Evolutionary trade-offs of insecticide resistance - The fitness costs associated with target-site mutations in the nAChR of Drosophila melanogaster.

The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field-evolved mutation being reported to date. This is an arginine-to-threonine substitution at position 81 of the nAChR_ß1 subunit in neonicotinoid-resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.

Voltage-gated sodium channels as targets for pyrethroid insecticides.

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

Influence of the RDL A301S mutation in the brown planthopper Nilaparvata lugens on the activity of phenylpyrazole insecticides.

We discovered the A301S mutation in the RDL GABA-gated chloride channel of fiprole resistant rice brown planthopper, Nilaparvata lugens populations by DNA sequencing and SNP calling via RNASeq. Ethiprole selection of two field N. lugens populations resulted in strong resistance to both ethiprole and fipronil and resulted in fixation of the A301S mutation, as well as the emergence of another mutation, Q359E in one of the selected strains. To analyse the roles of these mutations in resistance to phenylpyrazoles, three Rdl constructs: wild type, A301S and A301S+Q359E were expressed in Xenopus laevis oocytes and assessed for their sensitivity to ethiprole and fipronil using two-electrode voltage-clamp electrophysiology. Neither of the mutant Rdl subtypes significantly reduced the antagonistic action of fipronil, however there was a significant reduction in response to ethiprole in the two mutated subtypes compared with the wild type. Bioassays with a Drosophila melanogaster strain carrying the A301S mutation showed strong resistance to ethiprole but not fipronil compared to a strain without this mutation, thus further supporting a causal role for the A301S mutation in resistance to ethiprole. Homology modelling of the N. lugens RDL channel did not suggest implications of Q359E for fiprole binding in contrast to A301S located in transmembrane domain M2 forming the channel pore. Synergist bioassays provided no evidence of a role for cytochrome P450s in N. lugens resistance to fipronil and the molecular basis of resistance to this compound remains unknown. In summary this study provides strong evidence that target-site resistance underlies widespread ethiprole resistance in N. lugens populations.

Doublesex homolog is sex-specifically spliced and governs the sexual differentiation process in the whitefly Bemisia tabaci AsiaII-1.

The silverleaf whitefly Bemisia tabaci is one of the most destructive of crop pests globally. In Northern India cotton is predominately infested by the Asia II-1 species of B. tabaci. Though B. tabaci exhibits unique haplodiploidy in its reproductive behavior, to date very little is known regarding its sex determination mechanism. Here, an in-depth characterization of the AsiaII-1 doublesex (Btdsx) gene, which has been implicated in sex determination in B. tabaci, indicates the inclusion of six exons and five introns. The pre-mRNA is shown to sex-specifically splice, producing four male isoforms and one female isoform. These BtDsx proteins share common DNA binding (OD1) domains whereas they differ at their C-termini. RT-qPCR analysis revealed a significantly higher expression of Btdsx in female adults compared to that in male adults and earlier developmental stages. Functional characterization of Btdsx through RNA interference (RNAi) resulted in a significant reduction in its expression in both sexes. Btdsx knockdown concomitantly resulted in up-regulation of the expression of vitellogenin (vg) and vitellogenin receptor (vgr) genes in males and their down-regulation in females. Btdsx knockdown followed by mating resulted in reduced fecundity and percent egg hatching; however, no impact was observed on the female: male ratios in the progeny obtained from knockdown parents.