Complement factor D (CfD), a member of serine peptidases superfamily, acts as the rate-limiting enzyme of alternative complement pathway (AP) and is important for the AP-initiated C3 activation and subsequent amplification of complement cascade. The functions of the CfD in antibacterial and antiviral have been demonstrated; however, much less is known about the anti-parasite effect of CfD in teleost fish. In this study, we firstly demonstrated that the complements from goldfish serum showed high lethal effect on Gyrodactylus kobayashii acting as the AP. The serum activity against G. kobayashii was abolished by addition of EDTA, but not by EGTA + Mg²⁺. Subsequently, we cloned and characterized the goldfish (Carassius auratus) CfD (CaCfD), whose open reading frame consists of 753 bp, encoding 250 amino acids for a 48 kDa polypeptide. Bioinformatics analysis results showed that CfD had only one serine protease trypsin domain, and the serine active sites were completely conserved in teleost fish during evolution. The ubiquitous expression of CaCfD in all tested tissues was displayed, with the highest expression in the liver. After G. kobayashii infection, the mRNA levels of CaCfD were significantly up-regulated in blood, liver, spleen and kidney. The hemolysis percentage of the rabbit red blood cells (RRBCs) was enhanced by serum from G. kobayashii-infected goldfish, indicating that more complement components in serum were stimulated. Besides, we confirmed that recombinant CaCfD protein ((r)CaCfD) significantly improved the RRBCs lysis mediated by the complements, probably the increase of CaCfD level in complement system. (r)CaCfD also showed high anti-parasitic effect against G. kobayashii in vitro. Taken together, this study enriches the function of CfD participating in host defense against fish parasites, and the role of the complements in the protection of fishes against gyrodactylids infection deserves further investigation.

补体因子 D (CfD) 是丝氨酸肽酶超家族的成员，作为替代补体途径 (AP) 的限速酶，对于 AP 启动的 C3 激活和随后的补体级联放大很重要。已经证明了 CfD 在抗菌和抗病毒方面的功能；然而，关于 CfD 对硬骨鱼的抗寄生虫作用知之甚少。在这项研究中，我们首先证明了金鱼血清中的补体对作为 AP 的Gyrodactylus kobayashii显示出高致死作用。加入 EDTA 可消除针对G. kobayashii的血清活性，但 EGTA + Mg ^{2+ 则}不会。随后，我们克隆并表征了金鱼（Carassius auratus) CfD (CaCfD)，其开放阅读框由 753 bp 组成，为 48 kDa 的多肽编码 250 个氨基酸。生物信息学分析结果表明，CfD只有一个丝氨酸蛋白酶胰蛋白酶结构域，并且丝氨酸活性位点在进化过程中在硬骨鱼中是完全保守的。显示了CaCfD在所有测试组织中的普遍表达，在肝脏中表达最高。之后G. kobayashii感染的mRNA水平CaCfD均显著血，肝，脾，肾上调。G. kobayashii血清可提高兔红细胞 (RRBC) 的溶血百分比- 感染金鱼，说明血清中更多的补体成分受到刺激。此外，我们证实重组CaCfD蛋白（（r）CaCfD）显着改善了补体介导的RRBC裂解，可能是补体系统中CaCfD水平的增加。(r) CaCfD在体外还对G. kobayashii显示出高抗寄生虫作用。总之，这项研究丰富了 CfD 参与宿主防御鱼类寄生虫的功能，补体在保护鱼类免受 gyrodactylids 感染中的作用值得进一步研究。