There is an increasing resistance to insecticides in malaria-transmitting mosquitoes. This is particularly troubling because insecticide-treated bed nets are the main reason malaria-related deaths have decreased in the last decade. By using colonies of mosquitoes that originated from Mali in 1995 and 2014 and have since been maintained in a lab setting, we had the unique opportunity to study how the species evolved in the 19 years. We find that the 2014 colony is more resistance to insecticides, but this is decreased if you first expose the mosquitoes to a compound which inhibits their ability to break down toxins. This suggests that bed-nets treated with a combination of insecticides and this compound would be more effective. We also find that a particular set of variations in the mosquito DNA occurs more frequently in resistant mosquitoes. In the future, these markers could be used to track the spread of resistant mosquitoes.
In recent years, malaria mortality rates in sub-Saharan Africa have declined significantly, largely due to insecticide-treated bed net (ITN) campaigns. However, insecticide resistant malaria-transmitting mosquitoes, like Anopheles coluzzii and Anopheles gambiae, pose a major threat to this progress. A knock down resistance (kdr) allele, which reduces pyrethroid-binding affinity, has been increasing in geographical distribution and relative frequency throughout Africa, apparently in response to increased ITN usage. The kdr allele as well as known detoxification genes like cytochrome P450 genes can confer resistance to insecticides. Also following the increased ITN usage is a selective sweep of the cyp-1 haplotype on the X chromosome in Anopheles colonies. Here we leverage a cyp-1 2014 An. coluzzii colony and a cyp-2 1995 An. coluzzii colony to elucidate factors that could be driving this resistance.
Results indicate that the 2014 An. coluzzii colony is more resistant than the 1995 An. coluzzii colony in terms of knockdown time and mortality at 24 hours. Further, the increased mortality rate in the 2014 An. Coluzzii population following a pre-treatment with PBO, a P450 inhibitor, indicates that P450 genes may be a driving factor to their resistance. In order to capture transcriptomic signatures which may be highly tissue-specific or responsive to environmental stimuli, we compared transcriptomic profiles of Malphigian tubules (tissue important for detoxification) following a sublethal permethrin exposure in both colonies. The carboxylesterase gene COEAE5G is overexpressed in the resistant cyp-1 2014 colony compared to the relatively susceptible cyp-2 1995 An. coluzzii colony and the logFC increased with permethrin exposure.
This study also supports a link between the selected haplotype (cyp-1) and increased resistance. When comparing resistance rates between homozygous cyp-2 and heterozygous cyp-1/cyp-2 in both male and female individuals, the cyp-1 haplotype was associated with higher resistance in females (although not observed in males). Transcriptionally, the P450 gene CYP6Z1, which metabolizes permethrin, was upregulated in the cyp-1 resistant An. coluzzii colony following sublethal permethrin exposure, but not under control conditions. This indicates that genetic variation in the cyp-1 colony increases the responsiveness of CYP6Z1 to permethrin exposure. Further, this is the first study to examine structural variation in An. Coluzzii populations. Although there were polymorphic structural variants between the cyp-1 and cyp-2 haplotypes, none identified occurred on known detoxification genes.
This study suggests that a more effective bed net campaign could use PBO in combination with insecticides. Further, the identification of genetic markers linked to these regulatory alleles is an important next step in population genetic studies and insecticide resistance surveillance in this important vector species.