Table_2_Rhipicephalus bursa Sialotranscriptomic Response to Blood Feeding and Babesia ovis Infection: Identification of Candidate Protective Antigens.xlsx

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.

蜱虫（Ticks）是最为普遍的吸血节肢动物之一，可作为多种病原体的传播媒介与储存宿主。对蜱虫在吸血及病原体感染过程中的应答进行唾液转录组学（sialotranscriptomic）分析，可揭示蜱虫-宿主-病原体互作界面中的分子互作机制，为相关研究提供全新视角。本研究旨在鉴定并解析布氏扇头蜱（Rhipicephalus bursa）唾液腺（salivary gland, SG）中，在吸血以及绵羊巴贝斯虫（Babesia ovis）感染条件下发生差异表达的基因。本实验采用三组不同蜱虫样本的唾液腺进行RNA测序，分别为吸血感染组、吸血未感染组以及未吸血未感染组，用于基因表达特征分析与组间比较。最终从三组样本中分别获得7272条未吸血未感染蜱虫的表达序列标签（expressed sequence tags, ESTs）、13819条吸血未感染蜱虫的ESTs以及15292条吸血感染蜱虫的ESTs。本研究分别构建了针对吸血应答以及绵羊巴贝斯虫感染应答的差异表达转录本目录。研究选取了四个编码假定卵黄蛋白原-3（vitellogenin-3）、lachesin蛋白、甘氨酸富集蛋白以及分泌型胶样蛋白的基因，开展RNA干扰（RNA interference, RNAi）功能验证实验。实验结果显示，唾液腺中卵黄蛋白原-3、分泌型胶样蛋白以及lachesin的mRNA水平分别下调了92%、65%与51%。卵黄蛋白原-3的基因敲低会导致蜱虫死亡率升高，寄生后77%的蜱虫死亡。分泌型胶样蛋白的mRNA水平下调后，46%的蜱虫无法正常附着于宿主，且与对照组相比，吸血后的雌蜱体重显著降低。lachesin基因敲低则使布氏扇头蜱唾液腺中与绵羊巴贝斯虫感染相关的水平降低70%，同时蜱虫死亡率达70%。本研究结果加深了学界对蜱虫唾液腺基因在巴贝斯虫感染/增殖以及蜱虫吸血过程中作用的认知。此外，本研究验证了lachesin、卵黄蛋白原-3以及分泌型胶样蛋白可作为候选保护性抗原，用于开发新型蜱虫及蜱传疾病防控策略。