Sexual differences in the sialomes of the zebra tick, Rhipicephalus pulchellus

Abstract

Ticks rely exclusively on vertebrate blood for their survival. During feeding ticks inject into their hosts a sophisticated salivary potion that overcomes host hemostasis and adverse inflammatory responses. These mediators may also enhance pathogen transmission. Knowledge of the tick salivary protein repertoire may lead to vaccine targets to disrupt feeding and/or parasite transmission as well as to the discovery of novel pharmacological agents. Male saliva may also assist reproduction because males use their mouthparts to lubricate and introduce their spermatophores into the females' genital pore. The analyses of the sialomes of male and female ticks independently allow us to understand the strategy used by each gender to feed successfully. We sequenced cDNA libraries from pools of salivary glands from adult male and female Rhipicephalus pulchellus feeding at different time points, using the Illumina HiSeq protocol. De novo assembly of a total of 241,229,128 paired-end reads lead to extraction of 50,460 coding sequences (CDS), 11,277 of which had more than 75% coverage to known transcripts, or represented novel sequences, and were submitted to GenBank. Additionally, we generated the proteome, from the salivary gland extracts of male and female R. pulchellus, yielding a total of 454 and 2063 proteins respectively which were identified by one or more peptides with at least 95% confidence. The data set is presented as an annotated hyperlinked Excel spreadsheet, describing 121 putative secreted protein families. Female and male specific transcripts were identified.

Biological significance: This annotated R. pulchellus database represents a mining field for future experiments involving the resolution of time-dependent transcript expression in this tick species, as well as to define novel vaccine targets and discover novel pharmaceuticals. Gender specific proteins may represent different repertoires of pharmacological reagents to assist feeding by each sex, and in males may represent proteins that assist reproduction similarly to seminal proteins in other animals.

Keywords: Salivary glands; Tick; Transcriptome.

Figure 1. Anticoagulant activity of R. pulchellus salivary gland extracts

(A) The amidolytic activities of thrombin and FXa were measured in the presence of 15 ug of female or male crude salivary gland extracts. Female extracts were able to inhibit both thrombin and FXa to higher than 80% while male extracts inhibited the enzymes lower than 20%. (B) Crude salivary gland extracts from female ticks were subjected to fractionation by size exclusion chromatography. (C) Crude salivary gland extracts from male ticks were subjected to fractionation by size exclusion chromatography. Fractions from (B) and (C) were tested for their ability to inhibit the amidolytic activity of FXa and thrombin. Female extracts inhibited FXa (FXaI-1 and FXaI-2) and thrombin (TI-1) while male extracts only inhibited FXa to a small extent (FXaI-2).

Figure 2. Components of R. pulchellus transcriptome and proteome

Both transcriptome (A) and proteome (B) were classified into four categories – housekeeping, secreted, transposable elements and unknown. Secretory products are shown in more details, with further classification. The charts represent proportion of CDS and proteins in the transcriptome and proteome respectively.

Figure 3. Phylogenetics of the salivary metalloproteases of ticks

The evolutionary history was inferred using the Neighbor-Joining method [134]. The optimal tree with the sum of branch length = 75.51 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [135] and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). The analysis involved 170 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 823 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [25]. The bar at the top indicates 50% amino acid divergence. Sequences from GenBank are recognized by having the first 3 letters of the genus name, followed by the first 3 letters of the species name, followed by their accession numbers. Rhipicephalus pulchellus sequences start with RP. Other sequences were derived from a tick sialomes review [2]. Markers of the same color are used for genus, and marker shape for species differentiation.

Figure 4. Novel metastriate tick salivary family

(A) Clustal alignment. The symbols above the alignment indicates for * identity of residues, “:” similarity and “.” indicates less similarity. (B) Phylogenetic evolutionary history was inferred using the Neighbor-Joining method [134].The bootstrap consensus tree inferred from 10000. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates). All ambiguous positions were removed for each sequence pair. There were a total of 360 positions in the final dataset. The bar at the bottom indicates 50% amino acid divergence. Other conditions as in figure 1. The sequences were obtained from previous publications [2, 19]. The R. pulchellus sequences start with Rp and can be obtained from supplemental file 1.

Figure 5. Differential expression of secretory proteins

(A) The number of reads from male and female R. pulchellus belonging to the secretory class in increasing order. (B) Transcripts which are expressed more than 10 times in either sex, together with their classification. (C) Proteins which were found only in one sex in the proteome.

Figure 6. Five subclasses of bilaris proteins

(A) Schematic representation of the five subclasses of bilaris proteins. Subclass I has two full tandem Kunitz domains, each three conserved disulphide bonds, represented by the solid lines. A typical Kunitz domain disulphide bond pairing is as follows: C1-C6, C2-C4 and C3-C5. Subclass II and III has two missing cysteine residues C2, C4 and C3, C5, respectively, in the second Kunitz domain, resulting in a loss of a disulphide bond. Missing disulphide bonds are represented by the bold dashed line. Subclass IV and V has two extra cysteine residues in the second Kunitz domain, one located between the typical C4 and C5 cysteine residues, and the other after the C6 cysteine residue. This extra disulphide bond is represented by a bold solid line. However, subclass V contains a long inter-domain, proline-rich segment (~ 80 residues and represented by the bold dotted line. (B) Alignment of consensus sequences of the five bilaris subclasses showing conserved disulphide bridges.

Figure 7. Phylogenetics and associated number of reads of bilaris CDS

81 CDS belonging to the bilaris class were aligned with ClustalW and a phylogenetic tree was drawn using the Neighbor-Joining method in MEGA5 [25]. The subclass of each CDS is indicated by the symbol on the left of the accession number. The number of reads associated with each CDS is indicated by the bar chart, where red represents reads from the female library, and blue from the male library. Proteins that were identified in the female proteome are marked with a ♀ beside the bar chart.

Figure 8. Expression difference of bilaris proteins between male and female ticks

(A) Fold difference in the transcriptome is expressed over number of male reads. Seven transcripts were highly expressed in females while one was highly expressed in males. These sex biased transcripts which were more than 10 fold expressed in either sex are highlighted in grey. The subclass of the bilaris proteins are indicated beside the transcript name. It is noted that most of subclass I, III and IV were expressed by males, and subclass II and V in females, as tabulated in the inset. Transcripts marked with an asterisk (*) were used for RT-PCR studies. (B) RT-PCR was performed on selected bilaris proteins from subclass II, III and IV. Comparison of expression is in reference to females. Two independent biological samples were used to obtain the standard deviation.

Figure 8. Expression difference of bilaris proteins between male and female ticks

(A) Fold difference in the transcriptome is expressed over number of male reads. Seven transcripts were highly expressed in females while one was highly expressed in males. These sex biased transcripts which were more than 10 fold expressed in either sex are highlighted in grey. The subclass of the bilaris proteins are indicated beside the transcript name. It is noted that most of subclass I, III and IV were expressed by males, and subclass II and V in females, as tabulated in the inset. Transcripts marked with an asterisk (*) were used for RT-PCR studies. (B) RT-PCR was performed on selected bilaris proteins from subclass II, III and IV. Comparison of expression is in reference to females. Two independent biological samples were used to obtain the standard deviation.