Physiological,Immunological And Apoptotic Responses Of Sinonovacula Constricta To Ammonia Stress

The razor clam（Sinonovacula constricta）is a bivalve mollusk that lives in the low to medium intertidal zone along the western Pacific coast.Among the four major aquaculture shellfish in China,razor clams are a highly desirable and commercially valuable edible species.Ammonia is one of the most important toxic environmental factors in aquaculture and has a profound impact on the culture of razor clams and on the function they produce in ecological farming.However,razor clams,as a buried shellfish,are often subject to greater environmental ammonia stress due to their limited range and mobility.Therefore,this study measured the tolerance of razor clams to ammonia at three different growth stages（96 h semi-lethal concentration）;investigated the biochemical level changes of razor clams under ammonia stress from four perspectives:bioaccumulation,respiratory metabolism,tissue damage,and detoxification strategies;examined the dynamic changes of the antioxidant system of razor clams under ammonia stress;explored the balance between reactive oxygen species and antioxidant enzymes in time and space;detected the apoptosis in gills and hepatopancreas of razor clams,and initially confirmed the activation of the mitochondrial apoptotic pathway in razor clams.This study provides a reference for the study of the toxicological mechanism of ammonia on the antioxidant system and apoptosis and healthy culture of marine shellfish.1.The toxic effects of ammonia on three sizes of razor clams,including small（shell length 1.5±0.27 cm）,medium（shell length 3.5±0.41 cm）and large size（shell length 5.7±0.79 cm）,were studied using a biological toxicity assay.（1）Compared to other bivalve shellfish,razor clams did not have a generalized closed-shell behavior after ammonia poisoning.All three sizes of razor clams exhibited reduced shell closure muscle tone and delayed movement.Individuals of medium size and above showed atrophy and inactivation of the feet and water pipes.Hemolysis and swelling of the gills followed by atrophy after poisoning.（2）The 72 h and 96 h semi-lethal concentrations of non-ionic ammonia to small-sized razor clams were 3.689 mg/L and 2.501 mg/L,and the safe concentration was0.2501 mg/L.（3）The 72 h and 96 h semi-lethal concentrations of non-ionic ammonia for medium-sized razor clams were 4.217 mg/L,2.837 mg/L and the safe concentration was 0.2837 mg/L.（4）The 72h and 96h semi-lethal concentrations of non-ionic ammonia for large-sized razor clams were 10.860 mg/L,5.586 mg/L,and the safe concentration was0.5586 mg/L.2.Effects of ammonia stress on the physiological and biochemical levels of razor clamsMedium-sized razor clams were exposed to 0.25 mg/L,1.25 mg/L,2.5 mg/L of non-ionic ammonia for 72 h and recovered in ammonia-free seawater for 72 h,and the effects of ammonia on the respiratory metabolism level,tissue damage,and detoxification level conditions of razor clams were studied.（1）The ammonia concentration in hemolymph of razor clams in all three non-ionic ammonia concentrations increased significantly with time and reached a peak at72 h,with significant differences（P<0.05）among all groups,and the highest group could reach 247 times the initial value.After 72 h of recovery,all groups could not return to normal levels.（2）After 72 h of stress,the lactate（LD）content of the high concentration group reached its peak and was significantly higher than that of the low and medium concentration groups（P<0.05）.After 72 h of recovery,there was a significant decreasing trend in all three concentration groups,and the LD level in the high concentration group was always significantly higher than the initial value（P<0.05）.In the high concentration group,activities of lactate dehydrogenase（LDH）was consistently significantly higher than in the low and medium concentration groups from 24 h of ammonia nitrogen exposure（P<0.05）.Only the low concentration group returned to normal levels after 72h of recovery.（3）When the exposure time reached 72h,glutathione transaminase（AST）activities in both gill tissue and blood were significantly higher in the high concentration group than in the low and medium concentration groups（P<0.05）.There was no decreasing trend of AST activity in gill tissues of the high concentration group during the 72 h of recovery.（4）Glutamine（Gln）content in hepatopancreas tissues of razor clams was significantly higher in the medium and high concentration groups than the initial value and the low concentration group from 72 h of stress（P<0.05）.The glutamine synthetase（GS）activities in the high concentration group were significantly higher than that in the medium and low concentration groups after 48 h of stress and reached its peak at 72 h（P<0.05）.3.Effects of ammonia stress on the antioxidant system of razor clamsMedium-sized razor clams were exposed to 0.25 mg/L and 2.5 mg/L of non-ionic ammonia for 72 h and recovered in ammonia-free seawater for 72 h.The effects of ammonia on the accumulation of reactive oxygen species and the activities of antioxidant enzymes and related gene expressions in the gills and hepatopancreas of razor clams were investigated.（1）In the gills of razor clams,superoxide anion（₂^-）levels increased after exposure and peaked at 72 h,and also showed a tendency to decrease during the recovery phase.The trend of hydrogen peroxide（H₂O₂）levels was similar to that of₂^-and was significantly higher than the initial level at all time periods after the onset of stress（P<0.05）.The enzyme activities and gene expression of superoxide dismutase（SOD）and glutathione peroxidase（GPX）increased under ammonia-nitrogen stress in all groups.The enzyme activity and gene expression of CAT were inhibited in the high concentration group.（2）In the hepatopancreas of razor clams,the levels of₂^-and H₂O₂in the hepatopancreas of razor clams were significantly up-regulated after the onset of stress and the peak of both reactive oxygen species occurred at 72 h.Except for the H₂O₂content in the low concentration group,the ROS content in the hepatopancreas of razor clams was still significantly higher than the 0h（initial value）after 72 h of recovery（P<0.05）.The SOD-related indexes in the high concentration group decreased within 24h and turned to an increasing trend by 72 h.CAT enzyme activities in the high concentration group were inhibited during the stress phase.4.Effect of ammonia stress on the cell apoptosis of razor clamsMedium-sized razor clams（3.5±0.41 cm）were exposed to 0.25 mg/L and 2.5mg/L of non-ionic ammonia for 72 h and recovered in ammonia-free seawater for 72h.The effects of ammonia on the expression of genes related to the mitochondrial apoptotic pathway in razor clams were investigated,and the apoptosis of hepatopancreas and gill tissues was observed in situ using the TUNEL method.The results showed that:Ammonia stress significantly affected the expression of Bax,Bcl-2,Cyt-c,Caspase-7 genes and activated the mitochondrial apoptosis pathway.the expression of Bcl-2 showed different trends between the low and high concentration groups.The relative expression of Bcl-2 in the high concentration group peaked at 72 h and decreased significantly after recovery at 72 h（P<0.05）.Apoptosis plots showed that the degree of apoptosis was positively correlated with stress time and ammonia concentration,while apoptosis was more pronounced in constricted razor clam gill tissue than in hepatopancreas.