Toxicity Evaluation Of Chlorantraniliprole And Detoxifying Enzyme Response After Trace Exposure In Silkworm,Bombyx Mori

Silkworm(Bombyx mori),belongs to the Lepidoptera family Bombycidae,which has a history of domestication for more than 8,500 years,and it's an important economic insect.The silk produced is an important raw material for silk spinning.Mulberry leaves are polluted by pesticide residues,which leads to pesticide poisoning in silkworms,eventually leads to non-cocooning or death,and causes huge economic losses to silkworm farmers finally.As the main detoxifying metabolic organ,the silkworm fat body plays an important role in the process of pesticide metabolism Chlorantraniliprole is a new type of diamide insecticide that targets the fish nicotine receptor.It is widely used in the control of Lepidoptera pests.Foliar residues or airborne can cause pollution of mulberry leaves,which has a certain impact on the production.In this study,the silkworm variety "Jingsong × Haoyue" was used as the experimental material to evaluate the toxicity of chlorantraniliprole to fifth-instar silkworm.The pathological characteristics of fat body after chlorantraniliprole exposure were analyzed,and digital gene expression was used.The DGE technique was used to analyze the transcription characteristics of differentially expressed genes in fat bodies The detoxifying enzyme activity and transcriptional regulation of related genes were studied.The detoxification response of fat bodies to chlorantraniliprole was described The main findings are as follows:1.Toxicity evaluation of chlorantraniliprole to silkwormIn order to study the toxicity evaluation of chlorantraniliprole to fifth-instar silkworms,the effects of chlorantraniliprole on fifth-instar silkworms were studied by using continuous feeding and different feeding times.The results showed that after continuous feeding of chlorantraniliprole,the silkworm gradually developed symptoms of poisoning,slow movement,shortened body and refrained from vomiting.It will not die after refusing to eat.The traditional statistical LC50 method cannot accurately evaluate.According to the change law of weight gain at different processing times,a new evaluation method using median weight increase concentration(Wi50)is proposed Statistics show that Wiso at 24 h,48 h,72 h,and 96 h is 15.473 × 10-3 mg/L,0.369 × 10-3 mg/L,4.403 × 10-6 mg/L,and 0.112 × 10-6mg/LTrace chlorantraniliprole(0.01 mg/L)continuous feeding at different time periods showed that with the increasing of feeding time,the number of larval deaths increased,the mortality rate reached 96%at 96 h,and the pupation rate showed a decreasing trend.The pupation rate at 72 hours after feeding was 0%,and the effect on the cocoon layer rate showed a downward trend.After 48 h,the cocoon layer rate was significantly lower than that of the control group.The results showed that the long-term addition of chlorantraniliprole had a cumulative effect on the toxicity of silkworm.2.Pathological evaluation of fat body in silkworm and analysis of DGE dataIn order to study the damage of chlorantraniliprole to the fat body of silkworm,pathological analysis was performed on the fat body exposed to chlorantraniliprole.The results showed that the genetic material in the nucleus of the fat body was loosely arranged,the intercellular space increased,and the electron microscope The photos show that the distribution of rough endoplasmic reticulum in the fat body cells of the treatment group is reduced,the nuclear membrane of the nucleus is blurred,and the structure of the nucleoli and nucleolar body is loose,indicating that chlorantraniliprole caused some damage to the fat body cellsIn order to study the effect of damage caused by chlorantraniliprole on gene transcription levels,DGE analysis revealed that chlorantraniliprole was exposed for 48 h,and 960 up-regulated genes and 585 down-regulated genes were detected.GO analysis found that numerous genes involved in detoxification and nutrient metabolism were significantly up-regulated,and cell cycle and protein transcription-related genes were significantly down-regulated.KEGG Pathway data show that ion-related pathways and metabolic-related pathways are enriched with a large number of differential genes.The results showed that chlorantraniliprole affected the transcription of functional genes of the silkworm fat body.3.Responses of detoxifying enzymes and related genes in the fat bodyIn order to further study the response of detoxifying enzymes to chlorantraniliprole in fat body,the detoxifying enzyme activities and the transcription levels of related genes were examined.The results showed that after exposure to chlorantraniliprole,the detoxification-related genes CYP4M5,CYP6AB4,and CYP9G3 in the P450 family were significantly up-regulated(P?0.05),and the P450 enzyme activity increased significantly after 24 h(P ? 0.05).The peak of GST activity lags slightly behind the P450 enzyme activity,and is significantly higher than that of the control group at 72 h(P?0.05),related genes GSTel GSTe3 were increased at the same time.The transcription level of CarE gene increased,but the enzyme activity was lower than that of the control group after 48 h.Studies on the signal pathway PI3K/Akt/CucC that regulates detoxifying enzyme genes have found the transcription level of key genes in this pathway is significantly up-regulated,while Akt protein expression levels are suppressed.It was shown that after exposure to chlorantraniliprole,the PI3K/Akt pathway protein levels in the silkworm were inhibited,resulting in a decrease in the expression of keapl,which led to an increase in CncC and eventually an up-regulation of downstream related detoxifying enzyme activity.This article provides a new method for the toxicological evaluation of silkworm,and further studies the effects of trace chlorantraniliprole exposure on the growth and development of silkworm.It was found that exposure to chlorantraniliprole caused damage to the fat body of the silkworm,and affected the metabolism and transcription of protein-related genes.At the same time,detoxification enzymes in the fat body actively participate in metabolic detoxification after exposure to chlorantraniliprole.The results provided a reference for studying the toxicity of chlorantraniliprole to insects.