Screening And Application Of Immune Stimulants In Disease Control For Cultivated Mud Crab, Scylla Serreta

Mud crab,Scylla serrata,is the main cultivated marine crab in China,with production of 108,500 tons,88.14%of the total production of the world.Recently, serious diseases,including yellow hemolymph disease,vibriosis,eiliates disease,milk disease,brown spot disease etc,were recorded in the main provinces throughout China,caused by virus,vibrio,parasites,water pollution and unknown reasons. According to official report,the morbidity of cultivated mud crab in 2006 was about 13.77%in whole China,bringing 32.71 million dollars lost.We report the studies on the establishment and optical condition of phenoloxidase activity of hemocyte lysate.The phenoloxidase(PO) activity of hemocyte lysate supernatant(HLS) from mud crab,Scylla serrata was examined by treating HLS with various factors including temperature,pH,stimulants and inhibitors.The results showed that the optimum temperature and pH of PO was 25℃and 7.8.PO activity was increased by Sodium dodeeyl sulfate(SDS),Ca²⁺ and Mg²⁺,with the optimum concentration of 2.5 mg/mL,50 mmol/L,and 150 mmol/L,respectively.PO activity was inhibited by sodium diethyldithiocarbamate(DETC),ethylenediaminetetraacetic acid(EDTA),Cu²⁺ and sodium azide(NAN₃).In addition,the formalin killed bacteria of Vibrio minicus SS060818 and Aeromonas spp.MrM0602,the confirmed pathogenic strains isolated from mud crab and Macrobrachium rosenbergii,could obviously stimulate the PO activity,while Staphylococcus aureus ATCC25923 and Micrococcus lysoleikticus AS1.634 only showed slight stimulation.Both peptidoglycan(PG) and lipopolysaccharides(LPS) extracted from V.minicus SS060818 showed significant stimulation on PO activity,with the optimum concentration is 2.7 mg/mL and 0.5 mg/mL.Besides,the tests and optimal conditions for the main nonspecific immune index of S.serrata,including alkaline phosphatase (ALP),superoxide dismutase(SOD),phenoloxidase(PO),peroxidase(POD), anti-bacteria activities,phagocytic activity,were studied and established.More than thirty Chinese herbs were chosen for immune polysaccharide with water extraction followed by ethanol precipitation for polysaccharide.Stimulation on phenoloxidase(PO) activity was used for evaluation of immune polysaccharide.The results showed that polysaccharide from lagehead atractylodes,agaric,honeysuckle, rhubarb,grifola,dried orange peel,CIS-1 and CIS-2 can stimulate the PO activity remarkably,with the optimal concentration of 0.03125μg to 0.125 mg/mL.Other polysaccharide showed weak or no stimulation on PO.CIS-1,CIS-2 and forsythia polysaccharides were chose for mud crab injection at the concentration of 7.5×10^-3mg/mL,0.031 mg/mL and 1.25×10^-4 mg/mL,respectively,and haemolymph was collected at regular interval for superoxide Dismutase(SOD),alkaline phosphatase(ALP),peroxidase(POD),PO and phagocytosis rate(PR) of hemocytes tests.It showed that POD,PO and PR can be increased after injection of these three polysacchrides,compared with the control group.CIS-1 seemed the most effective polysaccharide with the haemolymph SOD increasing of 18.2%(from 12.76 U/mgprot to 15.08 U/mgprot),ALP increasing of 75%(from 0.52 U/L to 0.91 U/L). CIS-2 and forsythia did not increase the SOD and ALP of haemolymph after the crab was injection.The anti-bacteria activity was not improved after injection of polysaccharide.Mud crabs were challenged by dipping with Vibrio minicus SS060818,the virulent strain isolated from diseased crab,at the concentration of 1×10⁷ cfu/mL,after injecting with polysaccharides.The results showed that CIS-1,CIS-2 and forsythia polysaccharide can prolong the living period of experimental crab by 55.92%,139.35 %,12.43%,which indicating the polysaccharides injection can improve anti-vibrio ability of crab.CIS-1,CIS-2,peptidoglycan(PG) and lipopolysaccharides(LPS) were chosen for immunostimulants additives for mud crab feed.The results show that POD,PO, ALP of crab hemolymph were increased in the group of CIS-1,CIS-2,PG,LPS. CIS-2 group showed significantly increasing in ALP,POD and PO after 6 days.CIS-1 group reached the peak of ALP at 11d.SOD of PG and LPS group showed significantly higher than control group.Anti-bacteria activity in all immunol-stimulated groups did not give the significant difference from the control group.All groups showed more resistant to V.minicus SS060818 when the bacteria was used for dipping challenging at the concentration of 1×10⁷ cfu/mL 20 days after the immunol polysaccharide additives used.The mean survival time of CIS-1,CIS-2, PG and LPS group were 44.57%,104.51%,31.59%and 78.70%higher respectively,compared with that of control group.CIS-1,CIS-2,peptidoglycan(PG) and lipopolysaccharides(LPS) were used for stimulating the mud crab for over-winter cultivation,the highest risk period season for crab farming in Zhejiang Province with high profits.Mud crabs were injected intramuscularly with polysaccharide suspension and cultivated in cage with sand bottom.The results showed that CIS-2 gave the best effects,with the activity of 18.55U/mgprot,0.16 U/mL and 38.16 U/mL for ALP,PG and POD respectively,50 %,100%and 176%higher than that of control group.ALP activities in all stimulated groups were higher than that in control group.CIS-1 and LPS groups reached the peak of POD with the activity of 0.14 U/mL at 16d,75%higher than that of control group.ALP and PO of LPS group showed no significantly higher than control group.The survival rate of mud crab in CIS-1,CIS-2 and PG group was 73%, 82%and 70%,respectively,45 days after immuno-stimulated.The CIS-1 and CIS-2 were also used for over-winter fattening cultivation for mud crab in crab farm.ALP, PO and SOD showed higher than the control group 15 days alter injection intramuscularly of immunostimulants.The survival rate at 30 days in CIS-1 and CIS-2 group was 71%and 88%,20.33%and 49.15%higher than that of control group.Theses results indicated that the immunol polysaccharides can improve the immunity of mud crab,as well as the resistance against diseases.These immunol polysaccharide can be considered as the potential reagents for application of mud crab diseases control.