Toxcity Study Of Two Kinds Of Diamide Insecticides To Zebrafish Embryo

Diamide insecticides such as chlorantraniliprole and cyantranilipole are a new class of insecticide that selectively target insect ryanodine' receptors (RyR) which leads to uncontrolled calcium release in muscle, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera around the globe. The global turn-over of the whole chemical class was >$1.2 billion in 2013, representing approx. 8% of the insecticide market. The only report of diamide toxic evaluation on zebrafish (Danio rerio) was acute toxic, but the embryos toxic and toxicity mechanism remain unknown. Hence the toxicity of chlorantraniliprole and cyantranilipole on zebrafish embryos were conducted.Developmental toxicity and the mode of action concerning apoptosis for three formulations of chlorantraniliprole on zebrafish embryos were studied using the method of acridine orange staining and TUNEL assay, along with RT-qPCR. Developmental toxicity together with the whole mount and apoptosis at 24h for cyantranilipole on zebrafish embryos were investigated with acridine orange staining, TUNEL assay, Annexin V FITC/PI double-staining, JC-1 staining flow cytometry and RT-qPCR, meanwhile, studies on DNA damage, developmental toxicity and the genetic toxicity regulatory mechanism on zebrafish embryos were also applied using DAPI staining and single cell gel electrophoresis (SCGE). The results indicate that:1. Acute toxicity of the three formulations of chlorantraniliprole (96h-LC50) to zebrafish embryo are over 80 mg a.i.·L-1, 32.34 mg a.i.·L-1 (CL27.92-36.60 mg a.i.·L-1) and 25.96 mg a.i,·L-1 (CL24.59-33.59 mg a.i.-L-1) for the aqueous solution, suspension concentrate and granules respectively, which are all classified as low toxicity formulations.As a result, the toxicity of granules tops the three of them, suspension concentrate ranks the second and the aqueous solution is the least toxic one. It is also concluded that high concentration suspension concentrate and granules have significantly (p<0.05) induced the reduction in the body length of zebrafish embryos, while the aqueous solution does not. All of the three formulations of chlorantraniliprole can induce pericardial cyst and yolk cyst,with the granules making the biggest difference, followed by the suspension concentrate and the aqueous solution respectively.2. Both the results of acridine orange staining and TUNEL assay, indicate that apoptosis are the heart region, yolk and muscular cells with the granules and the suspension concentrate being equally toxic than the aqueous solution. Apoptosis pathway for the three formulations of chlorantraniliprole on zebrafish embryos were investigated using RT-qPCR, and the result of which shows that all the three formulations can induce apoptosis through the apoptosis pathway mediated by p53.3. The results indicate that cyantranilipole (95%) was classified as a medium toxic insecticide with 96h-LC50 being 1.57 mg·L-1 and the 95% confidence interval being 1.45-1.71 mg·L-1.4. When the concentration of cyantranilipole is over 1.00 mg·L-1, significant (p<0.05)reduction on the body length and heart rate can be detected and the number of embryos showing pericardial cyst, yolk cyst and spine deformity is more than that of the blank control group when the concentration goes up over 1.41 mg·L-1.5. The activity of CAT and SOD and the MDA content did not change significantly induced by cyantranilipole in zebrafish embryo.6. Embryonic DNA damage of cyantranilipole was detected using comet assay and DAPI staining micronucleus at the stage of 24hpf, the result indicates that the micronucleus rate, the DNA content on the comet tail and the tail distance are all significantly (p<0.05) increased when its concentration goes up,which signifies that there is a good dose-effect relation between cyantranilipole and the induction of the embryonic DNA damage.7. The results indicates that apoptosis were located mainly at the spine area at the stage of 24hpf and heart region and spine muscular area at the stage of 96hpf for cyantranilipole using acridine orange staining and TUNEL assay. Apoptosis rate and mitochondrial membrane potential movements are examed using flow cytometry at the stage of 24hpf,which suggests that apoptosis rate will go up and mitochondrial membrane potential significantly (p<0.05) down when the concentration of cyantranilipole rises.Moreover, when it hits 1.00 mg·L-1, signs showing the apoptosis rate is in negative correlation with the declining of the mitochondrial membrane potentail with the correlation coefficient being 0.784.8. Effects of cyantranilipole on genes concerning apoptosis were investigated using RT-qPCR, which indicates that cyantranilipole can induce embryonic apoptosis through mitochondrial apoptosis pathway,whose main pathway is that cyantranilipole induce embryonic DNA damage, causing p53 up-regulation, activating Bid in the cytoplasma and as a result, deducing the mitochondrial potential by transcending Bax to the mitochondrial membrane. Caspase-9 precursor protein is activated by the combination of Apafl and cytochrome, thus activating Caspase cascade reaction that induces apoptosis.Cyantranilipole induces apoptosis promoting genes Bax,Bid,p53,puma, Apaf1, Caspase-3,Caspase-8, Caspase-9 up-regulation and the anti-apoptosis gene Bcl-2 down-regulation.