| The silkworm(Bombyx mori,B.mori),an economically important agricultural insect,is a model species of lepidoptera.Silk has an important economic value and is widely used in the fields of agriculture,industry,medical treatment and biomaterial development.The silk gland of silkworm is an important organ for the synthesis and secretion of silk protein.The secreted silk fibroin synthesized by the posterior silk gland(PSG)accounts for 65%-75%of the total silk material.B.mori is extremely sensitive to chemical pesticides due to longterm domestication.Even trace amounts of pesticides can result in the failure of silkworms cocooning.Acetamiprid,a new neonicotinoid insecticide with good absorption activity and environmental compatibility,has been widely used in the control of agricultural and forestry pests.However,due to extensive use,its residues were found to appear in the environment,posing a threat to sericulture.The silkworm "Jingsong x Haoyue" was used as the material to explore the damage mechanism of trace amout of acetamiprid to the PSG of silkworm.The changes in epigenbiological characters of silkworm,pathological features of the PSG were studied after exposure to the trace acetamiprid.Differential gene expression in the PSG was analyzed after exposure to trace acetamiprid using digital gene expression profiling(DGE)and the transcription levels of silk protein synthesis and regulation related genes were invesgated.Moreover,the oxidative stress response of the PSG of B.mori after exposure to trace acetamiprid was described.The main results are as follows:1.Effects of trace acetamiprid on the growth and development of silkwormAfter continuous feeding the 3rd day of 5th instar silkworm larvae trace acetamiprid(0.15 mg/L)for 96 h,the larvae showed signs of mild poisoning,and the poisoned larvae became yellowish and were smaller in size.The consumption of mulberry leaves was reduced,and the body weight was decreased to 1.86 g(instead of gaining weight)at 96 h after exposure,and their survival rate was 96.67%.In the pupal stage,silkworm pupae became smaller and semi-molted pupae appeared,and the cocoon formation rate was significantly reduced to 58.00%compared to the control group(P<0.01).HPLC results showed that acetamiprid residues were 0.72 μg/mg and 1.21 μg/mg in the PSG after exposure to acetamiprid for 48 h and 96 h,respectively,acetamiprid residues were not detected in the PSG of the control larvae.The silk gland index(SGI)was significantly reduced(P≤0.05),and the SGI of female and male silkworms at 96 h decreased by 28.48%and 26.65%,respectively.The total cocoon mass,cocoon shell mass,and the ratio of cocoon shell were all found to be significantly decreased by 40.63%,51.28%and 20.14%,respectively(P≤0.01).Among them,the ratio of cocoon shell of female and male silkworms decreased by 18.35%and 21.56%respectively.The results showed that the growth and development of silkworms were hindered after exposure to trace acetamiprid,and that trace acetamiprid residues caused abnormal development of the PSG,which has a significant impact on the cocoon quality of the silkworm.2.The mechanism of damage to the PSG by trace amount of acetamiprid in silkworm,and DGE analysisTo explore the effect of trace acetamiprid exposure on the growth and development of PSG,pathological analysis of the PSG was carried out using histopathological sections in combination with transmission electron microscopy.Morphological and pathological comparisons at 96 h after exposure showed that the protein content of the PSG was decreased,with shrunk lumen and shortened length.The intracellular chromatin was unevenly distributed,endoplasma reticulum(ER)was less abundant and disintegrated,and nucleolar bodies were disintegrated and the number was reduced.Differentially expressed genes were analyzed by DGE to explore the effects of the PSG damage caused by trace acetamiprid exposure on gene transcription levels.At 96 h after trace acetamiprid exposure,a total of 4219 differentially expressed genes were identified,with 3893 up-regulated and 326 downregulated.GO(Gene Ontology)analysis found that a large number of differentially expressed genes are distributed in cell and tissue growth and development,detoxification,and nutrient metabolism.KEGG pathway analysis found that,with a large number of differentially expressed genes,metabolic response,stress response and inflammatory response-related signaling pathways are enriched.The transcript levels of the silk protein synthesis related genes Fib-H,Fib-L,P25,Bmsage and Bmdimm were all down-regulated,which were 0.75-,0.76-,0.65-,0.44-and 0.40-fold of the control group,respectively.Whereas the transcript levels of the silk protein trans-regulation factors FMBP-1 and FTZF1 were up-regulated by 2.55-and 1.56-fold,respectively.The results showed that the accumulation of trace acetamiprid caused damage to the PSG,leading to cell metabolism and dysfunction,affecting the transcription of silk protein synthesis-related genes,and blocking silk protein synthesis and secretion.3.Oxidative stress of the PSG after exposure to trace acetamipridIn order to further explore the effects of acetamiprid exposure on the level of oxidative stress in the PSG of the silkworm,DGE was used to obtain the gene transcription levels of oxidative stress-related signaling pathways in the PSG after acetamiprid exposure for 96 h.DGE analysis revealed that the differentially expressed genes of oxidative stress-related signaling pathways were enriched and significantly up-regulated.The contents of hydrogen peroxide(H2O2)and malondialdehyde(MDA)in the PSG were increased significantly,and the activities of superoxide dismutase(SOD)and catalase(CAT)enzymes were significantly increased,and all reached their maximum at 48 h.The contents of H2O2 and MDA were 10.46 mmol/gprot and 7.98 nmol/mgprot,respectively,and the SOD and CAT enzyme activities were 5.51 U/mgprot and 33.48 U/gprot,respectively.The transcription levels of antioxidant enzyme-related genes SOD,Mn-SOD,CuZn-SOD,CAT,TPX and GPX were upregulated,indicating that exposure to trace acetamiprid caused an antioxidant response in the PSG.The key genes FoxO,CncC,Keapl,NQO1,HO-1 and sMaf in the FoxO/CncC/Keap 1 signaling pathways(upstream signaling pathways that regulate antioxidant enzyme activity)showed an upward trend after exposure to trace acetamiprid for 0 h to 96 h,at 72 h reached the maximum value,which was increased by 2.91-,1.46-,1.82-,2.52-,2.32-and 4.01-fold,respectively.These results indicate that exposure to trace amount of acetamiprid caused oxidative stress in the PSG,which may be the cause of the cocoon formation disorder in silkworm.In this paper,we conducted an in-depth study on the effects of exposure to trace acetamiprid on silkworm growth and development,and revealed that treatment with trace acetamiprid caused damage to the PSG and affected the expression of silk protein synthesis and secretion related genes.Moreover,oxidative stress occured in the PSG,the peroxide content increased significantly,and both the transcription level of antioxidant-related genes and the activity of antioxidant enzymes increased significantly.The results provide a reference for the safety evaluation of acetamiprid residues on non-target insects. |
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