低温胁迫下虎纹蛙的生存力及免疫和抗氧化能力

Viability and changes of physiological functions in the tiger frog(Hoplobatrachus rugulosus) exposed to cold stress

全球气候变化是造成世界范围内两栖类种群衰退和灭绝的重要因素之一。随着极端天气出现变得日趋频繁,非季节性的、短期且剧烈的气温变化可能会严重干扰两栖类动物的生存与种群稳定。监测了浙江省金华市南山野生虎纹蛙(Hoplobatrachus rugulosus)分布区冬季的环境气温,并参考监测数据在实验室条件下研究了虎纹蛙在短期梯度降温(2℃/24 h)和急性冷暴露(即冷休克)(2℃)下的生存力及冷休克对机体免疫功能和抗氧化能力的影响。结果表明,虎纹蛙在冬季(2009-12-01—2010-03-31)经历的温度范围普遍在0—14℃之间,主要遭遇的低温区间在0—4℃,主要高温区间在10—14℃。通过梯度降温实验,发现温度降至12℃累积死亡率约为28.1%,10℃为87.5%,8℃为100%。在一定温度范围内,虎纹蛙死亡率与环境温度呈显著负相关(Pearson test,r=-0.952,P<0.05)。经曲线拟合,回归方程计算可得半数致死温度为11.5℃。虎纹蛙在冷休克处理下,在第6 h累积死亡率为45%,12 h为80%,24 h达到100%。虎纹蛙死亡率与冷休克时间呈显著正相关(Pearson test,r=0.91,P<0.05),半数致死时间为7.6 h。此外,冷休克(2℃,6 h)显著抑制了虎纹蛙脾脏巨噬细胞呼吸爆发强度(t=3.827,df=6,P<0.05)、全血吞噬活性(t=5.388,df=3.037,P<0.05)及胃溶菌酶活力(t=6.37,df=6,P<0.05);肝脏(t=0.773,df=8,P>0.05)和肾脏(t=0.164,df=4.542,P>0.05)组织脂质过氧化物产物丙二醛(MDA)的含量虽无明显变化,但肝脏(t=-2.817,df=6,P<0.05)和肾脏(t=-11.302,df=6,P<0.05)组织抗氧化物谷胱甘肽(GSH)含量及肝脏(t=-3.3,df=6,P<0.05)超氧化物歧化酶(SOD)活性均显著升高。上述结果表明虎纹蛙对低温较为敏感,冷休克能够诱导机体的免疫抑制,并导致机体对抗氧化物质需求的增加。可以推测,当遭遇极端低温天气时,低温胁迫可能会严重干扰虎纹蛙生理机能,加大种群的生存压力和疾病感染的风险。

Global climate change has been considered to be one of the key factors leading to declines and losses of amphibian populations. Though global warming is one of the acknowledged causes of fluctuations of amphibian population worldwide, unseasonal sharp fall in air temperature as result of global climate change may also affect the survival and stability of amphibian population severely. As amphibians are typical poikilothermic animals, environmental temperature can influence their physiological functions and enhance their susceptibility to infectious diseases. The immunological competence of amphibians may, therefore, make them vulnerable to parasites and disease particularly after discontinuous and extreme events （e.g. winter rainfall, late spring coldness）. In this article, we have investigated how cold stress affected the physiology and viability of tiger frogs （Hoplobatrachus rugulosus）. The winter temperatures experienced by wild tiger frogs in natural winter in the Southern Mountain of Jinhua in Zhejiang Province was monitored, and then replicated in subsequent experiments. The aim of the experiments was to determine how gradient cooling （2 ℃/24 h） and cold shock （2 ℃） affected the mortality rate of tiger frogs, and affected various aspects of their immune function and oxidation resistance. The results showed that the survival temperature range of wild overwintering was between 0-14 ℃ in 12/1/2009-3/31/2010, and minimum and maximum temperature ranged between 0-4 ℃ and 10-14 ℃ respectively. During the process of gradient cooling, we found that the accumulated mortality rate was 28.1% at 12 ℃, 87.5% at 10 ℃ and 100% at 8 ℃. A significant negative correction was found between environmental temperature and frog mortality within a certain temperature range （Pearson test, r=-0.952, P〈0.05）. And lethal temperature of 50% occurred at 11.2 ℃ based on a fitted curve regression equation. Under cold shock,45% mortality was observed after being directly stimulated at 2 ℃ for 6 h,80% mortality appeared at 2 ℃ for 12 h, and increased to 100% after 24 h. There appeared to be positive correction between cold shock time and mortality （Pearson test, r=0.91, P〈0.05）, and lethal time of 50% is 7.6 h at 2 ℃ according to fitted curve regression equation. Moreover, cold shock significantly inhibited the respiratory burst of spleen macrophages （t=3.827, df=6, P〈0.05）, peripheral blood phagocytic activity （t=5.388, df= 3.037, P〈0.05） and gastric lysozyme activity （t=6.37, df=6, P〈0.05） at 2 ℃ for （6 h）. Although there was no significant change in malonaldehyde （MDA） content, the glutathione （GSH） content of the liver （t=-2.817, df=6, P〈0.05） and kidney （t=-11.302, df=6, P〈0.05） increased. The activity of superoxide dismutase （SOD） also increased significantly in the liver （t=-3.3, df=6, P〈0.05）, while there was no apparent change within the kidney. The results indicate that tiger frogs are sensitive to low temperatures and that cold shock might lead to greater oxidative stress, more requirements for antioxidants and produce immunosuppression in tiger frogs. Consequently, low temperature stress will stimulate organism endocrine disorder and subject the frogs to a more intensive disturbance of physiological function, increase the survival pressure and susceptibility to infectious disease, which may affect development and dynamics of amphibian populations.