Location: Invasive Insect Biocontrol & Behavior Laboratory
Project Number: 8042-22000-315-030-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 31, 2024
End Date: Sep 30, 2025
Objective:
The objective is to examine roles of histone deacetylase inhibitors (HDACis) and histone acetylase inhibitors (HAis)- which may regulate the emergence of stress-induced insecticide resistance by testing the following questions: (Q1) Does treatment with HDACis and/or HAis alone affect survival and performance (i.e., larval mobility and herbivory) of CPB (in the absence of insecticide exposure)? (Q2) Do HDACis and/or HAis affect larval tolerance of insecticides? (Q3) Do HDACis and/or HAis affect insecticide tolerance similarly in susceptible and resistant CPB populations? (Q4) Does RNAi-based knockdown of HA and HDAC genes in highly susceptible Colorado Potato Bettle (CPB) populations affect resistance development?
Approach:
Design – To address Q1, we will conduct a factorial experiment, in which we expose CPB larvae to four different treatment groups with different combinations of HDACis and HAis without exposure to insecticide, to test for potential independent and synergistic toxic effects of these enzymes on CPB larval survival: 1) no HDACi, no Hai (insecticide-free cntrl); 2) HDACi only; 3) HAi only; and 4) HDACi + HAi. To address Q2, will use the same factorial design with identical enzyme treatments; but, all larvae will additionally be exposed to 10ppm dose of imidacloprid (Fig. 1B; see section on Treatments). To address Q3, we will replicate these experiments using CPB larvae collected from susceptible and resistant field populations (see Insects section). We will collect >100 adult beetles from fields in Long Island, New York (resistant) and central Vermont (susceptible) and rear them in the lab on potato plants. For all experiments, we will randomly select 30 third-instar larvae per treatment.
Treatments – For insecticide treatments, we will use aqueous industrial-grade imidacloprid (99%; in acetone) diluted to a 10 ppm dose. For HAi, we will use histone acetyltransferase p300 inhibitor C646 (50 µM dissolved in DMSO; Sigma-Aldrich). For HDACis, we will use the Class I-II deacetylase inhibitor trichostatin A (TSA; 50 µM dissolved in DMSO; Sigma-Aldrich). For dosing, we will place larvae individually into 6-well plates, randomizing in a full block design under a fluorescent light with a 16:8 light-dark photoperiod. We will pipette imidacloprid, C646, and TSA treatments in 1µL volumes onto the dorsal side of third-instar larvae.
Performance, survival, and histone acetylation assays – We will use larval behavior to assess resistance. After the treatment exposure, we will film larvae for three minutes (mobility assay) using high-resolution video cameras. The recordings will be analyzed using the animal tracking software ToxTrac8. Larvae will then be transferred to individual petri dishes with fresh potato leaf disk for one hour (herbivory assay). We will record measure leaf consumption with the application LeafByte9. We will then monitor them for 24 hours for survival. We will quantify total histone acetylation content and levels of epigenetic modifications at individual amino acid sites of larvae using Histone H3 Total Acetylation Assay Kits and other specific histone modification kits (H3K9ac,H3K18ac, and H3K27ac; EpigenTek).
DsRNA assays - To address Q4, field-collected CPB larvae (highly susceptible) from Beltsville, MD., will be treated separately with HA- and HDAC-specific dsRNAs designed here, using doses from 400ug-1mg/ml and delivered orally by potato leaf foliar spray, using ddH2O for untreated cntrl (n=30). CPB larvae will feed for 48h then moved to healthy plants. A subset of control and treated CPB larvae will be exposed to imidacloprid followed by leaf disc herbivory. All will undergo RNA extraction, cDNA production, and qRT-PCR analyses to examine differential expression. Experiment may be repeated for protein extracts to be sent to the Cooperator to examine differences in histone acylation or indications of resistance.
