Primary Study On The Immune Responses Of Portunus Trituberculatus Against The Hematodinium Parasite

The Chinese swimming crab, Portunus trituberculatus is an important commercial aquaculture crab species in China. In recent years, the aquaculture of the P. trituberculatus was seriously damaged by the epidemic disease “milk disease” caused by the parasitic dinoflagellate Hematodinium and the sustainable development of the crab aquaculture industry is under serious threat. The parasitic dinoflagellate Hematodinium could parasitize and infect many important economically crustacean species around the world with an expansion of the scope of its occurrence and the number of the host species, which affected not only the wild population of commercial valuable crustacean species, but also the sustainable aquaculture of major crustacean species. To date, most studies were focused on the species identification, detection technology and epidemiology of Hematodinium, while the basic research on the host-pathogen interaction between Hematodinium and its crustacean hosts is still very scarce. Thus, this dissertation systematically explored the interaction between the Hematodinium and its crustacean host P. trituberculatus from the perspective of the host immune responses so as to enrich the basic research of crustacean immune mechanism and provide theoretical basis for the prevention and control of the diseases caused by the pathogen of parasitic dinoflagellates in crab aquaculture. The major findings are listed as following:1. A novel Toll gene was firstly isolated and characterized from P. trituberculatus, with the full-length cDNA of 3745 bp and a 3012 bp open reading frame(ORF) encoding 1003 amino acid(aa). The amino acid sequence of P. trituberculatus Toll shared high similarity to Scylla paramamosain and Scylla serrata Tolls(88%), and was clustered with the counterparts of crustaceans in the phylogenetic tree. The Toll transcripts were extensively expressed in various tissues of P. trituberculatus, with the highest expression in hemocytes. During the early period(within 24 h) of the Hematodinium challenge, the Toll transcripts were inhibited significantly in hemocytes, gill, heart, and muscles of P. trituberculatus, suggesting that the parasite might suggest a transient inhibition of the Toll-mediated immune response in the crab host, which were potentially associated with the survival and parasitism of parasites in crustacean hosts. Then, the Toll transcripts were significantly up-regulated in hemocytes and heart at 48 h, and in hepatopancreas at 48 and 96 h post the parasitic challenge, which suggested an important role of Toll in crustacean immune defense against the parasites. Finally, the Toll transcripts were decreased significantly in hemocytes, hepatopancreas and muscles by 192 h post Hematodinium challenge, implying that the parasite might gradually destroy the defense function of the Toll-mediated signaling immune pathways during the host immune responses in late infection.2. The Hematodinium intrusion significantly influenced the host proPO system and could induce the increase of the proPO transcripts as well as the PO activity, which suggested the crustacean proPO system played an important role in the immune responses to the parasitic infection. Besides, the suppressed transcripts of the LGBP and PPAF genes by the Hematodinium challenge might imply an immunosuppressive effect to inhibit the function of hosts’ proPO system at the transcriptional level. In addition, the Hematodinium parasite could significantly decrease the number of hemocytes(THCs and DHCs) and result in obvious pathological alterations in hepatopancreas at 16 d and 24 d post infection.3. After the Hematodinium challenge, significant transcriptional changes of three important clip-SPs genes(PTcSP1-3) and two key proteinase inhibitors(a2m, serpin) was observed in P. trituberculatus, which might reflect that the serine proteinase cascade reactions were disturbed severely and potentially played vital roles in the crustacean immune responses to the Hematodinium parasite. Besides, distinct expression profiles of PTcSP1-3 transcripts suggested their diverse functions in P. trituberculatus immune responses to the parasitic challenge. In addition, combined with results of the changes of the proPO transcripts and PO activities, PTcSP1 and PTcSP3 were suggested to be important members of the P. trituberculatus proPO system and involved in regulating the activation of proPO system.4. Three novel genes(NOS, NOX and GPx) were firstly isolated from P. trituberculatus. Thereinto, the full-length cDNA of NOS was 4002 bp with a 3612 bp ORF encoding 1203 aa. The amino acid sequence of P. trituberculatus NOS shared high similarity to the counterparts of crustaceans(>79%) and was clustered with the crustacean NOS members in the phylogenetic tree. And the results of tissue distributions showed that all of the three novel genes showed high mRNA transcripts in hemocytes and hepatopancreas of P. trituberculatus. After challenged with the Hematodinium parasite(0-192 h), significant changes of the transcripts of the critical NO/O2·-- generating/scavenging related genes(NOS, NOX, CuZnSOD, CAT, GPx) as well as the corresponding enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in hemocytes and hepatopancreas of P. trituberculatus, which suggested that the NO/O2·-- generating and the antioxidant systems played vital roles in the crustacean innate immune responses to the parasitic intrusion. The NO/O2·-- generating system was likely to participate in supplementing the crustacean immune responses against Hematodinium. Meanwhile, the antioxidant system was importantly involved in regulating the levels of ROS and benefiting the host self-protection from oxidative damages during immune responses to the parasite.In conclusion, this dissertation shed the first and preliminary light on the host-parasite interaction between crustacean host P. trituberculatus and the parasitic dinoflagellate Hematodinium from the perspective of the hosts’ immune responses. The major finding of this dissertation will contribute to better understanding crustacean innate immune mechanisms and provide theoretical basis for the prevention and control of the diseases caused by the pathogen of parasitic dinoflagellates in aquaculture of the economic crustaceans.