Responses to Pyrethroids in Heavy metal tolerant Anopheles gambiae vector of Malaria

Abstract

Anopheles gambiae mosquito, the principle Afro-tropical vector of malaria, has been documented in habitats with heavy metals in excess of natural loads. The mosquito has also displayed resistance to most conventional insecticides, and potential to resist challenges by heavy metals. Investigations were conducted in strains of An. gambiae ss, in which tolerance to cadmium and lead heavy metals were established. This study was undertaken to determine any putative relationship between tolerance to (cadmium and lead) heavy metals, and resistance to pyrethroid insecticide in An. gambiae. Tolerance to cadmium and lead heavy metals was developed in larval stages and this development in tolerance was monitored using changes in lethal concentration (LC)50 of the individual metals between successive generations using bioassay technique. Heavy metal tolerant males and females individuals and relevant controls were separately screened for 1) expression of pyrethroid metabolic CYP6M2, CYP6P3 and CYP6Z1 genes of cytochrome p450 enzyme by quantitative PCR (qPCR) and 2) knockdown resistance gene mutation (kdr) conferring resistance to pyrethroids through standard PCR and sequencing. Phenotypic responses of the mosquitoes to pyrethroid insecticides were assessed by bioassay according to the standard WHO procedure for monitoring resistance to insecticides. Differences in expressions of the cytochrome p450 genes among the populations was determined by Analysis of Variance (ANOVA), and assessment of leucine to serine substitution that confer kdr gene mutation was conducted by multiple sequence alignment. The kdr mutation was not detected among the metal tolerant populations, and the LC50 responses to permethrin among the respective metal tolerant and control mosquito populations were similar. However, expressions of all the classes of cytochrome p450 genes were significantly lower in females than males (F(1, 46)=30.13 , p<0.0001 ) irrespective of treatment. Within gender, the reductions in expression were more pronounced in the male metal tolerant, relative to their respective controls populations. There were general reductions in expression levels of all the genes in metal tolerant relative to control population, where significantly lower expressions (F(2, 46)= 7.06, p= 0.0021) of the all genes were observed in the metal tolerant than in control male populations. The expression of all the genes in cadmium selected female populations was higher than in their respective controls, although the difference was not statistically significant (P > 0.05). These results suggest that adaptation to cadmium and lead heavy metals does not confer resistance to pyrethroid insecticides. Thus urban mosquitoes breeding in heavy metal polluted waters are susceptible to these insecticides, as long as the habitat is not compromised by any other sources of contamination from urban pollution. The understanding of the mechanism that results to emerging pyrethroid resistance is very important, since it will shade light on the necessary improvement strategies that are required to enhance its effectivity and thus ensure eradication of the vector and control of the disease.