| Global amphibian decline has led to public concern. Referring to relative studies about the effects of climate change on amphibain populations, we come up with two hypotheses to explain the causes of frog extinction. One hypothese is extreme low temperature stress mechanism hypothese. Frequent extreme weather is the main characteristic of global climate change, unseasonal or seasonal air temperature sharpfall may disturb the immune and antioxidant function, which increases the susceptibility to pathogenic microorganism and causes the populations decline. The other hypothese is interference mechanism hypothese of warm winter. The global climate change makes winter warm, which increases the overwintering temperature of frogs, and leads to temperature fluctuation in the daytime, and affects the normal hibernation. The frogs undergoing warm winter are possible much more sensitive to environment changes which is refered to hyperirritability. The hyperirritability could result in metabolic disturbance and immune function decrease which causes the populations recession.This paper selected the giant spiny frog (Paa spinosa), which was large economic species the population of which was shinking as research object, and monitored the winter temperatures experienced by wild tiger frogs in natural winter in the Southern Mountain of Jinhua in Zhejiang Province, and investigated the effect of acute cold stress and recovery and temperature fluctuant on viability, immune function, antioxidative defense, energy metabolism and relative Hsp70mRNA expression, the result of which could supply theoretical bases to investigate the cause of amphibian decline as result of global climate change. The main reasons are as follows:The control group was kept in22℃constantly, and the treatment group was maintained in4℃for12h/day and transferred to22℃for12h/day during the period of7days. The treatment samples were drawn discontinuously (0h、4h、12h、24h) on the first day and (12h、24h) on the seventh day, and detected the changes of the non-specific immunity, antioxidative defense and Hsp70mRNA expression. The results indicated that the respiratory burst of spleen macrophages and the peripheral blood phagocytic activity were both inhibited significantly after4℃and12℃(P<0.05), and restored to control level after recovery at22℃(P>0.05), while the inhibition effect disappeared on the seventh day; the gastric lysozyme activity was significantly inhibited for7days (P<0.05) and the malonaldehyde (MDA) content in liver and kidney were increased during the period of7days (P<0.05). Both the glutathione (GSH) content and the activity of superoxide dismutase (SOD) in liver and kidney were increased, and no significant differences were observed in glutathione (GSH) content (P<0.05). Moreover, Hsp70mRNA expression was significantly affected under the condition of cold stress(P<0.05). We can conclude that body immunity level and Hsp70mRNA expression were inhibited and body oxidative level was improved after cold stress, and confrontational capacitive change was detected in cellular immunity and antioxidative defense after cold stress repeatedly.The experiment animals were firstly acclimated at normal hibernation temperature (4℃) for one month to keep physical stability, which was thought to enter hibernation state. After acclimation, the group one was maintained in4℃, and the group two was set in temperature fluctuation by programming. The temperature in the group two was kept in4℃for5h, then gradually increased to12℃(2℃/h) for4h and finally decreased4℃(2℃/h) at every day, and repeated for30d. The aninals were sampled at initial stage, Id,7d and30d to detect the changes of immune function, energy metabolism and Hsp70mRNA expression. The results showed that six frogs in temperature fluctuation group were dead at the first week while only one was dead in constant temperature group. Non-specific immunity appeared to be in adaptation, for example peripheral blood phagocytic activity increase, and gastric lysozyme activity decresed significantly. In comparison to constant temperature group, temperature fluctuation resulted in increasing in varying degrees of blood phagocytic activity and gastric lysozyme activity. The liver glycogen content and the muscle glycogen content of temperature fluctuation group and constant temperature group were both obviously higher than that before hibernation, while blood glucose content was lower. Compared with constant temperature group, rising temperature in daytime at1d caused the significant increase of the liver glycogen content, and reasuted in massive consumption of the liver and muscles fat. In addition temperature fluctuation leaded to appearant high expression of Hsp70mRNA in liver. These results indicated that temperature fluctuation resulted in energy exhaust and enhance of immune fuction in frogs which possibly affect development and dynamics of amphibian populations. |
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