Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa.

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field-applicable markers to better track resistance Africa-wide

Anopheles gambiae distribution and insecticide resistance in the cities of Douala and Yaoundé (Cameroon): influence of urban agriculture and pollution

Background: Urban malaria is becoming a major health priority across Africa. A study was undertaken to assess the importance of urban pollution and agriculture practice on the distribution and susceptibility to insecticide of malaria vectors in the two main cities in Cameroon. Methods: Anopheline larval breeding sites were surveyed and water samples analysed monthly from October 2009 to December 2010. Parameters analysed included turbidity, pH, temperature, conductivity, sulfates, phosphates,nitrates, nitrites, ammonia, aluminium, alkalinity, iron, potassium, manganese, magnesium, magnesium hardness and total hardness. Characteristics of water bodies in urban areas were compared to rural areas and between urban sites. The level of susceptibility of Anopheles gambiae to 4% DDT, 0.75% permethrin, 0.05% deltamethrin, 0.1% bendiocarb and 5% malathion were compared between mosquitoes collected from polluted, non polluted and cultivated areas. Results: A total of 1,546 breeding sites, 690 in Yaoundé and 856 in Douala, were sampled in the course of the study. Almost all measured parameters had a concentration of 2- to 100-fold higher in urban compare to rural breeding sites. No resistance to malathion was detected, but bendiocarb resistance was present in Yaounde. Very low mortality rates were observed following DDT or permethrin exposure, associated with high kdr frequencies. Mosquitoes collected in cultivated areas, exhibited the highest resistant levels. There was little difference in insecticide resistance or kdr allele frequency in mosquitoes collected from polluted versus non-polluted sites. Conclusion: The data confirm high selection pressure on mosquitoes originating from urban areas and suggest urban agriculture rather than pollution as the major factor driving resistance to insecticide

Parallel evolution or purifying selection, not introgression, explains similarity in the pyrethroid detoxification linked GSTE4 of Anopheles gambiae and An. arabiensis

Insecticide resistance is a major impediment to the control of vectors and pests of public health importance and is a strongly selected trait capable of rapid spread, sometimes even between closely-related species. Elucidating the mechanisms generating insecticide resistance in mosquito vectors of disease, and understanding the spread of resistance within and between populations and species are vital for the development of robust resistance management strategies. Here we studied the mechanisms of resistance in two sympatric members of the Anopheles gambiae species complex – the major vector of malaria in sub-Saharan Africa – in order to understand how resistance has developed and spread in eastern Uganda, a region with some of the highest levels of malaria. In eastern Uganda, where the mosquitoes Anopheles arabiensis and An. gambiae can be found sympatrically, low levels of hybrids (0.4%) occur, offering a route for introgression of adaptively important variants between species. In independent microarray studies of insecticide resistance, Gste4, an insect-specific glutathione S-transferase, was among the most significantly up-regulated genes in both species. To test the hypothesis of interspecific introgression, we sequenced 2.3kbp encompassing Gste4. Whilst this detailed sequencing ruled out introgression, we detected strong positive selection acting on Gste4. However, these sequences, followed by haplotype-specific qPCR, showed that the apparent up-regulation in An. arabiensis is a result of allelic variation across the microarray probe binding sites which artefactually elevates the gene expression signal. Thus, facevalue acceptance of microarray data can be misleading and it is advisable to conduct a more detailed investigation of the causes and nature of such signal. The identification of positive selection acting on this locus led us to functionally express and characterise allelic variants of GSTE4. Although the in vitro data do not support a direct role for GSTE4 in metabolism, they do support a role for this enzyme in insecticide sequestration. Thus, the demonstration of a role for an up-regulated gene in metabolic resistance to insecticides should not be limited to simply whether it can metabolise insecticide; such a strict criterion would argue against the involvement of GSTE4 despite the weight of evidence to the contrary

Myxobolus makombensis n. sp. infection in African carp Labeobarbus batesii from the Makombè River, Cameroon: morphological and molecular characterization

In this study, we examined myxozoan infections of Labeobarbus batesii sampled from the Makombè River in Cameroon. Fish were infected with Myxobolus makombensis n. sp. in the gill filament and M. dibombensis in the fins. Mature myxospores of M. makombensis n. sp. are pyriform in frontal view and biconvex in lateral view, with a truncated and slightly narrow anterior end. Spore dimensions (mean ± SD, with range in parentheses) are 17.5 ± 0.22 (16.2-18.9) μm length, 13.4 ± 0.25 (12-14.9) μm width, and 7 ± 0.21 (6.7-7.5) μm thickness, and spores exhibit a conspicuous anterior intercapsular appendix of 4.4 ± 0.18 (3.9-5.5) µm length. Myxospores have 2 pyriform polar capsules of unequal size; the larger one is 9.8 ± 0. 22 (8.2-10.9) μm long × 4.7 ± 0.15 (3.5-5.2) µm wide, and the smaller one is 8.8 ± 0.22 (7-10) µm long × 4.3 ± 0.12 (3.5-5.2) µm wide. Polar filaments possess 10 to 11 coils in the large polar capsule and 8 to 10 coils in the small polar capsule. Phylogenetic analysis of SSU rDNA sequences showed clustering of M. makombensis n. sp. close to M. dibombensis recently reported from the fins of the same host within a clade composed exclusively of parasites infecting cyprinid fishes

Exposure to disinfectants (soap or hydrogen peroxide) increases tolerance to permethrin in Anopheles gambiae populations from the city of Yaounde, Cameroon

Background: The rapid expansion of insecticide resistance is limiting the efficiency of malaria vector control interventions. However, current knowledge of factors inducing pyrethroid resistance remains incomplete. In the present study, the role of selection at the larval stage by disinfectants, such as soap and hydrogen peroxide (H2O2), on adult mosquito resistance to permethrin was investigated. Methods: Field Anopheles gambiae sensu lato larvae, were exposed to variable concentrations of soap and H2O2. Larvae surviving to acute toxicity assays after 24 hours were reared to the adult stage and exposed to permethrin. The susceptibility level of adults was compared to the untreated control group. The effect of soap or hydrogen peroxide selection on the length of larval development and emergence rate was assessed. Result: Larval bioassays analysis showed a more acute effect of hydrogen peroxide on mosquito larvae compared to soap. The regression lines describing the dose mortality profile showed higher mean and variance to hydrogen peroxide than to soap. The duration of larval development (<5 days) and adults emergence rates (1 to 77%) were shorter and lower compare to control. Anopheles gambiae s.l. larvae surviving to selection with either soap or hydrogen peroxide or both, produced adults who were up to eight-times more resistant to permethrin than mosquitoes from the untreated control group. Conclusion: The present study shows that selective pressure exerted by non-insecticidal compounds such as soap and hydrogen peroxide affect An. gambiae s.l. tolerance to pyrethroids. This requires further studies with regard to the adaptation of An. gambiae s.l. to polluted habitats across sub-Saharan Africa cities