| The silkworm(Bombyx mori.)(B.mori)has been domesticated in China for about 8,500 years.It is an important economic insect,also is a model insect of the order Lepidoptera.Its life cycle includes four stages:eggs,larvae,pupae and adults.Due to long-term artificial selection,the silkworm is highly sensitive to adverse environments and many pesticides.The poisoning of silkworms caused by the pollution of mulberry leaves with pesticides has seriously hindered the healthy development of the silkworm and mulberry industry in China.Chlorantraniliprole is a new type of bisamide insecticide.It has a broad spectrum and high efficiency against Lepidoptera insects.Chlorantraniliprole has been widely used in agricultural and forestry production in recent years.Chlorantraniliprole exposure could affect the development and metamorphosis of silkworm.In order to investigate the mechanism of the trace chlorantraniliprole exposure on silkworms,and failed pupation,silkworm cultivar "Jingsong × Haoyue" was used as the material to study the trace chlorantraniliprole(0.01 mg/L)exposure for a period of time(24 h)in third day of fifth instar,the effects on the growth and development of the silkworm and the pupae metamorphosis were analyzed by digital gene expression profiling(DGE).The main research results are as follows:1.Residue of chlorantraniliprole in mulberry leaves and in silkworm bodiesIn order to study the chlorantraniliprole residues on mulberry leaves and in silkworm bodies,HPLC was used to study the residual characteristics of chlorantraniliprole on mulberry leaves and its metabolic kinetics in the hemolymph of silkworm.Our results showed that the residual half-life of chlorantraniliprole on mulberry leaves was 5.00 days,and the metabolic half-life in the hemolymph of silkworm was 2.79 days.2.Effects of trace chlorantraniliprole on biological characteristics of silkwormThis study investigated the symptoms of poisoning in silkworms after exposure to chlorantraniliprole,recorded and investigated the changes in body weight,cocoon quality index,and pupation rates of silkworms.The results showed that after exposure of trace chlorantraniliprole,the silkworm exhibited slow action.The body weight was significantly reduced at 48 hours after exposure(P ≤ 0.01),both the cocoon shell rate and cocooning rate were reduced.The pupation rate was 92.78%.3.Effects of trace chlorantraniliprole on the hormones and the effects of transcription of genes related in 20E signaling pathwaysIn order to study the mechanism of the reduction of the pupation rate of silkworms after exposure to trace chlorantraniliprole,the content of 20E in hemolymph was detected and it was found that the content of 20E was found to be significantly reduced(P≤0.01).The transcription levels of the key genes CYP302a1,CYP306a1,CYP314a1 and CYP315a1 related in 20E synthesis,were 91.04%,45.20%,68.81%and 98.08%respectively.The transcription levels of the 20E signaling pathway response genes EcR,USP,E74,and E75 were 56.86%,89.74%,73.82%and 35.41%respectively(P≤0.05).After in vitro injection of 20E,it was found that the epidermis was formed,but the metamorphosis could not be completed,indicating that the hindered formation of epidermis might be the cause of decreased 20E levels after chlorantraniliprole exposure.The transcription level of Ftz-f1 was significantly reduced(P≤0.01),and vitro injection of 20E failed to activate the transcription of Ftz-f1,which might be the reason cause pupation metamorphosis failure.4.Analysis of digital gene expression profiles of epidermis after chlorantraniliprole exposuredDGE results showed that,after trace chlorantraniliprole exposure,in the epidermis,4830 genes were significantly up-regulated(P≤0.001),and 993 genes were significantly down-regulated(P≤0.001).In the biological process GO classification label,DEGs are enriched in cellular processes and metabolic processes,with 361 and 330 genes.In the cellular component GO classification label,DEGs are enriched in cells,macromolecular complexes,membranes,membrane components and organelles,with 361,159,303,286,and 244 genes.Under the molecular function GO label,DEGs are enriched in binding and catalytic activity,with 492 and 449 genes.In the KEGG metabolic pathway,DEGs are enriched in environmental information processing classification label and metabolism with 729 and 752 differentially expressed genes.DEGs are enriched in multiple KEGG pathways,including oxidative stress,autophagy,apoptosis and energy metabolism,indicating that a range of physiological processes are in involved in pupation.In this study,the metabolism of trehalose in the KEGG energy metabolism pathway was investigated.Analysis of differentially expressed genes showed that the transcript level of the gene TRE encoding trehalase,the first enzyme in the chitin synthesis pathway was 0.11-fold of the control(P≤0.01),and that the transcript level of the gene TPS encoding trehalose synthase was 0.39-fold of the control(P ≤0.05).The synthesis of chitin is directly regulated by trehalose metabolism and supply balance of trehalose.Moreover,the transcript levels of the key genes GFAT and UAP involved in regulating the anabolism of chitin were also significantly reduced(P≤0.05).Notably,after chlorantraniliprole exposure,the activity of trehalase was significantly decreased(P≤0.001),and the content of trehalose was 108.18%of the control.After chlorantraniliprole exposure,the synthesis of trehalose was inhibited and the supply of trehalose was unbalanced,affecting the synthesis of chitin in the epidermis.Moreover,the transcription of the chitin synthase gene was inhibited,further affecting the accumulation of chitin in the epidermis.These combined factors might lead to the failure of the exposed silkworm larvae to pupate.The results showed that exposure to trace amounts of chlorantraniliprole resulted in a decrease in 20E levels and inhibition of chitin synthesis,affecting epidermal formation and that the under-expressed Ftz-f1 gene might be a important gene that is responsible for failed pupation metamorphosis. |
PDF Full Text Request