The JAK-STAT Pathway Controls Plasmodium vivax Load in Early Stages of Anopheles aquasalis Infection

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.疟疾每年在全球范围内影响约3亿人群，巴西境内年受累病例达45万。在巴西沿海地区，主要的疟疾传播媒介为海滨按蚊（Anopheles aquasalis），而间日疟原虫（Plasmodium vivax）是美洲绝大多数疟疾病例的致病原。昆虫拥有强大的抗感染免疫系统，其免疫应答由Toll、IMD及JAK-STAT三条通路调控。本研究针对间日疟原虫感染后海滨按蚊JAK-STAT通路的免疫调控作用展开分析。研究对三个基因进行了功能鉴定：转录因子信号转导与转录激活因子（Signal Transducers and Activators of Transcription，STAT）、活化STAT蛋白抑制剂（Protein Inhibitors of Activated STAT，PIAS）以及一氧化氮合酶（Nitric Oxide Synthase，NOS）。实验结果显示，STAT与PIAS的表达水平在雄虫中高于雌虫，在虫卵及一龄幼虫中的表达量亦高于幼虫与蛹期个体。在受到间日疟原虫攻击后24小时与36小时（h），STAT与PIAS的RNA转录水平显著升高。NOS的转录水平在感染后36小时（hpi）上升，而该蛋白在感染后24小时即可在部分中肠上皮细胞中被检测到。使用特异性抗体进行的免疫细胞化学实验表明：在未感染的蚊虫体内，STAT与PIAS主要分布于脂肪体中；而在感染间日疟原虫的蚊虫体内，这两种蛋白则出现在其他身体组织中。通过RNA干扰（RNAi）敲低STAT基因的表达后，海滨按蚊中肠内的卵囊数量显著增加。本研究首次明确证实，特定免疫通路参与了巴西疟疾传播媒介海滨按蚊与间日疟原虫的互作过程，为未来开发疟疾防控策略划定了潜在靶点。