Research On The Toxic Effects Of Nitenpyram To Silkworm

The silkworm（Bombyx mori L.）is an economically important insect in China,but it is sensitive to chemical pesticides.Mulberry leaves are the only food source for silkworms,and they are vulnerable to pesticide pollution during mulberry picking and silkworm rearing.In China’s planting mode,rice,fruit trees,and vegetables are often grown adjacent to mulberry fields.Nitenpyram is commonly used to control piercing-sucking mouthparts pests such as rice planthoppers,aphids,and thrips.During spraying,the liquid can easily drift to nearby mulberry fields with the wind,causing silkworm poisoning and affecting the safe production of sericulture.In this study,we evaluated the risk of nitenpyram to silkworms and studied the effects of low concentrations of nitenpyram on silkworm growth and development,economic indicators,DNA,detoxification enzymes and gene expression.The main findings are as follows:1.The LC₅₀ values of nitenpyram TC,AS,SP and WP against silkworms at 96 hours were6.935,16.284,1.208 and 13.550 mg/L,respectively,indicating high toxicity to silkworms.The risk coefficients of nitenpyram AS,SP and WP to the outermost and sub-outermost mulberry leaves were all greater than 1,indicating that the three formulations posed a risk to silkworms.2.The 1/10 LC₅₀,1/20 LC₅₀ and 1/40 LC₅₀ of nitenpyram had significant adverse effects on the body weight,cocoon weight and cocoon shell weight of silkworm,and the 1/10 LC₅₀concentration of nitenpyram was the most toxic to silkworm.The treatment of 1/10 LC₅₀,1/20LC₅₀ and 1/40 LC₅₀ of nitenpyram caused DNA damage in silkworm,and the degree of DNA damage increased with the increase of exposure time and concentration of nitenpyram.3.The activities of carboxylesterase（Car E）and glutathione-S-transferase（GST）were tested in silkworms.The results showed that the activities of Car E and GST in 2nd and 3rd instar silkworms were higher than those in the control group,and the activity at 1/10 of the LC₅₀of nitenpyram was the most significant.In the 4th instar,Car E activity was gradually inhibited,while GST activity returned to the normal level.The activities of Car E and GST returned to normal levels in the 5th instar.Real-time fluorescence quantitative tests showed that Car E-related genes（Car E,Car E-11）and GST-related genes（GSTd1,GSTz2,GSTe3）were affected to varying degrees by different nitenpyram concentrations,among which the expression of Car E-11 and GSTe3 was most significantly affected.4.The transcriptome and micro RNA expression profiles of silkworms exposed to different concentrations of nitenpyram were analyzed.In the transcriptome analysis,GO and KEGG analyses were performed under LC₅₀ nitenpyram exposure stress to determine their biological functions and related processes.The results showed that the most significant enrichment pathways were base excision repair and tyrosine metabolism.Under 1/40 of the LC₅₀nitenpyram exposure stress,the most significant enrichment pathways were neuroactive ligand-receptor interaction and purine metabolism.In the analysis of mi RNA expression profiles,GO and KEGG analyses were performed under LC₅₀ and 1/40 of the LC₅₀ nitenpyram exposure stress.The results showed that many differentially expressed genes were enriched in the m TOR,oxidative phosphorylation and Fox O signaling pathway,which were involved in the regulation of the immune process.Therefore,immune pathway-related genes were verified.The results showed that all immune-related genes were up-regulate,and the Imd gene was most significantly up-regulated.After exposure to 1/10,1/20 and 1/40 of the LC₅₀ nitenpyram,Fox O/Cnc C/Keap1 signaling pathway-related genes（Fox O,Cnc C,Keap1）and IIS signaling pathway-related genes（AKT,PDK,In R）were slowly expressed in the early stage,and the gene expression was gradually inhibited with the increase in the silkworm instar.5.After exposure to LC₅₀ nitenpyram,the expression of bmo-mi R-3378-5P,bmo-mi R-3326 and bmo-mi R-2761-3P was up-regulated,while the expression of bmo-mi R-2766-5P was down-regulated.Injecting bmo-mi R-3378-5P mimics,bmo-mi R-3326 mimics,bmo-mi R-2761-3P mimics and bmo-mi R-2766-5P into silkworm resulted in down-regulation of bmo-mi R-3378-5P,bmo-mi R-3326 and bmo-mi R-2761-3P expression and up-regulation of bmo-mi R-2766-5P.Similarly,after exposure to 1/40 LC₅₀ nitenpyram,the expression of bmo-mi R-3213-5P and bmo-mi R-133 was up-regulated,while the expression of bmo-mi R-3382-3P was down-regulated.Injecting bmo-mi R-3382-3P mimic,bmo-mi R-3213-5P inhibitor and bmo-mi R-133inhibitor into silkworm resulted in down-regulation of bmo-mi R-3213-5P and bmo-mi R-133expression and up-regulation of bmo-mi R-3382-3P.These findings suggested that the injection of mi RNA mimics/inhibitors could affect mi RNA and regulate the immune response.