Effects Of Heat Stress On Non-specific Immunity And HSP70Gene Expression In Three Koi Carp Strains

In order to explore the mechanism of koi’s anti-stress and the differences among different strains of koi, the study was performed to examine the effect of continuous heat stress on non-specific immune parameters and HSP70gene expression in Yamabuki Ogon, Showa Sanshoku, Kohaku. The fish were exposed to an increase in water temperature of3.5℃/h from25to32℃(The heating time was2h). Then the fish were continuously exposed to water temperature of32℃for24h. Samples for detections of plasma complement protein3(C3), plasma superoxide dismutase (SOD), plasma malondialdehyde (MDA), plasma alkaline phosphatase (AKP), plasma MPO (myeloperoxidase), respiratory burst (RB) of whole blood and relative expression content of HSP70were taken before stress, after stress0,4,8,12,16,20,24h, respectively.The results were as follows.(1) RB activity of three strains of koi had no significant changes after stress (P>0.05). In the whole course of experiment, RB activity of Kohaku and Showa Sanshoku were significantly higher than Yamabuki Ogon (P<0.05).(2) The levels of plasma complement C3increased in Yamabuki Ogon and Kohaku after heat stress, and it increased significantly after stress12-24h (P<0.05). The levels of plasma complement C3decreased in Showa Sanshoku after stress, and it decreased significantly after stress8-20h (P<0.05). The level of plasma complement C3of Showa Sanshoku was significantly higher than that of Yamabuki Ogon and Kohaku before stress (P<0.05), while the level of plasma complement C3of Showa Sanshoku was significantly lower than that of Yamabuki Ogon (P<0.05) and lower than Kohaku (P>0.05) at the end of stress.(3) Plasma SOD activities of three strains of koi had no significant changes after stress (P>0.05). In the whole experiment course, plasma SOD activities of Yamabuki Ogon were significantly higher than that of Showa Sanshoku and Kohaku (P<0.05).(4) Plasma MDA content decreased in Yamabuki Ogon, and it decreased significantly after stress8-24h (P<0.05). Plasma MDA content incresed at first and then decresed in Kohaku after stress, and it incresed significantly after stress16h (P<0.05). Plasma MDA content incresed in Showa Sanshoku, and it incresed significantly after stress 0,8,16-24h. Plasma MDA content of Yamabuki Ogon was significantly higher than that of Showa Sanshoku and Kohaku before stress (P<0.05), while plasma MDA content of Showa Sanshoku was significantly higher than that of Yamabuki Ogon and lower than Kohaku at the end of stress (P<0.05).(5) Plama AKP activities incresed in Yamabuki Ogon and Kohaku after stress, and increased significantly after stress8-24h and12-16h in Yamabuki Ogon and Kohaku, respectively. Plasma AKP activities of Showa Sanshoku decreased after stress, and it decreased significantly after stress20h (P<0.05). Before stress and after stress0-4h, the levels of plasma AKP of three strains koi were Showa Sanshoku>Kohaku>Yamabuki Ogon; after stress8-24h, the levels of plasma AKP of three strains koi were Kohaku>Showa Sanshoku>Yamabuki Ogon.(6) Compared to before stress, plasma MPO activities of Yamabuki Ogon and Kohaku had no significant changes (P>0.05) after stress; while plasma MPO activities of Showa Sanshoku increased after stress, and increased significantly after stress12,20,24h (P<0.05). In the whole experiment course, plasma MPO activities of Yamabuki Ogon were lower than that of Showa Sanshoku and Kohaku, except after stress4h and8h.(7) After heat shock, a similar time-dependent pattern was observed in the mRNA expression of HSP70in the three strains of koi. after stress Oh, the mRNA expression of HSP70increased significantly (P<0.05) and reached the peak, and increased by77.7,26.8,8.9-fold that of before stress in Yamabuki Ogon, Kohaku, Showa Sanshoku, respectively; after stress4-16h, the mRNA expression of HSP70recovered to the levels before stress; after stress20h, the mRNA expression of HSP70increased again. Among the three strains of koi, HSP70mRNA expression were Yamabuki Ogon>Kohaku>Showa Sanshoku after stress Oh and20h, Kohaku>Yamabuki Ogon>Showa Sanshoku after stress24h, and had no significant difference at other experiment time. These resullts indicated that the changes of non-specific immune parameters and HSP70mRNA expression were quite variable after heat stress in three strains of koi. After heat stress, HSP70mRNA expression was induced significantly in three strains of koi, and plasma Complement C3and AKP were induced significantly in Yamabuki Ogon and Kohaku, and plasma MPO in Showa Sanshoku. Considering of the differences of anti-stress ability in the three strains of koi in practice, it was deduced that complement C3, AKP, MPO and HSP70induced by heat shock played a considerably important role in coping with changes caused by heat stress.For further study the mechanisms of HSP70in antistress response in fish, our study cloned the full-length sequence of HSP70of koi using5’RACE and3’RACE. HSP70of koi was2289bp and encoded643amino acids with predictied molecular mass of7.046kD, and theoretical isoelectic point of5.47. HSP70was negatively charged, and stable in solution. Sequence analysis of the protein showed that HSP70of koi contained three signature sequences of HSP70family and a cytoplasm characteristic motif of EEVD and a tetrapeptide motif GGMP at C-terminus. The homology analysis indicated that HSP70of koi shared a highest identity of97.67%with the HSP70s of Ctenopharyngodon idella, and also shared as high as73.04%identity with the non-vertebrate of Drosophila auraria. HSP70of koi had no notable tansmembrane regions and signal peptide which indicated that the protein was non-transmenbane and non-secretory protein. HSP70of koi was hydrophilic, and had27potential antigenic epitopes. Secondary structurs of the protein were mainly alpha helixes and random coils, and little beta sheet and beta turn. We acquired dimensional structure of HSP70of koi by homology modeling.