An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus

Abstract

Background: Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R.sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences.

Results: Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained approximately 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as "unknown function". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library.

Conclusions: Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Figure 1

Relationship of Rhipicephalus sanguineus lipocalins to other related tick proteins. The circular phylogram is based on the alignment of sequences derived from this study and similar sequences obtained from the NR database from NCBI, and from tick sequences derived from dbEST. The red branches have bootstrap support above 75% (10,000 replicates). The bar at the center indicates 20% amino acid divergence. The R. sanguineus sequences are indicated by a circle, and start with RS-. The sequences obtained from the NR database are indicated by 5-6 letters related to the tick species followed by the NCBI accession number. Remaining sequences were deduced from dbEST and are available from Francischetti et al. [8].

Figure 2

Relationship of Rhipicephalus sanguineus Kunitz domain proteins to other related tick proteins. The circular phylogram is based on the alignment of sequences derived from this study and homologous sequences obtained from the NR database from NCBI, and from tick sequences derived from dbEST. The red branches have bootstrap support above 75% (10,000 replicates). The bar at the centre indicates 20% amino acid divergence. The R. sanguineus sequences are indicated by a circle, and start with RS-. The sequences obtained from the NR database are indicated by 5 letters (3 from the genus and 2 from the species name) followed by the NCBI accession number. Remaining sequences were deduced from dbEST and are available from Francischetti et al. [8].

Figure 3

The salivary basic tail and 18.3 kDa proteins of Rhipicephalus sanguineus. A) Clustal alignment with other tick proteins. The asterisk (*) indicates identical amino acids. B) Phylogram of the alignment. The branches shown in red have bootstrap support above 75%. The bar at the centre indicates 20% amino acid divergence. The R. sanguineus sequences are indicated by a circle, and start with RS-. The sequences obtained from the NR database are indicated by 5 letters (3 from the genus and 2 from the species name) followed by the NCBI accession number. Remaining sequences were deduced from dbESTand are available from Francischetti et al. [8].

Figure 4

The immunosuppressive protein related to other tick species. The R. sanguineus sequence is indicated by a circle, and start with RS-. The remaining sequences were derived from the NR database and are indicated by 5 letter followed by the NCBI accession number. The protein sequences were aligned using the Clustal program and the dendrogram was created using the Mega package after 10,000 bootstrap replicates using the neighbour joining (NJ) algorithm. The bar at the bottom represents 20% of amino acid substitution. Remaining sequences were deduced from dbEST and are available from Francischetti et al. [8].

Figure 5

The salivary defensins of Rhipicephalus sanguineus were aligned using ClustalW. The symbols at the top represent identity (*), conserved (:) and weakly conserved (.) amino acids. The bar below the alignment indicates the region where the Arg-Gly-Asp (RGD) triplet flanked by cysteines is found on RS-609.

Figure 6

Alignment of selected members of the 8.9 kDa family of hard ticks. The asterisk (*) shows the 6 conserved cysteines. The sequences obtained from the NR database are indicated by 5 letters (3 from the genus and 2 from the species name) followed by the NCBI accession number. Remaining sequences were deduced from dbEST and are available from Francischetti et al. [8].

Figure 7

Alignment of the one-of-each family of metastriate proteins. Blocks of identical amino acids are shown in yellow background; conserved cysteines are shown in black background; other conserved amino acids are shown in blue background. Sequences from R. s sanguineus start with RS-. Other sequences were obtained from Francischetti et al. [8].