Table_2_The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection.DOCX

The salivary glands (SG) of ixodid ticks play a pivotal role in blood feeding, producing both the cement and the saliva. The cement is an adhesive substance that helps the attachment of the tick to the host skin, while the saliva contains a rich mixture of antihemostatic, anti-inflammatory, and immunomodulatory substances that allow ticks to properly acquire the blood meal. The tick saliva is also a vehicle used by several pathogens to be transmitted to the vertebrate host, including various bacterial species from the genus Rickettsia. Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes the severe Rocky Mountain spotted fever. In Brazil, the dog yellow tick Amblyomma aureolatum is a vector of R. rickettsii. In the current study, the effects of an experimental infection with R. rickettsii on the global gene expression profile of A. aureolatum SG was determined by next-generation RNA sequencing. A total of 260 coding sequences (CDSs) were modulated by infection, among which 161 were upregulated and 99 were downregulated. Regarding CDSs in the immunity category, we highlight one sequence encoding one microplusin-like antimicrobial peptide (AMP) (Ambaur-69859). AMPs are important effectors of the arthropod immune system, which lack the adaptive response of the immune system of vertebrates. The expression of microplusin was confirmed to be significantly upregulated in the SG as well as in the midgut (MG) of infected A. aureolatum by a quantitative polymerase chain reaction preceded by reverse transcription. The knockdown of the microplusin expression by RNA interference caused a significant increase in the prevalence of infected ticks in relation to the control. In addition, a higher rickettsial load of one order of magnitude was recorded in both the MG and SG of ticks that received microplusin-specific dsRNA. No effect of microplusin knockdown was observed on the R. rickettsii transmission to rabbits. Moreover, no significant differences in tick engorgement and oviposition were recorded in ticks that received dsMicroplusin, demonstrating that microplusin knockdown has no effect on tick fitness. Further studies must be performed to determine the mechanism of action of this AMP against R. rickettsii.

Ixodid蜱虫的唾液腺（SG）在吸血过程中扮演着核心角色，分泌了凝固物质和唾液。凝固物质是一种粘附物质，有助于蜱虫附着于宿主皮肤，而唾液则富含抗凝血、抗炎和免疫调节物质，使得蜱虫能够有效地获取血液。蜱虫唾液亦成为多种病原体传播至脊椎宿主的载体，包括来自立克次氏菌属的多种细菌。立克次氏菌rickettsii是一种蜱虫传播的必需细胞内细菌，可导致严重的落基山斑点热。在巴西，犬黄蜱Amblyomma aureolatum是立克次氏菌rickettsii的传播媒介。在本次研究中，通过下一代RNA测序技术，确定了R. rickettsii实验感染对A. aureolatum SG的全基因表达谱的影响。共调节了260个编码序列（CDSs），其中161个上调，99个下调。针对免疫类别的CDSs，我们特别强调了一个编码微牛素样抗菌肽（AMP）的序列（Ambaur-69859）。AMPs是节肢动物免疫系统的重要效应因子，缺乏脊椎动物免疫系统的适应性反应。通过逆转录和定量聚合酶链反应，证实了微牛素在感染A. aureolatum的SG以及中肠（MG）中的表达显著上调。通过RNA干扰降低微牛素表达，导致感染蜱虫的流行率显著增加。此外，接受微牛素特异性dsRNA的蜱虫，其MG和SG中的立克次氏菌载量增加了十倍。微牛素敲低对R. rickettsii传播至兔子的效果没有观察到影响。此外，接受dsMicroplusin的蜱虫在吸血和产卵方面没有记录到显著差异，这表明微牛素敲低对蜱虫的适应性没有影响。为进一步确定此抗菌肽对R. rickettsii作用的机制，必须进行进一步的研究。