The Characteristics Of The Nonspecific Immune Responses Of Paa Spinosa Under Various Environmental Stresses

Declines of global amphibians, which are thought to be a bellwether for environmental degradation, have led to public concern. However, there is less agreement about the causes of these declines. Complex interactions of several factors, such as habitat loss, over-harvesting, pollution, increased ultra-violet irradiation as well as disease, are probably at work. Global climate changes, especially global warming, are also linked to the amphibian decline. As ectotherm, the physiology of amphibians is uniquely determined by external environment. Especially, environmental temperature has a significant effect on their immune system and the components of this system are highly temperature dependent. Hibernation, characterized by extensive time spent in a hypometabolic and low body temperature state and no food ingestions, is a useful strategy for surviving such temperatures. However, the temperature in winter becomes higher gradually because of global warming and the warm winters may affect the depth and time of torpor. Immune system plays an important role in protecting themselves from pathogeny invading, such as bacterial, virus as well as fungus. But up to now, the researches about immunity is still scarce, and the work on the dynamic inflammatory responses of amphibian immunity is less, even fewer studies to date offer insight into the effect of warming hibernating temperature on the immunity of amphibians.The presented work chose Paa spinosa, one of Chinese edible frogs whose wild population has declined sharply and the breeding work of which is still in the stage of exploration as our experimental animals. We established the methods to determine the nonspecific immunity indexes of the frogs and took them as the indexes to evaluate the habitat pressures of the wild and cultured giant spiny frog. We also studied the dynamic changes of the nonspecific immune responses after bacterial infection intraperitoneally. Further more, we did an artificial hibernation experiment under laboratory conditions to imitate a warm winter. The chief results were listed as follows.(1) The gastric lysozyme activity of the cultured giant spiny frog was significantly higher than that of wild ones (P<0.01), which means the bactericidal activity of the former was better than the latter. The results were in accordance with their different habitat pressures. Because of the high density, the reared giant spiny frog was crowded and stressed by the Suspended sediment in the water constantly, and the possibility of pathogen infection of the reared frogs was much more than the wild ones, too. So it is extremely important to the survival of the reared frogs to possess better bactericidal activity than the wild ones. Therefore, the difference in lysozyme activity reflected the various habitat pressures of the cultured and wild frogs and it could be used as the indexes to evaluate the habitat pressures of them.(2) In order to imitate the inflammatory response after bacterial infection, we injected 0.2 ml of heat-killed Escherichia coli cell suspension (approximately 8×108 cells/ml in sterile PBS) into the frogs intraperitoneally and sampled at 1,3,6,12 and 24 h following injection respectively to study the dynamic changes of the nonspecific immune responses. The respiratory burst activity increased significantly after 3h (p<0.05), maximized after 6h post-injection and got back into the initial level in 24 h. Gastric lysozyme activity was inhibited significantly by bacterial stimulation (p<0.05) and recovered to the initial level until 24 h later. These results indicated that the nonspecific immune could be triggered significantly by bacterial stimulation, but there was obvious timeliness and difference among the immune responses.(3) To imitate a warm winter, we induced giant spiny frog to hibernate at 12℃and 4℃, sampled on the 30th,60th and 90th day, respectively to assay the non-specific immunity. It turned out that 12℃has strong lethal effect on wild frogs, which showed that warm winter could interfere with the normal hibernation of wild frogs. However,12℃has no lethal effect on cultured frogs, which probably because of the acclimation. The respiratory burst activity and gastric lysozyme activity of both the cultured and the wild frogs decreased, which meant the immune function inhibition during hibernation. The peripheral blood phagocytic activity of wild frogs increased continuously and that of cultured ones increased after a transitory fall. We inferred that it was the result of the percentage of phagocytes increases in peripheral blood and it showed phagocytes played a more important role in the immune defense while the function of spleen weakened during hibernation. The spleen coefficient of the wild frog hibernating at 12℃was obviously greater than that hibernating at 4℃, and this difference embodied the temperature dependence of immune organs. Added LPS to the NBT reaction system to calculate the stimulation index, we found that the stimulation index of the wild frog hibernating at 12℃was obviously inferior to that hibernating at 4℃. This result implied that the wild frogs hibernating at higher temperature could not respond exogenous bacterial stimulation quickly, which may be one of the causes leaded to their higher mortality. Differed from the wild frogs, both the respiratory burst activity and the spleen coefficient of the cultured frog hibernating at 12℃was significantly greater than that hibernating at 4℃at the end of the first month, implied the spleen immune function of the former was stronger than the latter and this may be the reason for their higher survival rate. The cultured frogs could maintain relative stability of immune function by suppressing their spleen immune function decrease during hibernating at higher temperature, which differed from the wild frogs. The differences of the response to the same hibernation temperature between the wild and the cultured giant spiny frog may be caused by temperature acclimatization.Through the present study, the immune dynamic variation as well as the effect of warm winter on giant spiny frog's population decline was explored preliminarily, which has important significance in not only revealing the causes of amphibian population decline, but also enriching the frogs'immunology study data, especially for the research of the Chinese important economic frogs. Further more, this study can also provide technical guidance for the artificial breeding of giant spiny frog.