| Crustaceans in aquaculture occupy a very important position, and the knowledge of immune defense mechanisms as well as the development of novel immunostimulants is helpful for disease control in crustacean aquaculture. In the present study, the genes of LvIntegrin and LvLec from Litopenaeus vannamei, EsSOCS2 and EsALF-2 from Eriocheir sinensis were cloned by rapid amplification of cDNA ends (RACE) techniques. Their expression profiles in crustaceans challenged by several microorganisms were detected by RT-PCR technique, and their functions were examined by assaying their recombinant protein expressed in Escherichia coli BL21(DE3)-plysS. The immune protective effects of CpG ODNs for crustaceans against pathogenic microorganisms were also evaluated.The full-length cDNA of LvIntegrin and LvLec was 2621 and 618 bp, with typical domain ofβ-integrin subunit and CRD respectively. The mRNA expression of LvIntegrin was up-regulated in haemocytes of shrimps challenged by Listonella anguillarum. Recombinant LvIntegrin displayed agglutinative activities against E. coli, L. anguillarum, Micrococcus lysodeikticus and Pichia pastoris and promoted the proliferation of human fibroblasts. Recombinant LvLec represented agglutinative activity only aginst E. coli JM109. The agglutinative activities of LvIntegrin and LvLec were both of Ca2+ dependent. The mortality rates were significantly increased in LvIntegrin-RNAi-shrimps treated with L. anguillarum, and the EsproPO gene was inhibited in LvIntegrin-blocking-shrimps challenged by L. anguillarum.The full-length cDNA of EsSOCS2 and EsALF-2 was 2535 and 724 bp respectively, and EsSOCS2 contained three typical domains of ESS, SH2 and SOCS box. The mRNA expression of EsSOCS2 and EsALF-2 was both up-regulated in crabs challenged by the microorganisms. The recombinant EsALF-2 displayed antibacterial activity against L. anguillarum and P. pastoris, but no antibacterial activity against Gram-positive bacteria.The cellular and humoral immune responses were detected to evaluate the immune effects of CpG ODNs on innate immunity of crabs. The survival rates, WSSV copy numbers and several anti-virus factors were employed to evaluate the immune protective roles of CpG-C in shrimps against WSSV. 0.1μg of CpG-C could inhibit the ROS generation, enhance the activities of PO and lysozyme, up-regulate mRNA expression of Crustin. These results collectively suggested that CpG-C could activate innate immune responses of Chinese mitten crab, and might be used as a novel immunostimulant for disease control in E. sinensis. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in CpG-ODNs-pretreatment shrimps than that in control shrimps after WSSV infection. The ROS levels in CpG-ODNs-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection. These results together demonstrated that CpG-ODNs induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei.The results together indicated that LvIntegrin, LvLec, EsSOCS2 and EsALF-2 were crucial immune factors in crustaceans and all involved in the immune defense responses. CpG ODNs could activate the innate immunity of crustaceans and played important roles in protecting the shrimps against WSSV. CpG ODNs as immunostimulants could be used for immune prevention and disease control in crustacean species. The results were helpful for better understanding immune defense mechanisms and developing novel immunostimulants for disease control in marine invertebrates.
|
PDF Full Text Request