Location: Livestock Arthropod Pest Research Unit
Project Number: 3094-32000-041-012-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Jun 30, 2025
Objective:
The overall objectives of this project are to 1) evaluate levels of pyrethroid resistance in stable flies from geographically separated production facilities with different commodity focus in Texas, and 2) assess the role, if any, of metabolic pyrethroid resistance in the stable fly.
Approach:
Selection for Pyrethroid Resistant Lines. Multiple field collections of stable flies from Texas will be established and pressured with permethrin or deltamethrin over successive generations to select for survivors that display increased resistance to these pyrethroids. A previous permethrin selection regimen was used in Cooperator's laboratory, resulting in selection for a stable fly line exhibiting 15-fold resistance relative to the susceptible strain and a comparable or higher level of resistance will be the goal for this study. A subset of adult survivors and non-survivors (dead) at each generation will be snap frozen in liquid nitrogen and stored at -80 °C until processing. Synergist Bioassay. Upon development of the resistant line(s), bioassays will be completed with either permethrin or deltamethrin in combination with synergists that inhibit esterases (triphenyl phosphate, TPP), cytochrome P450 monooxygenases (piperonyl butoxide; PBO), and glutathione-S-transferases (diethyl maleate, DEM) to evaluate the role of metabolic detoxification, if any, in the resistant phenotype. A reduction of the resistance factor in the presence of the synergist will broadly indicate the metabolic pathway that may contribute to the observed resistance. Differential Expression of Metabolic Detoxification Pathway Genes. An RNAseq approach will be used to identify differentially expressed genes between resistant and susceptible flies, with a focus on genes annotated from the esterase, cytP450, and GST gene families. Vssc Screening. Phenotypic pyrethroid resistance can be simultaneously conferred by mutations in Vssc and modified expression of metabolic detoxification pathway genes. cDNAs will be synthesized from survivors/non-survivors, and the full-length coding sequence for Vssc will be isolated to evaluate whether additional polymorphisms that associate with increased resistance may be present at each generation of selection pressure.
