Multiplex PCR in the horn fly: Detection of pyrethroid, organophosphate and cyclodiene target site resistance

The horn fly, Haematobia irritans irritans, is an important pest to the livestock industry that causes economic losses of approximately US$1 billion in the U.S. and a similar value in Latin America. Horn fly control efforts still relies mainly on direct application of insecticides although horn fly populations from the U. S. and other countries have become resistant to some of these compounds. In this study, we developed a multiplex PCR assay to simultaneously detect target site resistance to pyrethroids, organophosphates and cyclodienes. The amino acid differences responsible for these resistance mechanisms are respectively: the kdr mutation, replacement of a leucine by phenylalanine in the S6 transmembrane fragment of domain II of the sodium channel, the G262A acetylcholinesterase (AChE) mutation, a substitution of a glycine for alanine at position 262 of the AChE protein, and the Rdl mutation, the replacement of an alanine with a serine at the position 302 at the GABA_A receptor locus. The multiplex PCR showed robust results in all our assays.  There was no gender bias either for the G262A AChE or the Rdl mutation, but the kdr mutation was more prevalent in females than males. The possibility of screening three different mutations in a single PCR reaction makes the multiplex PCR a useful and affordable tool that can assist in the early diagnosis of insecticide resistance and can contribute to development of rational insecticide usage to control horn flies and in designing novel strategies to prevent or minimize the spread and evolution of resistance in horn fly populations.