Global comparative analysis of ESTs from the southern cattle tick, Rhipicephalus (Boophilus) microplus
© Wang et al; licensee BioMed Central Ltd. 2007
Received: 16 February 2007
Accepted: 12 October 2007
Published: 12 October 2007
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.
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.
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.
Rhipicephalus (Boophilus) microplus, the tropical or southern cattle tick, is one of the most economically important tick vectors of pathogens that affect the global cattle population . 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 . 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 .
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  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 , Amblyomma variegatum  and Ixodes scapularis . 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 . 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 , 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  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  and SimiTri  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 . 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 sequencing of this pooled normalized cDNA library no longer seemed an efficient use of resources. Future EST sequencing would likely be more efficient if performed on libraries synthesized from targeted tissues of specific interest, such as synganglia, ovaries or salivary glands. Sequencing of several targeted libraries is underway.
The latest release of the annotation for the D. melanogaster genome sequence  notes 19,783 protein-coding transcripts. The latest genome assembly for the A. gambiae  has noted 14,089 gene transcripts. Assuming R. microplus has a similar number of transcripts as these two arthropods, the BmiGI set of 13,643 unique transcripts represents a significant fraction of the likely set of protein-coding transcripts in R. microplus. However, it is likely that BmiGI contains ESTs which are derived from non-coding RNAs, as EST databases have been shown to contain non-coding RNAs . Additionally, during use of BmiGI following annotation by BLAST analysis, it was noticed that some sequences had very high amino acid identity to bovine sequences. These likely resulted from bovine blood remaining in the gut of the adult ticks, one of the lifestages sampled and included in the pooled RNA used to synthesize the cDNA library. Additionally, some sequences appeared to be of protozoan origin and might have originated from commensual organisms within the tick or from a sample of Babesia bovis-infected larvae included during the library synthesis. The autoannotation pipeline used for assembling the gene index was not readily adaptable to remove bovine or protozoan sequences and this should be considered when using BmiGI. However, in our experience, these do not form an appreciable fraction of the BmiGI entries and should be easily identifiable by their high nucleotide identity to bovine or protozoan sequences in GenBank Blast search results.
Gene ontology assignment using different cutoffs
TIGR E < 1 × 10-27
PartiGene E < 1 × 10-25
PartiGene E < 1 × 10-8
Molecular function unknown
Structural molecule activity
Transcription regulator activity
Signal transducer activity
Enzyme regulator activity
Nucleic acid metabolism
Regulation of biological process
Biological process unknown
Cellular component unknown
We wished to attempt to predict gene function for TCs which were designated as unknowns and not assigned GO terms by the TIGR pipeline and to include GO annotation analysis for singletons when possible. Thus, we tried the software annot8r_blast2go in the PartiGene pipeline , using Blast E-values of 1 × 10-8 and 1 × 10-25 (Table 1). When the E-value is set at 1 × 10-25, 2,615 TCs (28% of the total TCs) and 730 singletons (17% of the total singletons) can be assigned a GO annotation. When the E-value is set at 1 × 10-8, 3,608 TCs (38%) and 1096 singletons (26%) can be assigned one or more GO terms. Thus, 66% of the members of BmiGI could not be assigned GO annotation, even using a relatively liberal E-value in the Blast. Singletons were annotated at a lower ratio of the total possible than TCs, most likely due to the singletons generally containing shorter sequence lengths compared to TCs. It is possible that some singletons represent transcripts from low copy number genes which might be unique to ticks or from genes with low sequence identity to those from organisms better represented in gene and protein sequence databases.
Global comparative genomics
We were interested in determining how related the genome of R. microplus is to other metazoan genomes. SimiTri  was developed for that purpose and is capable of globally comparing a target genome to three other genomes with the results displayed in an easily interpreted triangular graphic. In fact, SimiTri analysis was used to compare EST and whole genome databases from several nematode species, including C. elegans, Haemonchus contortus, and Nippostrongylus brasiliensis, and visualize evolutionary relationships between these nematodes . Hughes et al.  used SimiTri analysis for similar purposes in comparisons of translated ESTs from various beetles to the proteomes of D. melanogaster, H. sapiens, and C. elegans. However, since our research priorities are aimed at developing novel control technologies for cattle pests in general and R. microplus most specifically, our comparative analyses were guided by these priorities. We wished to use comparative genome analysis to help prioritize selection of possible gene or protein targets for developing novel control technologies, which could include vaccines or design of novel inhibitors aimed at selected gene products. Ideally, a control technology would present no toxicity to non-target organisms, with mammalian toxicity presenting greatest concern. Naturally, an anti-tick control technology which is highly toxic to cattle would be of limited use when applied to cattle compared to an effective approach with high target specificity. Thus we selected the genome of H. sapiens as the representative mammalian genome for comparative genome analysis with the BmiGI database, feeling that coding regions without orthologous members in mammals would provider better targets for further investigations. Likewise, as R. microplus is an arthropod, we selected the well-characterized genome of D. melanogaster for these comparisons. As cattle can be parasitized internally by nematodes, we selected C. elegans as a well-studied representative for the genome of that type of organism. Finally, the genome of A. gambiae was of interest as this organism is a blood-feeding arthropod vectoring a number of organisms which parasitize human red blood cells in a broadly similar fashion as B. bovis and B. bigemina parasitize cattle red blood cells.
Atyptical genes in SimiTri analysis
Dusty protein kinase (NP_991190)
COP1 protein (NP_071902)
Dusty protein kinase (NP_991190)
COP1 protein (NP_071902)
Dusty protein kinase (NP_991190)
Two other atypical genes in Table 2, TC7573 and TC9268 are related to actin binding. The top Blast hit for TC7573 is Destrin (E-Value = 1 × 10-78), an actin-depolymerizing factor , however, TC7573 shows over 99% nucleotide identity to ESTs from various Bos taurus cDNA libraries, including those from skin, liver, and placenta, and it is likely TC7573 is of bovine origin. TC9268 contains 2 PDZ (postsynaptic density protein, disc-large, zonulin-1) domains and has sequence similarity to syntenin (E-Value = 1 × 10-76), which is involved in diverse physiological processes resulting from its interaction with signaling and adhesion molecules . An EST [GenBank:CD791887] from R. appendiculatus has 85% nucleotide identity in the 5' region of TC9268, so this gene is present in at least two species of ticks. BlastP and phylogenetic tree analysis (Figure 2b) shows the coding region from TC9268 is very similar to coding regions from shrimp, honey bee, red fluor beetle, and several mosquitoes. A Drosophila gene does not appear in the phylogenetic tree (Figure 2b, Additional files 3 and 4), indicating a closely related sequence to TC9268 is not present in drosophilids, perhaps being lost from that group of species. In fact, C. elegans does not show a close relative of TC9268 and the absence of this gene coding region from both C. elegans and D. melanogaster would explain its atypical clustering in Figure 1b.
The top Blast hit for TC13445 is COP1 (constitutive photomorphogenic 1; E-Value = 1 × 10-91), a protein acting as an E3 ubiquitin ligase involved in light signaling in plants and tumorigenesis in mammals . A R. appendiculatus EST [GenBank:CD779568.1] has 94% nucleotide identity to TC13445. The BlastP and tree analysis (Figure 2c) shows that other than a Tribolium castaneum orthologue, sequences with high similarity to TC13445 seem to be generally absent from insects. The T. castaneum orthologue has the closest relationship to the R. microplus gene, and there is significant similarity to orthologues from organisms as diverse as fishes, primates, and several species of plants. Although D. melanogaster and Drosophila pseudoobscura do have sequences with limited similarity (E-Value = 1 × 10-13) to TC13445, the tree analysis showed both Drosophila sequences fell somewhat distant from the TC13445 (Figure 2c, Additional files 5 and 6). The nematodes do not appear to have a close relative of TC13445 either and, coupled with the absence of an orthologue in D. melanogaster with close similarity to TC13445, helps explain the atypical clustering of this sequence in Figures 1a and 1b.
TC12600 possesses significant sequence similarity to a protein (Q4RST5) that contains a Laminin_A domain (E-Value = 1 × 10-124), which exists in the extracellular space, functioning in the signaling process for morphogenesis and also playing a structural role . BlastP analysis shows the closest relatives to the R. microplus sequence are zebrafish, pufferfish and various mammals (Figure 2d), with an absence of insect sequences in the phylogenetic tree (Figure 2d, Additional files 7 and 8) indicating this gene may have been lost from most insects. It is possible these atypically clustering sequences, TC14523, TC9268, TC13445, and TC12600, could represent examples of convergent evolution resulting from the parasitic lifestyle of ticks on their mammalian hosts subjecting the tick to similar environmental and evolutionary pressures as mammals. However, the atypical clustering seen in the SimiTri analysis (Figure 1a–c, Table 2) appears to more likely result from gene loss in the arthropod selected as a query genome, an event more easily visualized by phylogenetic tree analysis of aligned sequences from many genomes than SimiTri analysis which is limited to three genomes. Additionally, the dipterans are better represented among arthropods having sequenced genomes or significant collections of ESTs. As more non-dipteran arthropod sequences become available, a better understanding of phylogenetic relationships will develop.
As the comparative analysis was performed with BmiGI, which is an incomplete database of tick gene coding regions, the SimiTri and BSR plots might present different results once the entire tick genome is available for analysis. In fact, once the I. scapularis genome is available and annotated , a more comprehensive SimiTri and BSR analysis could be easily done and the I. scapularis results compared to these presented in our study. It is our feeling that R. microplus gene coding regions not currently represented in BmiGI are genes expressed in either highly specialized tissues or at very low levels in the tick. A significant fraction of these genes might be involved in regulatory processes or gene cascades and could have conserved features across a number of arthropod, or even eukaryotic, classes of organisms. These genes would have plotted in the central portions of SimiTri plots and in Quadrant C of BSR plots. However, it is likely that a number of low abundance tick-specific genes have not been discovered during our EST sequencing and would not be in BmiGI. Genes with little or no similarity to those from organisms used in the SimiTri analysis would result in data points along the edges of the plot, while in BSR analysis, these individuals would plot in Quadrant A. Additionally, the Blast analysis E-value can be adjusted to act as a filter on the SimiTri and BSR results. As both SimiTri and BSR only plot sequences which pass the designated Blast E-value cutoff, these comparisons can be made more or less stringent by varying the E-value. If a query sequence does not have a Blast hit to any organism in GenBank, that sequence will not get plotted during either SimiTri or BSR analysis.
BmiGI is composed of 69% assembled TCs and 31% singletons. Thus the prot4EST translation data contains a significant proportion of small proteins resulting from translation of incomplete open reading frames and a 3' end bias is certainly present in EST databases generated from polyA RNA selection methodology as used in deriving BmiGI. In the prot4EST translations of BmiGI, 18% and 11% of the 9,494 TCs yielded translation products of < 80 and < 60 amino acids, respectively. The 4,238 singleton translation product set had 29% and 11% of its members with < 80 and < 60 amino acids, respectively. The combination of the likely 3' end bias of BmiGI and the generally less conserved nature of 3' untranslated regions could contribute to bias results of the BSR analysis toward proteins without matches to the other two queried organisms, thus plotting in Quadrant A. Although this possible bias should be kept in mind, the prot4EST polypeptide prediction pipeline contains ESTscan2.0 to recognize and separate probable protein coding regions from 5' and 3' untranslated regions , reducing their impact on the BSR analysis. As discussed in the previous paragraph, Blast E-value will also affect BSR. These small translation products are less likely to have Blast hits to any organism than longer proteins and, if this happens, would not appear on the BSR plots.
Protein domain analysis
The most abundant domains in BmiGI version 2
CDD Accession number
Number of hits
Percentage of the total hits
RNA recognition motif (RRM)
Trypsin like serine protease
Ubiquitin-conjugating enzyme E2, catalytic domain
Serine Proteinase Inhibitors (Serpin)
This study presents the analysis of BmiGI Version 2, which contains a significant fraction of the coding regions of the genome of R. microplus. Our results indicate that many genes of R. microplus are unique and have no homologs in other sequenced genomes. With E-value = 1 × 10-8, only 34% of the 13,765 members of BmiGI can be assigned one or more GO terms using this relatively liberal Blast E-value.
Among the BmiGI members which had Blast hits, BSR analysis found approximately 2,300 R. microplus sequences which did not have a close match to sequences from D. melanogaster, H. sapiens, C. elegans, or A. gambiae. This highlights there have been unique gene evolutionary processes in ticks and emphasizes the importance of sequencing a tick genome to better understand tick biology. In the absence of whole genome sequence , EST data is a good resource for gene discovery and will facilitate the study of acaricide resistance mechanisms in R. microplus. Our 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.
R. microplus EST sequences
The construction of the R. microplus normalized cDNA library, generation of ESTs, and assembly into the R. microplus gene index have been described . Briefly, a single normalized cDNA library was synthesized from pooled RNA samples which had been purified from ticks subjected to various environmental exposures, including heat shock, cold shock, host odor, infection with B. bovis, and various acaricides. The acaricide exposure experiments were performed with several strains of R. microplus which varied in their levels of susceptibility to pyrethroid, organophosphate and the formamidine amitraz. We also included RNA purified from eggs, nymphs, adults and dissected adult tick organs.
Comparative genomic analysis
The 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. GO terms  were assigned automatically using customized script based on BlastX search results. We also used PartiGene  as another pipeline for EST analysis, as this open source analytical package provided some powerful annotation options to compare to the TIGR autoannotation pipeline results. BmiGI Version 2 was analyzed by PartiGene and, to maintain consistency, the TC and singleton numerical designations in BmiGI were kept identical. The protein coding regions of R. microplus were determined by applying prot4EST  to BmiGI and using data from Uniprot for the Blastp . GO terms were assigned based solely on Blast E-values using the annot8r module from the PartiGene package.
For the SimiTri analysis , the program was downloaded  and BlastP searches were performed using the prot4EST translated sequences from BmiGI and a cutoff E-value < 1 × 10-8. Blosum62 was used as the matrix for these searches for its strength in detecting weak similarities between proteins. The predicted proteomes of S. cerevisiae, C. elegans, A. gambiae, D. melanogaster and H. sapiens were downloaded . TblastN searches were performed against EST databases from the ticks, R. appendiculatus and A. variegatum  and I. scapularis . The atypically clustering R. microplus genes were analyzed by BlastP analysis of the conceptual open reading frames as noted in BmiGI Version 2 followed by generation of phylogenetic trees using ClustalW and programs from the Phylip phylogeny inference package . In the Phylip package, SEQBOOT was used at default values except with 1000 replicates, PROTPARS at default values except with 10 jumbles, and CONSENSE and at all default values. The consensus trees were viewed using TREEVIEW . Subtrees containing sequences closely related to the BmiGI entry are displayed as figures while the entire tree and the alignment used to produce the tree are included as Additional files 1, 2, 3, 4, 5, 6, 7, 8.
In the BLAST Score Ratio (BSR) approach , the conceptual translation of the R. microplus reference genome (BmiGI Version 2) was compared with various proteome pairs of S. cerevisiae, C. elegans, A. gambiae, D. melanogaster and H. sapiens which served as Query1 and Query2. The graphical output files are plotted and divided into four quadrants using the BSR threshold value of 0.4.
Protein domain analysis were performed by RPS-Blast search against the Conserved Domain Database  using a cutoff E-value < 1 × 10-8. The translated sequences for the domains of interest were extracted from BmiGI and subjected to SimiTri analysis using the protocols described above.
M.W. was supported by the National Research Initiative of the USDA CSREES grant #2005-35604-15440 (to F.D.G.). The authors are grateful for the manuscript reviews and suggested revisions provided by Drs. Kelly Brayton and Carlos Suarez and the anonymous reviewers assigned by this journal. This article reports the results of research only. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation of endorsement by the U.S. Department of Agriculture.
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