MOLECULAR MECHANISM OF NON-SUSCEPTIBILITY TO PYRETHROIDS IN THE STABLE FLY, A SIGNIFICANT LIVESTOCK PEST
Tick and Biting Fly Research
2010 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.
Approximately 85% of the stable fly sodium channel coding sequence was isolated, and the region representing domain II was amplified from individual stable flies resulting from laboratory selection for pyrethroid resistance. A single base transversion was identified in domain II, resulting in an amino acid substitution of a leucine to a histidine that associates with an observed increase in pyrethroid resistance (approx. 15-fold resistance). The mutation was located in the same position as mutations accounting for the knockdown (kdr) resistance phenotype in other insects, thus the mutation was designated kdr-his. Frequency of the allele was assessed in field-collected stable fly specimens, and revealed that the kdr-his allele is prevalent. Insecticide use at the collection sites and stable fly dispersal patterns resulted in collections containing upwards of 60% homozygous mutants of the kdr-his allele. We will be screening field-collected stable fly specimens from other states to evaluate whether the kdr-his allele is present and whether prevalence of the allele may correlate with insecticide use, if any, in these states. A presentation documenting progress was delivered at the 57th Annual Entomological Society of America Meeting (2009), and a second presentation documenting recent results was delivered at the 54th Annual Livestock Insect Workers conference (2010).
ADODR monitors project activities by e-mail and phone correspondence with collaborators at University of Florida.