Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus

The cattle tick, Rhipicephalus (Boophilus) microplus, is a pest which causes multiple health complications in cattle. The G protein-coupled receptor (GPCR) super-family presents a candidate target for developing novel tick control methods. However, GPCRs share limited sequence similarity among orthologous family members, and there is no reference genome available for R. microplus. This limits the effectiveness of alignment-dependent methods such as BLAST and Pfam for identifying GPCRs from R. microplus. However, GPCRs share a common structure consisting of seven transmembrane helices. We present an analysis of the R. microplus synganglion transcriptome using a combination of structurally-based and alignment-free methods which supplement the identification of GPCRs by sequence similarity. TMHMM predicts the number of transmembrane helices in a protein sequence. GPCRpred is a support vector machine-based method developed to predict and classify GPCRs using the dipeptide composition of a query amino acid sequence. These two bioinformatic tools were applied to our transcriptome assembly of the cattle tick synganglion. Together, BLAST and Pfam identified 85 unique contigs as encoding partial or full length candidate cattle tick GPCRs. Collectively, TMHMM and GPCRpred identified 27 additional GPCR candidates that BLAST and Pfam missed. This demonstrates that the addition of structurally-based and alignment-free bioinformatic approaches to transcriptome annotation and analysis produces a greater collection of prospective GPCRs than an analysis based solely upon methodologies dependent upon sequence alignment and similarity

Analysis of Babesia bovis infection-induced gene expression changes in larvae from the cattle tick, Rhipicephalus (Boophilus) microplus

Background: Cattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world's tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, 'Babesia bovis', and transmitted to cattle through the bite of infected cattle ticks of the genus 'Rhipicephalus', with the most prevalent species being 'Rhipicephalus (Boophilus) microplus'. We studied the reaction of the 'R. microplus' larval transcriptome in response to infection by 'B. bovis'. Methods: Total RNA was isolated for both uninfected and 'Babesia bovis'-infected larval samples. Subtracted libraries were prepared by subtracting the 'B. bovis'-infected material with the uninfected material, thus enriching for expressed genes in the 'B. bovis'-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density micro arrays. The micro array probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR. Results: Ticks were allowed to feed on a 'B. bovis'-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted DNA libraries were synthesized from 'Babesia'-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a 'R. microplus' micro array designed from a 'R. microplus' gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of 'R. microplus' larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a 'B. bovis'-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a 'B. bovis'-infected calf contrasted to ticks feeding on an uninfected calf

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis

Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis