The Mechanism Of Damage In The Midgut Of Bombyx Mori After Chlorantraniliprole Exposure

The silkworm(Bombyx mori.)has been reared for more than 8500 years as one of the important economic insects.After the long-term artificial selection and indoor feeding,the resistance of silkworm to chemical pesticides in the environment is weak.Silkworm is poisoned by pesticide contamination on mulberry leaves,which leads to spinning obstacles and seriously affects the development of sericulture in China.Chlorantraniliprole(CAP)is a novel anthranilic diamide insecticide with high efficiency and broad spectrum for Lepidoptera,which is widely used in agricultural.B.mori is prone to poisoning by mistaking the mulberry leaves contaminated by CAP residues.The sublethal doses of CAP results in reduced growth and development of B.mori and failure in cocooning,which is a potential threat to sericulture.In order to study the effects of CAP exposure(0.01 mg/L)on the growth and physiological function of midgut in silkworm,the larvaes of B.mori(Jingsong × Haoyue)were used to investigate the body mass,damage to midgut and digestive enzyme activity of silkworm.Moreover,the differential genes in midgut were analyzed by digital gene expression(DGE)profile.The main results are as follows:1.The effects of CAP on biological characters of silkwormTo study the poisoning symptoms and body mass of silkworm after CAP exposure,the feed efficiency was investigated and the activity of digestive enzymes was detected.At 48 h after CAP exposure,the 5th-instar larvae showed head nystagmus,chaotic crawling and cramps.At 96 h,the larvae exhibited gastric juice spitting and retarded movement.The body mass of the larvae in the CAP-exposed group was significantly decreased by 64.46%at 96 h.The amount of ingested food and digestion were decreased by 0.47 and 0.44-fold,respectively.The activities of ?-amylase and trypsin were decreased by 0.69 and 0.20-fold,respectively.The results showed that CAP inhibited the feeding behavior of silkworm,weakened the digestion and absorption of food,which affected the physiological function of midgut and led to reduced body mass of B.mori.2.The damage to midgut of silkworm and transcriptional characteristics of midgut genes were analyzed by DGE after CAP exposureIn this study,the morphological changes of midgut after CAP exposure were observed by histopathological and ultrastructural analysis.The results showed that columnar cells and goblet cells were disintegrated,microvilli disappeared,the stroma became thinner and chromatin of nucleus became aggregated.To investigate how CAP caused damage to silkworm midgut,we identified differentially expressed genes(DEGs)between the CAP-exposed group and control group using DGE profiling analysis.Among 937 DEGs,there were 401 up-regulated and 536 down-regulated genes.Gene Ontology(GO)classification analysis revealed that DEGs were related to cell,cell part,binding,catalytic activity,organelle,cellular process,and metabolic process.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that DEGs involved in the major significantly enriched pathways included oxidative phosphorylation and metabolism-related pathways.The qRT-PCR results showed that the expressions of ?-amylase and trypsin-like protease at the mRNA level were greatly reduced by 0.31 and 0.50-fold,the expressions of the oxidative phosphorylation-related genes(NDUFV1,NDUFB7,NDUFC2,QCR7,QCR8,QCR9 and ATPeVOD)were decreased by 0.56,0.47,0.51,0.61,0.63,0.53 and 0.77-fold,respectively,and the expressions of antioxidant genes(SOD,GST,GPX and CAT)were reduced by 0.47,0.80,0.92 and 0.46-fold,respectively.The results of qRT-PCR were consistent with those of DGE data.3.Oxidative stress of midgut in silkworm after CAP exposureTo study the oxidative damage to midgut caused by CAP,the accumulation of reactive oxygen species(ROS)and the activities of antioxidant enzymes were detected.At 48 h,the level of O2-was 1.62-fold higher compared with the control group,the level of H2O2 was significantly increased by 1.87 and 2.21-fold,respectively,and the level of MDA was by 1.46 and 1.42-fold at 48 and 96 h,respectively.In the CAP-exposed group,the activity of CAT and CuZn/Mn-SOD were greatly decreased by 0.68 and 0.42-fold,respectively.The results indicated that CAP broke down the antioxidant defense system and caused oxidative damage to midgut,which affected the growth and development of silkworm.In this paper,the oxidative damage to midgut induced by sublethal dose of CAP is the key reason for the slow growth and changes in physiological function of midgut in B.mori.Our findings provided valuable insights into the standardized use of CAP on mulberry trees,the prevention and management of Lepidoptera by CAP.