Andrias O. O'Reilly

Nicotinic acetylcholine receptors (nAChRs) are the molecular targets of some important insecticides including triflumezopyrim and neonicotinoids. However, our understanding of insect nAChR pharmacology and the specific nAChR subunits targeted by these insecticides remains limited. Here, we cloned 11 nAChR subunit genes, comprising Nlα1 to Nlα8, Nlα10, Nlβ1 and Nlβ3, from Nilaparvata lugens, a highly damaging insect pest of rice crops worldwide. Analysis of the expression of these genes in different tissues of N. lugens by qPCR analysis identified the brain as the primary site of expression. Knock down of the expression of Nlα1, Nlα2, Nlα8 and Nlβ1 using RNAi reduced N. lugens sensitivity to triflumezopyrim, suggesting these genes encode potential target subunits for triflumezopyrim. Knock out of Nlα2 and Nlα8 nAChR subunits by CRISPR/Cas9 genome editing showed that their deletion significantly reduced the toxicity of triflumezopyrim toward N. lugens. Furthermore, the deletion of Nlα2 also increased N. lugens resistance to imidacloprid and dinotefuran. However, numerous attempts revealed that the Nlβ1 knockout was nonviable. In vitro expression of receptors composed of Drosophila homologous subunits showed that this all-insect nAChR was inhibited by nanomolar concentrations of triflumezopyrim. The present findings identify specific nAChR subunits that are important both as targets for monitoring resistance-associated mutations and as subjects for molecular studies aimed at developing novel insecticides targeting these essential ion channels.

The citrus red mite Panonychus citri has developed strong resistance to acaricides. Cytochrome P450 monooxygenases (P450s) can detoxify pesticides and are involved in pesticide resistance in many insects. Here, a pyridaben-resistant P. citri strain showed cross-resistance to cyenopyrafen, bifenazate, fenpyroximate, and tolfenpyrad. Piperonyl butoxide, a P450 inhibitor, significantly increased the toxicity of pyridaben to resistant (Pyr_Rs) and susceptible (Pyr_Control) P. citri strains. P450 activity was significantly higher in Pyr_Rs than in Pyr_Control. Analyses of RNA-Seq data identified a P450 gene (CYP4CL2) that is potentially involved in pyridaben resistance. Consistently, it was up-regulated in two field-derived resistant populations (CQ_WZ and CQ_TN). RNA interference-mediated knockdown of CYP4CL2 significantly decreased the pyridaben resistance in P. citri. Transgenic Drosophila melanogaster expressing CYP4CL2 showed increased pyridaben resistance. Molecular docking analysis showed that pyridaben could bind to several amino acids at substrate recognition sites in CYP4CL2. These findings shed light on P450-mediated pyridaben resistance in pest mites.

Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the ‘T1-chimera’ construct of the NaChBac sodium channel by truncating the C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N-terminus. Purified T1-chimera channels were tetrameric, conducted Na⁺ when reconstituted into proteliposomes and were blocked by the drug mibefradil. Both the T1-chimera and full-length NaChBac had comparable expression in the membrane whereas a NaChBac mutant lacking a cytoplasmic domain had greatly-reduced membranal expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerization. This phenomemon is found with other channels and thus our findings substantiate this as a common assembly mechanism.

The evolution of insecticide resistance mechanisms in natural populations ofAnophelesmalaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in theresistance to dieldrin locus(Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The twoRdlresistance mutations (296Gand296S) spread across West and Central AfricanAnophelesvia two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (fromA. gambiaeandA. arabiensistoA. coluzzii) and across non-concordant karyotypes of the 2La chromosomal inversion.Rdlresistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors.

Disulfide bridges are side-chain-mediated covalent bonds between cysteines that stabilize many protein structures. Disulfide mapping experiments to resolve these linkages typically involve proteolytic cleavage of the protein of interest followed by mass spectroscopy to identify fragments corresponding to linked peptides. Here we report the sequence-based “DIMPL” web tool to facilitate the planning and analysis steps of experimental mapping studies. The software tests permutations of user-selected proteases to determine an optimal peptic digest that produces cleavage between cysteine residues, thus separating each to an individual peptide fragment. The webserver returns fragment sequence and mass data that can be dynamically ordered to enable straightforward comparative analysis with mass spectroscopy results, facilitating dipeptide identification.