Evaluation Of The Acetamiprid Toxicity To Silkworm And Its Detoxification Response To The Midgut Under Trace Exposure

The silkworm(Bombyx mori)is a Lepidoptera model insect and an important economic insect.It is sensitive to chemical pesticides,and environmental residues of trace chemical pesticides will affect the safe production of sericulture.Acetamiprid has good root systemic,contact and stomach toxicity,and can effectively control pests such as Homoptera,Coleoptera,Diptera,and Lepidoptera,and is widely used in the control of agricultural and forest pests.In order to evaluate the toxicity of acetamiprid to silkworm and to explore the effect of acetamiprid stress on the physiological function of midgut of silkworm,silkworm "Jingsong × Haoyue" was used as the material and the Digital Gene Expression Profiling(DGE)was used to analysis the transcription of differential genes.The technology and system were used to systematically study the toxicity of acetamiprid to silkworm and the gene transcription characteristics of silkworm midgut after trace acetamiprid induction.The effects of trace acetamiprid on physiological function of midgut were analyzed.The main results are as follows:1.Toxicity evaluation of acetamiprid to silkwormAfter exposure to acetamiprid,the poisoned silkworm larvae eat mulberry slowly or stop eating mulberry.The breasts are significantly enlarged,the head and tail are upturned,the body swings left and right,twisted into a "C" or "S" shape,lying on the mulberry leaves,and vomiting.Intestinal fluid,the body shrinks and becomes soft.Statistics showed that the LC50(24 h)of acetamiprid to silkworm was 1.50 mg/L,indicating that acetamiprid was a high-risk grade for silkworm and had a strong lethal effect.2.Effects of ultra-trace acetamiprid on the growth and development of silkwormUltra-trace acetamiprid(0.01 mg/L,1/150 LC50)was used to continuously treat silkworms for 96 h.The growth of silkworms was hindered,no obvious symptoms of poisoning occurred,and no larval stage died.At 48 h,72 h,and 96 h,the body weights were decreased by 13.35%,15.75%,and 15.44%respectively;the food intake,digestion,and digestibility all decreased,and the food intake decreased significantly The cocooning rate and pupation rate were 90.00%and 55.00%,respectively;the total cocoon mass,cocoon shell mass,and the ratio of cocoon shell were all significantly reduced;among them,the female cocoon shell ratio decreased by 6.33%,and the male cocoon shell ratio decreased by 5.19%.The above results indicate that the exposure of ultra-trace acetamiprid reduced the feed efficiency of silkworm,hindered growth and development,and significantly affected the cocoon quality of silkworm.3.Ultra-trace acetamiprid damages the silkworm midgutPathological sections and electron microscopy analysis showed that after exposure to ultra-trace acetamiprid,the morphology of the cells was changed,the basal layer was thinned,the microvilli were sparse,the intestinal wall cells were disintegrated,the intracellular microvilli of the goblet cells were shed,and vacuoles were seen on the membrane of goblet cells.KEGG-Pathway enrichment analysis showed that 48 genes were significantly differentially expressed in nutrition metabolism,74 genes were significantly differentially expressed in stress response,and 38 genes were significantly differentially up-regulated related to inflammation.Trypsin activity in intestinal fluid was significantly reduced,amylase activity was significantly reduced at 24 h and 48 h(P ?0.05),and lipase activity was increased.24 h,48 h,and 72 h of exposure to acetamiprid increased the ROS levels in the midgut by 1.19,1.45,and 1.26 fold,respectively,and affected the expression of oxidative stress-related genes.The results showed that the exposure of ultra-trace acetamiprid caused oxidative stress in the midgut,causing damage to the midgut cells,activating inflammatory responses,affecting digestive enzyme activity,and affecting its physiological functions of digestion and absorption.4.Detoxification regulation mechanism of silkworm exposed to acetamipridAfter continuous exposure to trace amounts(0.15 mg/L,1/10 LC50)of acetamiprid for 48 h,the residual concentrations of acetamiprid in the hemolymph and midgut of the silkworm were 0.90 ?g/mg and 0.58 ?g/mg,respectively,at 96 h,Hemolymph and midgut acetamiprid residues were reduced.At 24 h and 72 h,the transcription levels of CYP4M5 and CYP6AB4 increased,while the transcription levels of CYP9A20 continued to be suppressed.In addition,P450 enzyme activity was significantly enhanced after exposure to acetamiprid for 24 h(P?0.05),and remained at the same level after 48 h.CarE and CarE-11 transcription levels and CarE activity were inhibited.From 24 h to 72 h,the transcription levels of GSTe3 and GSTd1 were significantly up-regulated(P?0.01),and from 48 h to 96 h,GST enzyme activity was significantly increased(P ? 0.05).In addition,the expression levels of FoxO,CncC,and Keap1,upstream genes of detoxifying enzymes,showed similar trends to GST.The results showed that after exposure to trace acetamiprids,the acetamiprid content in the midgut decreased,and the FoxO/CncC/Keap1 signaling pathway may activate GST expression and play a key role in the detoxification response process.This study indicates that trace acetamiprid induces damage to the midgut of the silkworm,affects the normal nutritional metabolism and growth and development of the silkworm,activates the midgut detoxifying enzyme response,and clarifies that GSTs expression may be regulated by the FoxO/CncC/Keap1 signaling pathway.It provides a reference for the toxicity evaluation of new tobacco pesticides to Lepidoptera insects,and has important reference value for further research on the mechanism of midgut damage and metabolic detoxification of silkworm under the stress of trace neonicotinoid pesticides.