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.

A Systems Level Analysis Reveals Transcriptomic and Proteomic Complexity in Ixodes Ricinus Midgut and Salivary Glands During Early Attachment and Feeding

“…Library Construction-Four samples mentioned above for 0 -12 h and 12-24 h from nymphal salivary glands (SG) 1 and midguts (MG), and six samples for 0 -12h, 12-24h and 24 -36 h for adult SG and MG were used to make Illumina Trueseq libraries which were sequenced using a Illumina Hiseq 2000 machine and the raw data deposited at the Sequence Read Archives (SRA) of the National Center for Biotechnology Information (NCBI) under accessions SRR641305, SRR641306, SRR641307, SRR641308, SRR641309, SRR641327, SRR641328, SRR641329, SRR641330, and SRR641331.…”

“…These 25,808 CDS supported the subsequent quantitative proteome analysis. 1 The abbreviations used are: SG, salivary glands; A, adult ticks; CDS, coding sequences; cs, cytoskeletal; det, detoxification; extmat, extracellular matrix and adhesion; Gb, giga base pairs; IEP, isoelectric point; imm, immunity related; met, metabolism; MG, midgut; N, nymphal ticks; ne, nuclear export; nr, nuclear regulation; ORF, open reading frames; pe, protein export machinery; pm, protein modification machinery; pr, proteasome machinery; ps, protein synthesis machinery; s, secreted proteins; SRA, Sequence Read Archives; st, signal transduction; tf, transcription factors; tm, transcription machinery; tr, transporters; uc/uk, unknowns; UPLC, ultra performance liquid chromatography.…”

“…In order to address this limitation, we herein extensively sequenced different transcriptome libraries to generate a sequence database which supported the application of a recently developed label-free data-independent acquisition based strategy (10) to study the proteome dynamics in our tissue collections. As a result of the study of the transcriptome and proteome dynamics simultaneously in the same tissue collections, we herein describe a systems-level view of gene expression in I. ricinus midgut and salivary glands over the first 24 h of tick attachment (for nymphs and adults) and up to 36 h for adults, because it is known that adult I. ricinus ticks feed slower than nymphal ticks (1). In doing so, we report the most comprehensive proteome coverage of this important class of pathogen vectors to date and begin to define the dynamic responses of these physiological compartments during the tick lifecycle.…”

De novo assembly and annotation of the salivary gland transcriptome of Rhipicephalus appendiculatus male and female ticks during blood feeding

“…Next generation sequencing, which produces millions of sequencing reads, can achieve the sequence depth required to elucidate even lowly expressed genes of complex protein families. To exploit these advances, a number of de novo sialotranscriptomes (sets of RNA molecules in tick salivary glands) have recently been generated using NGS technologies for a number of tick species; A. maculatum (Karim et al, 2011), Ixodes ricinus (Schwarz et al, 2013), Dermacentor andersoni (Mudenda et al, 2014), A. triste, A. parvum and A. cajennense (Garcia et al, 2014), Haemaphysalis flava (Xu et al, 2015) and R. pulchellus (Tan et al, 2015a). These transcriptomes highlighted the true expansion within salivary protein families in the different tick species and identified new candidate genes involved in feeding.…”

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…With the advent of next-generation sequencing (NGS) technologies, tick salivary gland transcriptomes have been described [23,[30][31][32][33][34][35][36][37][38][39]. However, the major limitation to these data is that it does not inform on which transcripts that encode for proteins are secreted in tick saliva.…”