De novo assembled salivary gland transcriptome and expression pattern analyses for Rhipicephalus evertsi evertsi Neuman, 1897 male and female ticks

Abstract

Ticks secrete proteins in their saliva that change over the course of feeding to modulate the host inflammation, immune responses, haemostasis or may cause paralysis. RNA next generation sequencing technologies can reveal the complex dynamics of tick salivary glands as generated from various tick life stages and/or males and females. The current study represents 15,115 Illumina sequenced contigs of the salivary gland transcriptome from male and female Rhipicephalus evertsi evertsi ticks of early, mid and late feeding stages from 1320 separate assemblies using three short read assemblers. The housekeeping functional class contributed to the majority of the composition of the transcriptome (80%) but with lower expression (51%), while the secretory protein functional class represented only 14% of the transcriptome but 46% of the total coverage. Six percent had an unknown status contributing 3% of the overall expression in the salivary glands. Platelet aggregation inhibitors, blood clotting inhibitors and immune-modulators orthologous to the ancestral tick lineages were confirmed in the transcriptome and their differential expression during feeding in both genders observed. This transcriptome contributes data of importance to salivary gland biology and blood feeding physiology of non-model organisms.

Figure 1

Comparison of BUSCO values of Rhipicephalus evertsi evertsi and transcriptomes published from other Rhipicephalus species. Indicated in parenthesis is the number of transcripts from each transcriptome submitted to BUSCO analysis. The percentage of single, duplicated, fragmented and missing genes are presented as a percentage fraction contributing to the whole transcriptome. The number of ORFs analysed are indicated in parenthesis.

Figure 2

Comparison of the Rhipicephalus evertsi evertsi (Reve) transcriptome with other publically available Rhipicephalus transcriptomes that include Rhipicephalus appendiculatus (Rapp), Rhipicephalus microplus (Rmic), Rhipicephalus pulchellus (Rpul), Rhipicephalus sanguineus (Rsan) and Rhipicephalus zambeziensis (Rzam). (A) A plot of the protein length in amino acid residues presented up to 2000 residues against the number of proteins. Proteins were binned in windows of 20 based on protein length. (B) Pairwise reciprocal best hit analysis for the publically available Rhipicephalus transcriptomes. (C) Venn diagram to show the number of shared orthologs found for the Rhipicephalus evertsi evertsi transcriptome based on reciprocal best hits. Similar diagrams can be found for the other publically available Rhipicephalus transcriptomes in the supplementary material. Numbers in parenthesis indicate total number of reciprocal best hits for each transcriptome and while those shared among transcriptomes are indicated in numbers.

Figure 3

Housekeeping proteins from Rhipicephalus evertsi evertsi that mapped to the five groups of known housekeeping pathways defined by Kegg orthology.

Figure 4

Expression analysis of the secretory (SEC) proteins in the Rhipicephalus evertsi evertsi transcriptome (n = 2040). The glycine-rich protein family (cement) accounted for 57% of the total expression within the class while contributing less than 10 percent of the transcripts in the class. Expression was measured by transcript per million (TPM).

Figure 5

Female and male housekeeping and secretory protein expression patterns per day expressed as percentage per day based on TPM ≥ 0.5 cut off for females and males. Protein families with a significant increase or decrease in expression are indicated with stars. Represented housekeeping proteins were: translation (TRSL), folding sorting and degradation (FSDE), transcription (TRSC), replication and repair (REPL), transport and catabolism (TRCA), signal transduction (STDU), carbohydrate metabolism (CMET), energy metabolism (EMET), amino acid metabolism (AMET), lipid metabolism (LMET), glycan biosynthesis and metabolism (GBME), nucleotide metabolism (NMET), signaling molecules and interaction (SIGN), cell motility (CMOT), digestive system (DIGS).

Figure 6

Illustrative effect of expression of the housekeeping (blue), secretory (orange) and unknown (grey) functional classes over time. Expression was measured as TPM ≥ 0.5 for both the male and female transcriptomes.

Figure 7

Principle component and subsequent expression analysis (volcano plot) of the male and female transcripts controlled for by day to identify differentially expressed genes over time in the male and female transcriptome. The scatter plot displays the largest variance between unfed male and female ticks (Reve Fem Day 0 and Reve Male Day 0) (31.29%) followed by the second largest variance between feeding time points of males (25.2%). The volcano plot represents the differentially expressed female and male transcripts over the feeding period as a fold change against absolute confidence. Differentially expressed transcripts were double filtered to have FDR value < 0.05 and a log2 fold change larger than 4 and smaller than − 4 (dots in blue). Those that are highly up- or downregulated are further to the right and left sides, while highly significant changes appear higher on the plot.

Figure 8

Differentially expressed secretory proteins in the male and female transcriptome with the highest significance as related to fold change over time compared to protein abundance as counts. Positive log2 fold changes are indicative of upregulation while negative log2 fold changes indicate downregulation.