Investigating Larval Ecology, Genetic Diversity and Insecticide Resistance Status of Anopheles Funestus in Different Ecological Landscapes in Western Kenya

Abstract

Background Malaria is the deadliest protozoan disease transmitted by female Anopheles mosquitoes. Africa region continues to lead the heaviest malaria burden globally. Africa region continues to lead the heaviest malaria burden globally. Anopheles funestus is one of the efficient vectors of Plasmodium. falciparum malaria exhibits high anthropophilic, endophilic and anthropophagic behaviours. Its competence to transmit P. falciparum outpaced other main malaria vectors in certain countries. An. funestus is a dominant vector in western Kenya responsible for year-round high malaria transmission. However, its larval ecological requirements, the genetic basis of adaptations and mechanisms of resistance to public health insecticides are poorly understood. Therefore, this study investigated its larval ecology and bionomics, population structure, diversity and molecular basis of insecticide resistance in western Kenya. Methods A larval survey was conducted, and all potential mosquito aquatic habitats were identified, georeferenced, characterized and carefully examined for the presence of mosquito larvae and predators. Endophilic mosquitoes were collected using pyrethrum spray catches, prokopack and mouth aspirators. Mosquito species were identified using molecular and morphological methods. Molecular techniques were used to detect sporozoite infections and sources of blood meals. WHO insecticide susceptibility test was employed to assess An. funestus resistant status to the insecticides. Sanger sequencing was employed to sequence the COII gene to determine genetic diversity and population structure. Whole transcriptomic analysis using RNA-sequencing was performed to investigate the molecular basis of insecticide resistance.Results An. funestus larvae were found in greater numbers in the highland site, Bungoma (85%, 95%, CI, 8.722–17.15) than in the lowland site, Kombewa (15%, 95%, CI, 1.33–3.91). Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. High P. falciparum sporozoite rate and human blood index were recorded. An. funestus populations from western Kenya had high haplotype diversity but low nucleotide diversity. No genetic differentiation or structure and a high level of gene flow were observed among the An. funestus populations. A high level of pyrethroid resistance was observed in various study sites (Bungoma, Teso, Siaya, Port Victoria and Kombewa) in western Kenya with Port Victoria having the highest level of resistance to both the type I (permethrin) and type II (deltamethrin) pyrethroids. The average mortality rate (MR) of pyrethroid resistance observed in all sites was 57.6%. Resistance to dichlorodiphenyltrichloroethane (DDT) was observed in Kombewa and Port Victoria but suspected resistance to DDT was observed in Siaya and Teso. However, An. funestus population was fully susceptible to 0.25% pirimiphos- methyl in all the sites. The preexposure to piperonyl butoxide (PBO) revealed high susceptibility to the pyrethroids in all the sites except for Port Victoria where suspected resistance for PBO + deltamethrin was observed (MR=96%). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by immunity proteins (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP binding cassettes (2%) and carboxylesterases (1%). The top cytochrome P450 genes that were overexpressed in the An. funestus in western Kenya include CYP6P9A, CYP6P9b, CYP6N1, CYP9J5, CYP49A1, AFUN020895, AFUN019365, CYP9K1, CYP304B. GSTs (GSTD1, GSTT2, GSTD7, GSTD11, GSTD3, GSTE6), peptidase S1 domain-containing protein (AFUN018482, AFUN018981, AFUN018580), cuticular protein (AFUN021427, AFUN021428, AFUN019106), UGTs (UGT310B2, UGT308D2, UGT306A3, AFUN003620) and sulfotransferase (AFUN016205, AFUN016207) were also overexpressed in An. funestus populations in western Kenya. Conclusions This study revealed the high adaptability of An. funestus to various breeding habitats and its larva co-exist with other mosquito larvae in the same aquatic habitats. An. funestus population is under selective pressure in western Kenya resulting in demographic expansion and the spread of variants through breeding. Population expansion and a high level of insecticide resistance suggest the high adaptability of An. funestus to various habitats, hence sustaining its vectorial capacity and malaria transmission. This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention developments could help in insecticide resistance management.