Antibacterial Analysis Of Coelomic Fluid From The Sea Cucumber(Apostichopus Japonicus)

Apostichopus japonicus belongs to Echinidermata, Holothuroidea, Aspidochirotidae,Stichopodidae, Apostichopus, and it is an economically important aquaculture species. Bacterial disease is one of the primary factors limiting the development of A. japonicus aquaculture. In order to study the antibacterial spectrum of coelomic fluid from A. japonicus and the effects of divalent metal ions on the antibacterial activities of coelomic fluid, using the method of bacterial growth curve determination, we determined the effects of coelomocyte lysate supernatant and coelomic fluid supernatant from A. japonicus on the growth of Vibrio harveyi, Vibrio splendidus,Shewanella baltica, Pseudoalteromonas nigrifacien, Staphyloccocus aureus, Micrococcus lysodeikticus, Streptococcus dysgadysgalactiae, Nocardiopsis sp., respectively, and then determined the effects of Mg2+, Mn2+, Ca2+, Fe2+, Zn2+, and Cd2+ that commonly found in marine environment on the antibacterial activities in A. japonicus coelomic fluid supernatant with M.lysodeikticus as tested bacteria. The results showed that the coelomocyte lysate supernatant had no obvious effects on the growth of all tested bacteria, while the coelomic fluid supernatant only showed strong inhibition on the growth of M. lysodeikticus. In addition, Mg2+ and Zn2+ increased the inhibition of coelomic fluid supernatant on the growth of M. lysodeikticus, while Mg2+, Ca2+,Fe2+ and Cd2+ showed no obvious effects on the antibacterial activities of coelomic fluid supernatant. The above results suggested that A. japonicus coelomic fluid in vitro only had limited-spectrum antibacterial activity, and the immune factors directly related to anti-bacteria were mainly distributed in coelomic fluid supernatant. Besides, these antibacterial factors in coelomic fluid supernatant developed a certain adaptability to the divalent metal ions that are commonly found in marine environment, and at certain concentrations, Mn2+ and Zn2+ might be beneficial to the promotion of A. japonicus antibacterial capacity.To further investigate the variation of immune-related enzyme activities in A. japonicus coelomic fluid after different bacterial challenge, V. splendidus, V. harveyi, P. nigrifacien, M.lysodeikticus and S. dysgadysgalactiae were injected into A. japonicus, respectively, and then the activities of acid phosphatase(ACP), alkaline phosphatase(AKP), superoxide dismutase(SOD),lysozyme(LYZ) and phenoloxidase(PO) were determined using p-nitrophenyl phosphate(p NPP)method, nitro blue tetrazolium chloride(NBT) method, M. lysodeikticus powder method and dopachrome formation method, respectively. The results showed that the challenge of V.splendidus induced significant increase of ACP and AKP activities, but great decrease of SOD,LYZ and PO activities; challenge with V. harveyi caused significant increase of ACP, SOD, LYZ and PO activities and irregular variation of AKP activity; after challenge with P. nigrifacien,ACP, LYZ and PO activities increased significantly, SOD activity increased firstly, and then decreased, and AKP activity exhibited irregular variation; after challenge with M. lysodeikticus,ACP and PO activities increased, SOD and LYZ activities increased firstly, and then decreased,however, LYZ activity returned to control level at 72 h post challenge, and AKP activity also exhibited irregular variation; the challenge of S. dysgadysgalactiae resulted in an inhibition in AKP activity at 4h, but notable promotion in the activities of other determined enzymes. The results suggested that PO was one of the most sensitive and efficient immune factors in A.japonicus innate immune system; no regular differences in the induction of A. japonicus immune factors were observed between Gram-positive bacteria and Gram-negative bacteria; between M.lysodeikticus and other four determined bacteria, the most significant difference was observed in the induction of LYZ, and LYZ might be the principle immune factor involved in the extermination of M. lysodeikticus; the variation of immune factors induced by V. splendidus differed greatly from those induced by other four bacteria, and the five immune factors selected in this study had potentials in the application for early warning of vibrio diseases; S.dysgadysgalactiae had potential in the application for immunostimulants in A. japonicus.