Location: Invasive Insect Biocontrol & Behavior Laboratory
Project Number: 8042-30400-003-030-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 31, 2024
End Date: Dec 31, 2026
Objective:
The objective is to reduce rapid insecticide resistance development in Colorado potato beetle. This will be accomplished by examining roles of histone deacetylase inhibitors (HDACis) and histone acetylase inhibitors (HAis)- which may regulate the emergence of stress-induced insecticide resistance. We propose that HAis and HDACis may influence transcriptional accessibility and overall gene expression in CPB following insecticide exposure by mediating histone acetylation. The following will be cnducted (modified for year 2): (1) Evaluate HDACis and/or HAis mechanism to affect gene expression in Colorado potato beetle exposed to a sublethal dose of insecticide; (2) Examine how Colorado potato beetle exposure to insecticides and different treatment combinations of HAi and/or HDACis influence the differential expression of specific genes; and (3) Determine if HDACis and/or HAis affect gene expression similarly (i.e., do they result in the same levels of expression in the same genes) in susceptible and resistant CPB populations?).
Approach:
The approach has been expanded based on promising year 1 laboratory results that demonstrated utility of histone deacetylases in reducing insecticide resistance in Colorado potato beetle pest. A factorial experiment will be conducted to test the effect of different combinations of HDACis, HAis, and a sublethal (LC25) concentration of insecticide on gene expression in an insecticide-susceptible population of CPB. Third-instar larvae (n=10) will be exposed from a lab-reared Colorado potato beetle lab colony (collected from organic farms in Vermont, USA) to one of four treatment combinations: 1)insecticide-only control); 2) insecticide + HDACi; 3) insecticide + HAi; and 4) insecticide + HDACi + HAi. For insecticide treatments, we will use aqueous industrial-grade imidacloprid (99%; dissolved in acetone) diluted to a 10 ppm dose. For HAi, histone acetyltransferase p300 inhibitor C646 (50 µM dissolved in DMSO) will be used. For HDACis, Class I-II deacetylase inhibitor trichostatin A (TSA; 50 µM dissolved in DMSO) will be used. For dosing, larvae will be placed individually into 6-well plates, randomizing in a full block design by treatment under a fluorescent light with a 16:8 light-dark photoperiod. Imidacloprid will be pipetted in 1 µL volumes onto the dorsal side of third-instar larvae. C646 and TSA will be administered orally by placing 1µL volumes directly on the mouthparts. These sets of experiments will all be replicated using Colorado potato beetle larvae collected from a resistant field population. Resistant beetles will be collected from potato fields in Long Island, NY., and reared for at least one generation in laboratory conditions prior to the experiment. RNA extraction and sequencing – Susceptible third-instar Colorado potato beetle larvae will be collected (ntreatment=10) from each of four treatments (ntotal=40) in a factorial experiment Larvae will be flash frozen in liquid nitrogen and stored at -80°C until processing. Total RNA will be extracted from thoracic tissue using Quick-RNA Tissue/Insect Microprep Kits (Zymo). RNA quantity and quality will be assessed using a Qubit 2.0 fluorometer.
Samples will be sent for RNA sequencing using a polyA-enrichment procedure to obtain total messenger RNA (mRNA). Transcriptomic data analyses – RNA sequence data will be processed via a bioinformatic pipeline previously developed in our lab, using FASTQC 0.11.4 to assess read quality; TRIMMOMATIC to trim adapter sequences and remove low quality bases7; and HISAT2 to align transcripts to the published CPB genome and count transcript copies per region of interest8. We will use DESEQ29 to identify differentially expressed genes between different treatments and use generalized linear models in R10 to test for significant differences in number of transcripts, setting a false discovery rate (FDR) of P < 0.05. Finally, we will use BLAST+ with online protein databases to characterized functions associated with differentially expressed genes and to determine whether expression may be linked to insecticide resistance traits.