Mining genes involved in indoxacarb resistance of Lobesia botrana (Denis and Schiffermüller) by de novo transcriptome assembly and differential expression analysis.

Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae) is one of the most important grapevine pests in Europe but, being a non-model organism, only limited genomic and transcriptomic resources are available for functional studies at the molecular level, such as those relevant to insecticide resistance and pest control. Hence, to gain insight into the mechanism of indoxacarb resistance, a blocker of insect voltage-gated sodium channels (NaV), we analysed the transcriptome and expression profile in 2nd instars of L. botrana from susceptible and field selected populations (LC50 resistance ratio 72). De novo transcriptome assembly using Trinity resulted in 141,581 isoforms clustered in 94,290 putative genes. The transcriptome completeness was supported by BUSCO: 92% of conserved orthologs (n= 1,658) were retrieved as a complete sequence, 6.3% displayed fragmented ORFs, and only 1.7% were missing. 36,250 genes were preliminary annotated relaying on the longest isoform per gene, by running Annocript pipeline against non-redundant protein databases (Nr), gene ontology (GO), cluster of orthologous groups of proteins (COG), KEGG orthology (KO) and long non-coding RNAs (lncRNAs). Conditional Reciprocal Best BLAST analysis of protein isoforms performed on Lepidoptera proteomes identified putative orthologs of multigene family members potentially involved in metabolic resistance (61 cytochrome P450 monooxygenases, 25 glutathione S-transferases, 13 carboxylesterases, 25 UDP-glucuronosyltransferases) as well as alternatively spliced isoforms of the NaV gene. Among 263 upregulated and annotated genes in the resistant population, functional GO enrichment analysis revealed overrepresentation of terms for cytochrome P450, due to up-regulation of CYP6B and CYP9A subfamily members as well as increased transcript level for UGT genes. Hydrolases were, on the contrary, overrepresented in 293 annotated genes, downregulated in the resistant population. These data tentatively suggest the reduced susceptibility to indoxacarb might be related to an increase of Phase I and II detoxification along with reduced bioactivation of the insecticide