| Pacific oyster Crassostrea gigas is one of the most important aquaculture species worldwide, and one of the most representative species among the mollusc, Lophotrochozoa. Along with the enlargement of the cultivation scale, high mortalities of hatchery-reared, juvenile oysters, has long been threatening the healthy and sustainable development of the oyster aquciculture industry. In the present study, using molecular immunology methods, the immunocompetence of different developmental stages during the ontogeny of oyster larvae was examined, and the ontogenesis and development of hematopoietic system was explored based on the expression pattern of conserved hematopoietic transcription factors. These results will deepen the study on the ontogenesis and development of immune system in marine invertebrates, as well as improve the diseases research work on the aquaculture development. 1. The immunological capacity in the larvae of Paci?c oyster C. gigasIn the present study, the development of immune system and its response against bacteria challenge were investigated in order to characterize the repertoire of immunological capacity of Paci?c oyster C. gigas during the ontogenesis. The phagocytosis was ?rstly observed in the early D-veliger larvae(17 hpf), especially in their velum site, which indicated the appearance of functional hemocytes during early D-veliger larvae stage. The whole-mount immuno?uorescence assay of three pattern recognition receptors(integrin β-1, caspase-3 and C-type lectin 3) and one immune effector gene(IL17-5) was performed in blastula, early D-veliger and umbo larvae, suggested that velum and digestive gland were the potential sites of immune system in the larvae. The lowest activities of antioxidant enzymes(superoxide dismutase and catalase) and hydrolytic enzyme(lysozyme), as well as descended expression levels of 12 immune genes at the transition between embryogenesis and planktonic, indicated that the larvae at hatching(9 hpf) were in hypo-immunity. While the ascending activities of enzymes and expression levels of seven immune genes during the trochophore stage(15 hpf) suggested the initiation of immune system. The steadily increasing trend of all the 12 candidate genes at the early umbo larvae(120 h) hinted that the immune system was well developed at this stage. After bacterial challenge, some immune recognition(TLR4) and immune effector(IL17-5 and defh2) genes were activated in blastula stage(4 hpf), and other immune genes were up regulated in D-veliger larvae, indicating that the zygotic immune system could respond earlier against the bacterial challenge during its development. These results overall indicated that the cellular and humoral immune components appeared at trochophore stage, and the cellular immune system was activated with its occurrence, while the humoral immune system executed until the early umbo larval stage. The immune system emerged earlier to aid larvae in defending bacterial challenge during the early stages of oyster development. 2. The identification and functional analysis of Cg-SCLIn the present work, a conserved haematopoietic transcription factor Tal-1/Scl(Stem Cell Leukemia) was identi?ed in Paci?c oyster(Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. In the adult oyster Cg-SCL was highly expressed in the hemocytes as well as gill and mantle. Among the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage(10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage(15 hpf) before resuming in umbo larvae(120 hpf). Whole-mount immuno?uorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were signi?cantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. The hemocyte speci?c genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. These results demonstrated that Cg-SCL could be used as haematopoietic speci?c marker to trace embryonic hematopoiesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae. 3. The identification and functional analysis of Cg-RuntA conserved hematopoietic transcription factor gene Cg-Runt was cloned from oyster C. gigas, which contains conserved runt homology domain, belonging to the Runx family. Cg-Runt was highly expressed in the hemocytes, gill, and mantle at both mRNA and protein level. Among the larval developmental stages, the mRNA transcripts of Cg-Runt could be detected after fertilization and reached to peak expression level at early trochophore larvae stage(10 hpf), then exhibited decreased trend after entered into D-veliger stage. Whole-mount immuno?uorescence assay further revealed that the immunoreactivity of Cg-Runt expressed throughout the whole blastula larvae, and gathered into vegetal pole in the gastrula larvae. By trochophore, the expression of Cg-Runt could be detected as a ring structure around the dorsal region, and finally formed sinus structure in the digestive gland region in the early D-veliger larvae. At the later D-veliger, the immunoreactivity of Cg-Runt exhibited three-bifurcation structure throughout the whole larvae, and the expression pattern lasted into the umbo larvae, with stronger immunoreactivity. The results suggested that Cg-Runt involved into the larval hematopoiesis. Besides, it maybe functions in the cell differentiation as well as histogenesis during the embryo period and later larval stages. 4. The identification and functional analysis of Cg-GATA-2/3A conserved hematopoietic transcription factor Cg-GATA-2/3 was cloned from the oyster C. gigas. Cg-GATA-2/3 contains conserved two tandem repeat zinc finger GATA domains, belonging to the GATA-123 family. Cg-GATA-2/3 was highly expressed in the hemocytes, gill, and mantle both at the mRNA and protein level. Whole-mount immuno?uorescence assay further revealed that the immunoreactivity of Cg-GATA-2/3 expressed throughout the whole blastula and gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae, which was similar with Cg-SCL. The distribution Cg-GATA-2/3 was also specialized into velum region with development into umbo larvae. When Cg-GATA-2/3 was interfered by dsRNA, the hemocyte speci?c gene Ec-SOD and haematopoietic transcription factor C-Myb were down-regulated, and the number of newborn cells was remarkably decreased both in the circulating hemocytes and gill tissue. These results suggested that Cg-GATA-2/3 played critical role in the production of hemocytes and the two waves of hematopoiesis in the oyster. In the embryonic hematopoiesis, it was speculated that Cg-GATA-2/3 indirectly functioned through the regulation of Cg-SCL, and directly functioned in the larval hematopoiesis together with Cg-SCL and Cg-Runt.In conclusion, during the early ontogenesis of larvae oyster, the maternal transferred immunity provides immune protection for the embryo of oyster, and the embryonic hematopoiesis occurred as early as the blastula stage. The occurrence of immune system started at the trochophore stage, followed by the larval hematopoiesis. Along with the development of the oyster larvae, the hemocytes in D-veliger larvae become functional and show signs of phagocytosis. The immune system should be well developed by the umbo larval stage. |
