Sidney E. Kunz

Toxicological and molecular characterization of pyrethroid-resistant horn flies, Haematobia irritans: identification of kdr and super-kdr point mutations.

Two pyrethroid-resistant strains of horn flies were found to be 17- and 688-fold more resistant to permethrin and 17- and 11,300-fold more resistant to cyhalothrin than a susceptible control strain. Synergism experiments with piperonyl butoxide showed that both target site insensitivity and metabolic resistance mechanisms were present in the Super Resistant strain. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), a 0.9 kb fragment of the putative sodium channel gene from susceptible and resistant flies was cloned and sequenced. Two sequence variants were detected, presumably arising from alternative splicing of transcripts. The amino acid sequences deduced from the resistant and susceptible fly gene fragments were identical except for three amino acid substitutions, two of which have been associated with resistance in house flies. A leucine to phenylalanine substitution associated with knockdown resistance (kdr) was found in both resistant strains. A methionine to threonine substitution associated with super-kdr was found in the Super Resistant strain. Translation of poly(A)+ RNA followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected translation products whose concentrations increased in association with pyrethroid resistance. Random-amplified polymorphic DNA (RAPD)-PCR of genomic DNA with over 260 DNA oligomers yielded one resistance-associated marker, designated HF-77, which was not detected in any susceptible flies but was present in 16% of the resistant individuals.

Role of the kdr and super-kdr sodium channel mutations in pyrethroid resistance: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies (Haematobia irritans)

The kdr and super-kdr point mutations found in the insect sodium channel gene are postulated to confer knockdown resistance (kdr) to pyrethroids. Using an allele-specific PCR assay to detect these mutations in individual horn flies, Haematobia irritans (L.), we determined the allelic frequency of the kdr and super-kdr mutations in several wild and laboratory populations. Wild populations with very similar allelic frequencies had resistance levels that ranged widely from 3- to 18-fold relative to a susceptible population. Conversely, the kdr allele frequency in a lab population with 17-fold resistance was nearly double that found in a heavily pressured wild population with 18-fold resistance. We conclude that, although the kdr mutation confers significant levels of pyrethroid resistance, a substantial component of resistance in insecticidally pressured populations is conferred by mechanisms that are PBO-suppressible. High super-kdr allele frequencies were detected in two resistant lab populations, but in wild populations with equivalent resistance the super-kdr allele frequency was very low. Interestingly, in over 1200 individuals assayed, the super-kdr mutation was never detected in the absence of the kdr mutation.