The emergence and spread of insecticide resistance compromises the efficiency of insecticides as prevention tool against the transmission of insect-transmitted diseases (Moyes et al. 2017). In this context, the understanding of the genetic mechanisms of resistance and the way resistant alleles spread in insect populations is necessary and important to envision resistance management policies. A common and important mechanism of insecticide resistance is gene amplification and in particular amplification of insecticide detoxification genes, which leads to the overexpression of these genes (Bass & Field, 2011). Cattel and coauthors (2020) adopt a combination of experimental approaches to study the role of gene amplification in resistance to organophosphate insecticides in the mosquito Aedes aegypti and its occurrence in populations of South East Asia and to develop a molecular test to track resistance alleles.
Their first approach consists in performing an artificial selection on laboratory Ae. Aegypti populations started with individuals collected in Laos. In the selected population, an initial 90% mortality by adult exposure to the organophosphate insecticide malathion is imposed. This population shows a steep increase in resistance to malathion and other organophosphate insecticides, which is absent in the paired control population. The transcriptomic patterns of the control and the evolved populations as well as of a reference sensitive population reveals, among other differences, the over-expression of five carboxy/choline esterase (CCE) genes in the insecticide selected population. These five genes happen to be clustered in the Ae. aegypti genome and whole genome sequencing of a highly resistant population combined to qPCR test on genomic DNA showed that the overexpression of these genes is due to gene amplification. Although it would have been more elegant to have replicate selected and control populations and to perform the transcriptomic and the genomic analyses directly on the experimental populations, the authors gather a set of experimental evidence which combined to previous knowledge on the function of the amplified and over-expressed genes and on their implication in organophosphate insecticide resistance in other species allow to discard the possibility that this gene amplification spread by drift in the selected population.
In a second part of the paper, copy number variation for CCE genes is checked in field sample populations. This test reveals the presence of resistance alleles in half of the fourteen South East Asia populations sampled. Very interestingly, it also reveals a high level of complexity and diversity among the resistance alleles: it shows first the existence, both in the experimental and the field populations, of at least two amplified alleles (differing by the number of genes amplified) and second a high variation in the copy number of amplified genes. This indicates that gene amplification as a molecular resistance mechanism has actually lead to a high diversity of resistance alleles. These alleles are likely to differ both by the level of resistance conferred and the fitness cost imposed in the absence of the insecticide and these two values are affecting the evolution of their frequency in the field and ultimately the spread of resistance.
The last part of the paper is devoted to the development of a high-throughput Taqman assay which allows to determine rapidly the copy number of one of the esterase genes amplified in the resistance alleles described earlier. This assay is nicely validated and will definitely be a useful tool to determine the occurrence of these resistance alleles in field population. The fact that it gives access to the copy number will also allow to follow its copy number across time and get insight into the complexity of resistance evolution by gene amplification.
To sum up, this paper studies the implication of carboxy/choline esterase genes amplification in organophosphate resistance evolution in Ae. aegypti, reveals the diversity among individuals and populations of this resistance mechanism, because of variation both in the identity of the genes amplified and in their copy number and sets up a fast and efficient tool to detect and follow the spread of these resistant alleles in the field. Additionally, the different experimental approaches adopted have generated genomic and transcriptomic data, of which only the part related to CCE gene amplification has been exploited. These data are very likely to reveal other genomic and expression determinants of resistance that will give access to an extra degree of complexity in organophosphate insecticide resistance determinism and evolution.

References

Bass C, Field LM (2011) Gene amplification and insecticide resistance. Pest Management Science, 67, 886–890. https://doi.org/10.1002/ps.2189
Cattel J, Haberkorn C, Laporte F, Gaude T, Cumer T, Renaud J, Sutherland IW, Hertz JC, Bonneville J-M, Arnaud V, Nous C, Fustec B, Boyer S, Marcombe S, David J-P (2020) A genomic amplification affecting a carboxylesterase gene cluster confers organophosphate resistance in the mosquito Aedes aegypti: from genomic characterization to high-throughput field detection. bioRxiv, 2020.06.08.139741, ver. 4 peer-reviewed and recommended by PCI Evolutionary Biology. https://doi.org/10.1101/2020.06.08.139741
Moyes CL, Vontas J, Martins AJ, Ng LC, Koou SY, Dusfour I, Raghavendra K, Pinto J, Corbel V, David J-P, Weetman D (2017) Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans. PLOS Neglected Tropical Diseases, 11, e0005625. https://doi.org/10.1371/journal.pntd.0005625