1a.Objectives (from AD-416):
The stable fly is a significant pest of confined and rangeland cattle in the United States. Control methods consist of chemical insecticide application and insecticide-treated fly targets. Despite use of insecticides for control, there have been a limited number of reports regarding stable fly resistance. This may be due to a lack of surveying natural field populations. The objectives of this project are to in vitro select for a colony of stable flies that demonstrates increased non-susceptibility to pyrethroids; to isolate and sequence the stable fly sodium channel coding sequence; to evaluate the sodium channel coding sequence from non-susceptible individuals in an effort to identify sequence polymorphisms associating with the phenotype; to develop a molecular assay to detect these sequence polymorphisms; and to assess sodium channel gene polymorphism in wild populations of stable flies.
1b.Approach (from AD-416):
The stable fly sodium channel coding sequence will be isolated and sequenced using fly specimens from a University of Florida susceptible colony. Individuals from this colony will be used to initiate a selection regime to identify those flies that exhibit a non-susceptible phenotype when exposed to pyrethroids. Non-susceptible flies from each generation will be crossed and used to propagate successive generations, pressuring at each generation to select for the most non-susceptible individuals. Flies from each generation will be archived, and live flies will be shipped to ARS for evaluation of polymorphisms within the sodium channel coding sequence that may associate with the non-susceptible phenotype. Should mutations of interest be identified, molecular assays will be developed to facilitate screening wild populations of stable flies for mutation frequency. It is conceivable that we will not identify sodium channel coding sequence mutations that associate with the non-susceptible phenotype. Should this occur, we will investigate alternative mechanisms of pyrethroid resistance, i.e., esterase mediated pyrethroid metabolism.
We isolated stable fly genomic DNAs from field collections made in 6 different states, and we screened these samples for the prevalence of the previously described kdr-His allele that is associated with increased resistance of stable flies to permethrin. Allele frequencies were reported to those collaborators that provided field specimens. Our collaborator at Univ of Florida is currently conducting a nationwide survey for permethrin resistance in stable flies, and live/dead specimens will be provided to us to screen for kdr-His allele frequency as well as to scan for the presence of other alleles that may associate with reduction in permethrin susceptibility.