Study On The Effects Of Environmental Stress On The Immune And Digestive Physiology Of Charybdis Japonica

Enzymatic, histological, and molecular methods were used to analyze the effectsof main pollutant factors in the aquaculture water (ammonia-N, nitrite-N, sulfide, andheavy metals e.g. cadmium, lead) on the immune and digestive physiology of marinecrab, Charybdis japonica in this paper, in order to study the toxic action ofenvironmental stress on C. japonica, and provide a theoretical basis for the control ofwater quality in artificial cultures and correlative toxicology research.The results showed that most immunity-related indicators (including density ofhemocyte (DHC), hemocyanin content, the activities of the phenoloxidase (PO),lysozyme (LSZ), superoxide dismutase (SOD) and catalase (CAT) declined after aninitial rise under environmental stress, with the exception of malondialdehyde (MDA)content that showed an overall increasing trend. After exposed for15days, themeasured indices were lower significantly in treatment groups than in the controlexcept the experimental groups at low stress concentrations. Significant negativecorrelations was found between stress concentration and the activities of SOD andCAT, while MDA content was found to be positively correlated with stressconcentration.The digestive enzyme activities in the hepatopancreas of C. japonica wereinduced to some degree after exposure to lower concentrations of stress (ammonia-N≤10mg/L, nitrite-N≤10mg/L, sulfide≤0.75mg/L). But higher concentration ofstress (ammonia-N≥20mg/L, nitrite-N≥20mg/L, sulfide≥1mg/L), resulted in asignificant inhibition of hepatopancreatic protease and amylase activities. On day7after exposure, digestive enzyme activities of the groups (ammonia-N≥10mg/L,nitrite-N≥2mg/L, sulfide≥1mg/L) decreased noticeably than in the control, and asignificant negative correlations was found between the amylase activities and stressconcentration.Noticeable changes were observed in the histological structure of some organs incrabs exposed to high concentrations of ammonia-N and sulfide. The chitin layer ofthe gills became thin and was partly ruptured, the epithelial lamina becamedisorganized, the organelles reduced and the chromatin condensed. The quantity ofhemolymph in the gill cavity was reduced and its density decreased. Hemocytes werecondensed with ruptured cytomembranes and abundant vacuoles in cytoplasm. Theglandular epithelium exhibited an irregular morphology, the number of secretory cells (B-cells) decreased and numerous vacuoles appeared. There were fewer organellesand the chromatin was condensed. The chitin layer of the stomach ruptured and thegastric epithelium became disordered, with abundant residual bodies formed in thecytoplasm.The expression of α-amylase (α-AMY), lactate dehydrogenase (LDH), malicdehydrogenase (MDH) and peroxidase (POD) isozymes was significantly inhibited byhigh concentration of the stress of ammonia-N, nitrite-N, and sulfide: the isozymeactivities declined or their expression bands reduced, and even disappeared. But themuscle was induced to express MDH-2and MDH-4with higher activities. Meaningwhile, the expression activities of MDH-1in gill and MDH-4in heart increasedobviously.The accumulation content of cadmium (Cd) and lead (Pb) in the hepatopancreasand gill were relatively higher，while minute amounts of Cd and Pb could be detect inthe ovary and muscle of the higher concentrations of group (Cd≥0.1mg/L, Pb≥5mg/L). Significant effect-dosage relationships were observed between theaccumulation content and Cd and Pb concentration in the hepatopancreas oftreatments.The full-length cDNA sequence of heat shock protein70(HSP70) was clonedfrom hepatopancreas of C. japonica consists of2203bp, containing an1953bp openreading frame (ORF), distributing108-2060bp, and encoding650amino acids,107bp5’UTR (untranslated region) and143bp3’UTR (untranslated region). The clusteranalysis showed the sequences obtained were highly conserved in Scyllaparamamosain, Scylla serrata, Portunus trituberculatus and Callinectes sapidus.The expression of HSP70of C. japonica were induced to some degree after thestress of ammonia-N, nitrite-N and sulfide, and the degree of induction showed asignificantly positively correlation with increasing concentrations. The HSP70expression reached a peak at3h or6h. However, HSP70mRNA expressions wereinhibited with longer stress time.In conclusion, high concentrations of environmental stress have significantinhibitory effect on the activities of immune-related enzymes, digestive enzymeactivities, the expression of isozymes and HSP70, and also damaged the organstructure. The activities of SOD and the content of MDA may be used as indices forevaluating the immune state of C. japonica under environmental stress. The amylaseactivity may be used as an index for evaluating the digestive state of C. japonica under stress. The accumulation content of Cd and Pb in the hepatopancreas may beused as biological indices for evaluating the pollution state of heavy metals. TheHSP70expression was affected quickly by the changes of environmental factors, andHSP70also could be used for an early indicator of environmental stress.