2012 Annual Report 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. 3.Progress Report: We confirmed that a previously reported mutation within the stable fly sodium channel, kdr-His, associated with increased resistance to permethrin, persisted in the laboratory-selected, colonized strain without any apparent fitness cost. In response to a request made to university and experiment station researchers, stable flies were received from various geographical locations within the United States and Canada. Isolation of genomic DNAs from these collections to evaluate kdr-His frequency has been initiated using the DNA-based assay designed as part of this subordinate project.