The Study On The Mechanisms Of Phoxim-induced Midgut Damage In Bombyx Mori

Silkworm(Bombyx mori.)belongs to Bombycidae of Lepidoptera,which originated from Chinese wild silkworm(Bombyx mandarina).Silkworm is a Lepidoptera model insect and is also an important economic insect.In the long artificial selection,the silk production performance of silkworm has been greatly improved.At the same time,due to long-term indoor feeding,the opportunity of silkworm exposure to chemical pesticides and wild bad environment is relatively few,its resistance to adverse environment is weak.Organophosphorus pesticides(OP),one kind of phosphorus-containing compound,are widely used to control the agricultural and forestry pests.As the organophosphorus pesticide residue period is longer,the production of silkworms often eat with organic phosphorus pesticide mulberry leaves and cause poisoning phenomenon.When silkworm contact with sub-lethal dose of pesticide,the mortality is very low or not dead,its normal growth and development and physiological metabolism affected,silkworm nutrition efficiency and economic traits are affected.Silkworm midgut is not only the main organ for food digestion and nutrient absorption but also the first natural barrier against foreign substances and chemical pesticides.Insect midgut epithelial cells secretion of the formation of the peritrophic membrane,so that digestion space compartmentalization,which is conducive to food digestion and absorption.Peritrophic membrane can also protect the midgut epithelium from food mechanical damage,to prevent foreign pathogens,heavy metals and chemical pesticides and other harmful substances into the midgut.Studies on the changes of midgut physiology and gene expression in silkworm after induction of pesticides show that midgut is also a toxic target organ exposure to insecticides.A lot of research on phoxim pesticides and silkworm toxicological aspects had studied in our laboratory.In oeder to study the mechanisms of sub-lethal dose phoxim-induced midgut damage in Bombyx mori,the toxic effects of sub-lethal dose of phoxim(4.0?g/mL)on the midgut of silkworm were studied with silkworm in this paper.The effects of sub-lethal dose of phoxim on the transcriptional characteristics of midgut and its physiological function were studied by digital gene expression profiling(DGE).The main results are as follows:1.The midgut damage of silkworm expouse to phoximIn order to investigate the effect of sub-lethal dose of phoxim on the growth of silkworm and its injury to midgut,this study calculated the body weight and survival rate of silkworm after treatment with phoxim,and the mesenteric lesions and cell ultrastructure were detected by pathological sections and transmission electron microscopy.After 24 h of phoxim exposure,the fifth-instar larvae of B.mori displayed apparent and typical symptoms of poisoning,including head nystagmus,chaotic crawling,cramps,and gastric juice spitting;at 48 h and 72 h weight decreased 25.85%and 30.36%,respectively;48 h mortality rate was 13.5%.After the induction of phoxim,the mesenteric tissue of silkworm was changed:the microvilli of the midgut decreased,the basal layer was thinner,the vacuolization of the goblet cells,the cell lysis and death;the ultrastructural damage of the cells:vacuolization of the midgut cells,Nuclear pyknosis,reduced microvilli,mitochondrial swelling.The results showed that sub-lethal dose of phoxim had damage to the midgut of silkworm and affected the growth of silkworm.2.The transcriptional characteristics of the midgut gene of silkworm after induction of phoxim were analyzed by DGEThe gene expression profile of midgut in silkworm was studied by DGE technique.The effect of phoxim treatment on the midgut of silkworm was analyzed from oxidative stress and nutrient metabolism.The results showed that there were 254 differentially expressed genes in the midgut of silkworm(FDR?0.001 and|log2Ratio|?1),148 genes were up-regulated and 106 genes were down-regulated.GO functional enrichment analysis showed that these significant differences were mainly involved in cell composition,molecular function and biological processes.KEGG-Pathway significant enrichment analysis showed that 12 Pathway genes were significantly different,and these pathways were mainly involved in oxidative phosphorylation and nutrient metabolism.3.Effects of phoxim on oxidative stress in midgut of silkwormWe used silkworm midgut as research materials,the effect of sub-lethal dose of phoxim on oxidative stress in midgut of silkworm was studied.The results showed that,after phoxim treatment,DGE analysis showed that 24 electron transport chain related genes were upregulated.The results of QRT-PCR showed that the electron transport chains of genes encoding NADH-CoQ1,Succinic-Q,cyt c reductase-S,cyt c oxidase-S,cytochrome c oxidase polypeptide IV,ATP synthase and vacuolar H~+-ATP synthase were significantly(P?0.05),respectively,up 1.50-,1.31-,1.42-,1.44-,1.70-,2.03-and1.43-fold.Phoxim-induced enhancement of the activity of the mitochondrial electron transport chain complexes and induced ROS accumulation in the midgut.The results showed that sublethal doses of phoxim enhanced the respiration of the midgut and the imbalance between the changes in the activity of the electron transport chain resulted in the accumulation of ROS.The electron transport chain of mitochondria may be a potential biomarker of intestinal toxicity in silkworms caused by exposure to phoxim.4.Effects of phoxim on nutrient metabolism in silkworm midgutIn this paper,the effects of sublethal doses of phoxim on nutrient metabolism in midgut of silkworm were studied.The result showed that the activity and expression of nutrition metabolism-related enzymes were dysregulated in midgut by phoxim exposure.DGE analysis revealed that 40 nutrition metabolism-related genes were differentially expressed.QRT-PCR results indicated that the expression levels of insulin/insulin growth factor signaling(IIS)pathway genes Akt,PI3K,PI3K60,PI3K110,IRS and PDK were reduced,whereas PTEN's expression was significantly increased in the midgut at 24 h after phoxim treatment.However,the transcription levels of Akt,PI3K60,PI3K110,IRS,In R and PDK were elevated and reached the peaks at 48 h,which were 1.48-,1.35-,1.21-,2.24-,2.89-,and 1.44-fold of those of the control,respectively.At 72 h,the transcription of these genes was reduced.Akt phosphorylation level was increasing along with the growth of silkworms in the control group.However,phoxim treatment led to increased Akt phosphorylation that surged at 24 h but gradually decreased at 48 h and 72 h.The results indicated that phoxim dysregulated the expression of IIS pathway genes and induced abnormal nutrient metabolism in silkworm midgut,which may be the reason of the slow growth of silkworms.