Global comparative analysis of ESTs from the southern cattle tick, Rhipicephalus (Boophilus) microplus

Minghua Wang 0 2 4 Felix D Guerrero 0 2 3 Geo Pertea 0 1 2 Vishvanath M Nene 0 1 2 0 ern cattle tick, is one of the most economically important tick vectors of pathogens that affect the global cattle pop- 1 The J. Craig Venter Institute , 9712 Medical Center Drive, Rockville, Maryland 20850 , USA 2 Background Rhipicephalus (Boophilus) microplus, the tropical or south- 3 USDA-ARS, Knipling Bushland U.S. Livestock Insect Research Laboratory; 2700 Fredericksburg Rd., Kerrville, TX 78028 , USA 4 Lorus Therapeutics Inc; 2 Meridian Road, Toronto, ON M9W 4Z7 , Canada Background: The southern cattle tick, Rhipicephalus (Boophilus) microplus, is an economically important parasite of cattle and can transmit several pathogenic microorganisms to its cattle host during the feeding process. Understanding the biology and genomics of R. microplus is critical to developing novel methods for controlling these ticks. Results: We present a global comparative genomic analysis of a gene index of R. microplus comprised of 13,643 unique transcripts assembled from 42,512 expressed sequence tags (ESTs), a significant fraction of the complement of R. microplus genes. The source material for these ESTs consisted of polyA RNA from various tissues, lifestages, and strains of R. microplus, including larvae exposed to heat, cold, host odor, and acaricide. Functional annotation using RPS-Blast analysis identified conserved protein domains in the conceptually translated gene index and assigned GO terms to those database transcripts which had informative BlastX hits. Blast Score Ratio and SimiTri analysis compared the conceptual transcriptome of the R. microplus database to other eukaryotic proteomes and EST databases, including those from 3 ticks. The most abundant protein domains in BmiGI were also analyzed by SimiTri methodology. Conclusion: These results indicate that a large fraction of BmiGI entries have no homologs in other sequenced genomes. Analysis with the PartiGene annotation pipeline showed 64% of the members of BmiGI could not be assigned GO annotation, thus minimal information is available about a significant fraction of the tick genome. This highlights the important insights in tick biology which are likely to result from a tick genome sequencing project. Global comparative analysis identified some tick genes with unexpected phylogenetic relationships which detailed analysis attributed to gene losses in some members of the animal kingdom. Some tick genes were identified which had close orthologues to mammalian genes. Members of this group would likely be poor choices as targets for development of novel tick control technology. - ulation [1]. The tick transmits protozoan (Babesia bovis and Babesia bigemina) and prokaryotic (Anaplasma marginale) organisms that cause babesiosis and anaplasmosis, which can result in severe agricultural losses in milk and beef production and restriction in traffic of livestock. The impact of R. microplus upon the US cattle industry was such that the US Department of Agriculture (USDA) led a campaign in the mid-20th century which eradicated the tick from the US [2]. The tick remains prevalent in Mexico and, since over a million cattle are imported annually into the US from Mexico, an extensive USDA quarantine program is in place to keep Boophilus ticks from reestablishing in the US [3]. Acaricides play a critical role in maintaining the success of the USDA quarantine program and in controlling tick infestations in Mexico and other parts of the world. However, reports of acaricide resistant R. microplus populations in Mexico [4,5] and R. microplus outbreaks in the US [6] highlight the need for development of novel tick control methodologies. Understanding the genome and the gene expression profile of the tick should facilitate the development of these control technologies. Several reports have described projects centered on the acquisition and analysis of tick expressed sequence tags (ESTs). Most of the reports focused on the genes transcribed in the salivary glands of ticks such as Rhipicephalus appendiculatus [7], Amblyomma variegatum [8] and Ixodes scapularis [9]. Additionally, the isolation of 1,344 ESTs from ovaries, salivary glands and hemocytes of R. microplus has been reported, however, the sequences have not been submitted to Genbank [10]. Genes expressed in salivary glands and ovaries are attractive targets for study because these tissues are involved in critical tick-host-pathogen interactions. In a more general approach, we have developed a R. microplus EST database, BmiGI [11], derived from various tissues, lifestages and tick strains, to facilitate research using molecular biological and genomic approaches to design novel tick control technologies. It is hoped the analysis of the database will lead to discovery of genes which can overcome tick control problems due to acaricide resistance and identify gene-based vulnerabilities in the processes involved in pathogen infection and transmission. In BmiGI Version 1, 53 putative acaricide resistance-associated sequences were identified. In the present study, we have assembled an updated gene index [12] which contains more than double the number of ESTs of Version 1. We present the Gene Ontology (GO) annotation analysis and RPS-Blast identification of conserved protein domains from BmiGI Version 2. Using the comparative genomics analytical tools Blast Score Ratio [13] and SimiTri [14] which provide visual outputs to allow global comparisons between genomes, we compared the proteome resulting from the conceptual translation of the R. microplus EST database with the proteomes from Homo sapiens, Anopheles gambiae, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae. We also performed more detailed SimiTri comparisons using several of the most abundant protein domains in the proteome of R. microplus. Results and discussion BmiGI statistics and GO annotation In the first version of BmiGI, ESTs were clustered and assembled into tentative consensus (TC) sequences using TIGR's autoannotation pipeline tools, and non-clustered, non-overlapping sequences defined as singleton sequences. A total of 20,417 ESTs were analyzed and the assembly yielded 8,270 unique members, including 5,760 TCs and 2,510 singleton ESTs [11]. In the second version of BmiGI, the total number of new ESTs sequenced was 22,095. These new sequences were combined with the ESTs in the BmiGI Version 1 for clustering to generate BmiGI Version 2, resulting in 9,403 TCs and 4,240 singletons. The number of novel sequences obtained significantly decreased as EST sequencing proceeded. The first 20,417 ESTs resulted in 8,270 unique members of BmiGI, a return rate of 41%. The second set, comprised of 22,095 ESTs, resulted in an additional 5,373 new members of BmiGI, a return rate of 24%. By the final stages of the second round of EST sequencing, a return rate of approximately 5% was being observed and further EST sequ (...truncated)