| The silkworm(Bombyx mori)is an important economic insect,and silk is widely used as a natural protein fiber in the textile and biomedical fields.Due to long-term artificial domestication,the silkworm is very sensitive to chemical pesticides,and pesticide pollution in mulberry gardens has caused silkworm poisoning,affecting the healthy development of China’s sericulture industry.Glyphosate is an organophosphorus herbicide,which is considered safe and non-toxic to animals because there is no glyphosate target 5enolpyruvylshikimate-3-phosphate synthase in animals,but with the large-scale use of glyphosate in agricultural,its safety to sericulture production has caused widespread concern and it is necessary to evaluate its safety.In this study,the commonly used strains "Jingsong×Haoyue" in sericulture production was used as the experimental object,the median lethal concentration(LC50)of glyphosate against silkworm was determined after 72 hours,and the effect of sublethal dose of glyphosate on biological characters of silkworm was studied.The digital gene expression profile of midgut tissues of silkworm treated with sublethal dose of glyphosate was constructed by RNA-Seq technology,the expression characteristics of differential genes were analyzed,and the antioxidant indexes in midgut tissues of silkworm treated with sublethal dose of glyphosate were determined,and the infection experiment of B.bassiana was conducted to explore the effects of sublethal dose of glyphosate on immune function of silkworm.The main results are as follows:1.Toxicity assessment of glyphosate to silkwormIn order to evaluate the toxicity of glyphosate to silkworm,glyphosate feeding was performed on silkworm at 3rd day of fifth instar(L5D3)with reference to the "Test guidelines on environmental safety assessment for chemical pesticides",standard number:GB/t 31270.11-2014.The results showed that glyphosate treated silkworm at high concentrations showed signs of toxicity such as stopped eating,sluggishness,softness,elongation of the body,and a few"C" or"S" shapes,and the LC50(72 h)of glyphosate for silkworm was 14875.98 mg/L,indicated that glyphosate is a low toxic pesticide for silkworm.The conventional use concentration of glyphosate in agricultural fields(2975.20 mg/L),one-fifth of the LC50(72 h),was used to continuously treated 3rd day of fifth instar(L5D3)silkworm larvae until cocooning,the results showed that the weight of silkworms in the glyphosate treated group was 3.81 g at 96 h,which was 9.07%lower than that in the control group(P<0.001);the digestibility of silkworms in the treated group was 34.1 1%,which was 2.79%lower than that in the control group(P<0.05);the α-amylase activity at 48 and 96 h was 0.92 and 0.89-fold that of the control group,respectively(P<0.05),the activity of trypsin at 48 and 72 h was 0.82 and 0.73-fold that of the control group(P<0.01),and the activity of lipase at 24 h increased significantly,which was 1.60-fold that of the control group(P<0.01).The whole cocoon volume of the treated group was only 1.72 g,which was 10.88%lower than that in the control group(P<0.01).The results indicated that the sublethal dose of glyphosate treatment in stunted growth and development of silkworm and affected economic traits.2.Damage to midgut of silkworm treated with sublethal dose of glyphosate and DGE analysisThe midgut is the first biological barrier of silkworm against exogenous harmful substances.In this study,the midgut tissues of the sublethal dose of glyphosate treated group for 48 h were observed by pathology and analyzed by transmission electron microscopy.The results showed that the basal layer of midgut tissue was thinner,microvilli were sparse,intestinal wall cells were shed,cytoplasmic membrane boundaries were blurred,and vacuoles appeared in the treatment group.The results indicated that sublethal doses of glyphosate could damage the midgut tissue structure of silkworm.In order to further investigate the cause of glyphosate-induced midgut injury of silkworm,Digital gene expression profiling(DGE)of midgut tissue of silkworm treated with sublethal dose of glyphosate was constructed by RNA-Seq technique.The results showed that a total of 125 differenced genes were produced in silkworm midgut after glyphosate treatment,of which 53 were significantly up-regulated and 72 were significantly downregulated(q<0.05).GO function and KEGG analysis showed that differenced genes were enriched in a variety of biological pathways,mainly related to oxidative stress,nutrient metabolism and immune defense pathways.The transcription levels of antioxidation-related gene Cat were up-regulated by 1.93-fold of the control group,while the transcription levels of Jafrac1 and FNSI were down-regulated by 0.71 and 0.56-fold of the control group.Fatp and Scpx transcription levels related to nutrient digestion and absorption were up-regulated,which were 1.80 and 1.60-fold that of control group,respectively.Immune-related UGT40B4 and UGT41A1 transcription levels were down-regulated by 0.60 and 0.23-fold of the control group,but serpin-2 and CTL11 transcription levels were down-regulated by 0.73 and 0.24-fold of the control group.The results indicated that the sublethal dose of glyphosate affected the transcription of several genes related to physiological functions in silkworm.3.Oxidative stress in midgut of silkworm treated with a sublethal dose of glyphosateIn order to further study the oxidative damage of silkworm midgut treated with sublethal dose of glyphosate,the content of peroxides in the midgut of silkworm treated with glyphosate was determined.The results showed that the content of O2-in midgut of silkworm treated with glyphosate was higher than that in control group,and the peak value at 24 h was 2.10-fold of that in control group(P<0.01).H2O2 content increased significantly at 24 and 48 h,which was 1.08 and 1.12-fold of that in control group(P<0.01).MDA content in the treatment group was higher than that in the control group at different time periods,and the peak value at 48 h was 4.53-fold that of the control group(P<0.001),indicated that glyphosate treatment in the accumulation of peroxide in the midgut tissue of silkworm.Antioxidant enzymes play an important role in peroxidase removal.In this study,the activity of antioxidant enzymes in midgut tissues of silkworms treated with glyphosate was further detected.The results showed that SOD activity in midgut tissues of silkworms treated with glyphosate at 24 and 48 h was higher than that of control group,and the activity at 48 h was 1.39-fold that of control group(P<0.01).The 96 h activity was 0.74-fold that of control group(P<0.01).CAT activity in glyphosate treated group was 2.07 and 1.53-fold of that in control group at 72 and 96 h,respectively(P<0.01).The Gpx activity in the 24,48 and 72 h treatment groups was lower than that in the control group,and reached a significant level at 48 and 72 h,which was 0.80 and 0.72-fold of that in the control group,respectively(P<0.01),indicating that glyphosate treatment resulted in the accumulation of peroxide in the midgut tissue of silkworm and the imbalance of antioxidant system.4.Effect of sublethal dose of glyphosate on immune function of silkwormIn order to study the effect of sublethal dose of glyphosate on immune defense ability of silkworm,the immune function was evaluated by B.bassiana infection.The results showed that the mortality rate of silkworm in glyphosate treatment group was significantly increased by 4.35-fold as much as that in control group(P<0.001).The morphology of mycelia of B.bassiana in hemolymph after infection by glyphosate treated group and control group was observed by microscopy.The extent of melanin coating of mycelia in glyphosate treated group was lower than that in control group,and mycelia proliferation was obvious.The activity of polyphenol oxidase(PO),a key enzyme in melanin synthesis,was significantly decreased by 0.75-fold of that in the control group(P<0.01),and the transcription levels of PAH,DDC,PPO1 and PPO2 genes associated with PO cascade reaction were significantly lower than those in the control group,suggesting that the sublethal dose of glyphosate interfered with the activity of PO and less melanin synthesis.Increased susceptibility to B.bassiana.The results of this study indicate that glyphosate has low toxicity to silkworm,and field dosage can cause oxidative damage to the midgut of silkworm,affecting the digestive capacity of the midgut and increasing susceptibility to B.bassiana.It provides a direct basis for the standardized use of glyphosate in mulberry gardens,and also provides an important reference for the safe production of silkworm. |
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