【作者】 周胜利；

【导师】 朱国念； 徐志宏；

【作者基本信息】 浙江大学 ， 环境生物学， 2010， 博士

【摘要】 沙蚕毒素类杀虫剂是应用最为广泛的一类杀虫剂之一。本论文以斑马鱼为模式生物,系统研究了沙蚕毒素类杀虫剂对成鱼的急性毒性以及对胚胎的发育毒性和致畸效应。试验结果表明：98%杀螟丹原药、18%杀虫双水剂、90%杀虫胺原药、83.7%杀虫环原药和沙蚕毒素对成鱼的96小时急性毒性LCso分别为0.280、28.8、36.7、0.172和0.444mg/L。98%杀螟丹原药、95.5%杀虫双纯品、18%杀虫双水剂、99%杀虫单纯品、90%杀虫胺原药、83.7%杀虫环原药和沙蚕毒素染毒都能抑制胚胎孵化,并且产生脊索弯曲、胚胎白化等相同表型,并且随着浓度的增加,各个部位异常症状出现的先后顺序具有很强的相似性。同时这几种杀虫剂都能抑制鱼鳔的正常充气膨大。对胚胎96小时致死中浓度分别为0.459、>128、12.5、>128、19.4、0.0879和0.119mg/L。对胚胎脊索形成抑制中浓度分别为0.0300、63.0、0.532、>128、0.578、0.00244和0.0127mg/L。脊索弯曲基本上是评价沙蚕毒素系列杀虫剂的最敏感指标,部分最低作用浓度甚至达到ppb级甚至更低,抑制中浓度相比于成鱼急性毒性致死中浓度普遍低两个数量级左右。因此根据成鱼急性毒性试验得到的结果是沙蚕毒素系列杀虫剂(包括沙蚕毒素)对鱼类普遍低毒,而据胚胎试验表明这类农药对鱼类普遍高毒.由于沙蚕毒素类农药本身分子结构的相似性和该类农药对斑马鱼胚胎造成相同的表型,作者认为该类农药具有相同的作用方式。故选用了杀螟丹作为典型进一步研究了其作用机制。脊索弯曲的试验结果表明,胚胎自发运动在脊索弯曲形成过程中起着重要作用,并且脊索弯曲部位和敏感性与染毒阶段相关。0.0250mg/L及以上浓度的杀螟丹抑制赖氨酰氧化酶活性,并呈剂量-效应关系。而0.100mg/L及以上浓度染毒上调了该基因的表达水平。由于本研究中脊索弯曲表型与文献中赖氨酰氧化酶基因沉默后表型相似,因此推测赖氨酰氧化酶是本论文中导致脊索弯曲的靶标。胚胎白化试验结果表明,0.0500mg/L及以上浓度的杀螟丹染毒抑制酪氨酸酶活性,并呈浓度-效应关系,并且在相同浓度下染毒,该基因表达水平上调。基于酪氨酸酶在黑色素形成过程中的关键作用,作者认为酪氨酸酶是本论文中导致胚胎白化的靶标。胚胎孵化试验结果表明,杀螟丹对胚胎孵化腺形成和孵化酶的活性无影响,而影响卵膜的消解,同时在胚胎孵化前去除杀螟丹可有效提高胚胎孵化率,因此作者认为本论文中胚胎孵化受阻是因为孵化酶的释放受抑制所导致。通过测定杀螟丹对斑马鱼一些重要器官形态发生的影响,结果表明,0.100mg/L及以上浓度的杀螟丹染毒影响斑马鱼胚胎中枢神经神经元的分化增殖,造成胚胎体节间血管变细并且排列混乱。0.0500mg/L及以上浓度杀螟丹处理抑制胚胎颅面软骨形成,造成部分软骨组成变得短小,并且影响胚胎肝脏和肠道的形态发生。本研究还测定了杀螟丹和沙蚕毒素对斑马鱼生殖的影响。试验结果表明,0.0150mg/L及以上浓度的杀螟丹和沙蚕毒素染毒轻微提高了雄鱼性腺指数,而0.0250mg/L及以上浓度的杀螟丹染毒降低了雄鱼精子活力三成以上,并且0.0150mg/L及以上浓度的杀螟丹降低亲鱼的繁殖力,对亲鱼交配次数和总产卵量都有明显影响。更多还原

【Abstract】 Nereistoxins are one of the widely used insecticidal classes. This thesis took the advantages of zebrafish model to investigate the developmental toxicity and its mechanisms of nereistoxins. The results of adult fish acute toxicity test showed that LC50 values of 98% Cartap Technical,18% Bisultap AS,90% Profurite-aminium Technical,83.7% Thiocyclam Technical and Nereistoxin were 0.280,28.8,36.7,0.172 and 0.444mg/L respectively. With exception of 99% Monosultap Standardal, embryos exposed to 98% Cartap Technical,95.5% Bisultap Technical,18% Bisultap AS,90% Profurite-aminium Technical,83.7% Thiocyclam Technical or Nereistoxin all exhibited similar defect phenotypes including wavy notochord, less melanin pigmentation, hatching failure and inflation failure of swimming bladder, and almost all of these abnormalities displayed a dose-dependent manner. Among these abnormalities, notochord was the most sensitive endpoint. The EC50 values for notochord defect were 0.0300,63.0,0.532,0.578,0.00244 and 0.0127mg/L respectively, comparing with the 96h LC50 values of embryo test which were 0.459, >128,12.5,19.4,0.0879 and 0.119mg/L respectively. These results indicated that most insecticides of nereistoxins were highly toxic to fish rather than lowly toxic as the adult fish acute toxicity test suggested.As these nereistoxins were with similar chemical structures, and for above defects, they also shared with the similar hierarchies of concentration thresholds, suggesting that they assaulted the same targets which resulted in the same defect phenotypes. Thus, the cartap was adopted as a typical drug to investigate the underlying mechanisms. The results showed that the wavy location of notochord was close related to the exposure period and the spontaneous movement of embryo was essential in the formation of notochord waviness. Cartap of 0.0250mg/L and above inhibited the activity of lysyl oxidase in a dose-dependent manner, whereas emryos exposed to concentrations of 0.100mg/L and above displayed an upregulated expression of lysyl oxidase gene. Considering on the notochord defect here being a phenocopy of morpholino knockdown of lysyl oxidase gene in the studies of some other researchers, nereistoxins were supposed to affect notochord morphogenesis via targeting this enzyme. The results also showed that embryos exposed to cartap at concentrations of 0.0500mg/L and above lost tyrosinase activity in a dose-dependent manner, whereas the expression of tyrosinase gene was upregulated. Given the essential role of tyrosinase in melanin pigmentation, the tyrosinase was supposed to be targeted by nereitoxins which resulted in less melanin pigmentation of embryos. For embyos of hatching failure, no digestion of chorion was observed during the whole exposure period, whileas, microscopic observation indicated that the formation of hatching gland seemed not to be affected and getting rid of cartap at pre-hatching stage could significantly rescue hatching failure. Besides, in vitro test indicated no effect of cartap on the activity of hatching enzyme, suggesting that nereitoxins arrested embryos from hatching via blocking the secretion of hatching enzyme.The effects of cartap on the morphogenesis of some organs were also examined. The results showed that cartap at concentrations of 0.100mg/L and above significantly reduced the proliferation of neurons in middle neural system and caused abnormal arrangement and thinning of intersegmental blood vessels. Moreover, the resulted also indicated that cartap at concentrations of 0.0500mg/L and above shortened some components of facial cartilage and inhibited the morphogenesis of liver and intestine.In this thesis, the author also investigated the effects of cartap and nereistoxin on the sperm quality of male fish and the effects of cartap on reproductivity. The results indicated that 0.0150 mg/L and above concentrations of both drug could slightly increase the GSI while, cartap of 0.0250 mg/L and obove could obviously reduced sperm activity by more than thirty percents. Moreover, parent fish exposed to cartap of 0.0150 mg/L and above became relative infertility by more than fourty percents.更多还原