Effects of hydroperiod duration on developmental plasticity in tiger frog(Hoplobatrachus chinensis) tadpoles

While developmental plasticity can facilitate evolutionary diversification of organisms,the effects of water levels as an environmental pressure on tiger frogs remains unclear.This study clarifies the relationship by studying the responses of tiger frog(Hoplobatrachus chinensis)tadpoles to simulated hydroperiods(i.e.,constant low water levels,constant high water levels,increasing water levels,decreasing water levels,rapid changes in water levels and gradual fluctuations in water levels)in a laboratory setting.ANOVA analysis showed that none of the water level treatments had any significant effect on the total length,body mass,or developmental stages of H.chinensis tadpoles half way through development(11 days old).Tadpoles raised in rapidly fluctuating water levels had protracted metamorphosis,whereas tadpoles raised under low and gradually fluctuating water levels had shortened metamorphosis.None of the water level treatments had a significant effect on the snout-vent length(SVL)or body mass of H.chinensis tadpoles at Gosner stage 42,or on the body mass of tadpoles at Gosner stage 45.However,the tadpoles raised in high levels and rapidly fluctuating water levels,significantly larger SVL at Gosner stage 45,while ones under gradually fluctuating water levels had smaller SVL than the other groups.Time to metamorphosis was positively correlated with body size(SVL)at metamorphosis in H.chinensis tadpoles.H.chinensis tadpoles under constant low water level had the highest mortality rate among all the treatments(G-test).Moreover,ANOVA and ACNOVA(with body length as the covariate)indicated that water levels had no significant effect on either the morphology(i.e.head length,head width,forelimb length,hindlimb length and body width)or the jumping ability of juvenile H.chinensis.These results suggest that the observed accelerated metamorphosis and high mortality of H.chinensis tadpoles under decreasing water level treatment was driven by density-induced physical interactions among increasing conspecifics.     

虎纹蛙蝌蚪对同域分布两种蝌蚪的食物选择

该文以虎纹蛙(Hoplobatrachus chinensis)蝌蚪为研究对象,检测其作为捕食者对同域分布两种不同类型(可口性和非可口性)猎物蝌蚪(黑眶蟾蜍和黑斑侧褶蛙)的食物选择及行为响应。结果表明,由于该两种被捕食物种在面对其共同捕食者或天敌时采取不同的反捕食策略(化学防卫及行为表现),即虎纹蛙蝌蚪在进食具有毒素的黑眶蟾蜍(Bufo melanostictus)蝌蚪后,能够在短时间内减少对这种可口性差的食物的选择,具有短期记忆行为,而黑斑侧褶蛙(Pelophylax nigromaculatus)蝌蚪能够采用行为防卫机制以减少被捕食风险,因此,虎纹蛙蝌蚪对两种猎物蝌蚪的总体选择不表现明显倾向。该结果为这3种两栖类动物的行为生态学研究提供了基础数据。     

Density But Not Kinship Regulates the Growth and Developmental Traits of Chinese Tiger Frog （Hoplobatrachus chinensis） Tadpoles

Kinship and density are believed to affect important ecological processes such as intraspecific competition, predation, growth, development, cannibalism, habitat selection and mate choice, In this work, we used Chinese tiger frog Hoplobatrachus chinensis tadpoles as an experimental model to investigate the effects of kinship and density on growth and development of this species over a 73 day period. The results showed that density can affect the growth and developmental traits （survival rate, larval period, size at the limb bud protrusion/metamorphic climax and body mass at different life stages） of H. chinensis tadpoles, while kinship does not. Tadpoles took longer to develop and potential metamorphosis was greater in high density groups of both sibling and non-siblings. The interaction of kinship and density did not significantly influenced growth traits of H. chinensis tadpoles during the experimental period. For coefficient variations of each growth trait, no differences were detected between sibling and non-sibling groups. These findings provide valuable information on the basic ecology of H. chinensis which will be helpful in future studies of other anuran species.     

杀虫剂硫丹对中国虎纹蛙的毒性效应： 存活率、红细胞核异常及酶活性

农药的使用对水生动物有直接和间接的影响。本文研究了有机氯杀虫剂硫丹对中国虎纹蛙（Hoplobatrachus chinensis）蝌蚪的毒性效应。具体而言,首先检测了硫丹的安全浓度（SC）,并在急性毒性实验中评估了硫丹的毒性等级。然后检测了外周血液中红细胞核形态异常,并在慢性毒性实验中测定了酸性磷酸酶（ACP）、碱性磷酸酶（AKP）和乳酸脱氢酶（LDH）的活性。结果表明,在急性毒性中,随着硫丹溶液浓度的增加和染毒时间的延长,蝌蚪的平均死亡率显著增加,即蝌蚪存活率取决于农药剂量和染毒的时长。硫丹对中国虎纹蛙蝌蚪的96 h半致死浓度LC50值为23.38 μg/L,安全浓度为2.34 μg/L,为剧毒性农药。在血液红细胞核异常实验中,观察到5种不同类型的细胞核异常：核碎裂、双核、不等分裂、核凹陷和核空洞。血液红细胞核异常率与农药浓度呈正相关。在酶活性检测中,三种酶的活性均受硫丹浓度的影响。与对照组相比,酸性磷酸酶（ACP）和碱性磷酸酶（AKP）活性随硫丹浓度的增加而降低,乳酸脱氢酶（LDH）活性随着硫丹浓度的增加呈先升高后降低。研究结果表明,硫丹对中国虎纹蛙蝌蚪具有很高的毒性,并证明了血液红细胞核异常和特定的代谢酶可以作为环境监测的生物标志物。     

异丙甲草胺、醚菌酯和咪鲜胺锰盐对中华大蟾蜍蝌蚪的急性致死效应

采用静态换水方法研究了异丙甲草胺、醚菌酯和咪鲜胺锰盐3种农药对中华大蟾蜍(Bufo gargarizans)蝌蚪的急性毒性及其联合毒性.结果表明,在急性毒性和两两联合毒性实验中,不同浓度农药对蝌蚪的死亡率影响均显著.在24 h、48h及72 h急性毒性实验中,异丙甲草胺对中华大蟾蜍蝌蚪的半致死浓度(LC50)分别为29.81、28.81和25.83mg/L,醚菌酯为1.72、1.46和1.41 mg/L,咪鲜胺锰盐为7.43、3.75和3.22 mg/L.异丙甲草胺、醚菌酯和咪鲜胺锰盐3种农药的安全浓度(SC)分别为8.07、0.32和0.29 mg/L.异丙甲草胺为中等毒性农药,而醚菌酯和咪鲜胺锰盐均为剧毒性农药.在两两联合的毒性实验中,异丙甲草胺-醚菌酯和异丙甲草胺-咪鲜胺锰盐均表现为中等毒性,醚菌酯-咪鲜胺锰盐表现为剧毒性.在24 h、48 h及72h两两联合毒性实验中,异丙甲草胺-醚菌酯对中华大蟾蜍蝌蚪的半致死浓度(LC50)分别为18.41、15.69和13.38 mg/L,异丙甲草胺-咪鲜胺锰盐为15.56、10.45和8.11 mg/L,醚菌酯-咪鲜胺锰盐为4.17、2.84和2.00 mg/L,且其相对应的安全浓度分别为3.42、1.41和0.40 mg/L.在联合毒性评价中,异丙甲草胺-醚菌酯联合组在24 h和48 h表现为拮抗作用,醚菌酯-咪鲜胺锰盐联合组在48 h表现为加和作用,其余均表现为协同作用.同样,重复方差分析结果表明,农药浓度、染毒时间及两者相互作用对蝌蚪的存活率影响均显著.与前期发表数据进行比较,表明中华大蟾蜍蝌蚪对污染物具有很强的敏感性.这些结果将为无尾类动物的毒理学研究提供科学依据.     

Isolation and characterization of microsatellite loci in tiger frog (Hoplobatrachus rugulosus)

Nine novel microsatellite loci were developed from the tiger frog (Hoplobatrachus rugulosus) using enrichment methods. The number of alleles ranged from 6 to 11 with an average of 8.56 alleles per locus. The observed and expected heterozygosity ranged from 0.571 to 0.935 (average 0.796) and 0.765 to 0.888 (average 0.837) respectively. These polymorphic loci can be used in population structure, gene flow, and population differentiation.     

低温胁迫下虎纹蛙的生存力及免疫和抗氧化能力

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。     

虎纹蛙选择体温和热耐受性在个体发育过程中的变化

体温是影响变温动物表现的最重要生理学变量。检测了国家二级保护动物虎纹蛙的雌性亚成体、雄性亚成体、幼体和蝌蚪这4个发育阶段的选择体温和热耐受性。单因子方差分析表明,虎纹蛙选择体温、耐受低温、耐受高温和温度耐受范围的组间差异均显著,幼体的选择体温(24.13℃)显著低于雌性亚成体(28.06℃)、雄性亚成体(29.27℃)和蝌蚪(28.23℃),雌性亚成体、雄性亚成体和蝌蚪之间差异不显著;幼体的耐受低温(13.85℃)显著高于雌性亚成体(11.27℃)、雄性亚成体(10.84℃)和蝌蚪(10.74℃),雌性亚成体、雄性亚成体和蝌蚪之间差异不显著;幼体具有显著低的耐受高温(35.48℃)、蝌蚪具有显著高的耐受高温(43.31℃),雌性亚成体(39.55℃)和雄性亚成体(39.02℃)的耐受高温差异不显著;幼体(21.62℃)具有显著小的温度耐受范围、蝌蚪(32.58℃)具有显著大的温度耐受范围,雌性亚成体(28.28℃)和雄性亚成体(28.18℃)的温度耐受范围差异不显著。虎纹蛙幼体和亚成体体温和水温之间在降温速度和升温速度的相关关系均显著。用回归剩余值去除水温变化速度对体温变化的影响,双因子方差分析(降温和升温速度为重复检验设置)表明,幼体的体温变化速度显著大于亚成体,两性亚成体间差异不显著;温度变化类型(降温和升温)和两因子的交互作用对体温变化的影响不显著。基本热生态位分离和体温调节能力的发育限制是形成上述现象的最可能的原因。     

浙江地区虎纹蛙肥满度研究

分别于2007年6～10月采集了浙江金华、临安和丽水三个地区的虎纹蛙(Hoplobatrachus rugulosus)共102只进行肥满度研究,以期为我国唯一的国家二级无尾两栖类的保护提供一定的参考依据。本研究结果显示:浙江地区虎纹蛙的肥满度指标K在夏秋季节、雌雄个体以及成幼体之间均没有显著性差异(P>0.05);而重/长指标Kwl在夏秋季节和成幼体之间则存在着极显著的差异(P<0.01),而在性别间的差异性却并不显著(P>0.05)。通过对虎纹蛙样本肥满度指标K和重/长指标Kwl的比较,得知Kwl具有更高的灵敏度,并建议在无尾两栖类的肥满度研究中将两种指标结合使用,以便得到更为详尽的研究结果。     

非离子氨和亚硝酸氮对虾虎鱼仔鱼的急性毒性及安全浓度评价

目的检测非离子氨和亚硝酸氮对鱼类的生态毒性效应。方法在水温（25±1）℃、溶氧（6.07～6.77）mg/L、盐度30～31、pH8.0～8.2的条件下,采用半静水式生物毒性试验方法研究了非离子氨和亚硝酸氮对裸项栉虾虎鱼（Ctenogobius gymnauchen）仔鱼的急性毒性效应（非离子氨浓度梯度设置为0、0.413、0.735、1.309、2.329、4.147 mg/L,亚硝酸氮浓度梯度设置为0、3.0、4.14、5.713、7.884、10.88 g/L）。结果非离子氨和亚硝酸氮暴露后仔鱼出现呆滞、侧游、呼吸困难、体色变白、身体弯曲等中毒症状,且随着暴露浓度的升高与暴露时间的延长,死亡率逐渐增加,存在明显的剂量效应关系和时间效应关系;非离子氨和亚硝酸氮对裸项栉虾虎鱼仔鱼96 hLC50分别为9.1 mg/L和12.405 g/L,其安全浓度分别为0.91 mg/L和1.2405 g/L。结论裸项栉虾虎鱼对非离子氨和亚硝酸氮具有较强的耐受力,非离子氨对裸项栉虾虎鱼仔鱼的毒性显著大于亚硝酸氮。     

Evolutionary significance of oral morphology in the carnivorous tadpoles of tiger frogs, genus Hoplobatrachus (Ranidae)

We describe the larval stages of three species of the Asian-African tiger frogs Hoplobatrachus chinensis, H. occipitalis and H. tigerinus . The tadpoles of all three species are very similar, with peculiar oral features: (1) double rows of needle-like labial teeth, (2) strong emarginations on the large jaw sheaths and (3) keratinized spurs on the buccal floor. Characters 1 and 2 (and perhaps 3) are probably related to the carnivorous habits of these tadpoles. A molecular phylogeny based on 2430 base pairs of two nuclear and four mitochondrial genes corroborated monophyly of Asian and African Hoplobatrachus, and identified Euphlyctis as their sister group. Tadpoles of the latter genus lack buccal spurs and double labial tooth rows but share large jaw sheaths, the upper with a medial projection. Therefore, the common ancestor of Euphlyctis and Hoplobatrachus probably was also characterized by this state, and may have been facultatively carnivorous. Further carnivorous specializations in Hoplobatrachus could explain why tiger frogs have been so successful in populating arid environments where ponds are at high risk of desiccation. Larval morphology may prove to be the key innovation which enabled them to disperse, in the Late Cenozoic, into their current very wide distribution area in Asia and Africa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 171–181.     

3种酰胺类稻田除草剂对克氏原螯虾的急性毒性

[目的] 研究稻虾共作模式条件下,稻田封闭除草剂对克氏原螯虾产生的急性毒性。[方法] 采用半静态试验方法,研究3种酰胺类除草剂（乙草胺、丙草胺和丁草胺）对克氏原螯虾的急性毒性,计算3种酰胺类除草剂对克氏原螯虾的安全浓度,并分析其LC₅₀衰减规律。[结果] 随着暴露时间和除草剂浓度的增加,虾体侧躺,步行足和游泳足活动频率降低,最终死亡。乙草胺对克氏原螯虾的96 h半致死浓度（LC₅₀）和安全浓度（SC）分别为0.0707和0.0146 mL·L^-1;丙草胺对克氏原螯虾的96 h LC₅₀和SC分别为0.0119和0.0021 mL·L^-1;丁草胺对克氏原螯虾的96h LC₅₀和SC分别为0.0073和0.0014 mL·L^-1。乙草胺、丙草胺及丁草胺除草剂LC₅₀随着暴露时间延长呈下降趋势,符合双曲线衰减模型,回归方程分别为：① y₁=2.0840x^-0.7380（R²=0.9973）;② y₂=0.1106x^-0.4930（R²=0.9872）;③ y₃=0.2236x^-0.7480（R²=0.9990）。克氏原螯虾对3种除草剂的敏感性由高到低依次为：丁草胺 > 丙草胺 > 乙草胺。[结论] 乙草胺和丙草胺可在稻田综合种养生产过程中按常规剂量使用,而丁草胺按常规剂量使用可能存在较大的风险。     

三种农药对斑马鱼的急性毒性和生物浓缩系数

采用斑马鱼(Brachydanio rerio)为试验生物,研究了吡虫啉、三唑磷和哒螨灵的急性毒性和生物浓缩系数．通过毒性试验,获得了这3种农药对斑马鱼的24、48、72和96h LC50值．这3种农药的96h LC50值分别为281．37mg·L^-1、8．37mg·L^-1和11．66μg·L^-1．根据急性毒性数据和有关技术指南,确定生物浓缩试验浓度和持续暴露时间为14d．经0．5和5mg·L^-1吡虫啉暴露后的生物浓缩系数(BCFs)分别为1．52和0．97．经0．02和0．2mg·L^-1三唑磷暴露后的BCFs分别为9．00和8．45．经0．1和1μg·L^-1哒螨灵暴露后的BCFs分别为5600和4920．     

三聚氰胺对花鲈的急性毒性实验研究

本文以花鲈(Lateolabrax japonicus)为实验对象,采用接触、腹腔注射及口服三种致毒方式,进行了非蛋白氮物质-三聚氰胺对花鲈的急性毒性实验研究。口服急性毒性实验中,通过在饲料中添加不同浓度的三聚氰胺（0、500、2000、5000及10000 mg/kg）,进行了21d的花鲈养殖实验。实验结果表明：三聚氰胺溶解度较低,其水溶液没有表现出急性毒性,LC50 > 3500 mg/L;而在腹腔注射致毒方式下,三聚氰胺对花鲈的半致死剂量LD50 = 890.07 mg/kg•w;LD5095%可信限为：778.63－1017.45 mg/kg•w;在口服急性毒性实验中,10000 mg/kg组三聚氰胺降低了花鲈的摄食与生长(p<0.05),饲料系数显著升高 (p<0.05);三聚氰胺对花鲈的存活率、肥满度、肝体比及脏体比均无显著影响 (p>0.05)。饲料中添加三聚氰胺没有显著影响花鲈血清的丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总蛋白、葡萄糖、胆固醇、甘油三酯及尿素氮 (p>0.05),但显著影响了花鲈血清的碱性磷酸酶活性,10000 mg/kg组的碱性磷酸酶活性显著高于其他各处理组 (p<0.05),其他各组间无显著差异(p>0.05)。三聚氰胺对花鲈21d的最大未观察到有害作用剂量（NOAEL, no-observed-adverse-effect-level）为131.99 mg/kg•w•d。     

Acute toxicity tests on three species of the genus Lecane (Rotifera: Monogononta)

Three rotifer species, Lecane hamata L. luna, and L. quadridentata, were submitted to acute toxicity tests to compare their susceptibility to 11 toxicants. In acute tests with 48-h exposure of neonates of less than 24 h old, copper was most toxic with LC₅₀ values in the range of 0.06–0.33 mg l^–1, while acetone was the least toxic with LC₅₀ values in the range of 5000–7000 mg l^–1. Differences in LC₅₀ value of up to 22-fold were found in the susceptibility to lead between the three species. These data indicate large differences in toxicity among members of the same genus, and point out that it is necessary to submit several species to toxicity tests in order to assess the potential effects of toxicants to rotifers. The commonly used Brachionus calyciflorus cannot be considered representative of all freshwater rotifers in this respect.     

虎纹蛙皮肤分泌物抗氧化二肽——Gln/Lys-Met的纯化及鉴定

目前已自青蛙皮肤分泌物中提取出多种属于“第三套抗氧化系统”的多肽,它们具有较强的抗氧化作用.本文从虎纹蛙皮肤分泌物中提取具有抗氧化活性的多肽物质,并检测其活性.利用电刺激虎纹蛙背部腺和耳后腺获得其皮肤分泌物,利用凝胶过滤色谱Sephadex G-50和反相高效液相色谱（reverse-phase high performance liquid chromatography, RP-HPLC）进行分离纯化.以2,2-二苯基-1-苦肼基（2,2-diphenyl-1- picrylhydrazyl, DPPH·）清除率为指标,测定多肽的抗氧化活性,经ESI- MS（electrospray ionization mass spectrometry）质谱测定该多肽的相对分子质量.结果显示,自虎纹蛙皮肤分泌物中获得一种具有抗氧化活性的二肽,其分子质量为0.277 kD,可能的氨基酸序列为Gln/Lys-Met.虎纹蛙皮肤分泌物中的该二肽是发挥抗氧化作用的重要组成部分.     

生姜精油对黑腹果蝇的急性毒性分析

在蔗糖水溶液中加入不同浓度的生姜精油饲喂野生型黑腹果蝇成虫,用时间-剂量-死亡率模型对96h内各处理果蝇成虫累计死亡率随时间、剂量的变化进行拟合.结果表明:较高浓度的生姜精油对果蝇成虫具有较强的急性毒性.0.5%生姜精油作用72h,果蝇成虫的累计死亡率分别为雌88%、雄79%;1%和10%生姜精油作用72h,雌雄成虫的累计死亡率均达100%.随着处理时间的延长,达到50%致死水平所需生姜精油浓度(即LC50)逐渐降低.Tween-20对果蝇成虫(尤其是雄虫)也有较强的毒性,高浓度时作用尤为明显.     

Cu对中国林蛙蝌蚪的急性毒性

试验测定了不同温度条件下Ｃｕ２＋对中国林蛙蝌蚪的急性毒性．结果表明,在水温为９～１３℃、１８℃和２４℃条件下,Ｃｕ２＋对１０ｄ龄中国林蛙蝌蚪９６ｈ的ＬＣ５０分别为５．０１、３．８０和２．９９ｍｇ·Ｌ－１,致死阈浓度分别为４．０、２．５和１．６ｍｇ·Ｌ－１,而在９６ｈ内的无可观察效应浓度分别为２．５、１．６和１．０ｍｇ·Ｌ－１．１８℃条件下Ｃｕ２＋对２０ｄ龄中国林蛙蝌蚪的９６ｈＬＣ５０为２．１７ｍｇ·Ｌ－１．     

3种除草剂对七星瓢虫的急性毒性与初级风险评估

为了评估除草剂对七星瓢虫Coccinella septempunctata的毒性影响和初级风险,采用药膜法进行了3种除草剂的急性接触毒性测试,并评估其初级风险。结果表明：10%噁唑酰草胺乳油、10%噁嗪草酮悬浮剂和20%异噁唑草酮悬浮剂对七星瓢虫的急性接触毒性分别为LR₅₀>3.61×10² g a.i./ha, LR₅₀>1.485×10² g a.i./ha, LR₅₀>3.60×10² g a.i./ha。10%噁唑酰草胺乳油、10%噁嗪草酮悬浮剂和20%异噁唑草酮悬浮剂的相对安全系数<5,属于中等及以下风险农药,对七星瓢虫的农田内和农田外喷雾场景风险均可接受(危害商分别为HQ_in<0.997,HQ_in<2.000,HQ_in<1.000,HQ_in均<5;HQ_off<0.020,HQ