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The horn fly, Haematobia irritans, is a blood-feeding parasitic fly with a global distribution that includes Europe, Africa, Asia, and the Americas. The fly has a major detrimental economic impact upon cattle production, with losses estimated at over $800 million annually in the United States and $2.5 billion in Brazil alone. Insecticide resistance in specific horn fly populations has been a problem for many years and there are several mechanisms whereby resistance develops. Little is known about the complement of metabolic enzymes encoded by the horn fly's genome that might provide the fly with detoxification or sequestration pathways to survive insecticide treatments. The cytochrome P450, glutathione S-transferase, and esterase enzyme families contain members that are capable of sequestering and/or detoxifying xenobiotic molecules such as insecticides. We sought to develop a comprehensive dataset of metabolic enzyme-encoding transcript sequences from the adult horn fly, as this is the life stage whose actions directly impose the economic costs to cattle producers. We used an Illumina paired-end read RNA-Seq approach to determine the adult horn fly transcriptomes from laboratory and field populations of horn flies with varying levels of pesticide resistance, including untreated and pyrethroid-treated newly eclosed adult flies. We followed with bioinformatic analyses to discern sequences putatively encoding cytochrome P450, esterase, and GST enzymes. We utilized read-mapping of RNA-Seq data and quantitative real-time polymerase chain reaction (qRT-PCR) to examine gene expression levels of specific P450 transcripts in several fly populations with varying degrees of pesticide resistance.

Ticks, as blood-feeding ectoparasites, affect their hosts both directly and as vectors of viral, bacterial and protozoal diseases. The tick's mode of feeding means it must maintain intimate contact with the host in the face of host defensive responses for a prolonged time. The parasite-host interactions are characterized by the host response and parasite counter-response which result in a highly complex biological system that is barely understood. We conducted transcriptomic analyses utilizing suppressive subtractive hybridization (SSH) to identify transcripts associated with host attachment and feeding of larval, adult female and adult male ticks. Five SSH libraries resulted in 511 clones (assembled into 36 contigs and 90 singletons) from differentially expressed transcripts isolated from unattached frustrated larvae (95), feeding larvae (159), unattached frustrated adult female ticks (68), feeding adult female ticks (95) and male adult ticks (94 clones). Unattached 'frustrated' ticks were held in fabric bags affixed to cattle for up to 24 h to identify genes up-regulated prior to host penetration. Sequence analysis was based on BLAST, Panther, KOG and domain (CDD) analyses to assign functional groups for proteins including: cuticle proteins, enzymes (ATPases), ligand binding (histamine binding), molecular chaperone (prefoldin), nucleic acid binding (ribosomal proteins), putative salivary proteins, serine proteases, stress response (heat shock, glycine rich) and transporters. An additional 63% of all contigs and singletons were novel R. microplus transcripts or predicted proteins of unknown function. Expression was confirmed using quantitative real time PCR analysis of selected transcripts. This is the first comprehensive analysis of the R. microplus transcriptome from multiple stages of ticks and assists to elucidate the molecular events during tick attachment and development.

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.

Between 1999 and 2002, the effect of mid-season doramectin treatments on the level of resistance in pyrethroid-resistant horn fly populations was examined at three separate Louisiana State University Agricultural Center research stations. The cattle were treated with pyrethroid ear tags in all years at all farms, and each farm received a mid-season doramectin treatment in 1 year. The number of weeks of control at Red River was 11 weeks higher in the year following the mid-season treatment of doramectin. At Macon Ridge, the number of weeks of control was 2 weeks higher in the year following the doramectin treatment. No change was observed at St. Joseph. The LC50s for fly populations tested at Macon Ridge and St. Joseph were found to increase for pyrethroids from the spring populations to the fall populations between 2000 and 2002. The LC50s for fly populations at Red River followed the same trends except in 2000, the year when the doramectin treatment was administered. Flies collected pre and post-treatment each year from St. Joseph and Red River were assayed for two alleles (kdr and skdr) associated with target site resistance to pyrethroids. Flies collected pretreatment at Macon Ridge in 1999 also were assayed for the kdr and skdr, and this population of flies had a frequency of 85.6% R-kdr alleles. At St. Joseph and Red River there was a general decline in the frequency of homozygous susceptible skdr (SS-skdr) and homozygous susceptible kdr (SS-kdr) individuals, as well as a general increase in homozygous resistant skdr (RR-skdr) and homozygous resistant kdr (RR-kdr) individuals, during the 4-year study. At both sites, the frequency of R-kdr alleles increased significantly in flies collected in the fall compared to flies collected in the spring with the exception of Red River in 2000, when dormacetin was applied. The frequency of the R-kdr alleles was significantly higher in flies collected in the fall compared to flies collected in the spring in the following year at both sites in two out of three comparisons. The frequency of R-skdr alleles was significantly lower in fly populations tested in the spring compared to fly populations tested in the fall at both farms in years when doramectin was not applied but there were no differences in the years when doramectin was applied. The frequency of R-skdr alleles was significantly higher in fly populations tested in the fall compared to in the spring the following year during all three comparisons at Red River and in one of three comparisons at St. Joseph.

The horn fly, Haematobia irritans irritans, is an obligate blood-feeding parasite of cattle that causes significant economic impact in many countries. We investigated the resistance of a horn fly population from Louisiana/USA to endosulfan, a cyclodiene insecticide. Bioassays were performed in 2010 and 2011 in order to determine the resistance ratio of the population to endosulfan and a PCR assay was developed to detect the Rdl mutation which is the replacement of an alanine with a serine at the GABA receptor locus that has been associated with resistance to cyclodienes in other insect species. Endosulfan tags had provided 8 weeks of effective control in 2010 but only 1 week in 2011. After only one summer (June-September/2010) of exposure to the endosulfan tagged cattle, there was a significant increase in the resistance ratio for endosulfan in the fly population. Most flies surveyed by the PCR diagnostic assay were homozygous susceptible at the Rdl locus, the resistant (R) allele was mainly present in the heterozygous state and there was no difference in the frequency of the R allele between female and male flies. After the first year's exposure of the horn flies to the endosulfan tags, the frequency of the R allele increased significantly. However, after one year without endosulfan treatment (2011-2012), the frequency of the R allele significantly dropped. These results indicate that target site resistance was responsible, at least in part, for the resistance and that a fitness cost is possibly associated with the Rdl mutation.

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.

Pyrethroid resistance in three horn fly populations in Louisiana was monitored by weekly fly counts, filter paper bioassays, and diagnostic PCR assays for the presence of pyrethroid resistance-associated mutations in the sodium channel gene coding region. The PCR assay for the knockdown resistance (kdr) and superkdr sodium channel mutations was used to determine the frequency of the target site insensitivity mechanism in the populations of horn flies, which possessed varying degrees of insecticide resistance. The bioassays and frequency of homozygous-resistant (RR) kdr genotypes were relative predictors of the fly control subsequently observed. Flies exposed to filter paper impregnated with a discriminating concentration of one of four different insecticides were collected when 50% mortality was estimated. Genotypes for the dead flies and the survivors were determined by the PCR assay. The results of the PCR assays indicated that the genotype at the kdr locus of the flies exposed to the two pyrethroids had an effect upon whether the flies were considered to be alive or dead at the time of collection. The kdr genotype of flies exposed to either diazinon or doramectin was unrelated to whether the flies were considered to be alive or dead, except for a single comparison of flies exposed to diazinon. When possible interactions of the kdr and superkdr mutations were compared, we found that there were no associations with the response to diazinon and doramectin. For one location, there were no survivors of the 75 flies with the SS-SS (superkdr-kdr) homozygous susceptible wild type genotype exposed to pyrethroids, while there were survivors in all of the other five genotypes. The SS-RR genotype flies were more susceptible to the pyrethroids than the SR-RR flies, but that was not the case for exposure to diazinon or doramectin. In the St. Joseph population, there were an adequate number of flies to demonstrate that the SS-SR genotype was more susceptible to pyrethroids than the SS-RR and that flies with the SR-SR genotype were more susceptible to pyrethroids than the flies with the SR-RR genotype.