Gene expression during the early stages of host perception and attachment in adult female Rhipicephalus microplus ticks.

The cattle tick, Rhipicephalus microplus, is a serious pest of cattle, with significant economic consequences to the livestock industries of tropical and semitropical countries. Rhipicephalus microplus belongs to the Metastriata group of the Ixodidae family known as hard ticks. When adult hard ticks feed, mating has not yet occurred and an initial host attachment phase of 1-2 days is followed by a slow feeding phase that can last several days. Once mating occurs, feeding concludes with a rapid engorgement phase that is completed in 12-36 h. Our group's interest in mining the genome and transcriptome of R. microplus for novel targets for development of tick control technologies led us to investigate the early transcriptional events occurring upon tick attachment and subsequent feeding. We placed newly molted unfed adult R. microplus females upon a bovine host and harvested the attached ticks after 3, 6, 12, and 24 h. We also placed a group of these ticks in a gas-permeable tube taped onto the side of the bovine host. These ticks were able to sense the host but unable to penetrate the tube to begin attachment and were ultimately harvested after 3 h. This study produced a comprehensive transcriptome from newly molted adult ticks and will provide a useful resource for studies of tick feeding and host perception and also assist genome annotation refinements.

The adult horn fly transcriptome and its complement of transcripts encoding cytochrome P450s, glutathione S-transferases, and esterases.

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.

Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer.

BACKGROUND: Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus). METHODS: Using 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections. RESULTS: LPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00-0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas. CONCLUSIONS: Two dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established.

Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico.

BACKGROUND: Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. METHODS: We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. RESULTS: We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). CONCLUSIONS: These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.

Distribution patterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia.

Resistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with target site pyrethroid resistance. We developed a multiplex PCR using allele-specific primers to amplify wild type or mutated genotypes of the three mutations simultaneously. This assay was used to screen tick samples originating from Brazil, Argentina, Mexico, South Africa and Australia whose phenotype to flumethrin and cypermethrin had been determined by the use of the Larval Tarsal test (LTT) or the Larval Packet Test (LPT). These mutations were found to have distinct geographical distributions and result in different resistance phenotypes. The L64I Domain II mutation conferring resistance to several SP compounds was found in all the Brazilian, Argentinean and Australian populations and in one South African population, with frequencies between 38% and 100% in flumethrin and cypermethrin resistant populations. In contrast, this mutation was not found in samples from Mexico, while the Domain III mutation was found exclusively in this country. The G72V Domain II flumethrin-specific mutation was found in a single Australian population, with a very low resistant allele frequency (3%). The homozygous resistant RR genotype of the L64I Domain II mutation correlated significantly with the survival rates at the discriminating doses of flumethrin and cypermethrin. This survey shows the widespread distribution of the L64I Domain II mutation and provides evidence of its geographic separation from the Domain III mutation.