| Global amphibian population decline has become a focus issue in recent years. The complex reasons include global warming, infectious disease and so on. As the transitional population from aquatic to terrestrial, amphibians are considered as reliable indicators of environmental quality, because they are characterized by some unique biochemical and ecological features and particularly sensitive to environmental changes. Ascorbic Acid (AA), a vital and multifunctional micronutrient, plays an important role in improving skeletal development, resisting stress and oxidation as well as regulating immunity. Moreover, AA is also a typical representative of antioxidants in amphibians and reptiles during evolution. Considering the importance of AA in biological function and evolution, this article chose AA levels in tissues and the key enzyme in AA biosynthesis—L-gulono-1,4-lactone oxidase activity (GLO) as biomarkers to evaluate the health condition and survivability of the amphibians, and studied their response efficiency and sensitivity in stress condition.Giant spiny frogs (Paa spinosa) usually inhabit subtropical or tropical high-altitude montanes. They are very sensitive to the changes of environmental factors, such as pathogenic microorganisms and climate factor. The resistance to environmental stressor is also relatively weak, so giant spiny frogs can be used as one of the indicators in monitoring environmental changes. In addition, there is high morbidity in giant spiny frogs'breeding, which explains its sensitivity to the environment factors further. So this article chose giant spiny frogs as the research objects to study the changing regularity of AA level in tissues and GLO activity by simulating bacteria invasion and warmer winter state. The main results were listed as follows: 1. The determining method of giant spiny frogs' GLO activity was established and optimized. AA levels in tissues and GLO activity were taken as evaluation indexes of habitat stress to study the health condition and survivability of both the wild and cultured giant spiny frogs. The GLO activity was determined using the DNPH colorimetric method and optimized by evaluating substrate (L-gulonolactone) and glutathione (GSH) concentration, incubation temperature and time. The results presented that adding 10 mM substrate and no GSH into the reaction medium with 60 minutes' incubation at 25℃was the enzymatic optimal reaction condition, and the enzyme activity amounted to (17.71±0.08)μg TAA/mg soluble protein/h. Compared with wild giant spiny frogs, GLO activity in cultured population was higher but AA levels in tissues were less. In other words, cultured giant spiny frogs consumed more AA in survival environment stress, which was consistent with their survival pressure. These results implied that AA level and GLO activity can be served as the indexes to evaluate the habitat stress of giant spiny frogs.2. To imitate the inflammatory response and oxidative stress after bacterial infection, cultured giant spiny frogs were injected 0.2 ml of heat-killed Escherichia coli (E.coli) suspension intraperitoneally and sampled at 1,3,6,12 and 24h following injection respectively to study the dynamic changes of the respiratory burst activity of spleen macrophagocyte, AA level in tissues and GLO activity. The present results indicated that after injecting heat-killed E.coli, NBT reaction intensity and renal AA concentrations increased obviously(P<0.05). On the contrary, GLO activity decreased significantly(P<0.05). However, all of the indexes recovered to the initial level within 24h. These results confirmed that injecting heat-killed E.coli could stimulate giant spiny frogs'spleen macrophagocyte to produce more oxygen free radicals and increase the organismal oxidation degree. Moreover, to a certain extent, it also showed that AA levels in tissues and GLO activity could reflect frogs'health condition and survivability effectively under the bacteria stress.3. The research on the effect of different cold exposure temperatures on AA level, GLO activity as well as energy utilization of wild and cultured giant spiny frogs:wild and cultured frogs were exposed at 12℃and 4℃at the same time,12℃has obvious lethal effect on wild frogs, which showed that warmer winter could interfere with the normal hibernation of wild frogs and cause them to death. However,12℃has no lethal effect on cultured frogs, which probably because the acclimation enhanced their tolerance to warmer winter. Compared with pre-exposure, hepatic and renal AA concentrations as well as GLO activity of wild giant spiny frog decreased, but only hepatic AA level of frogs hibernating at 12℃declined significantly (P<0.05). In contrast, hepatic AA level of cultured frogs hibernating at either 12℃or 4℃descended a little(P>0.05), but renal AA concentrations increased significantly first and then markedly reduced(P<0.05). GLO activity was inhibited obviously(P<0.05), which reflected the temperature dependence of enzymes. These results indicated that higher hibernation temperature made AA consumption increase, suggesting that ascending hibernation temperature would trigger greater oxidative stress to frogs. Therefore, the AA levels in tissues could reflect the oxidative stress caused by cold exposure and could be served as the biomarker. Wild giant spiny frogs hibernating at 4℃showed cryogenic tolerability, for example, the glycogen level in liver increased (P<0.05), and the consumption of dry matter(excluding fat) in muscle of frogs hibernating at 12℃ascended significantly(P<0.05). But cultured giant spiny frogs hibernating at 4℃did not appear glycogen level elevation and showed no cryogenic tolerability, consuming more hepatic dry matter(excluding fat), which indicated that the adaption ability of cultured giant spiny frogs to low temperature declined.In conclusion, hepatic and renal AA level as well as GLO activity of giant spiny frogs could show the corresponding changes in bacteria invasion or warmer winter state, which reflected the stress state of organism. Therefore, AA concentrations in tissues and GLO activity could be served as the biomarker to reflect the health condition and survivability of organism. Because of its sensitivity to the environment factors, giant spiny frogs could be used as one of the indicators in monitoring environmental changes and their threatened status could forecast the environmental quality. In addition, this study could offer reference for the breeding and protection of giant spiny frogs. |
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