The Immunoresponse Characteristics And Molecularly Resistant Mechanism Of Apostichopus Japonicus Subjected To The Marine Environment And Pathogens

In order to supplement the theory that diseases occurred to sea cucumber, and to explore the technique of safe prevention and cure for its diseases, the pathogens are isolated from diseased sea cucumbers and primarily assayed its pathogenicity by general methods. Moreover, the change of some related non-specific immune indexes about sea cucumbers exposed to germs is detected under the simulated culture surroundings. Several genes related to the immune response are identified by analyzing constructed cDNA library and EST data in bacterial infected sea cucumbers. And then, the antagonistic bacterium is chosen from prepared various bacteria, but also its antagonistic characteristics are tested. The most results are as follows:The pathogens are identified as two psedoaltermonas bacteria and one undetailed virus respectively by microbiological assay. As virus particle, the bacterium Pseudoalteromonas sp shows more severe infectiosity and pathogenicity. The LSZ activity and AKP activity, induced SOD activity, CAT activity and PO activity, complement component C3contents and respiratory burst activity all significantly decrease, and that ACP activity and complement component C4contents show a little increase in the coelomic fluid of sea cucumbers suffering from bacterial infection with elevated concentration. However, the SOD activity and LSZ activity in the epimucus are sensitive to and obviously suppressed by bacterial infection. And furthermore, the epimucous CAT activity, ACP activity and complement component C4contents all rise when infecting bacteria is under low concentrations, but that decline during bacterial infecting with high concentrations. Under virus infecting conditions, the alteration of immune enzymes activity in the coelomic fluid is more evident than that in the epimucus, and the LSZ activity, ACP activity, SOD activity and PO activity are all reduced, and only CAT activity enhanced.The SOD activity and ALP activity are depressed, and but the LSZ activity raised in the coelomic fluid under high ammonia-N concentrations, and yet the above enzymes activity decline, the GPx activity continuously increase with additional bacterial infection, and as well the altering amplitude of coelomic enzymes activity decrease least or increase most under medium ammonia-N concentrations. The humoral SOD activity, GPx activity, LSZ activity, PO activity and ALP activity are raised with the increasing Na2SeO3concentrations under low Na2SeO3conditions, and that the humoral SOD activity, GPx activity and LSZ activity firstly ascend then decline at certain time, and yet PO activity and ALP activity keep rise during the additional bacterial infecting treatments. The induced SOD, CAT, AKP and PO activity decrease, and GPx activity and LSZ activity are markedly suppressed in the coelomic fluid under high nitrite-N concentrations, and additional hypoxia or bacterial infection aggravate depressing the CAT, GPx, LSZ and PO activity, but may induce SOD activity in the coelomic fluid. And besides, additional hypoxia treatments slow down induced ACP activity, but promote AKP activity, and that additional bacterial infection strengthen the induced ACP activity and obviously counteracts the suppression of AKP activity by high nitrite-N concentrations. The inducement of humoral SOD, GPx and AKP activity in the sea cucumbers is facilitated by additional bacterial infection under moderately low salinity. However, the reduction of above enzymes activity is quickened by additional bacterial infection under high salinity. Moreover, optimized saline treatments may induce humoral LSZ activity, but bacterial infection impairs the induced humoral LSZ activity. The induced humoral SOD, GPx, LSZ and PO activity all are mutually promoted by elevated temperature and bacterial infection during the seawater is below23℃, and that the above enzymes activity is apparently suppressed by bacterial infection under seawater high temperature stress. And then, the humoral AKP activity is repressed with risen seawater temperature and induced by bacterial infection respectively. The humoral SOD and PO activity may be reinforced, and the humoral GPx and LSZ activity alters insignificantly when the seawater temperature is slowly raised, but high temperature pretreatments lead to obvious decrease of humoral SOD and LSZ activity. And so, proper stimulus of temperature can alleviate disease severity of sea cucumbers despite the low stability of inducement on sea cucumbers.The cDNA library with an inserted fragment of0.8-2.5kb and also high quality is constructed, and its capacity is3.24×105cfu/mL.1106ESTs with high quality are obtained according to the results of library sequences assaying and elementarily bioinformatics processing.533single gene clusters including165overlapping sequence groups and368single sequences are confirmed by using related software to splice the EST.25immunity-related genes are found after analyzing the genes with homologous sequences. The results of analyzing immunity-related genes by real-time PCR technique indicate that genes encoding serum lectin isoform, complement component C3and complement component3-like protein in the coelomic fluid of infected A. japonicus showed most remarkable increase, which demonstrates their important effect in the immune defending of holothurian A. japonicus.Five antagonistic bacteria are selected by solid culture of dot-inoculating and filter paper piece-inoculating, and then further research by Niujin cup method validates the bacterium both YAAJ6and YASM12show highest antagonistic activity. Furthermore, the test in vivo demonstrates the above bacteria enhance disease resistance of A. japonicus to pathogenic infection by increasing SOD, ALP and LSZ activity in coelomic fluid and promoting the phagocytic activity of coelomocytes. Besides, the ECP of antagonistic bacteria shows the most resistant activity to pathogens at28℃or pH8.0, and yet the ECP is sensitive to protease K or streptomycin-protease.In conclusion, bacteria and virus can lead to the decrease of non-specific immunity in A. japonicus., and so cause the related symptoms about "skin ulceration" in holothurian, and unmoderate change of seawater environment advances the possibility of bacterial infecting on A. japonicus., but immune response is induced under reasonable environmental conditions, and also acclimatization treating may temporarily enhance the immune capacity. Otherwise, selecting and then applying the antagonistic bacteria on holothurian is feasible measures of disease prevention and cure.