The Anopheles gambiae detoxification chip: a highly specific microarray to study metabolic-based insecticide resistance in malaria vectors

Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN/U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroid-resistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed.     

Multiple insecticide resistance in Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo

Successful implementation of an integrated vector control program will rely on availability of accurate vector information in the specific location. However, such information can be limited in some countries. The aim of this study was to obtain baseline vector information from Pointe Noire on the Congo coast (Republic of the Congo). Field sampling was conducted during April 2009 in the village of Boutoto and its surrounds, close to the city of Pointe Noire. Anopheles gambiae sensu lato mosquitoes were collected resting indoors. Samples were analyzed for insecticide susceptibility, species identification, and Plasmodium sporozoite infection. Molecular and biochemical assays were conducted to characterize insecticide resistance mechanisms. The malaria vector A. gambiae S-form was the only mosquito species identified, and it had a high Plasmodium falciparum infection rate (9.6%). Multiple insecticide resistance was detected in this population with full susceptibility to only one insecticide class, the organophosphates. Dieldrin and DDT resistance was mainly attributed to target-site resistance (the Rdl and L1014F/L1014S kdr mutations respectively), whereas pyrethroid resistance was mainly attributed to P450 metabolic enzyme-mediated detoxification in addition to kdr. The role of various insecticide resistance mechanisms revealed a complex association between metabolic detoxification and reduced target-site sensitivity.     

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

One of the challenges faced in malarial control is the acquisition of insecticide resistance that has developed in mosquitoes that are vectors for this disease. Anopheles gambiae, which has been the major mosquito vector of the malaria parasite Plasmodium falciparum in Africa, has over the years developed resistance to insecticides including dieldrin, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pyrethroids. Previous microarray studies using fragments of 230 An. gambiae genes identified five P450 loci, including CYP4C27, CYP4H15, CYP6Z1, CYP6Z2, and CYP12F1, that showed significantly higher expression in the DDT-resistant ZAN/U strain compared with the DDT-susceptible Kisumu strain. To predict whether either of the CYP6Z1 and CYP6Z2 proteins might potentially metabolize DDT, we generated and compared molecular models of these two proteins with and without DDT docked in their catalytic sites. This comparison indicated that, although these two CYP6Z proteins share high sequence identity, their metabolic profiles were likely to differ dramatically from the larger catalytic site of CYP6Z1, potentially involved in DDT metabolism, and the more constrained catalytic site of CYP6Z2, not likely to metabolize DDT. Heterologous expressions of these proteins have corroborated these predictions: only CYP6Z1 is capable of metabolizing DDT. Overlays of these models indicate that slight differences in the backbone of SRS1 and variations of side chains in SRS2 and SRS4 account for the significant differences in their catalytic site volumes and DDT-metabolic capacities. These data identify CYP6Z1 as one important target for inhibitor design aimed at inactivating insecticide-metabolizing P450s in natural populations of this malarial mosquito.     

Distribution of the molecular forms of Anopheles gambiae and pyrethroid knock down resistance gene in Nigeria

We investigated the distribution of the molecular M and S forms of Anopheles gambiae and the knock down resistance (kdr) gene associated with pyrethroid and DDT resistance in A. gambiae s.s. at 13 localities across Nigeria. Two-three days old adult female mosquito reared from larval collections were tested using standard WHO procedures, diagnostic test kits and impregnated papers to assess their pyrethroid resistance status. Specimens were identified by PCR assays and characterized for the kdr gene. DNA from adult A. gambiae s.s. collected from human dwellings were also tested for the presence of the kdr gene. The overall collection was a mix of the molecular M and S forms across the mangrove (63:37%), forest (56:44%), and transitional (36:64%) ecotypes, but almost a pure collection of the S form in the Guinea and Sudan-savanna. Results of insecticide susceptibility tests showed that mosquitoes sampled at seven localities were susceptible to permethrin, deltamethrin, and DDT, but populations of A. gambiae resistant to these insecticides were recorded at six other localities mainly in the transitional and Guinea-savanna ecotypes. The kdr gene was found only in the molecular S forms, including areas where both forms were sympatric. The overall kdr frequency was low: <47% in forest, 37-48% in the transitional, and 45-53% in Guinea-savanna. The data suggest that pyrethroid resistance in A. gambiae in Nigeria is not as widespread when compared to neighbouring West African countries.     

Insecticide resistance in Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and Anopheles gambiae Giles (Diptera: Culicidae) could compromise the sustainability of malaria vector control strategies in West Africa

Insecticides from the organophosphate (OP) and pyrethroid (PY) chemical families, have respectively, been in use for 50 and 30 years in West Africa, mainly against agricultural pests, but also against vectors of human disease. The selection pressure, with practically the same molecules year after year (mainly on cotton), has caused insecticide resistance in pest populations such as Bemisia tabaci, vector of harmful phytoviruses on vegetables. The evolution toward insecticide resistance in malaria vectors such as Anopheles gambiae sensus lato (s.l.) is probably related to the current use of these insecticides in agriculture. Thus, successful pest and vector control in West Africa requires an investigation of insect susceptibility, in relation to the identification of species and sub species, such as molecular forms or biotypes. Identification of knock down resistance (kdr) and acetylcholinesterase gene (Ace1) mutations modifying insecticide targets in individual insects and measure of enzymes activity typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transferase) are indispensable in understanding the mechanisms of resistance. Insecticide resistance is a good example in which genotype–phenotype links have been made successfully. Insecticides used in agriculture continue to select new resistant populations of B. tabaci that could be from different biotype vectors of plant viruses. As well, the evolution of insecticide resistance in An. gambiae threatens the management of malaria vectors in West Africa. It raises the question of priority in the use of insecticides in health and/or agriculture, and more generally, the question of sustainability of crop protection and vector control strategies in the region. Here, we review the susceptibility tests, biochemical and molecular assays data for B. tabaci, a major pest in cotton and vegetable crops, and An. gambiae, main vector of malaria. The data reviewed was collected in Benin and Burkina Faso between 2008 and 2010 under the Corus 6015 research program. This review aims to show: (i) the insecticide resistance in B. tabaci as well as in An. gambiae; and (ii) due to this, the impact of selection of resistant populations on malaria vector control strategies. Some measures that could be beneficial for crop protection and vector control strategies in West Africa are proposed.     

Dynamics of knockdown pyrethroid insecticide resistance alleles in a field population of Anopheles gambiae s.s. in southwestern Nigeria

BACKGROUND & OBJECTIVES: Pyrethroid insecticide resistance in the malaria vector Anopheles gambiae Giles is mainly associated with reduced target site sensitivity arising from a single point mutation in the sodium channel gene, often referred to as knockdown resistance (kdr). This resistance mechanism is widespread in West Africa and was reported for the first time in Nigeria in 2002. Here we present changes in the susceptibility/resistance status of the molecular 'M' and 'S' forms of An. gambiae and the frequency of the kdr alleles from 2002-05. METHODS: Adult anophelines were sampled quarterly inside human dwellings from January 2002 to December 2005 and adults reared from wild larvae were identified using morphological keys. Samples belonging to the An. gambiae complex were subjected to PCR assays for species identification and detection of molecular 'M' and 'S' forms. Insecticide susceptibility tests were carried out using standard WHO procedures and test kits only on 2-3 days old adult An. gambiae s.s. reared from larval collections. The kdr genotypes were determined in both live and dead specimens of An. gambiae s.s. using alleles-specific polymerase chain reaction diagnostic tests. RESULTS: The overall collection showed that the molecular 'S' form was predominant (> 60%) but the proportions of both forms in the mosquito populations from 2002-05 were not statistically different. Both forms also occurred throughout the period without apparent relationship to wet or dry season. Insecticide susceptibility tests did not show any significant increase in the resistance status recorded for either Permethrin or DDT from 2002-05, rather, an improvement in the susceptibility status of the mosquitoes to these insecticides was observed from 2004-05 relative to the tests performed in 2002-03. CONCLUSION: The proportion of the molecular 'M' and 'S' form of An. gambiae and the kdr frequencies have not increased significantly from 2002 when it was first reported in Nigeria. However, the findings on susceptible mosquitoes exhibiting the kdr gene need further investigation. Further monitoring of this may provide additional information on the ongoing debate on the possibility of restriction in gene flow and reproductive barriers in these sympatric taxa.     

First report of the L1014S kdr mutation in wild populations of Anopheles gambiae M and S molecular forms in Burkina Faso (West Africa)

We investigated the occurrence of the L1014F and L1014S kdr mutations in malaria vector populations in Burkina Faso (West Africa). A cross-sectional survey was conducted at 10 sites all located in cotton cultivation areas which are assumed to be the major insecticide resistance selection foci in Burkina Faso. The hot ligation method was used to detect the two kdr mutations in field collected Anopheles gambiae s.l. samples. For the first time in Burkina Faso the L1014S mutation was identified in both M and S forms of An. gambiae s.s. populations collected from the site of Koupela in the central-eastern region at low frequency. Furthermore, the L1014S mutation was also found in one specimen of An. arabiensis collected from the Dano site. The data generated in this study provides additional evidence of the spread of the L1014S mutation into An. gambiae s.l. populations in West Africa. It is now important to evaluate the role of the L1014S mutation in the pyrethroid resistance phenotype and assess its potential impact on the efficacy of pyrethroid-based control measures in West Africa where several resistance mutations now coexist.     

Anopheles gambiae resistance to pyrethroid-treated nets in cotton versus rice areas in Mali

The rise and spread of Anopheles gambiae s.l. (the major malaria vector sub-Saharan Africa) resistance to pyrethroids is of great concern owing to the predominant role of pyrethroid-treated nets in the WHO global strategy for malaria control. Use of pyrethroids for agricultural purposes may exert a strong selection pressure, favouring the emergence of insecticide resistance. The objective of this study was to evaluate the efficacy of alpha-cypermethrin treated nets in settings where insecticides are used against pests. This was assessed in two ways, i.e. under laboratory conditions using the WHO standard cones test technique and in experimental huts, on Anopheles gambiae s.l. collected in two Malian rural sites, Koumantou characterised by cotton crops and high insecticide use and Sélingué, a rice field area with low insecticide use. According to the WHO standard cones test technique, there was no difference between mosquitoes collected in the two sites; KD50 time was less than 3 min and the KD95 time below 30 min. Nevertheless, in the experimental huts with alpha-cypermethrin treated bed nets, the mosquito mortality rate was significantly lower in Koumantou (102/361, 28.2%) than in Sélingué (122/233, 52.3%) (RR: 0.65, 95%CI: 0.56-0.76) (p<0.001). In addition, in Koumantou the percentage of unfed mosquitoes found in the veranda was much lower in the huts with untreated (26.0%, 33/127) than in those with treated nets (92.2%, 118/128) (p<0.01) while in Sélingué there was no difference between huts with treated and untreated bed nets. Alpha-cypermethrin treated bed nets had a significant effect on mortality and repelling behaviour of Anopheles gambiae s.l. though in Koumantou treated bed nets were less efficacious, possibly due to the intense use of pesticide for agriculture.     

Breakpoint structure reveals the unique origin of an interspecific chromosomal inversion (2La) in the Anopheles gambiae complex

Paracentric chromosomal inversions are major architects of organismal evolution and have been associated with adaptations relevant to malaria transmission in anopheline mosquitoes. The processes responsible for their origin and maintenance, still poorly understood, can be illuminated by analysis of inversion breakpoint sequences. Here, we report the breakpoint structure of chromosomal inversion 2La from the principal malaria vector Anopheles gambiae and its relatives in the A. gambiae complex. The distal and proximal breakpoints of the standard (2L+a) arrangement contain gene duplications: full-length genes and their truncated copies at opposite ends. Intact genes without pseudogene copies in the alternative arrangement (2La) imply that 2L+a is derived and was viable despite damage to genes, because duplication preserved gene function. A unique origin for the interspecific 2La inversion was challenged previously by indirect genetic evidence, but breakpoint sequences determined from members of the A. gambiae complex strongly suggest their descent from a single event. The derived position of 2L+a, long considered ancestral in this medically important group, has significant implications for the phylogenetic history and the evolution of vectorial capacity in the A. gambiae complex.     

A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum

Cytochrome P450-mediated detoxification is one of the most important mechanisms involved in insecticide resistance. However, the molecular basis of this mechanism and the physiological functions of P450s associated with insecticide resistance remain largely unknown. Here, we exploited the functional genomics and reverse genetic approaches to identify and characterize a P450 gene responsible for the majority of deltamethrin resistance observed in the QTC279 strain of Tribolium castaneum. We used recently completed whole-genome sequence of T. castaneum to prepare custom microarrays and identified a P450 gene, CYP6BQ9, which showed more than a 200-fold higher expression in the deltamethrin-resistant QTC279 strain when compared with its expression in the deltamethrin-susceptible Lab-S strain. Functional studies using both double-strand RNA (dsRNA)-mediated knockdown in the expression of CYP6BQ9 and transgenic expression of CYP6BQ9 in Drosophila melanogaster showed that CYP6BQ9 confers deltamethrin resistance. Furthermore, CYP6BQ9 enzyme expressed in baculovirus metabolizes deltamethrin to 4-hydroxy deltamethrin. Strikingly, we also found that unlike many P450 genes involved in insecticide resistance that were reported previously, CYP6BQ9 is predominantly expressed in the brain, a part of the central nervous system (CNS) containing voltage-gated sodium channels targeted by deltamethrin. Taken together, the current studies on the brain-specific insect P450 involved in deltamethrin resistance shed new light on the understanding of the molecular basis and evolution of insecticide resistance.     

The role of agricultural use of insecticides in resistance to pyrethroids in Anopheles gambiae s.l. in Burkina Faso

Agricultural use of insecticides is involved in the selection of resistance to these compounds in field populations of mosquitoes in Burkina Faso. Anopheles gambiae s.l. was resistant to permethrin and DDT in cotton-growing and urban areas, but susceptible in areas with limited insecticide selection pressure (rice fields and control areas). Nevertheless, resistance to these insecticides was observed in a village on the outskirts of the rice fields at the end of the rainy season, suggesting that the latter population of mosquitoes had migrated from the surrounding cotton villages into the rice fields. A seasonal variation of resistance observed in the cotton-growing area is related to the distribution of the molecular M and S forms of An. gambiae, since resistance to pyrethroids has so far only been reported in the S form. Pyrethroid resistance in west African An. gambiae was conferred by target site insensitivity through a knockdown resistance (kdr)-like mutation, which was present at high frequencies in mosquitoes in the cotton-growing and urban areas.     

Adaptive divergence between incipient species of Anopheles gambiae increases resistance to Plasmodium

The African malaria mosquito Anopheles gambiae is diversifying into ecotypes known as M and S forms. This process is thought to be promoted by adaptation to different larval habitats, but its genetic underpinnings remain elusive. To identify candidate targets of divergent natural selection in M and S, we performed genomewide scanning in paired population samples from Mali, followed by resequencing and genotyping from five locations in West, Central, and East Africa. Genome scans revealed a significant peak of M-S divergence on chromosome 3L, overlapping five known or suspected immune response genes. Resequencing implicated a selective target at or near the TEP1 gene, whose complement C3-like product has antiparasitic and antibacterial activity. Sequencing and allele-specific genotyping showed that an allelic variant of TEP1 has been swept to fixation in M samples from Mali and Burkina Faso and is spreading into neighboring Ghana, but is absent from M sampled in Cameroon, and from all sampled S populations. Sequence comparison demonstrates that this allele is related to, but distinct from, TEP1 alleles of known resistance phenotype. Experimental parasite infections of advanced mosquito intercrosses demonstrated a strong association between this TEP1 variant and resistance to both rodent malaria and the native human malaria parasite Plasmodium falciparum. Although malaria parasites may not be direct agents of pathogen-mediated selection at TEP1 in nature--where larvae may be the more vulnerable life stage--the process of adaptive divergence between M and S has potential consequences for malaria transmission.     

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid‐resistant Aedes aegypti?

Background Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non‐specific esterases, and to enhance cuticular penetration of the insecticide. Objective To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. Methods Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti‘detox chip’. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM‐resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM‐treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. Conclusion Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.     

Insecticide treated nets: impact on vector populations and relevance of initial intensity of transmission and pyrethroid resistance

Insecticide treated bednets locate a deposit of a quick-acting insecticide of low human toxicity between a sleeper and host-seeking mosquitoes. Thus a chemical barrier is added to the often incomplete physical barrier provided by the net. Treated nets may be considered as mosquito traps baited by the odour of the sleeper. Trials in Assam, Tanzania and elsewhere have shown that when a whole community is provided with treated nets, so many mosquitoes of anthropophilic species are killed by contact with the nets that the density and/or sporozoite rate of the vector population is reduced. In order to gain this "mass" or community effect, in addition to widespread personal protection, and thus to achieve the full potential of the treated net method, a high per cent coverage of the community is needed. This suggests that organised free provision of treated nets, comparable to a house spraying programme, is likely to be more cost-effective than trying to market nets and insecticide to very poor rural people. In areas with high malaria transmission, where acquisition of immunity to malaria is very important, it has been argued that vector control (without vector eradication) could, in the long run, make the situation worse by preventing the normal build-up of immunity. However, our data from Tanzania do not support this idea--3-4 years after provision of nets (which are re-treated annually) young children are still showing clear health benefits; older children are not "paying" for this by showing worse impact of malaria. There is less malaria morbidity in a highland area where malaria transmission is about 15x less intense than in a nearby lowland area. The per cent impact of treated nets malaria morbidity in both area was very similar. At present only pyrethroids are used for net treatment which suggested that emergence of pyrethroid resistance would have a disastrous effect. However, in West Africa, where there is now a high frequency of the kdr resistance gene in Anopheles gambiae, it is reported that treated nets continue to have a powerful impact on vector populations. In Tanzania, pyrethroid resistance has not been detected in malaria vectors, but it has emerged in bedbugs after seven years use of treated nets.     

Population genetic structure of Plasmodium falciparum in the two main African vectors, Anopheles gambiae and Anopheles funestus

We investigated patterns of genetic diversity of Plasmodium falciparum associated with its two main African vectors: Anopheles gambiae and Anopheles funestus. We dissected 10,296 wild-caught mosquitoes from three tropical sites, two in Cameroon (Simbock and Tibati, separated by 320 km) and one in Kenya (Rota, >2,000 km from the other two sites). We assayed seven microsatellite loci in 746 oocysts from 183 infected mosquito guts. Genetic polymorphism was very high in parasites isolated from both vector species. The expected heterozygosity (H(E)) was 0.79 in both species; the observed heterozygosities (H(O)) were 0.32 in A. funestus and 0.42 in A. gambiae, indicating considerable inbreeding within both vector species. Mean selfing (s) between genetically identical gametes was s = 0.33. Differences in the rate of inbreeding were statistically insignificant among sites and between the two vector species. As expected, because of the high rate of inbreeding, linkage disequilibrium was very high; it was significant for all 21 loci pairs in A. gambiae and for 15 of 21 pairs in A. funestus, although only two pairwise comparisons were between loci on the same chromosome. Overall, the genetic population structure of P. falciparum, as evaluated by F statistics, was predominantly clonal rather than panmictic, a population structure that facilitates the spread of antimalarial drug and vaccine resistance and thus may impair the effectiveness of malaria control efforts.     

Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation

Attempts to reconstruct the phylogenetic history of the Anopheles gambiae cryptic species complex have yielded strongly conflicting results. In particular, An. gambiae, the primary African malaria vector, is variously placed as a sister taxon to either Anopheles arabiensis or Anopheles merus. The recent divergence times for members of this complex complicate phylogenetic analysis, making it difficult to unambiguously implicate interspecific gene flow, versus retained ancestral polymorphism, as the source of conflict. Using sequences at four unlinked loci, which were determined from multiple specimens within each of five species in the complex, we found contrasting patterns of sequence divergence between the X chromosome and the autosomes. The isolation model of speciation assumes a lack of gene flow between species since their separation. This model could not be rejected for An. gambiae and An. arabiensis, although the data fit the model poorly. On the other hand, evidence from gene trees supports genetic introgression of chromosome 2 inversions between An. gambiae and An. arabiensis, and also points to more broad scale genetic exchange of autosomal sequences between this species pair. That such exchange has been relatively recent is suggested not only by the lack of fixed differences at three autosomal loci but also by the sharing of full haplotypes at two of the three loci, which is in contrast to several fixed differences and considerably deeper divergence on the X. The proposed acquisition by An. gambiae of sequences from the more arid-adapted An. arabiensis may have contributed to the spread and ecological dominance of this malaria vector.