Role of CYP9E2 and a long non-coding RNA gene in resistance to a spinosad insecticide in the Colorado potato beetle, Leptinotarsa decemlineata.

Spinosads are insecticides used to control insect pests, especially in organic farming where limited tools for pest management exist. However, resistance has developed to spinosads in economically important pests, including Colorado potato beetle (CPB), Leptinotarsa decemlineata. In this study, we used bioassays to determine spinosad sensitivity of two field populations of CPB, one from an organic farm exposed exclusively to spinosad and one from a conventional farm exposed to a variety of insecticides, and a reference insecticide naïve population. We found the field populations exhibited significant levels of resistance compared with the sensitive population. Then, we compared transcriptome profiles between the two field populations to identify genes associated primarily with spinosad resistance and found a cytochrome P450, CYP9E2, and a long non-coding RNA gene, lncRNA-2, were upregulated in the exclusively spinosad-exposed population. Knock-down of these two genes simultaneously in beetles of the spinosad-exposed population using RNA interference (RNAi) resulted in a significant increase in mortality when gene knock-down was followed by spinosad exposure, whereas single knock-downs of each gene produced smaller effects. In addition, knock-down of the lncRNA-2 gene individually resulted in significant reduction in CYP9E2 transcripts. Finally, in silico analysis using an RNA-RNA interaction tool revealed that CYP9E2 mRNA contains multiple binding sites for the lncRNA-2 transcript. Our results imply that CYP9E2 and lncRNA-2 jointly contribute to spinosad resistance in CPB, and lncRNA-2 is involved in regulation of CYP9E2 expression. These results provide evidence that metabolic resistance, driven by overexpression of CYP and lncRNA genes, contributes to spinosad resistance in CPB.

Exposure to Temperature and Insecticides Modulates the Expression of Small Noncoding RNA-Associated Transcripts in the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can adapt to various challenges, including temperature fluctuations or select insecticide treatments. This pest is also an ongoing threat to the potato industry. Small noncoding RNAs such as miRNAs, which can control posttranscriptionally the expression of various genes, and piRNAs, which can notably impact mRNA turnover, are modulated in insects under different conditions. Unfortunately, information regarding the expression status of key players involved in their synthesis and function is for the most part lacking. The current study thus aims at assessing the levels of such targets in L. decemlineata exposed to hot and cold temperatures as well as treated to the insecticides chlorantraniliprole, clothianidin, imidacloprid, and spinosad. Transcript expression levels of Ago1, Ago2, Ago3, Dcr2a, Dcr2b, Expo-5, Siwi-1, and Siwi-2, components of pathways associated with small noncoding RNA production or function, were measured by qRT-PCR and revealed modulation of select transcripts in response to temperature challenges and to select insecticides. RNAi-mediated reduction of Ago2 transcript levels in L. decemlineata injected with Ago2-targeting dsRNA and exposed to cold and warm temperatures was also conducted. Changes in survival rates were observed for the latter condition in dsRNA- versus saline-injected insects. These results showcase the differential expression of select targets involved in small noncoding RNA homeostasis and provide leads for the subsequent assessment of their involvement during stress response in L. decemlineata using RNAi-based approaches.