De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies

Abstract

Tick salivary gland (SG) proteins possess powerful pharmacologic properties that facilitate tick feeding and pathogen transmission. For the first time, SG transcriptomes of Ixodes ricinus, an important disease vector for humans and animals, were analyzed using next-generation sequencing. SGs were collected from different tick life stages fed on various animal species, including cofeeding of nymphs and adults on the same host. Four cDNA samples were sequenced, discriminating tick SG transcriptomes of early- and late-feeding nymphs or adults. In total, 441,381,454 pyrosequencing reads and 67,703,183 Illumina reads were assembled into 272,220 contigs, of which 34,560 extensively annotated coding sequences are disclosed; 8686 coding sequences were submitted to GenBank. Overall, 13% of contigs were classified as secreted proteins that showed significant differences in the transcript representation among the 4 SG samples, including high numbers of sample-specific transcripts. Detailed phylogenetic reconstructions of two relatively abundant SG-secreted protein families demonstrated how this study improves our understanding of the molecular evolution of hematophagy in arthropods. Our data significantly increase the available genomic information for I. ricinus and form a solid basis for future tick genome/transcriptome assemblies and the functional analysis of effectors that mediate the feeding physiology and parasite-vector interaction of I. ricinus.

Keywords: cDNA; high-throughput annotation; molecular evolution; public database; tick feeding; tick life stages.

Figure 1.

Functional classification of contig-encoded I. ricinus proteins. Pie chart represents the functional classification of all contig-encoded I. ricinus proteins from the combined assembly of the Illumina and 454 sequencing datasets of all 4 SG libraries (Supplemental File S1). The overall contig proportion of the different protein families (%) is shown, and all secreted SG proteins were further divided into different subfamilies.

Figure 2.

Phylogram of the basic tail family. Basic tail-encoding protein sequences of different hematophagous arthropods (defined by GenBank accession numbers) and of all 4 SG Illumina libraries that were significantly ≥10-fold more frequent in a certain SG library compared to a second SG library (EN vs. LN, LN vs. EN, EA vs. LA, LA vs. EA) were used for phylogenetic reconstruction by ML and bayesian methods. Abundance levels of each protein in a certain SG library are also shown. The presented ML tree was rooted with Aedes aegypti (GenBank: AAY41832), and only strongly supported nodes with bootstrap values ≥ 50% are given. Bayesian posterior probabilities ≥ 0.5 are also displayed for clade nodes that showed the same topology in the ML and bayesian trees. Scale bar (mean amino acid substitution/site) in the ML tree is at the bottom center of the figure.

Figure 3.

Phylogram of the Kunitz domain family. Kunitz domain-encoding proteins of hematophagous arthropods, of nematodes (defined by GenBank accession numbers), and of all 4 SG Illumina libraries that were significantly ≥10-fold more frequent in a certain SG library compared to a second SG library (EN vs. LN, LN vs. EN, EA vs. LA, LA vs. EA) were used for phylogenetic reconstruction by ML and bayesian analyses. Abundance levels of each protein from a certain SG library are displayed. The presented ML tree was rooted with Caenorhabditis elegans (GenBank: CAA98467), and only strongly supported nodes with bootstrap values ≥ 50% are shown. Bayesian posterior probabilities ≥ 0.5 are also given for clade nodes that had the same topology in the ML and bayesian phylograms. Scale bar (mean amino acid substitution/site) of the ML tree is at the bottom center of the figure.