Table_3_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.XLSX

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.

蜱虫的唾液腺（SG）在吸血过程中扮演着至关重要的角色，它们不仅产生黏合剂，还分泌唾液。黏合剂是一种具有粘附性的物质，有助于蜱虫附着于宿主皮肤；而唾液则含有抗凝血、抗炎和免疫调节物质的丰富混合物，这些物质使得蜱虫能够顺利获取血液。蜱虫的唾液还充当了多种病原体传播至脊椎动物宿主的载体，其中包括来自立克次体属（Rickettsia）的各种细菌。立克次体属的立克次体（Rickettsia rickettsii）是一种蜱传的严格细胞内寄生的细菌，可导致严重的落基山斑点热。在巴西，狗黄蜱（Amblyomma aureolatum）是立克次体属立克次体的传播媒介。在当前研究中，通过下一代RNA测序技术，确定了R. rickettsii实验感染对A. aureolatum唾液腺的全局基因表达谱的影响。感染过程中共调节了260个编码序列（CDS），其中161个上调，99个下调。在免疫类别的CDS中，我们强调了一种编码一个微牛素样抗菌肽（AMP）（Ambaur-69859）的序列。AMPs是节肢动物免疫系统的重要效应分子，它们缺乏脊椎动物免疫系统中的适应性反应。通过逆转录和定量聚合酶链反应证实，微牛素的表达在感染A. aureolatum的唾液腺以及中肠（MG）中显著上调。通过RNA干扰敲低微牛素的表达，导致感染蜱虫的流行率显著增加。此外，在接收到微牛素特异性双链RNA的蜱虫中肠和唾液腺中记录到了一个数量级更高的立克次体负荷。观察不到微牛素敲低对立克次体属立克次体传播至兔子的影响。此外，在接收到双链微牛素的蜱虫中，吸血和产卵行为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。为进一步确定该抗菌肽对立克次体属立克次体的作用机制，必须进行进一步的研究。