丽鱼三代虫感染对尼罗罗非鱼补体系统的激活途径研究

China is the largest tilapia breeding and exporting country in the world. During the cultivation process of Nile tilapia (Oreochromis niloticus), the monogenean Gyrodactylus cichlidarum can cause heavy mortalities of juvenile tilapia. While the study on the immune mechanism of tilapia against Gyrodactylus infection is still scarce. Our transcriptome analysis of Oreochromis niloticus infected with the Gyrodactylus cichlidarum shows that the complement system (important component of fish innate immune system) plays an important role in anti-Gyrodactylus cichlidarum infection. Other in vitro studies have proved that fish complements have lethal effect on Gyrodactylus, but it is still not clear which pathway take major responsibility for the activation of fish complement system after Gyrodactylus infection. This project according to the genetic information of complement factor B (OnBf) and complement C4 (OnC4) that we have identified in Oreochromis niloticus, proposed through the establishment of tilapia oral expression/RNAi system mediated by liposome, study the function of OnBf during the process of Gyrodactylus cichlidarum infection, interference OnBf and OnC4 in vivo to inhibit complement alternative pathway and classical pathway respectively, then combining the laboratory infection experiment and in vitro experiment, to determine the activation pathway of tilapia complement system after Gyrodactylus cichlidarum infection. The results of this study will help to deeply analyze the immune defense mechanism of tilapia against Gyrodactylus cichlidarum infection, and provide a theoretical basis for the prevention and control of Gyrodactyliasis.

我国是全球最大的罗非鱼养殖与出口国，在尼罗罗非鱼养殖过程中丽鱼三代虫感染造成了幼鱼大量死亡，目前关于罗非鱼对三代虫免疫防御机制的研究还很缺乏。补体是鱼类先天性免疫系统的重要部分，本团队通过转录组分析发现，罗非鱼补体参与了三代虫感染引起的免疫应答。也有研究通过体外实验证明鱼类补体对三代虫具有杀伤作用。然而，在三代虫感染后，鱼类补体系统通过哪种途径被激活目前尚无定论。本项目拟根据我们在尼罗罗非鱼中鉴定获得的补体B因子（OnBf）与补体C4（OnC4）基因信息，通过建立脂质体介导的罗非鱼口服过表达/RNAi模型，研究OnBf在罗非鱼抗三代虫感染过程中的作用，在体内干扰补体OnBf与OnC4来分别抑制补体替代途径与经典途径，再结合实验室三代虫感染实验与体外实验，以确定丽鱼三代虫感染对尼罗罗非鱼补体系统的激活途径。本研究将有助于深入解析罗非鱼对三代虫感染的免疫机制，为鱼类三代虫病害防控提供理论依据。

三代虫主要寄生在鱼的体表，给养殖鱼类带来了持续危害。已有研究表明补体在鱼类对抗三代虫过程中发挥重要作用。本研究主要通过一系列在鱼类血清与体表粘液中的杀虫实验，揭示了尼罗罗非鱼补体杀灭丽鱼三代虫的作用与机制。罗非鱼血清和体表粘液对丽鱼三代虫具有很强的杀灭作用，并且这种杀灭作用在热灭活补体或者抗体中和C3之后被显著抑制。扫描电镜结果显示，罗非鱼血清中的丽鱼三代虫表皮出现了严重的损伤。加入酵母聚糖抑制补体替代途径之后，罗非鱼血清对于丽鱼三代虫的杀灭作用显著被抑制，而且补体经典途径的抑制（加入EGTA或者L赖氨酸）对于杀虫作用也有微弱的抑制。这些结果证明，罗非鱼补体对于丽鱼三代虫具有杀灭作用，并且这种杀灭作用主要通过补体替代途径激活，同时经典途径也有参与。除此之外，我们意外的发现草鱼（非宿主）血清中的补体对于丽鱼三代虫的杀灭作用显著弱于罗非鱼（宿主）血清中的补体，这可能与单殖吸虫宿主特异性的识别机制有关。以上结果有助于揭示鱼类对三代虫的免疫防御机制，为鱼类三代虫病的防治提供新的思路与理论基础。