Research On The Effects Of The Toxic Dinoflagellate Alexandrium Tamarense And Its Mechanisms On Disease Resistance Of The Shrimp Fenneropenaeus Chinensis

Dinoflagellate Alexandrium tamarense, a producer of paralytic shellfishpoison, is often found in farming ponds. Here we researched on the effects of the toxicdinoflagellate Alexandrium tamarense and its mechanisms on disease resistance of theshrimp Fenneropenaeus chinensis, an important mariculture species in China. The studyconsists of three parts:The first part: Studies on the toxicity of A. tamarense to F.chinensis.The growth of A. tamarense was studied under laboratory conditions. Mousebioassay (AOAC) was used to determine the toxicity of the extract from species of A.tamarense (mouse units, MU/cell) at different growth stages and culture temperatures.And the acute toxic effect of A. tamarense on F. chinensis was studied by exposure to A.tamarense. In addition, histopathology observation was conducted in gill andhepatopancreas after96h exposure and injection.The results showed that A. tamarense(ATHK) was moderate temperature strain and the growth rate at optimal temperature of20°C was0.24d-1。 The toxicity of the isolate culture was low, which was1.85×10-5-4.57×10-5MU/cel1(3.50-8.64pg STX Equal/cell) at optimal temperature of20°C. The toxicity of the isolate culture reached the maximum value at the logarithmicgrowth stage.The toxicity of the isolate culture decreased with temperature increasedand had highest value in the lowest temperature of16°C; In acute experiment, the96hLC50of A. tamarense was1.0×104cells/mL to F. chinensis, respectively. Based on the96h LC50, the safe concentrations were obtained, which was1.0×103. Histopathologyobservation revealed that A. tamarense and the extract could cause some abnormalhistological changes in gill and hepatopancreas, respectively, such as cellular swellingand vacuolated. The research results above indicate that pond productivity and qualityof the harvested organisms may be affected by A. tamarense. The second part: Effects of A. tamarense on MDA, SOD, GST and apoptosisin gill and hepatopancreas of the shrimp F. chinensis.The shrimp were intramuscularly injected the crude toxin extracted from A.tamarense cells. The dose injection was carried out only one time during the experiment,using extracted solution from1.4×103algae cells. Superoxide dismutase (SOD)activity,glutathione-S-transferase (GST) activity and malonaldehyde (MDA) contentwere analyzed in hepatopancreas and gill at1,3,6,12,24and48h after stress.Theindividuals of F. chinensis were exposed to200and1000cells/mL A. tamarense,followed by determination of the superoxide dismutase (SOD) activity, glutathioneS-transferase (GST) activity, malonyldialdehyde (MDA) concentration, and caspasegene (FcCasp) expression of gill and hepatopancreas. In addition, apoptosis inhepatopancreas of F. chinensis at96h after exposure was determined by terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.Theresults showed that SOD activity and GST activity in hepatopancreas and gill increasedwithin6h. However, they in gill were inhibited at12h and48h after stress. MDAcontent in hepatopancreas was no significant change except at1h after stress, butincreased in gill with time prolonging. Superoxide dismutase (SOD) activity,glutathione S-transferase (GST) activity, malonyldialdehyde (MDA) content andFcCasp expression of gill were analyzed at3,6,12,24,48,72and96h after exposure.The results showed SOD activity, GST activity, MDA content and FcCasp expressionin gill of F. chinensis exposed to200cells/mL A. tamarense as a whole first increasedand then decreased with increasing exposure time. However, exposed to1000cells/mLA. tamarense, SOD activity in gill of F. chinensis increased and then decreased withincreasing exposure time, and was significantly (P<0.05) inhibited between24and96h.GST activity in gill of F. chinensis was significantly (P<0.05) inhibited except at3and48h post treatment, The changes in SOD and GST activities in gill of F. chinensis inthis study suggest that these enzymes were actively involved in the detoxificationprocess in gill of F. chinensis. MDA content and FcCasp expression in gill of F.chinensis exposed to1000cells/mL A. tamarense as a whole increased with increasedexposure time, and displayed a time-response relationship.The FcCasp transcript levelin gill of F. chinensis exposed to A. tamarense was positively and linearly correlated tothe MDA content. Both the hepatopancreatic SOD and GST activities of F. chinensisexposed to1000cells/mL A. tamarense showed a bell-shaped response with increasing exposure time. The hepatopancreatic MDA concentration of F. chinensis exposed to1000cells/mL A. tamarense increased gradually between48and96h, and theincreasing trend of MDA concentration was corresponding to the decreasing trend ofGST activity. The hepatopancreatic FcCasp transcript level of F. chinensis exposed to1000cells/mL A. tamarense was positively and linearly correlated to the MDAconcentration. Results of the TUNEL assay showed that exposure to1000cells/mL A.tamarense induced apoptosis in hepatopancreas of F. chinensis. The current studyrevealed that A. tamarense exposure treatment influenced the antioxidative status of F.chinensis and caused lipid peroxidation and apoptosis in gill and hepatopancreas of theshrimp. The findings from this study demonstrated that SOD, GST and MDA weresensitive and suitable to be used as potential oxidative biomarkers of F. chinensis andother aquatic invertebrates exposed to A. tamarense in the short term. Further studies arestill needed to clarify the mechanism of toxicity of A. tamarense to aquaticinvertebrates.The third part: Effects of A. tamarense on relative expression of immune relatedgenes and disease resistance of F. chinensisNitric oxide (NO) signaling is involved in many physiological processes invertebrates and invertebrates. In crustaceans, nitric oxide synthase (NOS) plays asignificant role in innate immunity. To evaluate the roles of NOS in response toVibrio parahaemolyticus and toxic dinoflagellate Alexandrium tamarense challenge inChinese shrimp Fenneropenaeus chinensis. An NOS gene (FcNOS) was cloned fromthe hepatopancreas of F. chinensis by reverse transcription polymerase chain reaction(RT-PCR) coupled with rapid amplification of cDNA ends (RACE) methods. Thefull-length of FcNOS cDNA was4616bp, with an open reading frame (ORF) of3582bp, encoding a polypeptide of1193amino acids with the predicted molecular weight of134.7kDa and the estimated isoelectric point of7.01. FcNOS contained the conserveddomains and binding motifs of NOS found in a variety of organisms. Quantitativereal-time RT-PCR analysis revealed that FcNOS transcript could be detected in mostshrimp tissues, and strongly expressed in gill and hepatopancreas of F. chinensis. AfterV. parahemolyticus injection, FcNOS transcript in gill increased in the early stage assame as in hepatopancreas. The results suggested that FcNOS might be associated withthe immune defenses to V. parahemolyticus in F. chinensis. After200and1000cells/mLA. tamarense exposure, FcNOS expression under1000cells/mL A. tamarense treatment was significantly down-regulated in gill and hepatopancreas from12h and72h,respectively, thus demonstrating the inhibition of FcNOS expression by highconcentration of A. tamarense exposure. After the shrimp were exposed to A. tamarense(100,200,500,1000,2000and4000cells/mL treatments) for6days and thenchallenged by injection with V. parahaemolyticus, the cumulative mortality as a wholeincreased as the concentration of A. tamarense increased at the same time, and thecumulative mortality of the shrimp under1000cells/mL A. tamarense treatment wassignificantly greater than that under200cells/mL A. tamarense treatment at the sametime, suggesting that the exposure to A. tamarense dropped the ability ofvibrio-resistance of F. chinensis. Individuals of F. chinensis were exposed to200cells·mL-1and1000cells·mL-1A. tamarense, and their TLR gene and Relish geneexpressions were then determined at3,6,12,24,48,72,96h and144h. Underexposure to200cells·mL-1A. tamarense, the relative expressions of TLR gene andRelish gene were not consistently inhibited in the hepatopancreas, gill and hemolymphof the shrimp between72h and144h. However, such expressions were significantlyinhibited under exposure to1000cells·mL-1A. tamarense in the gill and hemolymphwithin48-144h and in the hepatopancreas between72h and144h. After theindividuals of F. chinensis pre-exposed to A. tamarense (200cells·mL-1and1000cells·mL-1) were artificially infected with WSSV, a positive infection by the WSSV wasdetected after3days. The cumulative mortality of F. chinensis was100%after7daysfor exposure to both A. tamarense and seawater control, but was higher in the A.tamarense group than in the seawater control group at the same sampling time. Inaddition, when shrimps were exposed to A. tamarense for6days, followed by infectionwith Vibrio anguillarum, their cumulative mortality at the same sampling time was stillhigher in the A. tamarense group than that in the seawater control. Therefore, it issuggested that A. tamarense affected the relative expressions of NOS, TLR and Relishgenes of the shrimp and the disease resistance in F. chinensis.