Molecular characterization of insecticide resistance mechanisms in populations of Leptinotarsa decemlineata: Potential for pest control using RNA interference and understanding natural phenology of insecticide resistance

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including imidacloprid, and demonstrated that the median lethal concentration to kill 50% of the test organisms (LC50) in different field populations varies greatly, which may suggest that resistance of L. decemlineata is heritable and involves genetic changes. Furthermore, L. decemlineata possesses multiple mechanisms of resistance, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases to combat an insecticide insult.;We examined imidacloprid resistance among different field populations in the Central Sands region of Wisconsin. The LC50 values collected in 2008-2011, and 2013-2014, indicate some field locations remain susceptible, while nearby fields (<100km) have developed resistance. We compiled a transcriptome for 'susceptible' and 'resistant' populations by isolating mRNA from adult beetles and analyzed differences in transcript abundance to uncover potential mechanisms of resistance. Differences were observed in constituently up- and down-regulated transcripts among different populations, including the up-regulation of 3 cytochrome p450s and a glutathione synthetase related protein in multiple resistant populations.;We further hypothesize that a portion of the up-regulated transcripts encode for pesticide resistance and may be suppressed to re-establish a susceptible phenotype. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein using RNA interference, we observed reductions in measured resistance to imidacloprid.;Finally, we examined temporal changes in phenotypic response to imidacloprid between a susceptible and an imidacloprid-resistant population. Estimated LC50 values varied throughout the growing season in the resistant population, with increased susceptibility among over-wintered, recently emerged adult beetles compared with heightened resistance in the second generation. The abundance of mRNA transcript was compared between multiple time points, and showed that transcripts encoding cuticular proteins and cytochrome p450s were highly up-regulated during peaks of resistance.