Rhipicephalus bursa Sialotranscriptomic Response to Blood Feeding and Babesia ovis Infection: Identification of Candidate Protective Antigens

Abstract

Ticks are among the most prevalent blood-feeding arthropods, and they act as vectors and reservoirs for numerous pathogens. Sialotranscriptomic characterizations of tick responses to blood feeding and pathogen infections can offer new insights into the molecular interplay occurring at the tick-host-pathogen interface. In the present study, we aimed to identify and characterize Rhipicephalus bursa salivary gland (SG) genes that were differentially expressed in response to blood feeding and Babesia ovis infection. Our experimental approach consisted of RNA sequencing of SG from three different tick samples, fed-infected, fed-uninfected, and unfed-uninfected, for characterization and inter-comparison. Overall, 7,272 expressed sequence tags (ESTs) were constructed from unfed-uninfected, 13,819 ESTs from fed-uninfected, and 15,292 ESTs from fed-infected ticks. Two catalogs of transcripts that were differentially expressed in response to blood feeding and B. ovis infection were produced. Four genes coding for a putative vitellogenin-3, lachesin, a glycine rich protein, and a secreted cement protein were selected for RNA interference functional studies. A reduction of 92, 65, and 51% was observed in vitellogenin-3, secreted cement, and lachesin mRNA levels in SG, respectively. The vitellogenin-3 knockdown led to increased tick mortality, with 77% of ticks dying post-infestation. The reduction of the secreted cement protein-mRNA levels resulted in 46% of ticks being incapable of correctly attaching to the host and significantly lower female weights post-feeding in comparison to the control group. The lachesin knockdown resulted in a 70% reduction of the levels associated with B. ovis infection in R. bursa SG and 70% mortality. These results improved our understanding of the role of tick SG genes in Babesia infection/proliferation and tick feeding. Moreover, lachesin, vitellogenin-3, and secreted cement proteins were validated as candidate protective antigens for the development of novel tick and tick-borne disease control measures.

Keywords: Babesia spp.; RNA interference; Rhipicephalus bursa; sialotranscriptomics; vaccine; vector-pathogen interactions.

Figure 1

Radar plots of the three transcriptomes per represented molecular functions (A) and biological processes (B). The lines represent a pattern of the three transcriptomes unfed-uninfected, fed-uninfected and fed-Babesia ovis infected, allowing a visual comparison between conditions.

Figure 2

Rhipicephalus bursa SG transcriptional response to blood meal based on Gene Ontology functional classes assignments of encoded proteins. Yellow bars represent down regulated genes, orange bars represent up regulated genes with statistical significance (P < 0.05).

Figure 3

Rhipicephalus bursa SG transcriptional response to Babesia ovis infection based on Gene Ontology functional classes assignments of encoded proteins. Gray bars represent down regulated genes and blue bars represent up regulated genes with statistical significance (P < 0.05).

Figure 4

Differentially gene expression of Rhipicephalus bursa SG in response to blood feeding evaluated by qPCR. Red bars represent SG from fed R. bursa ticks and green bars represent the SG from unfed R. bursa ticks. ^*P < 0.05.

Figure 5

Differentially gene expression of Rhipicephalus bursa SG in response to Babesia ovis infection evaluation by qPCR. Red bars represent the B. ovis infected SG and green bars represent the SG from uninfected R. bursa ticks. ^*P < 0.05.

Figure 6

Proposed model of putative vitellogenin-3, cement protein and lachesin functions and its impact on Rhipicephalus bursa SG during feeding and Babesia ovis infection. (A) Vitellogenin-3 described function relates to heme detoxification and lipid storage contributing for cell survival. A decrease of the expression of putative vitellogenin-3 leads to deficient heme seizure, increasing the formation of reactive oxygen species (ROS) as well as cellular toxicity. Lipid storage is also compromised leading to an unbalance in the production of energy. (B) Putative cement protein is a component of the cement cone, which facilitates the tick attachment and feed on the host. An impact in the production of cement proteins leads to an incapacity of ticks to correctly attach and subsequently feed on the host, resulting in tick death and reduced blood ingestion. (C) Lachesin is a cell surface protein that as a potential role in cell adhesion, maintaining apical-basal polarity, vesicle trafficking, cell growth and survival, as well as parasite invasion. A negative manipulation of the expression of lachesin results in an abnormal cell growth and ultimately cell apoptosis, and also a decrease of Babesia spp. infection.