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.

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BackgroundThe susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.Methodology/Principal FindingsA new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F1 adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.ConclusionThis study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.

BackgroundAlthough Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations.Methodology/Principal Findings3,000 F1 adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1h30min exposure and less than 50% mortality at 3h30min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved.Conclusion/SignificanceThe high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.

Wolbachia is a common maternally inherited bacterial symbiont able to induce crossing sterilities known as cytoplasmic incompatibility (CI) in insects. Wolbachia-modified sperm are unable to complete fertilization of uninfected ova, but a rescue function allows infected eggs to develop normally. By providing a reproductive advantage to infected females, Wolbachia can rapidly invade uninfected populations, and this could provide a mechanism for driving transgenes through pest populations. CI can also occur between Wolbachia-infected populations and is usually associated with the presence of different Wolbachia strains. In the Culex pipiens mosquito group (including the filariasis vector C. quinquefasciatus) a very unusual degree of complexity of Wolbachia-induced crossing-types has been reported, with partial or complete CI that can be unidirectional or bidirectional, yet no Wolbachia strain variation was found. Here we show variation between incompatible Culex strains in two Wolbachia ankyrin repeat-encoding genes associated with a prophage region, one of which is sex-specifically expressed in some strains, and also a direct effect of the host nuclear genome on CI rescue.