Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2−- generating and antioxidant systems in host immune responses to Hematodinium

• A full-length cDNA of NOS gene and the partial cDNAs of NOX, GPx genes was acquired from Portunus trituberculatus.
• Extensive distributions of NOS, NOX and GPx genes were observed in the major tissues of P. trituberculatus.
• The NO/O2·−- generating system participated in hosts' immune responses against the Hematodinium parasite.
• The antioxidant system was important to alleviate oxidative damages during hosts' immune responses against the parasite.

Increasing evidences have established that the nitric oxide synthase (NOS) and NADPH oxidase (NOX) play important roles in host defense system by catalyzing the production of nitrogen oxide (NO) and superoxide anions (O2−), respectively. While, there are limited studies to explore the roles of NOS/NOX enzymes in crustacean immunity, and no studies as yet were attempted to elucidate their functions in host immune responses to parasites. In the present study, we cloned a full-length cDNA of NOS and two partial cDNA fragments of NOX and GPx from the economic valuable crab Portunus trituberculatus. The full-length cDNA of NOS was 4002 bp in length that encoded 1203 amino acids containing motifs of the NOS protein and conserved domains. The phylogenetic analysis showed that the NOS protein sequence was clustered together with those of crustacean species in the phylogenetic tree. All of the three novel genes showed high mRNA transcripts in the immune-related tissues (e.g. hemocytes, hepatopancreas) of P. trituberculatus. Striking fluctuation in the transcripts of the critical NO/O2−- generating/scavenging related genes (NOS, NOX, CuZnSOD, CAT, GPx) as well as in the enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in the hemocytes and hepatopancreas of P. trituberculatus post challenged with the parasitic dinoflagellate Hematodinium, indicating that the NO/O2−- generating and the antioxidant systems played vital roles in the crustacean innate immunity against the parasitic intrusion. The results indicated a novel respect of the host-parasite interaction between the crab host and the parasitic dinoflagellate Hematodinium.