Sequence and transcript expression of the super-kdr locus of the horn fly, Haematobia irritans.

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.

Detection of target site resistance to pyrethroids and organophosphates in the horn fly using multiplex polymerase chain reaction.

The horn fly, Haematobia irritans L., is an obligate blood-feeding fly and the primary insect pest parasitizing cattle in the United States. Pesticide resistance has become a substantial problem for cattle producers, and although several mechanisms of resistance are possible, target site resistance is the most important mechanism preventing control of this fly in the United States and possibly other countries. We developed a multiplex polymerase chain reaction assay to detect the known target site, pyrethroid resistance-associated mutation in the horn fly and a recently reported G262A mutation in the horn fly acetylcholinesterase, the target site for organophosphates. As expected, the pyrethroid resistance target site mutation was found in fly populations from Texas, Louisiana, Washington, Georgia, Mexico, and Brazil. This mutation was found to have a gender bias as it was more prevalent in females than males. The G262A acetylcholinesterase mutation was found in Texas, Louisiana, Washington, Georgia, and Mexico, but not Brazil. There was no gender bias in the occurrence of this mutation, and there was no correlation between the occurrence of the kdr and the G262A mutations. Unlike the case with the pyrethroid target site mutation, the presence of G262A did not appear to exclusively provide the level of resistance required to account for bioassay results. It is likely an additional mutation(s) occurs in the target site and/or a metabolic resistance mechanism exists in organophosphate-resistant horn fly populations.

Microarray analysis of acaricide-inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus.

Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13 000 probes, having been derived from a previously described R. microplus gene index (BmiGI Version 2; Wang et al., 2007). Relative quantitative reverse transcriptase-PCR, real time PCR, and serial analysis of gene expression data was used to verify microarray data. Among the differentially expressed genes with informative annotation were legumain, glutathione S-transferase, and a putative salivary gland-associated protein.

Serial analysis of gene expression in the southern cattle tick following acaricide treatment of larvae from organophosphate resistant and susceptible strains.

Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos. Serial analysis of gene expression (SAGE) was used to analyse differential gene expression in response to OP treatment and to compare the responses of OP-treated and -untreated resistant and susceptible tick larvae. An R. microplus Gene Index was used as an EST database to identify genes which corresponded to SAGE tags whose abundance changed in response to acaricide exposure. Relative quantitative RT-PCR was used to confirm the differential expression results from the SAGE experiments. Of particular interest is a SAGE tag which corresponds to a cytochrome P450-like EST in the Gene Index which was more abundant in untreated OP resistant larvae compared to untreated OP susceptible larvae. This SAGE tag was also more abundant in OP resistant larvae treated with OP compared to OP susceptible larvae treated with OP.

Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas.

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.