Detecting Presence or Absence of Alternative Splicing at the Superkdr Locus in Horn Fly, Haematobia irritans

Solis, Gabriela Dolores
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Changes to pest control efforts are dependent on knowing the interaction that occurs between insecticides and the pest in question. Horn flies (Haematobia irritans) are agricultural pests that have had negative economic consequences due to their detrimental effects on cattle and their development of insecticide resistance. The superkdr locus has a single base change, from a thymine to a cytosine, distinguishing it from the kdr gene and resulting in replacement of methionine with threonine; this mutation has been associated with pyrethroid-resistant horn flies. In houseflies (Musca domestica), the superkdr locus was discovered to occur on mutually exclusive exons, exon C and exon D. Exon C was found in place of exon D in the cDNA from an adult horn fly head, and encodes a truncated, and presumably non-functional, sodium channel α subunit isoform. The purpose of this research is to determine if alternative splicing is occurring at the superkdr locus of horn flies. Two different methods were performed to determine the presence or absence of alternative splicing. The first method involved sequence analysis of the sodium channel gene containing the superkdr locus. cDNA and genomic DNA were cloned, sequenced, and compared using MacVector. The second method used a SNP assay to perform genotyping via real-time PCR. With the SNP assay, one can genotype the superkdr locus in cDNA and genomic DNA, and simultaneously detect alternative splicing if the locus was not detected in cDNA. I determined that there is no significant difference between the genotype of the superkdr locus in cDNA compared to genomic DNA. If alternative splicing of the superkdr is occurring, it seems to be rare. Overall, this shows genomic DNA can be used to determine the prevalence of pyrethroid-resistance population. The SNP assay used to genotype is both time and cost-effective, and it can be used to determine if pyrethroid is the best choice of insecticide to use depending on the prevalence of pyrethroid-resistance in a wild population.
Horn flies, Alternative splicing, Superkdr
Solis, G. D. (2017). <i>Detecting presence or absence of alternative splicing at the superkdr locus in Horn Fly, Haematobia irritans</i> (Unpublished thesis). Texas State University, San Marcos, Texas.