Effects Of Sublethal Concentration Imidacloprid On Toxicological Response And Gene Expression Profile Of Silkworm（Bombyx Mori） Silk Gland

Silkworm（Bombyx mori）is a model insect of Lepidoptera as well as an important economic insect in China since ancient times.Due to long-term artificial domestication,silkworms was highly sensitive to adverse environments and insecticides.Imidacloprid,as a representative of neonicotinoid insecticides,is widely used in the agricultural and forestry production industry in controlling pests such as whitefly,planthopper and aphid.In sericulture production,the incident that silkworms eat mulberry leaves contaminated with imidacloprid by mistake was often occurred.When silkworms were poisoned by imidacloprid,the initial traits of which were not obvious,then the growth and development slowing down with no cocooning or thin cocooning after mature silkworms,which resulting in serious economic losses.In this study,the silkworm strain“Jing song×Hao yue”was used and the effects of sublethal concentration of imidacloprid on the growth and development of silkworm,silk gland tissue damage,and the content of acetylcholinesterase,detoxification enzymes,antioxidant enzymes and metal ions were investigated.Further more,the expression characteristics of genes in silk gland were analyzed by RNA-seq high-throughput sequencing.The main research results are as follows:（1）The LC₅₀ of imidacloprid to silkworm was 7.36 mg/L by the toxicity test using different concentrations of imidacloprid solutions on the third day of the fifth instar larvae.The sublethal concentration of 0.8 mg/L was determined as the follow-up test concentration combined with the growth and development of silkworm.（2）After treatment with sublethal concentration of imidacloprid,the body weight of male and female silkworms were significantly decreased,and the female silkworms were more obvious as the less smaller silk gland volume,lighter weight and was significantly decreased silk gland index.Transmission electron microscopy showed that with the imidacloprid treatment,the cell structure was destroyed with the nucleus of the posterior silk gland cells were enlarged,the branches of nucleus were not obvious,the rough endoplasmic reticulum was reduced,the free ribosomes were increased,the golgi apparatus was abnormal,some vacuoles appeared,which affected the normal development of silk gland tissue.（3）The oxidative stress induced by sublethal concentration of imidacloprid solution in silk gland tissue of silkworm resulted in great changes in enzyme activity in silk gland tissue,mainly manifested in decreased Ach E activity,increased detoxification enzymes Car E and GST activities,and decreased antioxidant enzymes SOD and CAT activities.（4）After sublethal concentration of imidacloprid treatment,the concentration of Fe²⁺and Na⁺in the middle silk gland decreased significantly,while the concentration of Mn²⁺,Cu²⁺,Zn²⁺,Mg²⁺and K⁺increased significantly;however,the concentration of Fe²⁺,Cu²⁺,Mg²⁺,Na⁺in the posterior silk glands increased significantly,and Mn²⁺,Zn²⁺,Ca²⁺and K⁺decreased significantly.This indicated that imidacloprid treatment caused damage to silk gland tissue,which was characterized by disorder of metal ions in tissue.（5）Through transcriptome sequencing,the digital gene expression profiles of silk gland in the imidacloprid treatment group and the control group on the fifth day of the 5th instar silkworm were analyzed.A total of 442 differentially expressed genes were obtained,of which170 genes were significantly up-regulated and 272 genes were significantly down-regulated in the imidacloprid treatment group.GO functional enrichment analysis showed that 43 Go terms reached significant enrichment（P≤0.05）,including 18 biological processes,16 cell components and 9 molecular functions.The cell components were mainly enriched in extracellular region,extracellular space,myosin complex,nucleolus,and catalase.Molecular functions are mainly enriched in motor activity,monooxygenase activity,serine endopeptidase inhibitor activity,and iron ion binding.Biological processes are mainly enriched in innate immune response,antibacterial humoral response,defense response to bacteria,ion transport and so on.KEGG pathway analysis is mainly enriched in eukaryotic ribosomes,ECM receptor interaction,galactose metabolism,ascorbic acid and aldose metabolism,PPAR signaling pathway and other related pathways.The q PCR verification test of its key genes showed that the transcriptome analysis results were accurate and reliable.（6）The transcription levels of the key silk gland silk protein genes were determined by qPCR.The results showed that the m RNA expressions levels of silk fibroin genes Fib-H,Fib-L and P25 were significantly down-regulated at 48 h and 72 h after imidacloprid treatment.The m RNA expressions level of sericin gene Ser1 was down-regulated to 0.11 times of the control group at 48 h.Ser2 was significantly down-regulated at 48 h and 72 h.Ser3 was significantly up-regulated to 4.12 times and 2.17 times at 24 h and 48 h,respectively,and only 0.22 times of the control group at 72 h.