Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

Bioassay and biochemical analyses of insecticide resistance in southern African Anopheles funestus (Diptera: Culicidae)

Anopheles funestus Giles has been implicated as a major malaria vector in sub-Saharan Africa where pyrethroid insecticides are widely used in agriculture and public health. Samples of this species from northern Kwazulu/Natal in South Africa and the Beluluane region of southern Mozambique showed evidence of resistance to pyrethroid insecticides. Insecticide exposure, synergist and biochemical assays conducted on A. funestus suggested that elevated levels of mixed function oxidases were responsible for the detoxification of pyrethroids in resistant mosquitoes in these areas. The data suggested that this mechanism was also conferring cross-resistance to the carbamate insecticide propoxur.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

Can anything be done to maintain the effectiveness of pyrethroid-impregnated bednets against malaria vectors?

Pyrethroid-treated bednets are the most promising available method of controlling malaria in the tropical world. Every effort should be made to find methods of responding to, or preventing, the emergence of pyrethroid resistance in the Anopheles vectors. Some cases of such resistance are known, notably in An. gambiae in West Africa where the kdr type of resistance has been selected, probably because of the use of pyrethroids on cotton. Because pyrethroids are irritant to mosquitoes, laboratory studies on the impact of, and selection for, resistance need to be conducted with free-flying mosquitoes in conditions that are as realistic as possible. Such studies are beginning to suggest that, although there is cross-resistance to all pyrethroids, some treatments are less likely to select for resistance than others are. Organophosphate, carbamate and phenyl pyrazole insecticides have been tested as alternative treatments for nets or curtains. Attempts have been made to mix an insect growth regulator and a pyrethroid on netting to sterilize pyrethroid-resistant mosquitoes that are not killed after contact with the netting. There seems to be no easy solution to the problem of pyrethroid resistance management, but further research is urgently needed.     

Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non–alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.     

Resistance to carbosulfan in Anopheles gambiae from Ivory Coast,based on reduced sensitivity of acetylcholinesterase

Resistance to carbosulfan, a carbamate insecticide, was detected in field populations of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) from two ecologically contrasted localities near Bouaké, Ivory Coast: rural M'bé with predominantly M form of An. gambiae susceptible to pyrethroids; suburban Yaokoffikro with predominantly S form of An. gambiae highly resistant to pyrethroids (96% kdr). The discriminating concentration of 0.4% carbosulfan (i.e. double the LC100) was determined from bioassays with the susceptible An. gambiae Kisumu strain. Following exposure to the diagnostic dosage (0.4% carbosulfan for 1 h), mortality rates of female An. gambiae adults (reared from larvae collected from ricefields) were 62% and 29% of those from M'bé and Yaokoffikro, respectively, 24 h post-exposure. Exposure for 3 min to netting impregnated with the operational dosage of carbosulfan 200 mg/m2 gave mortality rates of 88% of those from M'bé and only 12.2% for Yaokoffikro. In each case the control untreated mortality rate was insignificant. Biochemical assays to detect possible resistance mechanism(s) revealed the presence of insensitive AChE in populations of An. gambiae at both localities, more prevalent in the S form at Yaokoffikro than in M form at M'bé, as expected from bioassays results. Our study demonstrates the need to monitor carbamate resistance among populations of the An. gambiae complex in Africa, to determine its spread and anticipate vector control failure if these insecticides are employed.     

Anopheles gambiae genome: perspectives for malaria control

Malaria, a disease that infects 300 million people throughout the world and kills more than a million people, mostly children in sub-Saharan Africa, involves three organisms. The human host where the disease is seen, the protozoan Plasmodium parasite and the mosquito. The parasite is transmitted to humans only by the mosquito vector, which in sub-Saharan regions is generally Anopheles gambiae. Malaria along with AIDS and tuberculosis are killing large numbers of people and crippling the economies of the affected African countries. Though an enormous effort has been made during the past twenty years to develop vaccines to block malaria in humans, the incidence of the disease is increasing in Africa. The reasons for this development include a breakdown in mosquito control related to increased insecticide resistance, as well as increased parasite resistance to antimalarial drugs. It is clear that new methods of Anopheles mosquito control are needed to ameliorate the medical and economic situation in sub-Saharan Africa. As a step toward new malaria control methods, the international Plasmodium falciparum and Anopheles gambiae consortia have carried out the full genome sequencing of the most deadly malaria parasite and the most efficient vector. These, combined with the human genome sequence, provide the genomic infrastructure for a better understanding of the complex interactions within the malaria triad. This essay discusses possible strategies as to how the Anopheles genome can contribute to malaria control.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

Insecticide mixtures for mosquito net impregnation against malaria vectors

Insecticides belonging to the pyrethroid family are the only compounds currently available for the treatment of mosquito nets. Unfortunately, some malaria vector species have developed resistance to pyrethroids and the lack of alternative chemical categories is a great concern. One strategy for resistance management would be to treat mosquito nets with a mixture associating two insecticides having different modes of action. This study presents the results obtained with insecticide mixtures containing several proportions of bifenthrin (a pyrethroid insecticide) and carbosulfan (a carbamate insecticide). The mixtures were sprayed on mosquito net samples and their efficacy were tested against a susceptible strain of Anopheles gambiae, the major malaria vector in Africa. A significant synergism was observed with a mixture containing 25 mg/m2 of bifenthrin (half the recommended dosage for treated nets) and 6.25 mg/m2 of carbosulfan (about 2% of the recommended dosage). The observed mortality was significantly more than expected in the absence of any interaction (80% vs 41%) and the knock-down effect was maintained, providing an effective barrier against susceptible mosquitoes.     

Combined pyrethroid and carbamate 'two-in-one' treated mosquito nets: field efficacy against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus

A new approach is proposed in the treatment of mosquito nets, using a 'two-in-one' combination of pyrethroid and non-pyrethroid insecticides applied to different parts of bednets. The objectives are mainly to overcome certain limitations of pyrethroid-impregnated bednets currently recommended for malaria control purposes. Apart from developing alternatives to pyrethroid dependency, we sought to counteract pyrethroid irritant effects on mosquitoes (excito-repellency) and resistance to pyrethroids. The idea takes advantage of the presumed host-seeking behaviour of mosquitoes confronted by a net draped over a bed, whereby the mosquito may explore the net from the top downwards. Thus, nets could be more effective if treated on the upper part with residual non-irritant insecticide (carbamate or organophosphate) and with a pyrethroid on the lower part. Sequential exposure to different insecticides with distinct modes of action is equivalent to the use of a mixture as a potential method of managing insecticide resistance. We also intended to improve the control of nuisance mosquitoes, especially Culex quinquefasciatus Say (Diptera: Culicidae) that often survive pyrethroids, in order to encourage public compliance with use of insecticide-treated nets (ITNs). Polyester bednets were pretreated with residual pyrethroid (bifenthrin 50 mg/m2 or deltamethrin 25 mg/m2) on the lower half and with carbamate (carbosulfan 300 mg/m2) on the upper half to minimize contact with net users. Unreplicated examples of these 'two-in-one' treated nets were field-tested against wild mosquitoes, in comparison with an untreated net and bednets treated with each insecticide alone, including PermaNet wash-resistant formulation of deltamethrin 50 mg/m2. Overnight tests involved volunteers sleeping under the experimental bednets in verandah-trap huts at Yaokofikro, near Bouaké in C te d'Ivoire, where the main malaria vector Anopheles gambiae Giles, as well as Culex quinquefasciatus Say, are highly resistant to pyrethroids. Efficacy of these ITNs was assessed in the huts by four entomological criteria: deterrency and induced exophily (effects on hut entry and exit), blood-feeding and mortality rates (immediate and delayed). Overall, the best impact was achieved by the bednet treated with carbosulfan alone, followed by 'two-in-one' treatments with carbosulfan plus pyrethroid. Blood-feeding rates were 13% An. gambiae and 17% Cx. quinquefasciatus in huts with untreated nets, but only 3% with carbosulfan ITNs, 7-11% with combined ITN treatment, 6-8% An. gambiae and 12-14% Cx. quinquefasciatus with pyrethroid alone. Mosquitoes that entered the huts were killed sooner by nets with combined treatment than by pyrethroid alone. Mortality-rates in response to ITNs with carbosulfan (alone or combined with pyrethroid) were significantly greater for Cx. quinquefasciatus, but not for An. gambiae, compared to ITNs with only pyrethroid. About 20% of sleepers reported potential side-effects (headache and/or sneezing) from use of ITN treated with carbosulfan alone. Further development of this new 'two-in-one' ITN concept requires a range of investigations (choice of effective products, cost-benefit analysis, safety, etc.) leading to factory production of wash-resistant insecticidal nets treated with complementary insecticides.     

Impact of environment on mosquito response to pyrethroid insecticides: Facts,evidences and prospects

By transmitting major human diseases such as malaria, dengue fever and filariasis, mosquito species represent a serious threat worldwide in terms of public health, and pose a significant economic burden for the African continent and developing tropical regions. Most vector control programmes aiming at controlling life-threatening mosquitoes rely on the use of chemical insecticides, mainly belonging to the pyrethroid class. However, resistance of mosquito populations to pyrethroids is increasing at a dramatic rate, threatening the efficacy of control programmes throughout insecticide-treated areas, where mosquito-borne diseases are still prevalent. In the absence of new insecticides and efficient alternative vector control methods, resistance management strategies are therefore critical, but these require a deep understanding of adaptive mechanisms underlying resistance. Although insecticide resistance mechanisms are intensively studied in mosquitoes, such adaptation is often considered as the unique result of the selection pressure caused by insecticides used for vector control. Indeed, additional environmental parameters, such as insecticides/pesticides usage in agriculture, the presence of anthropogenic or natural xenobiotics, and biotic interactions between vectors and other organisms, may affect both the overall mosquito responses to pyrethroids and the selection of resistance mechanisms. In this context, the present work aims at updating current knowledge on pyrethroid resistance mechanisms in mosquitoes and compiling available data, often from different research fields, on the impact of the environment on mosquito response to pyrethroids. Key environmental factors, such as the presence of urban or agricultural pollutants and biotic interactions between mosquitoes and their microbiome are discussed, and research perspectives to fill in knowledge gaps are suggested.     

Distribution of a knockdown resistance mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in western and southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future.     

Determination of the resistance to organophosphate, carbamate, and pyrethroid insecticides in Panamanian Anopheles albimanus (Diptera: Culicidae) mosquitoes

Introduction. The susceptibility of Anopheles albimanus to organophosphates, carbamates and pyrethroid insecticides was unknown in the Panama communities of Aguas Claras, Pintupo and Puente Bayano, located in the Amerindian Reservation of Madungandi. This region is considered a malaria transmission area, where An. albimanus is the main vector. Objective. The resistance to organophosphate insecticides, carbamates and pyrethroids was evaluated in field populations of the Anopheles albimanus in Panama. Materials and methods. Progeny of An. albimanus collected in three localities in the indigenous Madugandi region were exposed to bioassays of susceptibility to organophosphate insecticides (fenitrothion, malathion and chlorpyrifos), the carbamate (propoxur) and pyrethroids (deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin). The protocols were in accordance with those established for adult mosquitoes by World Health Organization. Results. The three strains of the An. albimanus were resistant to the pyrethroid insecticides deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin. Susceptibility remained for the organophosphate insecticides fenitrothion, malathion, chlorpyrifos, and the carbamate insecticide propoxur. Conclusion. The results provided important information to the vector control program, contributing to the application of new strategies on the use of insecticides, and thereby lengthening the life of the insecticide in use.     

Population structure, speciation, and introgression in the Anopheles gambiae complex

We review here what is known about the population structure and evolutionary dynamics of members of the Anopheles gambiae complex with emphasis on the situation in West Africa. First, the importance of the 2nd chromosome inversion polymorphism is demonstrated especially in adaptation to levels of aridity, a major environmental variable in Africa. This affects the distribution of karyotypes on both a macro- and micro-geographic scale as well as temporally. Such differentiation leads to karyotypes being differentially effective transmitters of malaria and differentially susceptible to indoor residual spraying of insecticides. Second, we review the evidence that cryptic taxa, especially in An. gambiae s.s., exist. This observation stems from both karyotype studies and molecular studies. It is abundantly clear that West African populations of An. gambiae s.s. are often not panmictic units, with premating factors evidently acting to maintain distinct genetic forms. Third, we review phylogenetic studies that have revealed the presence of introgression between the two most important vectors, An. gambiae and An. arabiensis. This is most evident for the 2nd chromosome inversions. This interpretation of phylogenetic data is consistent with a direct laboratory study indicating inversions in this chromosome are stably maintained in back-crossed populations. All of this information has led to the view that members of the An. gambiae complex are highly variable with an abundance of adaptive genetic variation. This presents a significant challenge to vector control programs designed to reduce malaria in sub-Saharan Africa.     

Resistance to dieldrin + fipronil assorts with chromosome inversion 2La in the malaria vector Anopheles gambiae

Cyclodiene insecticide resistance in malaria vector mosquitoes of the Anopheles gambiae species complex (Diptera: Culicidae) has been reported previously from several parts of Africa. We report resistance to dieldrin, a cyclodiene, in two laboratory strains of An. gambiae Giles sensu stricto code-named Ian P20 from Nigeria (1979) and CIG from Cote d'Ivoire (1997). Dieldrin resistance levels were high in adult female mosquitoes (40-75% survived exposure to 4% dieldrin for 1 h) and was closely linked with chromosomal paracentric inversion 2La. This inversion did not occur in Hardy-Weinberg proportions, but showed an excess of heterozygotes in both strains, which may account for the high levels of resistance. This linkage also suggests that dieldrin resistance in Ian P20 is dominant. After subsamples of strain Ian P20 were exposed for 1 h to dieldrin 4% or fipronil 2% (discriminating concentrations), the resultant mortality-rates (61% and 65%) were not significantly different. Most survivors after fipronil treatment also survived subsequent exposure to dieldrin (46/50=92%). This apparent cross-resistance between insecticides of two classes (cyclodiene and phenyl pyrazole) has implications for the management of insecticide resistance in wild populations of the An. gambiae complex.     

Limburger cheese as an attractant for the malaria mosquito Anopheles gambiae s.s

In the process of bloodfeeding, female Anopheles can transmit malaria parasites to humans. At night, while searching for blood, these insects respond to visual, physical and chemical properties of humans. Current research concentrates on the identification of kairomones, which guide mosquitoes to humans. Earlier observations on the biting behaviour of Anopheles gambiae s.s. on humans have now resulted in the discovery of a remarkable attractant for this important malaria vector, and it is thought that this will accelerate the development of odour-baited traps for malaria mosquito surveillance and control in sub-Saharan Africa, as discussed here by Bart Knols and Ruurd De Jong.     

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.