Developmental And Reproductive Toxicity Of Nereistoxins On Zebrafish

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.