Physiological Response To Stress Of Nitrite,Ammonia And Temperature In Grass Carp,Ctenopharyngodon Idella

Grass carp is one of the most important cultured fishes in China，but they may faceto various environmental stress in high density culturing. We preliminarily discussedthe physiological response to environmental stress in grass carp by the study ofseveral stress-related gene expressions under nitrite, ammonia and temperature stress.1. Effect of high concentration exposure of nitrite on expression of sod and hsp90in grass carp, Ctenopharyngodon idellaThe histological and molecular responses of grass carp Ctenopharyngodon idella tohigh concentration nitrite exposure were studied in the present study by determiningthe lethal concentration, and expression patterns of sod and hsp90. The24h LC₅₀fornitrite exposure was determined:445mg/L. Grass carps were treated at fiveconcentrations （50,87,151,263,400mg/L） of nitrite for24h, and three tissues （gill,intestine and liver） were collected to measure the expression of sod and hsp90. Afterhigh concentration of nitrite exposure, the two genes （sod and hsp90） in differenttissues unregulated significantly, indicating that high concentration of nitrite caninduce the expression of stress protein. These results also suggest that sod and hsp90are appropriate biomarkers for nitrite exposure assessment in grass carp2. Effect of high concentration exposure of ammonia on gill structure alternationand expression of sod and hsp90in grass carp, Ctenopharyngodon idellaThe histological and molecular responses of grass carp Ctenopharyngodon idella tohigh concentration ammonia exposure were studied in the present study bydetermining the lethal concentration, gill cellular structure and expression patterns ofsod and hsp90. Grass carps were treated at five concentrations （50,72,104,151,220mg/L） of ammonia for24h, and then gill tissue was collected for histological analysis,and three tissues （gill, intestine and liver） were collected to measure the expression of sod and hsp90. After high concentration of ammonia exposure, the arrangement andstructures of gill cells changed dramatically. Edema and fusion of secondary lamellaecould be observed, and edge of cell and nuclear could not be clearly defined.Meanwhile, the two genes （sod and hsp90） in different tissues unregulatedsignificantly, indicating that high concentration of ammonia can impair the cellularstructures and induce the expression of stress protein. These results also suggest thatsod and hsp90are appropriate biomarkers for ammonia exposure assessment in grasscarp3. Effect of rapid temperature change on expression of hsp70and hsp90in grasscarp, Ctenopharyngodon idellaGrass carp is one of the most important cultured fishes in China. Duringtransportation in summer, massive mortality often occurs. As molecular chaperones,Hsps assist cells in their recovery from stress and promote cytoprotection. Limitedreports described the expression of Hsps in grass carp at high temperatures, and therelationship between oxygen consumption, expression of heat shock protein andmortality need to be investigated based on the oxygen-and capability-limited thermaltolerance theory （OCLTT） to further elucidate the thermal adaptation of this speciesagainst high temperature in a view of integrated biology. In the present study, theexpressions of hsp70and hsp90after rapid temperature increase were studied. Grasscarps were acclimated under20C and then exposed in designated temperatures（20,22,24,26，28,30,32,34C） for3h heat shock. After2h-recovery at20C, liver,muscle and gill of tested fish were sampled and hsp70and hsp90were determinedusing semi-quantitive real-time PCR. Expression of hsps increased with temperatureincrease until32C in muscle and gill, and then decreased at34C. In liver, expressionof hsps kept increasing in all temperature treatments. These results indicated thathsp70and hsp90were sensitive to temperature increase in grass carp. Analyzed heatshock proteins data with previous results of oxygen consumption and lethaltemperatures of grass carp, the physiological adaptations of grass carp could beexplained by oxygen-and capability-limited thermal tolerance theory （OCLTT）,which pointed out that the imbalance between oxygen demand and tissue oxygen supply ability was the primary factor which limited organisms’ thermal tolerance andthe ability of aerobic metabolism greatly limited the survival of organism beyondcritical temperatures. With temperature increase, the maximum value of oxygenconsumption in grass carp occurred at²8C, and then the oxygen consumptiondecreased when temperature was beyond28C, indicating a transformation fromaerobic metabolism to anaerobic metabolism at this critical temperature. Theexpressions of heat shock proteins kept increasing until32C in muscle and gill ingrass carp, and provided defense against high temperature. However, this protectionof heat shock proteins was limited and then death occurred when temperature wasbeyond32C. The change of metabolism, increase of oxygen free radicals and theconsumption of energy due to stress proteins synthesis of grass carp were closelyrelate to the decrease of capability of immune response when water temperature wasbeyond28C. In general, high water temperature could alter the metabolic pathway,induce heat shock protein expression, reduce immune response, and then causelarge-scale mortality. Therefore, appropriate ways should be applied to maintain watertemperature under28C to avoid large-scale mortality during transportation.4. Molecular cloning and sequence analysis of hsp70in common carp, CyprinuscarpioTotal RNA was extracted from grass carp （Cyprinus carpio）, hsp70gene wasisolated by using RT-PCR and RACE. The full length cDNA sequence was2303bpwhich contained a137bp5’ UTR, a234bp3’ UTR, and a1932bp putative ORFwhich encodes a polypeptide of643amino acids with an estimated molecular mass of70.5kDa and the theoretical isoelectric point of5.42. Semi RT-PCR analysis showedexpression of hsp70in all tissues examined and there was no significant difference.Molecular cloning and sequence analysis of hsp70will be helpful to clarify thephysiological adaption of common carp （Cyprinus carpio） and adaption differencebetween other species to environment...