| Temperature is regarded as an important abiotic variable influencing almost theentire life history of fish, including survival, ingestion, growth, development andreproduction. Temperature tolerance of fish limits its distribution, both in time andspace. As global climate change has increased extreme weather events, the study ofthermal tolerance mechanism and heat-tolerance breeding of aquatic organismbecomes a work of great value both in scientific research and aquaculture industry.However, works in thermal tolerance of marine fish are limited, and there lacksevaluation indicators of heat tolerance. Paralichthys olivaceus (Temminck&Schlegel,1846) is an ecologically and economically valuable flatfish species in China, whoseculture is widely distributed along the coast of North China, and is expanded to theSouth China. However, the aquaculture of this species has been suffered from the hotsummer, and is frequently shocked by mass mortality. Paralichthys dentatus (L.,1766)is a high temperature resistant species, which is also an important commercial flatfishalong the Atlantic coast. The hybrids of these two species (P. olivaceus×P.dentatus) inherits the excellent economic characters and performs well both ingrowth and thermal tolerance. We investigated the tissue specific responses in thehistological alteration, enzymic changes (superoxidase dismutase, SOD; catalase,CAT; reduced glutathione, GSH; lysozyme, LZM and pyruvate kinase, PK) and theexpression of hsp70of P. olivaceus to elevated temperature. We compared these twospecies under different heat modes. We also cloned the hsp70/hsp90genes of P.olivaceus, P. dentatus and the hybrids. Furthermore, we investigated the the changesof P. olivaceus, P. dentatus and the hybrids in related enzymes and hsp70/hsc70/hsp90expressions at29℃, with the purpose of clarifying the effect of elevated temperature to these species, which may provide us a better understanding of themechanism of heat tolerance and hybrid vigor in marine fishes. The main results areas follows:1) The tissues of P. olivaceus responded differently to the elevated temperature.There were a range of histological changes in liver, kidney and gill, including theedema, epithelial lifting, desquamation and necrosis. The SOD, GSH and LZMshowed relatively higher activity in liver. According to the time point of highestexpression of hsp70, the tissues could be divided into two groups: the2-h groupcomprised the spleen, liver, gill and kidney, while the6-h group comprised thegut, stomach, muscle, heart and brain. When investigated the tissue specificexpression of hsp70at respective time points, we found that the brain and gillwere tissues of rich hsp70expression before heat shock. However, the liver, gilland muscle had much higher hsp70expression under high temperature, with thetop expression at the time point of1h,2h, and6h (and12h).It was worth mentioning that, the tissues with the most significant lesions (gill,liver and kidney) also responded much earlier (2h) in hsp70expression than othertissues, especially the gill and liver, since they have shown the most outstandingmanifestations both in histological, enzymic and mRNA levels, which made themideal candidate tissues in the further thermal study of this important species.2) In chronic heat mode, the cumulative survival rate (CSR) of P. olivaceus wasmuch lower than that of the hybrids, with the gill structure damaged moreseriously. In P. olivaceus, the activity of SOD, CAT, LZM and PK fluctuateddramatically, all of which declined around the temperature of29℃and32℃.Moreover, the hsp70expression in P. olivaceus also increased significantly at29℃and32℃, whose up-regulated ranges were higher than those in the hybrids.All of these corresponded to the changes of CSR, which suggested the P.olivaceus was more sensitive to the temperature of29℃and32℃.However, in the hybrids, the CSR was higher, the gill structure under hightemperature was relatively more complete, the enzymic changes were more stable,and the response of hsp70was later than that in P. olivaceus, with a lower up-regulation and a trend of fall back above29℃. These all implied us the betterthermal tolerance of this species.So far, we could synthesize the characters of heat sensitive species, which mightbe revealed in many levels, such as lower CSR, severe damages in tissuesstructure, tremendous changes in enzymic activity and the earlier and strongerreaction in hsp70expression. In contrast, a thermal tolerance species alwaysshows better CSR and stability in other levels.3) In acute heat mode (26℃,29℃and32℃), the CSR in the hybrids was higher,with the lag of death occurrence (4h) and mass mortality (12h) compared to P.olivaceus (1.5h,4h), and the gill structure in the hybrids was more completed.The changes in enzymic activity reflected that, the oxidation resistance andimmune defenses were weakened by the extreme temperature both in P. olivaceusand the hybrids, with the increase of energy expenditure. The hsp70expressionincreased significantly at0.5h both in these two species. However, these reactionsof hsp70in P. olivaceus were more violent and persistent, which expressed infaster and higher response, as well as slower fall after rise. We found the resultsin acute heat mode were largely consistent with those in chronic heat mode,which suggested the hybrids had a better thermal tolerance. However, thisadvantage was more remarkable in the chronic mode.4) The full-length cDNA of hsp70and hsp90were cloned from P. olivaceus, P.dentatus and the hybrids. The analysis indicated these two genes were both highlyconserved. The hsp90isolated in our study was more closed to hsp90-β.When been heat shocked at29℃, the changes in enzymic activity (SOD, GSH,LZM and PK) showed relatively similar trends, especially in the SOD and LZM,whose curve turned out to be an “M”. Analogous curve has been found in chronicheat mode. The expression of hsp70got more tremendous and regular changesthan hsc70and hsp90, which made it an ideal candidate gene in the study ofthermal tolerance in marine fish. |
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