Project Number: 3020-43000-033-08-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2015
End Date: Aug 31, 2020
Determine the molecular factors that contribute variation in susceptibility of red flour beetle and confused flour beetles to aerosol insecticides and improve the effectiveness of aerosol insecticides for the management of stored product insect populations.
1. Determining metabolic-based detoxification mechanisms contributing to variation in susceptibility to different aerosol insecticides. Laboratory bioassay will be carried out to compare the susceptibility of the red flour beetle and the confused flour beetle to selected aerosol insecticides (e.g., dichlorvos, esfenvalerate, methoprene, pyrethrins, and pyriproxyfen). At the same time, commonly used insecticide synergists (e.g., diethyl maleate or DEM, piperonyl butoxide or PBO, and triphenyl phosphate or TPP) will be used to assess the major metabolic mechanisms leading to different susceptibilities to different aerosol insecticides in the two insect pests. Results from the insecticide bioassay incorporated with each of the three synergists will help us reveal the importance and role of the major detoxification enzyme families (e.g., cytochrome P450 monooxygenases, esterases, and glutathione S-transferases) in causing different insect susceptibilities to different aerosol insecticides. 2. Identifying and characterizing candidate genes likely involved in metabolic detoxification of aerosol insecticides. Genome-wide search for specific genes encoding each of three major detoxification enzyme families will be conducted in the red flour beetle by using well-established bioinformatics approaches. Because of large gene number in each major detoxification enzyme family in insects, only candidate genes which are most likely to be involved in insecticide detoxification will be selected for this study. The selection of the candidate genes will be based on the published literature in the red flour beetle and other insect species. Because genome sequence of the confused flour beetle is not available, the cDNA of each candidate gene will be sequenced by using standard molecular biology protocols. The development- and tissue-specific expression patterns of each candidate gene will be determined by PCR. The reveal of the expression patterns of the candidate gene can help us design the experiment to evaluate insect development and tissue-specific role of each candidate gene in insecticide detoxification. 3. Revealing role of candidate genes in causing different susceptibility to different aerosol insecticides. The role of each candidate gene in metabolic detoxification of aerosol insecticides will be assessed by the RNA interference (RNAi)-mediated gene silencing technique. Specifically, double-stranded RNA (dsRNA) will be constructed and synthesized based on the DNA sequence of each candidate gene. The gene-specific dsRNA will be injected into the hemocoel of the larvae of each insect species. The suppression of the candidate gene expression will be monitored by using reverse transcription quantitative PCR (RT-qPCR) at different time points. Change in larval susceptibility to selected aerosol insecticides will be examined by bioassay in dsRNA-treated larvae. An increase in the susceptibility to a given aerosol insecticide after RNAi of a specific gene suggests the role of the gene in detoxification in the insect.