Search

Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males Anopheles funestus, African malaria vector

There has been a renewed interest for larviciding during the recent decade. Although biological larvicides are considered not to be harmful to non-target organisms, there is still not sufficient data on the effect of new long lasting larvicide formulations such as VectoMax G combining Bacillus thuringiensis israelensis and B. sphaericus on the environment especially on non-target organisms. The present study aimed to assess the possible influence of VectoMax G on the diversity and abundance of the aquatic fauna cohabiting with mosquito larvae in breeding habitats during a larviciding trial in the city of Yaoundé. Twelve districts of the city of Yaoundé divided into 6 intervention and 6 control sites were chosen for the study. In each district 4 semi-permanent or permanent aquatic habitats were followed. VectoMax G application was done once every two weeks during 6 months and aquatic organisms were collected 48h after each treatment. All collected organisms were brought to the laboratory for identification. Physico-chemical parameters were recorded as well. A high diversity of the zooplankton was recorded in the intervention areas with 28 species collected against 14 species in the control areas. Cladocerans were the most represented group in both sites while Oestracods were found only in control sites. A total of 19 macro-invertebrates species were recorded in the control areas vs 16 species in the intervention areas. Gasteropods were the most represented groups of macro-invertebrates. Vertebrates such as larvivorous fishes and amphibians larvae were also found in approximately similar densities in both sites. The study indicated no significant influence of larviciding with VectoMax G on the diversity and abundance of the non-target aquatic fauna in the city of Yaoundé.

Constant assessment of insecticide resistance levels is mandatory to implement adequate malaria control tools, but little information is available on the annual dynamics of resistance. We, therefore, monitored variations in resistance in Anopheles gambiae s.l. over four seasons during 2 years in two localities of Yaoundé: urban Etoa‐Meki and peri‐urban Nkolondom. Mosquitoes were collected seasonally at larval stage and reared to adults for insecticide susceptibility tests and molecular analysis of resistance mechanisms. Anopheles coluzzii was found in Etoa‐Meki and An. gambiae in Nkolondom. Low mortalities to pyrethroids were observed (permethrin

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α cypermethrin with comparable affinities (KM of 3.3 μM ± 0.4 and 3.6 μM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min− 1 ± 0.2) versus deltamethrin (2.68 min− 1 ± 0.09). Timecourse assays revealed progressive depletion of the above pyrethroids with production of four HPLCdetectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas.

Additional file 1. Sequence alignment of the Ace-1 gene at the G119S point mutation in live and dead An. gambiae mosquitoes 24 h after exposure to carbamate.

Additional file 1. Logistic regression of Aedes aegypti mortality to insecticides across cities and habitat types. Suburban habitat, city of Douala, and Bendiocarb were reference levels.

Additional file 2: Table S2. Trade names, types, active ingredients, WHO toxicity and chemical classes, and dose of pesticides used by farmers in Yaoundé, Cameroon.

Additional file 1: Table S1. Frequency of target site mutations characterized in Anopheles gambiae (s.l.) F0 from Atatam. Table S2. Frequency of L119F-GSTe2 resistance markers among dead and alive An. funestus exposed to DDT, deltamethrin 1x, 5x and 10x. Table S3. Differential expression of metabolic resistance genes among F1 Deltamethrin 1X, 5x, 10x and unexposed mosquitoes from Atatam compared with the FANG susceptible strain. Table S4. Differential expression of metabolic and cuticular resistance genes among the An. gambiae sl population from Atatam as compared with the Kisumu susceptible strain.

Additional file 1: Table S1. Household indices and brand nets from the collection sites.

Additional file 1: Table S1. List of primers used for the nested PCR.

Additional file 1: Table S1. Survey sheet on pesticide vendors and farmers in the city of Yaoundé, Cameroon.

Additional file 2. Number of An. gambiae s.l. mosquitoes identified among those alive and dead after exposure to insecticides.

The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.