudon, Mon State, Myanmar. J Vector Ecol. 2002;27(1):44-54.

[154] Htay A, Minn S, Thaung S, Mya MM, Than SM, Hlaing T, Soe S, Druilhe P, Queuche F. Well-breeding *Anopheles dirus* and their role in malaria transmission in Myanmar.

[155] Harbach RE, Garros C, Manh ND, Manguin S. Formal taxonomy of species C of the *Anopheles minimus* sibling species complex (Diptera: Culicidae). Zootaxa.

[156] Harbach RE, Parkin E, Chen B, Butlin RK. *Anopheles*(*Cellia*) *minimus* Theobald (Diptera: Culicidae): neotype designation, characterization, and systematics. Proc Entomol Soc

[157] Somboon P, Rory A, Tsuda Y, Takagi M, Harbach RE. Systematics of *Anopheles* (*Cellia*) *yaeyamaensis* sp. n., alias species E of the *An. minimus* complex of southeastern Asia

[158] Farid MA, Chen CT, Hsu TC, Liu SY. Report of WHO evaluation team on malaria

[159] Miyagi I, Toma T, Malenganisho WL, Uza M. Historical review of mosquito control as a component of malaria eradication program in the Ryukyu Archipelago. Southeast

[160] Garros C, Van Bortel W, Trung HD, Coosemans M, Manguin S. Review of the Minimus Complex of *Anopheles*, main malaria vector in Southeast Asia: from taxonomic issues

[161] Rongnoparut P, Ugsang DM, Baimai V, Honda K, Sithiprasasna R. Use of a remote sensing-based geographic information system in the characterizing spatial patterns for

to vector control strategies. Trop Med Int Health. 2006 Jan;11(1):102-14.

Southeast Asian J Trop Med Public Health. 1988;19(4):667-80.

Southeast Asian J Trop Med Public Health. 1999;30(3):447-53.

Environ Biol. 2002 Jan;23(1):95-100.

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

1997;28(3):610-4.

2007;1654:41-54.

Wash. 2006;108(1):198-209.

(Diptera: Culicidae). Zootaxa. 2010;2651:43-51.

eradication in the Ryukyu Islands, 1965. Geneva1966.

Asian J Trop Med Public Health. 1996;27(3):498-511.

Asia: a challenge for vector control. Trop Med Int Health. 2005;10(3):251-62.


[186] Bangs MJ, Subianto DB. El Nińo and associated outbreaks of severe malaria in highland populations in Irian Jaya, Indonesia: a review and epidemiological perspective. Southeast Asian J Trop Med Public Health. 1999 Dec;30(4):608-19.

[174] Cooper RD, Waterson DG, Frances SP, Beebe NW, Sweeney AW. Speciation and distribution of the members of the *Anopheles punctulatus* (Diptera: Culicidae) group in

[175] Cooper RD, Waterson DG, Frances SP, Beebe NW, Pluess B, Sweeney AW. Malaria vectors of Papua New Guinea. Int J Parasitol. 2009 Jun 5;39(13):1495-501.

[176] Bugoro H, Cooper RD, Butafa C, Iro'ofa C, Mackenzie DO, Chen CC, Russell TL. Bionomics of the malaria vector *Anopheles farauti* in Temotu Province, Solomon Islands:

[177] Ebsworth P, Bryan JH, Foley DH. Ecological distribution of mosquito larvae of the *Anopheles punctulatus* group on Niolam (Lihir) Island, Papua New Guinea. J Am Mosq

[178] Cooper RD, Frances SP. Malaria vectors on Buka and Bougainville Islands, Papua New

[179] Beebe NW, Bakote'e B, Ellis JT, Cooper RD. Differential ecology of *Anopheles punctula‐ tus* and three members of the *Anopheles farauti* complex of mosquitoes on Guadalcanal, Solomon Islands, identified by PCR-RFLP analysis. Med Vet Entomol. 2000;14(3):

[180] Charlwood JD, Graves PM, Alpers MP. The ecology of the *Anopheles punctulatus* group of mosquitoes from Papua New Guinea: a review of recent work. P N G Med J.

[181] Charlwood JD. The influence of larval habitat on the ecology and behavior of females of the Punctulatus Group of *Anopheles* mosquitoes from Papua New Guinea. In: Lounibos LP, Rey JR, Frank JH, editors. Ecology of mosquitoes: Proceedings of a workshop. Vero Beach: Florida Medical Entomology Laboratory; 1985. p. 399-406.

[182] Beebe NW, Cooper RD, Morrison DA, Ellis JT. A phylogenetic study of the *Anopheles punctulatus* group of malaria vectors comparing rDNA sequence alignments derived from the mitochondrial and nuclear small ribosomal subunits. Mol Phylogenet Evol.

[183] Lee DJ, Hicks MM, Griffiths M, Debenham ML, Bryan JH, Russell RC, Geary M, Marks EN. The Culicidae of the Australasian Region. Volume 5. Nomenclature, synonymy, literature, distribution, biology and relation to disease. Genus *Anopheles*. Subgenera

*Anopheles*, *Cellia*. Canberra: Australian Government Publishing Service; 1987.

[184] Charlwood JD. A differential response to mosquito nets by *Anopheles* and *Culex* mosquitoes from Papua New Guinea. Trans R Soc Trop Med Hyg. 1986;80(6):958-60.

[185] Spenser T, Spenser M, Venters D. Malaria vectors of Papua New Guinea. P N G Med

Papua New Guinea. J Med Entomol. 2002;39(1):16-27.

issues for malaria elimination. Malar J. 2011;10:133.

Guinea. J Am Mosq Control Assoc. 2002;18(2):100-6.

Control Assoc. 2001;17(3):181-5.

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

308-12.

1986;29(1):19-26.

2000 Dec;17(3):430-6.

J. 1974;17(1):22-30.


**Chapter 5**

## **Phylogeography, Vectors and Transmission in Latin America**

Jan E. Conn, Martha L. Quiñones and Marinete M. Póvoa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55217

## **1. Introduction**

The overall focus of this chapter is the impact of phylogeographic studies on information pertinent to vector control, and an update on the relative importance and taxonomic status of five malaria vectors, some of which are species complexes, in the subgenus *Nyssorhynchus*: *Anopheles albimanus* Wiedmann, Albitarsis Complex, *Anopheles aquasalis* Curry, *Anopheles darlingi* Root, and *Anopheles nuneztovari* s.l. Gabaldón, considering literature predominantly since 2000. This cut-off date is to avoid repetition or overlap with some of the same subjects that have been covered in other places [1-4].

It is also of interest to vector control and elimination programs that, since 2000 and a more recent compilation that included a list of confirmed or potential Latin American malaria vectors [5], some vector species have been implicated in additional regions or countries by enzyme-linked immunosorbent assay [ELISA; 6], PCR techniques [7], VecTest [8] or more definitive biological and epidemiological evidence has been provided. Examples of these include *An. rangeli* Gabaldón, Cova Garcia and Lopez, initially implicated in Amapá state, Brazil [9] and subsequently in Putumayo, southern Colombia [10]; and *An. triannulatus*, (Neiva and Pinto) incriminated more broadly from Amazonian Brazil [11], then locally from Amapá, Brazil [12]. Furthermore, based on high frequency, biting behavior, seasonality, ELISA and nested-PCR, for the first time, *An. rondoni* (Neiva and Pinto) has been implicated in Matapá, Pará state, Brazil [13]. This is an understudied species, and its potential as a vector in other localities and regions in its distribution (Argentina, Bolivia, Brazil) is worth investigating.

A relatively early summary of information on the five most important malaria vectors in Latin America was published in 1986 [14]. This publication focused on four species in the *Nysso‐*

© 2013 Conn 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 Conn 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.

*rhynchus* subgenus: *Anopheles albimanus*, *Anopheles aquasalis*, *Anopheles darlingi*, *Anopheles nuneztovari* and one in the *Anopheles* subgenus, *Anopheles pseudopunctipennis* Theobald. Naturally, more than 20 years later, this list of five is debatable, although most researchers would still consider *An. darlingi* to be the primary vector overall, and *An. albimanus* to be one of the most important. Nevertheless, some aspects of this publication are still relevant, and it serves as a useful historical introduction.

A review published in 2012 [15] summarized the overall findings of much of the available literature on genetic diversity of malaria vectors, including those in Latin America, and concluded that Pleistocene (0.01-2.6 mya; 16) environmental changes have been the primary drivers of divergence, at least at the species and population levels. These changes and earlier ones during the Miocene (2.6-5.3 mya)/Pliocene (5.3-23.0 mya) were hypothesized to have influenced the phylogeography of some co-distributed neotropical vector species, including *Anopheles darlingi* and selected Albitarsis Complex members [17].

## **2. Biology and vector status**

#### **2.1.** *An. albimanus*

Throughout its broad, mostly coastal distribution (Figure 1), *Anopheles albimanus* is an impor‐ tant local vector and is considered to be ecologically adaptable (18). In general this species is crepuscular, zoophilic, exophagic, exophilic and seasonally abundant (19-20). Despite heteo‐ geneity of several attributes, such as host-feeding behaviour, longevity, insecticide resistance and susceptibility to Plasmodium species, thoughout its distribution, it has maintained single species status (20).

#### *2.1.1. Colombia*

*An. albimanus* is distributed in Colombia along the Atlantic and Pacific coasts (Figure 1). It is the main malaria vector on the Pacific coast, but its presence is considered a risk factor in other regions, even where malaria transmission is low [22]. Adult abundance of *An. albimanus* is associated with malaria transmission. The El Niño-Southern Oscillation Event (ENSO), that affects global climatic conditions every 2 to 7 years, has been strongly associated with increases in malaria cases, particularly in areas where *An. albimanus* is the main malaria vector, such as the Pacific coast [23]. *An. albimanus* breeding sites are very diverse, ranging from temporary small ponds to lagoons, and even include artificial containers. Its human biting rates can range between a few specimens per night night up to thousands, depending on the availablity of breeding sites in and around villages. Despite its considerable distribution on both coasts, *An. albimanus* has been found naturally infected with *P. vivax* only along the Pacific [24] (Table 1). In this region, its biting activity shows at least two peaks, one around midnight and a second one of less intensity before dawn, both indoors and outdoors [4]. Consistent use of insecticidetreated nets (ITNs) in this region could potentially reduce malaria transmission risk. In the Buenaventura peri-urban area, around 20% of the bites occur indoors and the main biting activity is outside houses between 18-21h. Then, at midnight, there is a second peak inside houses. Finally, between 05-06h, activity increases again outdoors [4].

*rhynchus* subgenus: *Anopheles albimanus*, *Anopheles aquasalis*, *Anopheles darlingi*, *Anopheles nuneztovari* and one in the *Anopheles* subgenus, *Anopheles pseudopunctipennis* Theobald. Naturally, more than 20 years later, this list of five is debatable, although most researchers would still consider *An. darlingi* to be the primary vector overall, and *An. albimanus* to be one of the most important. Nevertheless, some aspects of this publication are still relevant, and it

A review published in 2012 [15] summarized the overall findings of much of the available literature on genetic diversity of malaria vectors, including those in Latin America, and concluded that Pleistocene (0.01-2.6 mya; 16) environmental changes have been the primary drivers of divergence, at least at the species and population levels. These changes and earlier ones during the Miocene (2.6-5.3 mya)/Pliocene (5.3-23.0 mya) were hypothesized to have influenced the phylogeography of some co-distributed neotropical vector species, including

Throughout its broad, mostly coastal distribution (Figure 1), *Anopheles albimanus* is an impor‐ tant local vector and is considered to be ecologically adaptable (18). In general this species is crepuscular, zoophilic, exophagic, exophilic and seasonally abundant (19-20). Despite heteo‐ geneity of several attributes, such as host-feeding behaviour, longevity, insecticide resistance and susceptibility to Plasmodium species, thoughout its distribution, it has maintained single

*An. albimanus* is distributed in Colombia along the Atlantic and Pacific coasts (Figure 1). It is the main malaria vector on the Pacific coast, but its presence is considered a risk factor in other regions, even where malaria transmission is low [22]. Adult abundance of *An. albimanus* is associated with malaria transmission. The El Niño-Southern Oscillation Event (ENSO), that affects global climatic conditions every 2 to 7 years, has been strongly associated with increases in malaria cases, particularly in areas where *An. albimanus* is the main malaria vector, such as the Pacific coast [23]. *An. albimanus* breeding sites are very diverse, ranging from temporary small ponds to lagoons, and even include artificial containers. Its human biting rates can range between a few specimens per night night up to thousands, depending on the availablity of breeding sites in and around villages. Despite its considerable distribution on both coasts, *An. albimanus* has been found naturally infected with *P. vivax* only along the Pacific [24] (Table 1). In this region, its biting activity shows at least two peaks, one around midnight and a second one of less intensity before dawn, both indoors and outdoors [4]. Consistent use of insecticidetreated nets (ITNs) in this region could potentially reduce malaria transmission risk. In the Buenaventura peri-urban area, around 20% of the bites occur indoors and the main biting

serves as a useful historical introduction.

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

**2. Biology and vector status**

**2.1.** *An. albimanus*

species status (20).

*2.1.1. Colombia*

*Anopheles darlingi* and selected Albitarsis Complex members [17].

**Figure 1.** Distribution of *Anopheles albimanus* highlighted in green [21].



\*, Reference 27 refers to *Anopheles albitarsis* E (now *An. janconnae*) and Póvoa 2010 (unpublished data) refers to the first evidence of *An. oryzalimnetes* as a potential vector. References 12 and 25 refer to *An. marajoara* as a vector, while the remaining references refer to *An. albitarsis* s.l. as vectors.

**Table 1.** Regional South American vectors subgenus *Nyssorhynchus*: evidence for malaria vector status.

#### *2.1.2. Peru*

**Taxon Country Local transmission Regional**

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

Brazil Marabá, Pará; Matapi River, Amapá; Amazon region; Macapá, Amapá; N. Amazon region, Roraima; Boa Vista, Roraima; Serra do Navio, Amapá

Colombia Puerto Carreño, Vichada

Belém, Pará

Pará; Matapi River, Amapa; Macapá, Amapá; Boa Vista, Roraima; Serra do Navio, Amapá; Pará, N. Brazil; Anajás, Pará; Goianesia do Para,

Suriname Brokobondo; Galibi; Paramaribo

Pará

Vichada; Quibdó, Chocó; Dibulla, Guajira

Camopi; Saint Georges de l'Oyapock; Maroni River; Upper Maroni Amazonian forest; Village of Loca; Village of Twenke

Colombia Puerto Carreño,

*An. aquasalis* Brazil São Luis, Maranhão;

*An. darlingi* Brazil Marabá, Pará; Belém,

French Guiana

Albitarsis Complex*\**

*An. albimanus* Colombia Pacific region ELISA, PCR 24

**transmission**

Venezuela Sifontes, Bolivar ELISA 30-31

Guyana Mahdia VecTest 34

Venezuela coastal areas 1

Córdoba; Villavicencio, Meta; Putumayo

Peru eastern region ELISA 46 Suriname Maroni River ELISA 43; 47

Amazon region ELISA, PCR,

VecTest

ELISA 29

ELISA 32-33

ELISA 35

Amazon region ELISA, PCR 9; 11-13; 25; 27-28; 33;

ELISA 4; 10; 29; 38-41

ELISA 42-45

36-37; Póvoa 2012 (unpub. data)

**Evidence Reference**

9; 11-13; 25-28; Póvoa 2010 (unpub. data)

> *An. albimanus* in Peru is considered the main malaria vector along the Pacific coast, particularly in the north, where it is seasonal, and linked to agriculture [50]. Due to high insecticide application, mainly in rice fields, *An. albimanus* is resistant to all insecticides used in public health in this area [51]. Flooded rice fields provide ideal mosquito breeding habitat and *An*. *albimanus* density is associated with rice crops. Since 2005, the Peruvian Minister of Health, together with the Agricultural sector, implemented a modified irrigation system, so that the fields are dry for a week, and then intermittently irrigated, resulting in a decrease in mosquito larvae by 87% [52]. An important follow-up question would be whether this *An*. *albimanus* larval control has actually resulted in a decrease in malaria incidence rate (MIR), or in local *An. albimanus* adult female abundance, as measured by human biting rate (HBR) and ento‐ mological inoculation rate (EIR).

> In several other South American countries (Ecuador, Panama, Venezuela) where *An. albimanus* is a malaria vector, as determined by sporozoite detection or other comparable information in earlier studies, data based on newer techniques are not available. Howev‐ er, some recent investigations have drawn attention to new distributions or larval habitat characterizations that pinpoint areas of fruitful potential research and possible targets for control measures [53-56].

#### **2.2. Albitarsis Complex**

Presently, there are eight recognized species (*An*. *albitarsis* s.s., *An. albitarsis* F, *An. albitarsis* G, *An. albitarsis* I, *An*. *deaneorum*, *An. janconnae*, *An. marajoara*, *An. oryzalimnetes)* and one lineage (*An. albitarsis* H) in the Albitarsis Complex [57]. The species described as near *An. janconnae* from Colombia [58] is now considered to be *An. albitarsis* I [57]. The overall distribution of members of this complex is wide-ranging, including both Central and South America, as well as some Caribbean Islands [59] (Figure 2).

**Figure 2.** This map shows the predicted probability of occurrence of *An. albitarsis* in the Americas [2]

#### *2.2.1. Brazil*

Six species of the Albitarsis Complex are known from Brazil to date: *An*. *albitarsis* s.s., *An. albitarsis* G, *An. deaneorum*, *An. janconnae*, *An. marajoara*, and *An. oryzalimnetes*, [57,60] (Figure 3). The most broadly distributed member of this complex is *An. marajoara* Galvão and Dam‐ asceno [57,59]. It can be very abundant locally [12], and its breeding site types vary from swampy shores of lakes and ponds to small road puddles; it is generally associated with sunlight and often with aquatic or semi-aquatic vegetation [59]. It has been found infected by *Plasmodium falciparum* (Welsh), *Plasmodium vivax* (Grassi & Feletti) 210 and *P. vivax* 247, and *Plasmodium malariae*(Grassi & Feletti), and is a peri-urban as well as a rural vector, depending on locality, availability of breeding sites and hosts [12, 25]. It is also associated with deforested areas of the Vale do Ribeira in the southeastern Atlantic Forest of Brazil [61]. Although the EIR in Amapá state, Brazil was found to be lower than that of *An. darlingi*, it is an important local vector, at least in lowland rainforest in parts of the eastern Amazon [12, 62].

The distribution of *An. janconnae* Wilkerson and Sallum [classified previously as *An. albitarsis* E; [27, 60] appears to be limited to northern Amazonian Brazil, including along the Amazon River [57, 60]. Larval habitat types in several localities in Roraima state ranged from marsh to seepag‐ es to stream margins, and, based on analyses of several environmental variables*, An. janconnae* could be classified as a habitat specialist [McKeon, Conn & Povoa, unpublished data, 2012]. *An. janconnae*wasincriminatedasalocalmalariavectoraroundBoaVista,thecapitalofRoraimastate [27]. It is likely that the infected specimens identified as *An. albitarsis* s.l. from this region [26, 28] (Table 1), are *An. janconnae*, at least according to the geographic distribution [57].

*An.oryzalimnetes* (Wilkerson and Motoki), previously *An. albitarsis* B [60] has a broad distribu‐ tion in Brazil that includes the Amazon region and southern Brazil [57]. It is frequently associated with rice fields, and is anthropophilic [60]. It was determined to be positive by ELISA for *Plasmodium* in Pará state [M.M. Povoa 2010, unpublished data], and may play a role in local transmission.

#### *2.2.2. Colombia*

from Colombia [58] is now considered to be *An. albitarsis* I [57]. The overall distribution of members of this complex is wide-ranging, including both Central and South America, as well

**Figure 2.** This map shows the predicted probability of occurrence of *An. albitarsis* in the Americas [2]

Six species of the Albitarsis Complex are known from Brazil to date: *An*. *albitarsis* s.s., *An. albitarsis* G, *An. deaneorum*, *An. janconnae*, *An. marajoara*, and *An. oryzalimnetes*, [57,60] (Figure 3). The most broadly distributed member of this complex is *An. marajoara* Galvão and Dam‐ asceno [57,59]. It can be very abundant locally [12], and its breeding site types vary from

*2.2.1. Brazil*

as some Caribbean Islands [59] (Figure 2).

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

At least three members of *An. albitarsis* s.l. are present in Colombia. *Anopheles marajoara* (some collections of which, according to the map [57], may be *An. albitarsis* I) is widely distributed [22], and its biology is similar to that described above under *An. marajoara* in Brazil. It is conisidered to be a regional vector in Colombia [22, 39, 63]. In the municipality of Puerto Carreno in eastern Colombia near the Venezuelan border, it was detected infected with *P. falciparum* [29] at a surprisingly high rate (1.92%; 3/152 specimens infected). Here, its peak biting time was 18-19h, with a minor peak from 20-21h, and it was collected both indoors and outdoors. It is suggested that together with *An. darlingi*, the dominant vector in the area, it is responsible for maintaining local malaria transmission in this municipality [29].

The second member of the complex is sympatric with *An. darlingi* in the east, and probably involved in malaria transmission. This species has been identified by various names, including *An. allopha, An. marajoara*, near *An. janconnae* [58], and most recently as *An. albitarsis* I [57]. Its known distribution thus far is restricted to Colombia. Relatively little is known about its biology, because of species identification issues, and there is no direct evidence yet for its involvement in malaria transmission. It appears that the specimens from Vichada, Colombia, identified as *An. marajoara* [64], are *An. albitarsis* I [57], so the distribution of *An. marajoara* in Colombia, and its involvement in malaria transmission, need re-evaluation.

The third species, *An. albitarsis* F, was first described from Puerto Carreño, near the Venezuelan border, [65]. In this locality it was found in sympatry with *An. darlingi* and a species in the Albitaris Complex now defined as *An. albitarsis* I. Its distribution is hypothesized to include Colombia, Venezuela and Trinidad [57]. Because it can easily be confused morphologically with *An. marajoara*, and it is found in regions of malaria endemnicity, this species is of some epidemiological importance.

#### *2.2.3. Venezuela*

It now appears that at least *An. albitarsis* I and possibly also *An. albitarsis* F are present is Venezuela [57]. Furthermore, *An. albitarsis* I could be sympatric in some regions with *An*. *marajoara* (the identification of which needs to be confirmed in Venezuela using molecular techniques), a local vector of *P. vivax* in western Venezuela [66]. *An. marajoara* also plays a significant role, together with *An. darlingi*, in malaria transmission in five localities in southern Venezuela [30-31]. In this gold mining region, where transmission is yearlong, the peak biting time, (19-21h, mostly before midnight), was comparable with most other reports of *An. marajoara*. Although *An. marajoara* was feeding both indoors and outdoors, it was significantly exophagic.

#### **2.3.** *Anopheles aquasalis*

This brackish-water breeder is found along the Pacific as far south as Ecuador and along the Atlantic to southern Brazil [1,14] (Figures 3,4). It is rarely found far from the ocean, but it can tolerate quite low salt ion concentrations, and has been detected in freshwater springs. It can be present in enormous numbers in marshy coastal areas, so that even if it is not extremely susceptible to *Plasmodium*, it can maintain malaria transmission when its abundance is high, especially during the rainy season [67]. It persists as an important local vector in Sucre state, eastern Venezuela, where a series of pioneering studies have identified hotspots of local transmission that are very useful for prevention and control efforts [68-69]. It has also been incriminated as a vector of *P. falciparum* and [or] *P. vivax* in Maranhão [32] and Pará states in Brazil [33, 70]. In Linden and Madia, Guyana [34], *An. aquasalis* was detected infected with *P. vivax* using VecTest, but the total sample size of anophelines collected was very small (*n*=45). In three towns in Suriname, Paramaribo, Brokopondo and Galibi, *An. aquasalis* was also detected infected with *Plasmodium* by ELISA [35], and is likely responsible for local, coastal transmission in this region.

#### **2.4.** *Anopheles darlingi*

The species considered to be the most important vector in the Amazon basin is *Anopheles darlingi* [2,3] (Figure 5). It is anthropophilic, adaptive and it has been incriminated in many localities in many countries, where it is often labeled a national vector (Table 1). As such, it has been the focus of a very wide range of research, monitoring and control efforts, and the publication for the first time of its complete genome is an exciting new development (GenBank accession number ADMH00000000).

#### *2.4.1. Bolivia*

The third species, *An. albitarsis* F, was first described from Puerto Carreño, near the Venezuelan border, [65]. In this locality it was found in sympatry with *An. darlingi* and a species in the Albitaris Complex now defined as *An. albitarsis* I. Its distribution is hypothesized to include Colombia, Venezuela and Trinidad [57]. Because it can easily be confused morphologically with *An. marajoara*, and it is found in regions of malaria endemnicity, this species is of some

It now appears that at least *An. albitarsis* I and possibly also *An. albitarsis* F are present is Venezuela [57]. Furthermore, *An. albitarsis* I could be sympatric in some regions with *An*. *marajoara* (the identification of which needs to be confirmed in Venezuela using molecular techniques), a local vector of *P. vivax* in western Venezuela [66]. *An. marajoara* also plays a significant role, together with *An. darlingi*, in malaria transmission in five localities in southern Venezuela [30-31]. In this gold mining region, where transmission is yearlong, the peak biting time, (19-21h, mostly before midnight), was comparable with most other reports of *An. marajoara*. Although *An. marajoara* was feeding both indoors and outdoors, it was significantly

This brackish-water breeder is found along the Pacific as far south as Ecuador and along the Atlantic to southern Brazil [1,14] (Figures 3,4). It is rarely found far from the ocean, but it can tolerate quite low salt ion concentrations, and has been detected in freshwater springs. It can be present in enormous numbers in marshy coastal areas, so that even if it is not extremely susceptible to *Plasmodium*, it can maintain malaria transmission when its abundance is high, especially during the rainy season [67]. It persists as an important local vector in Sucre state, eastern Venezuela, where a series of pioneering studies have identified hotspots of local transmission that are very useful for prevention and control efforts [68-69]. It has also been incriminated as a vector of *P. falciparum* and [or] *P. vivax* in Maranhão [32] and Pará states in Brazil [33, 70]. In Linden and Madia, Guyana [34], *An. aquasalis* was detected infected with *P. vivax* using VecTest, but the total sample size of anophelines collected was very small (*n*=45). In three towns in Suriname, Paramaribo, Brokopondo and Galibi, *An. aquasalis* was also detected infected with *Plasmodium* by ELISA [35], and is likely responsible for local, coastal

The species considered to be the most important vector in the Amazon basin is *Anopheles darlingi* [2,3] (Figure 5). It is anthropophilic, adaptive and it has been incriminated in many localities in many countries, where it is often labeled a national vector (Table 1). As such, it has been the focus of a very wide range of research, monitoring and control efforts, and the publication for the first time of its complete genome is an exciting new development (GenBank

epidemiological importance.

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

*2.2.3. Venezuela*

exophagic.

**2.3.** *Anopheles aquasalis*

transmission in this region.

accession number ADMH00000000).

**2.4.** *Anopheles darlingi*

*An. darlingi* is distributed in the northeastern Bolivian Amazon, in the departments of Pando, Beni and Santacruz de la Sierra, along the border with Brazil [71]. It shows a biting peak between 19-21h, with 83% of the bites occurring before 22h, when most local people go to bed. After this time, numbers decline, with little or no activity between 02:30-05h [72]. There have been relatively few studies on this species in Bolivia, and data are very scarce.

**Figure 3.** South American localities where malaria vectors have been incriminated by various methods since the year 2000. Species codes: ALB, *Anopheles albimanus*; ALC, Albitarsis Complex; AQU, *An. aquasalis*; DAR, *An. darlingi*; NUN, *An. nuneztovari*. The darker grey area is Brazil.

**Figure 4.** Distribution of *Anopheles aquasalis* highlighted in orange [21]

#### *2.4.2. Brazil*

Most of the newest incriminations of the continued involvement of *An. darlingi* in malaria transmission originate in Amazonian Brazil (Table 1, Figure 3). Rather than summarizing each new investigation, this section focuses on the findings on *An. darlingi* in a longitudinal study that investigated bloodmeal hosts, transmission, and seasonal abundance in three riverine villages along the Matapí River in Amapa state, northern Amazonian Brazil [12, 62, 73]. An ELISA analysis for IgG of common vertebrates found that the highest human blood indices (HBI) were in *An. darlingi* and *An. marajoara*. What was unexpected was that the HBIs of *An. darlingi* varied significantly among the three villages, which are only 1.5-7.0 km apart, likely because of host availability. It was found to be important to conduct a census of animals in each locality to be able to interpret the HBI results correctly. Even though *An. darlingi* was the most abundant species collected at human landing catches in each village, the HBI of *An. darlingi* resting collections, from under houses or in vegetation, ranged from 0.017-0.405, demonstrating how opportunistic this species can be, despite its anthropophily [12]. From the same study sites 113,117 mosquitoes collected from 2003-2005 were analyzed by ELISA. For this part of the study, *An. darlingi* and *An. marajoara* had the highest proportion of positives and also the highest EIRs, and thus the highest human-vector contact. Nevertheless, *An. darlingi* is still considered to be more important in this study area than *An. marajoara* because of its higher EIR [12]. Seasonal abundance was measured for 32 consecutive months of collection and showed that *An. darlingi* was most abundant during the wet-dry transition period between June and August, and that a strong positive correlation of *An. darlingi* abundance with rainfall lagged by several months. The latter finding may indicate that rainfall could be an important factor in predicting vector abundance, at least locally.

#### *2.4.3. Colombia*

**Figure 4.** Distribution of *Anopheles aquasalis* highlighted in orange [21]

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

Most of the newest incriminations of the continued involvement of *An. darlingi* in malaria transmission originate in Amazonian Brazil (Table 1, Figure 3). Rather than summarizing each new investigation, this section focuses on the findings on *An. darlingi* in a longitudinal study that investigated bloodmeal hosts, transmission, and seasonal abundance in three riverine villages along the Matapí River in Amapa state, northern Amazonian Brazil [12, 62, 73]. An ELISA analysis for IgG of common vertebrates found that the highest human blood indices (HBI) were in *An. darlingi* and *An. marajoara*. What was unexpected was that the HBIs of *An. darlingi* varied significantly among the three villages, which are only 1.5-7.0 km apart, likely because of host availability. It was found to be important to conduct a census of animals in each locality to be able to interpret the HBI results correctly. Even though *An. darlingi* was the most abundant species collected at human landing catches in each village, the HBI of *An. darlingi* resting collections, from under houses or in vegetation, ranged from 0.017-0.405, demonstrating how opportunistic this species can be, despite its anthropophily [12]. From the

*2.4.2. Brazil*

The distribution of *An. darlingi* in Colombia is widespread but heterogeneous, and hypothe‐ sized to be interrupted by the Andes. It is found mainly south and east of the Andes, including the Amazon region, bordering Brazil and Peru, but also north and west of the Andes, along

**Figure 5.** Distribution of *Anopheles darlingi* highlighted in pink [21].

the main Colombian rivers (such as Magdalena, Cauca and Atrato) [22, 74]. *An. darlingi* is the main malaria vector throughout its distribution. Although associated with forest environ‐ ments, it was also detected in the peri-urban area of the cities of Quibdó (Chocó) and Villavi‐ cencio (Meta), where malaria transmission occurs [40]. Breeding sites are the typical streams with slow water movement, but stationary water bodies such as natural and constructed fish ponds also provide good habitats. Similar to Peru (see below), the biting behaviour in Colom‐ bia is mostly before midnight, from 18-24h, with a smaller peak at sunrise (05-06h), but there is also persistant biting activity throughout the night [39].

#### *2.4.4. French Guiana*

In French Guiana, there has been renewed activity on malaria vectors, with most findings incriminating*An. darlingi* as theprimaryvector(Table 1).Between2000-2002 inthreeAmerindi‐ an villages in the Upper-Maroni region of the Amazon forest, *An. darlingi* bit throughout the night, withpeaks at 21:30-03:30handagainafter 05:30h[44].The biting rate was veryhigh(255.5 bites/person/night) and specimens were infected with *P. falciparum*, *P. vivax* and *P. malariae*. Behavior was characterized as endo-exophagic and exophilic. The malaria transmission risk exists all year but probably it is greater during the rainy season when vectorial capacity was estimated to be higher [44]. A combination of ITNs and repellent is recommended; IRS is not efficient because of the housing materials and relative inaccessibility of this region. A second study in the Maroni area compared villages of Amerindian Wayanas and the Aloukous [45]. Significantfindings include:thepeaklocalmalariacasereportingis thesametimeframe(August to October) as the highest IMT (numbers of infected mosquitoes surviving long enough to transmit) of *An. darlingi*; the possibility that the persistent yearlong transmission is focused or perhaps limited to the Amerindian villages; and different bionomics of *An. darlingi* in the two villages which lead investigators to conclude that in this region there may exist two distinctive subspecies of*An. darlingi*.Ananalysis of collections from2006-2011,inseveralregions ofFrench Guiana, detected *An*. *darlingi* infected by *P. vivax* from Camopi and Saint Georges de l'Oya‐ pock, both near the Oyapock River along the eastern border with Brazil [42].

#### *2.4.5. Peru*

*An. darlingi* is the main malaria vector species in eastern Amazonian Peru, the area with the highest malaria transmission in the country. It invaded this region in the 1990s [75], and its distribution now includes peri-urban settlements around the city of Iquitos, Loreto province. This change has been attributed to logging, agriculture and urban expansion, associated with deforestation [76-77]. To date, its greatest abundance is associated with areas of at least partial deforestation [78]. The main breeding sites in the Peruvian Amazon are streams and river margins in forested areas [77], however, the density of fish ponds has shown a positive association with malaria cases along roads in Loreto, suggesting that such ponds could be important local sources of this species [79]. *An. darlingi* was found naturally infected with *P*. *vivax* and *P. falciparum* in Loreto [46]. The human biting activity, which is similar indoors and outdoors, peaks two hours after sunset [77]. Because of the early evening biting peak, personal protection may be necessary to supplement bed-net use.

#### *2.4.6. Panama*

the main Colombian rivers (such as Magdalena, Cauca and Atrato) [22, 74]. *An. darlingi* is the main malaria vector throughout its distribution. Although associated with forest environ‐ ments, it was also detected in the peri-urban area of the cities of Quibdó (Chocó) and Villavi‐ cencio (Meta), where malaria transmission occurs [40]. Breeding sites are the typical streams with slow water movement, but stationary water bodies such as natural and constructed fish ponds also provide good habitats. Similar to Peru (see below), the biting behaviour in Colom‐ bia is mostly before midnight, from 18-24h, with a smaller peak at sunrise (05-06h), but there

In French Guiana, there has been renewed activity on malaria vectors, with most findings incriminating*An. darlingi* as theprimaryvector(Table 1).Between2000-2002 inthreeAmerindi‐ an villages in the Upper-Maroni region of the Amazon forest, *An. darlingi* bit throughout the night, withpeaks at 21:30-03:30handagainafter 05:30h[44].The biting rate was veryhigh(255.5 bites/person/night) and specimens were infected with *P. falciparum*, *P. vivax* and *P. malariae*. Behavior was characterized as endo-exophagic and exophilic. The malaria transmission risk exists all year but probably it is greater during the rainy season when vectorial capacity was estimated to be higher [44]. A combination of ITNs and repellent is recommended; IRS is not efficient because of the housing materials and relative inaccessibility of this region. A second study in the Maroni area compared villages of Amerindian Wayanas and the Aloukous [45]. Significantfindings include:thepeaklocalmalariacasereportingis thesametimeframe(August to October) as the highest IMT (numbers of infected mosquitoes surviving long enough to transmit) of *An. darlingi*; the possibility that the persistent yearlong transmission is focused or perhaps limited to the Amerindian villages; and different bionomics of *An. darlingi* in the two villages which lead investigators to conclude that in this region there may exist two distinctive subspecies of*An. darlingi*.Ananalysis of collections from2006-2011,inseveralregions ofFrench Guiana, detected *An*. *darlingi* infected by *P. vivax* from Camopi and Saint Georges de l'Oya‐

pock, both near the Oyapock River along the eastern border with Brazil [42].

protection may be necessary to supplement bed-net use.

*An. darlingi* is the main malaria vector species in eastern Amazonian Peru, the area with the highest malaria transmission in the country. It invaded this region in the 1990s [75], and its distribution now includes peri-urban settlements around the city of Iquitos, Loreto province. This change has been attributed to logging, agriculture and urban expansion, associated with deforestation [76-77]. To date, its greatest abundance is associated with areas of at least partial deforestation [78]. The main breeding sites in the Peruvian Amazon are streams and river margins in forested areas [77], however, the density of fish ponds has shown a positive association with malaria cases along roads in Loreto, suggesting that such ponds could be important local sources of this species [79]. *An. darlingi* was found naturally infected with *P*. *vivax* and *P. falciparum* in Loreto [46]. The human biting activity, which is similar indoors and outdoors, peaks two hours after sunset [77]. Because of the early evening biting peak, personal

is also persistant biting activity throughout the night [39].

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

*2.4.4. French Guiana*

*2.4.5. Peru*

*An. darlingi* was recently detected in Panamá for the first time, in the eastern Darien region, near the border with Colombia, associated with the highest prevalence of drug-resistant *P. falciparum* [80]. This species was only collected by human landing catch, and not recorded in light traps, confirming its high anthropophily. Despite the extension and increased frequency of deforestation in Panama, *An. darlingi* has been detected only in the east. Its discovery suggests that unplanned deforestation should be avoided to prevent further expansion of this very anthropophilic species, and hence potential *P. falciparum* transmission, to other regions in the country.

#### *2.4.7. Suriname*

A timely and important new development in Suriname is the apparent collapse of populations of *An. darlingi* in the sparsely inhabited interior, in and around three study communities, correlated with two main factors: the introduction of ITNs and climatic events, *i.e.*, unusual flooding which coincided with the beginning of the control activities in 2006 [47]. However, it should be noted that indoor residual spraying (IRS), active case detection (ACD), and a public awareness campaign also were implemented throughout the interior in 2006. As the authors point out, for Suriname, the next challenge is to try to find ways to use these methods to reduce or eliminate transmission among the gold-mining communities, where people are very mobile, and often active (not using ITNs) during potential biting times of *An. darlingi*. The latter are notoriously plastic, and vary locally and regionally. An important determination will be whether these results [47] can serve as a model for some communities where *An. darlingi* and malaria transmission are endemic in other countries.

#### *2.4.8. Venezuela*

Investigations along the Upper Orinoco River, southern Venezuela from 1994-1995 confirmed that *An. darlingi* was responsible for most, if not all of the local transmission of *P. falciparum*, *P. vivax* and *P. malariae* [48], that children under the age of 10 were at greatest risk, and that the EIR of *An. darlingi* was 129 positive bites/person/year. In a gold-mining region in southern Venezuela, studies from 1999-2000 [30-31] also determined that *An. darlingi* was one of two main vectors (the other was *An*. *marajoara* but see above under Albitarsis Complex). Surpris‐ ingly, many of the bionomic aspects of the two species in the five localities studied were quite similar (both more abundant during the rainy season, both biting indoors and outdoors with pronounced endophagic behavior), although *An. marajoara* was more abundant overall. The most striking bionomics difference between the two species was the peak biting time: *An. darlingi* bit throughout the night with two minor peaks (23-0h and 03-04h), whereas *An. marajoara* had a peak from 19-21h [30-31].

#### **2.5.** *Anopheles nuneztovari* **s.l.**

*Anopheles nuneztovari* s.l. is restricted to northern and Amazonian South America (Figure 6) and has been considered to be two genetically, ecologically and epidemiologically distinc‐ tive geographic populations, with the perception that the Colombian/western Venezuelan population was a regional vector (anthrophophilic and endo-exophagic) and the Amazoni‐ an population, mostly zoophilic and exophagic, was not [1, 5, 11]. Initially, evidence for malaria transmission by *An. nuneztovari* s.l. was found predominantly in Colombia and western Venezuela [22, 49]. However, a series of positive ELISA results and incrimina‐ tions of malaria transmission involvement from localities in the Brazilian Amazon (Table 1; Figure 3) since 2000 soon undermined this relatively simple view. *An. goeldii*, which had been synonymized with *An. nuneztovari*, was resurrected as a valid species [81]. This work proposed different geographical distributions for each species, with *An. goeldii* in the Amazon region and *An. nuneztovari* more restricted to Colombia and Venezuela. The report of the discovery of *An. nuneztovari* infected with *Plasmodium* from Saint Georges de l'Oyapock, French Guiana, using results from a longitudinal study (2006-2011) is of at least local relevance, but it will be taxonomically important to determine whether this species is actually *An. nuneztovari*, or might possibly be *An. goeldii*, since susceptibility of *An. goeldii* to *Plasmodium* has not yet been tested [42].

**Figure 6.** Distribution of *Anopheles nuneztovari* s.l. highlighted in blue [21].

*An. nuneztovari* s.l. is widely distributed in Colombia [22], particularly in the east, along the Venezuelan frontier, in the northwest region (Departments of Córdoba and Antioquia), where approximately 50% of the malaria cases occur, and in some areas along the Pacific Coast in the west, notably along the San Juan river (Chocó), and in the Buenaventura area (Valle). Specimens from Tierralta, Córdoba, a region of crop and livestock production, where *An. nuneztovari* s.l. was the most abundant species collected by human landing catch‐ es, were infected by *P. vivax* [38]. The breeding sites include small, permanent ponds, sunlit flooded pastures, and it has been determined that aquaculture ponds are one of this species' most frequent breeding places. In the west, in Cimitarra (Santander), such ponds, character‐ ized as permanent, completely exposed to sun and containing emerging vegetation, particu‐ larly grasses, represent approximately 81% of the breeding sites [63]. In Colombia*, An. nuneztovari* s.l. shows differing biting behavior by region. An exophagic tendency has been described in the northwest (Córdoba) [82], whereas in the east (Santander), a more endopha‐ gic behavior has been described [63]. The endophagic-exophilic variability makes control by residual insecticides very difficult.

## **3. Phylogeography**

tive geographic populations, with the perception that the Colombian/western Venezuelan population was a regional vector (anthrophophilic and endo-exophagic) and the Amazoni‐ an population, mostly zoophilic and exophagic, was not [1, 5, 11]. Initially, evidence for malaria transmission by *An. nuneztovari* s.l. was found predominantly in Colombia and western Venezuela [22, 49]. However, a series of positive ELISA results and incrimina‐ tions of malaria transmission involvement from localities in the Brazilian Amazon (Table 1; Figure 3) since 2000 soon undermined this relatively simple view. *An. goeldii*, which had been synonymized with *An. nuneztovari*, was resurrected as a valid species [81]. This work proposed different geographical distributions for each species, with *An. goeldii* in the Amazon region and *An. nuneztovari* more restricted to Colombia and Venezuela. The report of the discovery of *An. nuneztovari* infected with *Plasmodium* from Saint Georges de l'Oyapock, French Guiana, using results from a longitudinal study (2006-2011) is of at least local relevance, but it will be taxonomically important to determine whether this species is actually *An. nuneztovari*, or might possibly be *An. goeldii*, since susceptibility of *An. goeldii*

to *Plasmodium* has not yet been tested [42].

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

**Figure 6.** Distribution of *Anopheles nuneztovari* s.l. highlighted in blue [21].

*An. nuneztovari* s.l. is widely distributed in Colombia [22], particularly in the east, along the Venezuelan frontier, in the northwest region (Departments of Córdoba and Antioquia), where approximately 50% of the malaria cases occur, and in some areas along the Pacific

### **3.1.** *Anopheles albimanus*

An exemplary study, based on large sample sizes using microsatellite markers and a mtDNA *ND5* gene fragment, laid the groundwork for several *Anopheles albimanus* phylogeographic ideas [83]. These researchers detected restricted gene flow that they hypothesized to be the result of the physical barrier of the Central American Cordillera. Recent work, more geo‐ graphically focused on one country or a region, with additional local sampling, provides additional insights into phylogeography in Central America [20, 85] and Colombia [84].

A mitochondrial DNA *COI* gene fragment and microsatellites were used to test for congruence with biogeographical provinces [86] in Colombia. In this case [84], one population, Turbo, was from Magdalena (Caribbean), three were from Maracaibo (Caribbean), and the four were from Choco (Pacific). The eight populations tested were clearly differentiated into two coastal regions, Caribbean and Pacific, with evidence for a late Pleistocene expansion (estimated to 21,994 years ago) or a selective sweep. Even though there was evidence for historical restric‐ tions to gene flow (*COI* data), the microsatellites detected contemporary gene flow between the regions. Interestingly, a SAMOVA analysis found an unusual division. Only the three most easterly populations along the Caribbean coast grouped together. The fourth and most western Caribbean population, Turbo, was consistently more closely related to the four Pacific populations. Taken together, these data suggest possible semi-permeable boundaries among the three biogeographical provinces tested. Most relevant to malaria is the fact that the evidence for contemporary gene flow indicates that insecticide resistance genes, for example, could spread readily in these Colombian regions [84].

*An. albimanus* from Central America was examined using a fragment of the mtDNA *COI* gene to test the original hypothesis [83]. Physical barriers to gene flow were not detected (i.e., the Central America Cordillera was porous for *An. albimanus*) and contemporary isolation by distance was not supported [20]. Three divergent, co-occuring haplotype groups were detected using a statistical parsimony network, and these were not evenly distributed across Costa Rica

and Panama. A new hypothesis suggested that they could be the result of multiple introduc‐ tions into the region, probably caused by historical fragmentation and subsequent secondary contact. A more wide-ranging study incorporated the samples from Colombia [84], Ecuador, and Nicaragua with those from Costa Rica and Panama [20] and added two molecular fragments: the nuclear *white* gene and the ITS2. A SAMOVA analysis defined three large population demes, one from Nicaragua, Costa Rica and the Atlantic coast of western Panama; a second one incorporating the Pacific coast of western Panama, central-eastern Panama and the Caribbean Colombian coast; and a third one restricted to the Pacific coast of Colombia and Ecuador [85]. There were also four haplogroups, based on the *COI* fragment, which differed little from those found in the earlier Panamanian study [20] except for the addition of a fourth, restricted to the Pacific coast of Colombia and Ecuador, and separated by 18 mutation steps from its nearest haplogroup. Interestingly, because it tracks an earlier history, the *white* gene network showed much less divergence, supporting the overall conclusion that the primary time-frame for anopheline divergence at the species level is Pleistocene [15]. In summary, the combined *An. albimanus* data set strongly supported the presence of a single species in this region, which was expected, but also found very robust evidence for Pleistocene geographic fragmentation followed by range expansion across southern Central America [85].

#### **3.2. The Albitarsis Complex**

Following the newest revelations about the number of species (eight) plus a novel lineage (*An. albitarsis* H) in *An. albitarsis* s.l. [57], parts of an earlier study on the biogeography and popu‐ lation genetics of this complex [17] need to be reconsidered and modified. This is particularly the case for *An*. *janconnae*, which is more restricted than thought (under the taxonomic name of *An. albitarsis* E), the expanded distribution of *An. albitarsis* F (which now includes Venezuela and Trinidad as well as Colombia), the complexities of the distribution of *An. marajoara*, which really may have a very broad range, newly described *An. albitarsis* G, distributed along the Brazilian Amazon, and *An. albitarsis* I, restricted to northwestern Colombia [57]. Despite these problems, one recent study can be used to illustrate the phylogeography of at least *An. albitarsis* G [as far as it is known; 57] and part of the range of *An. marajoara* [87]. *An. albitarsis* G [lineage 2 in reference 87] may be restricted to localities near the Amazon River or its tributaries. It has little population structure and the small subdivisions that were detected in haplotype networks were unrelated to geographic locality. The evidence from the mtDNA *COI* fragment used in this study indicates that this lineage is older than *An*. *marajoara* [lineage 1 in reference 87]. On the other hand, the *white* gene and ITS2 data detected a single network between *An. albitarsis* G and *An. marajoara*, indicating that the divergence is recent. The most compelling result in this study concerning *An. marajoara* is that SAMOVA defined two population demes along the Amazon River, splitting this species into western and eastern entities with differing genetic characteristics. The boundary is located near Rio Jari in Amapá state, not far from one detected in *An. darlingi* [88] and an earlier one seen in a study of *An. nuneztovari* s.l. using restriction fragment length analysis of the mtDNA genome [89]. For *An. marajoara*, this boundary is permeable, since there were shared haplotypes on either side [87]. A denser sampling of all three species could more rigorously test whether this is the result of underlying geological boundaries or perhaps more recent climatic events.

#### **3.3.** *An. darlingi*

and Panama. A new hypothesis suggested that they could be the result of multiple introduc‐ tions into the region, probably caused by historical fragmentation and subsequent secondary contact. A more wide-ranging study incorporated the samples from Colombia [84], Ecuador, and Nicaragua with those from Costa Rica and Panama [20] and added two molecular fragments: the nuclear *white* gene and the ITS2. A SAMOVA analysis defined three large population demes, one from Nicaragua, Costa Rica and the Atlantic coast of western Panama; a second one incorporating the Pacific coast of western Panama, central-eastern Panama and the Caribbean Colombian coast; and a third one restricted to the Pacific coast of Colombia and Ecuador [85]. There were also four haplogroups, based on the *COI* fragment, which differed little from those found in the earlier Panamanian study [20] except for the addition of a fourth, restricted to the Pacific coast of Colombia and Ecuador, and separated by 18 mutation steps from its nearest haplogroup. Interestingly, because it tracks an earlier history, the *white* gene network showed much less divergence, supporting the overall conclusion that the primary time-frame for anopheline divergence at the species level is Pleistocene [15]. In summary, the combined *An. albimanus* data set strongly supported the presence of a single species in this region, which was expected, but also found very robust evidence for Pleistocene geographic

fragmentation followed by range expansion across southern Central America [85].

underlying geological boundaries or perhaps more recent climatic events.

Following the newest revelations about the number of species (eight) plus a novel lineage (*An. albitarsis* H) in *An. albitarsis* s.l. [57], parts of an earlier study on the biogeography and popu‐ lation genetics of this complex [17] need to be reconsidered and modified. This is particularly the case for *An*. *janconnae*, which is more restricted than thought (under the taxonomic name of *An. albitarsis* E), the expanded distribution of *An. albitarsis* F (which now includes Venezuela and Trinidad as well as Colombia), the complexities of the distribution of *An. marajoara*, which really may have a very broad range, newly described *An. albitarsis* G, distributed along the Brazilian Amazon, and *An. albitarsis* I, restricted to northwestern Colombia [57]. Despite these problems, one recent study can be used to illustrate the phylogeography of at least *An. albitarsis* G [as far as it is known; 57] and part of the range of *An. marajoara* [87]. *An. albitarsis* G [lineage 2 in reference 87] may be restricted to localities near the Amazon River or its tributaries. It has little population structure and the small subdivisions that were detected in haplotype networks were unrelated to geographic locality. The evidence from the mtDNA *COI* fragment used in this study indicates that this lineage is older than *An*. *marajoara* [lineage 1 in reference 87]. On the other hand, the *white* gene and ITS2 data detected a single network between *An. albitarsis* G and *An. marajoara*, indicating that the divergence is recent. The most compelling result in this study concerning *An. marajoara* is that SAMOVA defined two population demes along the Amazon River, splitting this species into western and eastern entities with differing genetic characteristics. The boundary is located near Rio Jari in Amapá state, not far from one detected in *An. darlingi* [88] and an earlier one seen in a study of *An. nuneztovari* s.l. using restriction fragment length analysis of the mtDNA genome [89]. For *An. marajoara*, this boundary is permeable, since there were shared haplotypes on either side [87]. A denser sampling of all three species could more rigorously test whether this is the result of

**3.2. The Albitarsis Complex**

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

Several studies on the phylogeography of *An. darlingi* have been undertaken. The earliest one [90] used the mtDNA *COI* fragment and detected a significant genetic division between Central America/northwestern Colombia, and the rest of South America. According to the statistical parsimony network, the more widespread and ancestral haplotypes were in Amazonian and southern South America, suggesting that the Central American/Colombian haplotypes may have originated there. This division was also supported by sequences of the *white* gene, which found two genotypes, genotype I, restricted to the Amazon, and genotype II, in northwestern Colombia and Venezuela, and Central America [91]. A microsatellite analysis of 1,376 samples also strongly supported the initial *COI* genetic division, and found substantial structure within the Amazon Basin [91]. The conclusion was that there were two main drivers for this division: differences in effective population size among the divisions, and physical distances between the populations. A more sophisticated analysis of the mtDNA *COI* fragment included addi‐ tional Brazilian samples and excluded the Central American samples [88]. These researchers detected six main population groups in South America, and found ancestral distribution to be central Amazonia. They proposed that populations became isolated by three barriers: the Amazon River, the Andes and the southeastern Brazilian coastal ranges. They also found that limited dispersal across some landscape types has promoted differentiation between other proximate populations. A local study of *An. darlingi* in Córdoba and Antioquia, Colombia, using mtDNA *COI*, microsatellites and the *white* gene [74] supported the earlier geographic hypothesis [90], discovering that the five populations tested were more closely related to the Central American populations of *An. darlngi* that they were to South American *An*. *darlingi*. Because of local high gene flow among the five populations, similar control strategies could be implemented in these two contiguous Colombian states. Similarly, newly detected *An. darlingi* from Panama were most closely related to Colombian and Central American *An. darlingi* [80]. Concordant phylogeographies were determined for the two neotropical vectors *An. darlingi* and *An*. *triannulatus*[92]. With the mtDNA *COI* fragment, SAMOVA detected four similar population subdivisions: one in southern coastal Brazil, two in central Brazil and one northeast of the Amazon. Both species originated south of the Amazon River and seem to have followed a similar expansion pathway to their present-day distributions. Other neotropical anophelines with similar distributions may share a common spatial and demographic history with these species, and remain to be evaluated.

#### **3.4.** *An. aquasalis*

The only study that attempted to analyse *An. aquasalis* within a phylogeographic framework was conducted using a fragment of the mtDNA *COI* gene with specimens from five localities on either side of the Amazon, in Amapá and Pará states, Brazil [93]. The most important findings from this study inferred that despite the width of the mouth of the Amazon, this freshwater delta was not a barrier for the salt-water tolerant *An. aquasalis*, likely because of so much tidal mixing, and the numerous islands and channels in the region. However, gene flow was restricted, based on isolation by distance that was detected using a Nested Clade Analysis [94]. The relative regional importance of *An. aquasalis* as a malaria vector has waned since the earlier publications [1, 14], so there may be fewer opportunities to pursue phylogeographic questions, especially because the distribution is relatively limited. However, no one has compared specimens from the Atlantic and Pacific coasts, and it is possible that population structure similar to that found for *An. albimanus* [85], could be detected in *An. aquasalis*, considering that both species share a relatively narrow coastal distribution in South America, and were subjected to the same kinds of Pleistocene environmental changes.

#### **3.5.** *An. nuneztovari* **s.l.**

The revision of the taxonomic status of *An. nuneztovari* s.l., that now includes *An. nuneztovari* s.s. and *An*. *goeldii* [81] has implications for the interpretation of the first study of *An. nunez‐ tovari* phylogeography, which focused on the nuclear *white* gene [95]. Five lineages were detected [95], 2 and 3 in Colombia/Venezuela and 1, 4 and 5 in Amazonian Brazil. The earliest divergence, during the Pliocene (5.3-23.0 mya), is between Colombia and Venezuela west of the Andes (lineage 3) and Amazonian lineage 4. The most likely hypothesis to explain this divergence is an early uplift of the East Andean Cordillera [96]. Curiously, the levels of genetic divergences among the five lineages were high, although the minimum spanning network of the haplotypes connected all of them. There were five localities where two lineages were sympatric: in Brazil - Boa Vista, Roraima state; Altamira, Pará state and near Pôrto Velho, Rondonia state; Guayaramerín, Beni, Bolivia; and Rio Socuavó, Zulia, Venezuela. These localities are of special interest, since they may be admixture zones or hotspots of divergence. The simplest hypothesis to explain the five lineages taxonomically is that the two in Colombia/ Venezuela are *An. nuneztovari* s.s. and the three in Brazil are *An. goeldii*. The sharing of haplotypes across the Andes, between eastern and western Venezuela [95], is congruent with and supports findings for *An. albimanus* [83], The Albitarsis Complex [57] and *An. darlingi* (Conn, unpublished data) that have hypothesized that the eastern Andean Cordillera is only a partial barrier for anopheline mosquitoes.

A second phylogeographic study was undertaken with some of the same samples, plus new ones from Amazonian Brazil, using a mtDNA *COI* fragment [97], which charts a more recent history of divergence, all within the Pleistocene, compared with the *white* gene fragment. In this work, there were two major monophyletic clades, I and II. Specimens from Bolivia/ Colombia/Venezuela represent the most basal subclade, IIC; whereas the Amazonian speci‐ mens were found in clades I and II-A and II-B. There were also several localities of sympatry among the clades: five in Amapá, Amazonas and Pará states, Amazonian Brazil, and one in Suriname. None of these are the same as the ones detected by the *white* gene study, perhaps suggesting that these were later areas of sympatry. There was an intriguing connection detected between the specimens from Colombia/Venezuela and those from Amazonian Bolivia, which had previously been seen when sequences of the rDNA ITS2 were used [98]. This may be the signature of the marine incursion hypothesis [95]. One of clades I, II-A or II-B likely represents *An. goeldii*, but additional analyses are needed to determine which one, and also to test the hypothesis of multiple species in the Amazon.

## **4. Conclusions**

earlier publications [1, 14], so there may be fewer opportunities to pursue phylogeographic questions, especially because the distribution is relatively limited. However, no one has compared specimens from the Atlantic and Pacific coasts, and it is possible that population structure similar to that found for *An. albimanus* [85], could be detected in *An. aquasalis*, considering that both species share a relatively narrow coastal distribution in South America,

The revision of the taxonomic status of *An. nuneztovari* s.l., that now includes *An. nuneztovari* s.s. and *An*. *goeldii* [81] has implications for the interpretation of the first study of *An. nunez‐ tovari* phylogeography, which focused on the nuclear *white* gene [95]. Five lineages were detected [95], 2 and 3 in Colombia/Venezuela and 1, 4 and 5 in Amazonian Brazil. The earliest divergence, during the Pliocene (5.3-23.0 mya), is between Colombia and Venezuela west of the Andes (lineage 3) and Amazonian lineage 4. The most likely hypothesis to explain this divergence is an early uplift of the East Andean Cordillera [96]. Curiously, the levels of genetic divergences among the five lineages were high, although the minimum spanning network of the haplotypes connected all of them. There were five localities where two lineages were sympatric: in Brazil - Boa Vista, Roraima state; Altamira, Pará state and near Pôrto Velho, Rondonia state; Guayaramerín, Beni, Bolivia; and Rio Socuavó, Zulia, Venezuela. These localities are of special interest, since they may be admixture zones or hotspots of divergence. The simplest hypothesis to explain the five lineages taxonomically is that the two in Colombia/ Venezuela are *An. nuneztovari* s.s. and the three in Brazil are *An. goeldii*. The sharing of haplotypes across the Andes, between eastern and western Venezuela [95], is congruent with and supports findings for *An. albimanus* [83], The Albitarsis Complex [57] and *An. darlingi* (Conn, unpublished data) that have hypothesized that the eastern Andean Cordillera is only

A second phylogeographic study was undertaken with some of the same samples, plus new ones from Amazonian Brazil, using a mtDNA *COI* fragment [97], which charts a more recent history of divergence, all within the Pleistocene, compared with the *white* gene fragment. In this work, there were two major monophyletic clades, I and II. Specimens from Bolivia/ Colombia/Venezuela represent the most basal subclade, IIC; whereas the Amazonian speci‐ mens were found in clades I and II-A and II-B. There were also several localities of sympatry among the clades: five in Amapá, Amazonas and Pará states, Amazonian Brazil, and one in Suriname. None of these are the same as the ones detected by the *white* gene study, perhaps suggesting that these were later areas of sympatry. There was an intriguing connection detected between the specimens from Colombia/Venezuela and those from Amazonian Bolivia, which had previously been seen when sequences of the rDNA ITS2 were used [98]. This may be the signature of the marine incursion hypothesis [95]. One of clades I, II-A or II-B likely represents *An. goeldii*, but additional analyses are needed to determine which one, and

and were subjected to the same kinds of Pleistocene environmental changes.

**3.5.** *An. nuneztovari* **s.l.**

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

a partial barrier for anopheline mosquitoes.

also to test the hypothesis of multiple species in the Amazon.

There have been many changes in the incrimination, identification and several new insights into the phylogeography of the species discussed in this chapter. The most important taxo‐ nomic changes are those in the Albitarsis Complex, with the discovery of two new species, *An. albitarsis* G and I, and a new lineage, *An. albitarsis* H. Nothing is known about their involvement in malaria transmission, although their ranges all include malaria endemic areas, or their local contribution to diversity or to a better understanding of the complex patterns of Amazonian biogeography and phylogeography. The relative paucity of new work on *An*. *aquasalis* is a reminder that its relative importance appears to be lessening, although it is still likely to be important locally, particularly when in high abundance. Obviously, the importance of *An. darlingi* in still on the rise in several localities in many countries, attributable mainly to its remarkable adaptability and association with landscape changes. The resurrection of *An. goeldii* from synonymy is also a milestone, because it provides a first step toward resolving a longstanding discussion about the possible importance of *An. nuneztovari* s.l. in local trans‐ mission in Amazonian Brazil. It may also clarify some aspects of the recent phylogeographic inferences based on *white* and the mtDNA *COI* genes. Lastly, the detection of concordant phylogeographies, one of which is *An. darlingi* in Brazil, depict a clear path towards future research which will have important epidemiological consequences.

## **Acknowledgements**

We thank Ricardo Guimarães (LabGeo/IEC/SVS) for creating the map in Figure 3. We thank Sara Bickersmith for tireless work on the table, editorial suggestions, and slight modifications to the map. Some of the unpublished work cited here was funded by a grant from the National Institutes of Health (USA) to JEC (AI R01 54139-02) and a grant from CNPq (Brazil) to MMP.

## **Author details**

Jan E. Conn1\*, Martha L. Quiñones2 and Marinete M. Póvoa3

\*Address all correspondence to: jconn@wadsworth.org

1 Department of Biomedical Sciences, School of Public Health, State University of New York-Albany and Griffin Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA

2 Martha L. Quiñones, Faculty of Medicine, National University of Colombia, Bogotá D.C., Colombia

3 Section of Parasitology, Instituto Evandro Chagas, Secretariat of Health Surveillance, Min‐ istry of Health, Belém, Pará, Brazil

## **References**


[11] Tadei WP, Dutary Thatcher B. Malaria vectors in the Brazilian amazon: *Anopheles* of the subgenus *Nyssorhynchus.* Revista do Instituto de Medicina Tropical de São Paulo 2000;42(2) 87-94.

**References**

tomologist 2000;46 238-249.

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

mology 1987;24(4) 433-437.

Parasitology 1993;58(2) 283-292.

2006;101 617-623.

do Instituto Oswaldo Cruz 2001;96(2) 179-184

cis. Parasites & Vectors 2010;3 72.

tance: a review. Parasites & Vectors 2011;4 177.

do Instituto Oswaldo Cruz 2011;106: 223-238.

[1] Lounibos LP, Conn JE. Malaria vector heterogeneity in South America. American En‐

[2] Sinka ME, Rubio-Palis Y, Manguin S, Patil AP, Temperley WH, Gething PW, Van Boeckel T, Kabaria CW, Harbach RE, Hay SI. The dominant *Anopheles* vectors of hu‐ man malaria in the Americas: occurrence data, distribution maps and bionomic pré‐

[3] Hiwat H, Bretas G. Ecology of *Anopheles darlingi* Root with respect to vector impor‐

[4] Montoya-Lerma J, Solarte YA, Giraldo-Calderón GI, Quiñones ML, Ruiz-López F, Wilkerson RC, González R. Malaria vector species in Colombia: a review. Memórias

[5] Marrelli MT, Sallum MAM, Marinotti O. The second internal transcribed spacer of nuclear ribosomal DNA as a tool for Latin American anopheline taxonomy – a criti‐

[6] Wirtz RA, Burkot TR, Graves PM, Andre RG. Field evaluation of enzyme-linked im‐ munosorbent assays for *Plasmodium falciparum* and *Plasmodium vivax* sporozoites in mosquitoes (Diptera: Culicidae) from Papua New Guinea. Journal of Medical Ento‐

[7] Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Molecular and Biochemical

[8] Ryan JR, Davé K, Collins KM, Hochberg L, Sattabongkot J, Coleman RE, Dunton RF, Bangs MJ, Mbogo CM, Cooper RD, Schoeler GB, Rubio-Palis Y, Magris M, Romer LI, Padilla N, Quakyi IA, Bigoga J, Leke RG, Akinpelu O, Evans B, Walsey M, Patterson P, Wirtz RA, Chan AS. Extensive multiple test center evaluation of the VecTest ma‐ laria antigen panel assay. Medical and Veterinary Entomology 2002;17 321-332.

[9] Póvoa MM, Wirtz RA, Lacerda RN, Miles MA, Warhurst D. Malaria vectors in the municipality of Serra do Navio, State of Amapá, Amazon Region, Brazil. Memorias

[10] Quiñones ML, Ruiz F, Calle DA, Harbach RE, Erazo HF, Linton YM. 2006. Incrimina‐ tion of *Anopheles (Nyssorhynchus) rangeli* and *An. (Nys.) oswaldoi* as natural vectors of *Plasmodium vivax* in Southern Colombia. Memórias do Instituto Oswaldo Cruz

cal review. Memórias do Instituto Oswaldo Cruz 2006;101(8) 817-832.


[36] dos Santos RL, Padilha A, Costa MD, Costa EM, Dantas-Filho Hde C, Póvoa MM. Malaria vectors in two indigenous reserves of the Brazilian Amazon. Revista de Saúde Pública 2009;43(5) 859-868.

[25] Conn JE, Wilkerson RC, Segura MN, de Souza RT, Schlichting CD, Wirtz RA, Póvoa MM. Emergence of a new neotropical malaria vector facilitated by human migration and changes in land use. American Journal of Tropical Medicine and Hygiene

[26] de Barros FS, Honório NA, Arruda ME. Mosquito anthropophily: implications on malaria transmission in the Northern Brazilian Amazon. Neotropical Entomology

[27] Póvoa MM, de Souza RT, Lacerda RN, Rosa ES, Galiza D, de Souza JR, Wirtz RA, Schlichting CD, Conn JE. The importance of *Anopheles albitarsis* E and *An. darlingi* in human malaria transmission in Boa Vista, state of Roraima, Brazil. Memórias do In‐

[28] da Silva-Vasconcelos A, Kató MY, Mourão EN, de Souza RT, Lacerda RN, Sibajev A, Tsouris P, Póvoa MM, Momen H, Rosa-Freitas MG. Biting indices, host-seeking ac‐ tivity and natural infection rates of anopheline species in Boa Vista, Roraima, Brazil

[29] Jiménez P, Conn JE, Wirtz R, Brochero H. *Anopheles* (Diptera: Culicidae) vectores de malaria en el municipio de Puerto Carreño, Vichada, Colombia. Biomedica 2012;32

[30] Moreno JE, Rubio-Palis Y, Páez E, Pérez E, Sánchez V, Vaccari E. Malaria entomolog‐ ical inoculation rates in gold mining areas of Southern Venezuela. Memorias do Insti‐

[31] Moreno JE, Rubio-Palis Y, Páez E, Pérez E, Sánchez V. Abundance, biting behavior and parous rate of anopheline mosquito species in relation to malaria incidence in gold-mining areas of southern Venezuela. Medical and Veterinary Entomology

[32] Ribeiro MCT, Goncalves EDD, Tauil PL, da Silva AR. Epidemiological aspects of a malaria focus in the districts of São Luis, MA. Revista da Sociedade Brasileira de Me‐

[33] Póvoa MM, Conn JE, Schlichting CD, Amaral JC, Segura MN, da Silva AN, dos San‐ tos CC, Lacerda RN, de Souza RT, Galiza D, Santa Rosa EP, Wirtz RA. Malaria vec‐ tors, epidemiology, and the re-emergence of *Anopheles darlingi* in Belém, Pará, Brazil.

[34] Laubach HE, Validum L, Bonilla JA, Agar A, Cummings R, Mitchell C, Cuadrado RR, Palmer CJ. Identification of *Anopheles aquasalis* as a possible vector of malaria in

Guyana, South America. West Indian Medical Journal 2001;50(4) 319-21.

[35] Póvoa MM. 2002. Report on Malaria transmission in Suriname. 5p

from 1996 to 1998. Memórias do Instituto Oswaldo Cruz 2002;97(2) 151-161.

2002;66(1) 18-22.

13-21.

2007;21(4) 339-349.

2010;39(6) 1039-1043.

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

stituto Oswaldo Cruz 2006;101(2) 163-168.

tuto Oswaldo Cruz 2009;104(5) 764-768.

dicina Tropical 2005;38(3) 272-274.

Journal of Medical Entomology 2003;40(4) 379-386.


sis Group (*Anopheles: Nyssorhynchus*) of Neotropical malaria vectors. Parasites & Vec‐ tors 2012;5:44.

[58] Gutiérrez LA, Orrego LM, Gómez GF, López A, Luckart S, Conn JE, Correa MM. A new mtDNA COI gene lineage closely related to *Anopheles janconnae* of the Albitarsis complex in the Caribbean region of Colombia. Memórias do Instituto Oswaldo Cruz 2010;105 1019-1025.

[47] Hiwat H, Mitro S, Samjhawan A, Sardjoe P, Soekhoe T, Takken W. Collapse of *Anopheles darlingi* populations in Suriname after introduction of insecticide-treated nets (ITNs); malaria down to near elimination level. American Journal of Tropical

[48] Magris M, Rubio-Palis Y, Menares C, Villegas L. Vector bionomics and malaria trans‐ mission in the Upper Orinoco River, Southern Venezuela. Mem Inst Oswaldo Cruz

[49] Rubio-Palis Y. 2000. *Anopheles* (*Nyssorhynchus*) de Venezuela: taxonomia, bionomía, ecología e importancia médica. Escuela de Malariología y Saneamiento Ambiental "Dr. Arnoldo Gabaldon" y Proyecto Control de Enfermedades Endémicas, Maracay,

[50] Guthmann JP, Hall AJ, Jaffar S, Palacios A, Lines J, Llanos-Cuentas A. Environmental risk factors for clinical malaria: a case-control study in the Grau region of Peru. Transactions of the Royal Society of Tropical Medicine and Hygiene 2001;95(6)

[51] Vargas F, Córdova O, Alvardo A. Determinación de la Resistencia a insecticidas en *Aedes aegypti, Anopheles albimanus* y *Lutzomyia peruensis* procedentes del Norte Perua‐ no. Revista Peruana de Medicina Experimental y Salud Publica 2006;23(4) 259-264.

[52] DIGESA-Direccion General de Salud Ambiental, Ministerio de Salud de Perú. Plan de Implementación de la Estrategia de Riego con Secas Intermitentes en el Cultivo de Arroz para el Control Vectorial de la Malaria en Regiones Priorizadas del Perú. Lima,

[53] Loaiza JR, Bermingham E, Scott ME, Rovira JR, Conn JE. Species composition and distribution of adult *Anopheles* (Diptera: Culicidae) in Panama. Journal of Medical

[54] Pinault LL, Hunter FF. New highland distribution records of multiple *Anopheles* spe‐

[55] Pinault LL, Hunter FF. Larval habitat associations with human land uses, roads, riv‐ ers, and land cover for *Anopheles albimanus, A. pseudopunctipennis,* and *A. punctimacu‐ la* (Diptera: Culicidae) in coastal and highland Ecuador. Frontiers in Physiology

[56] Pinault LL, Hunter FF. Characterization of larval habitats of *Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula,* and *Anopheles oswaldoi* s.l. popu‐ lations in lowland and highland Ecuador. Journal of Vector Ecology 2012;37(1)

[57] Ruiz-Lopez F, Wilkerson RC, Conn JE, McKeon SN, Levin DM, Quiñones ML, Póvoa MM, Linton YM. DNA barcoding reveals both known and novel taxa in the Albitar‐

cies in the Ecuadorian Andes. Malaria Journal 2011;11(10) 236.

Medicine and Hygiene 2012;86(4) 649-655.

2007;102(3) 303-311.

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

Venezuela.

577-583.

Perú, 2011. 33p.

2012;3 59.

124-136.

Entomology 2008;45(5) 841-851.


neous environment. American Journal of Tropical Medicine and Hygiene 2010;82(2) 194-201.


[81] Calado DC, Foster PG, Bergo ES, dos Santos CLS, Galardo AKR, Sallum MAM. Res‐ urrection of *Anopheles goeldii* from synonymy with *Anopheles nuneztovari* (Diptera, Culicidae) and a new record for *Anopheles dunhami* in the Brazilian Amazon. Memó‐ rias do Instituto Oswaldo Cruz 2008;103(8) 791-799.

neous environment. American Journal of Tropical Medicine and Hygiene 2010;82(2)

[70] da Silva Ade N, Fraiha-Neto H, dos Santos CC, Segura Mde N, Amaral JC, Gorayeb Ide S, Lacerda RN, Sucupira IM, Pimentel LN, Conn JE, Póvoa MM. Anophelines in Belém, Pará, Brazil: current and retrospective data. Cadernos de Saude Publica

[71] Lardeux F, Chávez T, Rodríguez R, Torrez L. *Anopheles* of Bolivia: new records with an updated and annotated checklist. Comptes Rendus Biologies 2009;332(5) 489-499.

[72] Harris AF, Matias-Arnéz A, Hill N. Biting time of *Anopheles darlingi* in the Bolivian Amazon and implications for control of malaria. Transactions of the Royal Society of

[73] Zimmerman RH, Galardo AK, Lounibos LP, Arruda M, Wirtz R. Bloodmeal hosts of *Anopheles* species (Diptera: Culicidae) in a malaria-endemic area of the Brazilian Am‐

[74] Gutiérrez LA, Gómez GF, González JJ, Castro MI, Luckart S, Conn JE, Correa MM. Microgeographic genetic variation of the malaria vector *Anopheles darlingi* Root (Dip‐ tera: Culicidae) from Córdoba and Antioquia, Colombia. American Journal of Tropi‐

[75] Aramburu Guarda J, Ramal Asayag C, Witzig R. Malaria reemergence in the Peruvi‐

[76] Turell MJ, Sardelis MR, Jones JW, Watts DM, Fernandez R, Carbajal F, Pecor JE, Klein TA. Seasonal distribution, biology, and human attraction patterns of mosquitoes (Diptera: Culicidae) in a rural village and adjacent forested site near Iquitos, Peru.

[77] Reinbold-Wasson DD, Sardelis MR, Jones JW, Watts DM, Fernandez R, Carbajal F, Pecor JE, Calampa C, Klein TA, Turell MJ. Determinants of *Anopheles* seasonal distri‐ bution patterns across a forest to periurban gradient near Iquitos, Peru. American

[78] Vittor AY, Gilman RH, Tielsch J, Glass G, Shields T, Lozano WS, Pinedo-Cancino V, Patz JA. The effect of deforestation on the human-biting rate of *Anopheles darlingi*, the primary vector of Falciparum malaria in the Peruvian Amazon. American Journal of

[79] Maheu-Giroux M, Casapía M, Soto-Calle VE, Ford LB, Buckeridge DL, Coomes OT, Gyorkos TW. Risk of malaria transmission from fish ponds in the Peruvian Amazon.

[80] Loaiza J, Scott M, Bermingham E, Rovira J, Sanjur O, Conn JE. *Anopheles darlingi* (Diptera: Culicidae) in Panama. American Journal of Tropical Medicine and Hygiene

Tropical Medicine and Hygiene 2006;100(1) 45-47.

cal Medicine and Hygiene 2010;83(1) 38-47.

azon. Journal of Medical Entomology 2006;43(5) 947-956.

an Amazon region. Emerging Infectious Diseases 1999;5 209-215.

Journal of Medical Entomology 2008;45(6) 1165-1172.

Tropical Medicine and Hygiene 2006:74(1) 3-11.

Acta Tropica. 2010 115(1-2);112-118.

2009;81(1) 23-26.

Journal of Tropical Medicine and Hygiene 2012;86(3) 459-463.

194-201.

2006;22(8) 1575-1585.

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

