Toxicity And Molecular Mechanism Of Pyriproxyfen On Different Tissues Of Silkwrom,bombyx Mori

Silkworm（Bombyx mori）is a model organism of Lepidoptera,which is a silk secreting insect with high economic value.Because the silkworm has been growing under the good conditions suitable for its growth and development for a long time,the silk production performance of the silkworm has been improved,at the same time,the silkworm is extremely sensitive to pesticides,viruses and other adverse factors.Mulberry leaves,as the main natural food of silkworm,are easily polluted by drifting in nearby farmland,residual spraying equipment or pesticides directly used for mulberry pest control during cocoon production season.Once the polluted mulberry leaves are fed to the silkworm,they can cause acute or chronic poisoning and damage various tissues of the silkworm,thus affecting the growth and development of the silkworm and the yield of cocoon silk.Pyriproxyfen is a kind of insect growth regulating pesticide with high insecticidal activity and strong target specificity.It has strong juvenile hormone activity,and has highly effective killing effect on some pests of Homoptera,Thysanoptera,Diptera,Lepidoptera,etc.It is widely used in the control of health pests and agricultural and forestry pests at present.In recent years,the phenomenon of cocoon free in many silkworm areas is caused by the chronic poisoning of mulberry leaves polluted by pyriproxyfen.The toxicity of pyriproxyfen to silkworm has been reported,but the damage to main organs of silkworm,such as silk gland,midgut,fat body and ovary,and the molecular mechanism of toxicity to various organs have not been reported.In this study,the silkworm variety 7031 was used as material.On the 4^thday of 5^thinstar,the silkworm larvae were fed with mulberry leaves treated with 0.001 mg/L pyriproxyfen（treatment group）and dd H₂O（control group）respectively,and then fed normally.The symptoms of larva poisoning were observed,and the indexes of larva weight,cocooning rate,dead cage rate,cocoon layer rate,number of eggs laid,hatching number and hatching rate were investigated by means of Biostatistics;Hematoxylin-Eosin Staining（HE staining）and Quantitative Real-time PCR（q RT-PCR）were used to observe and detect the histopathological changes and the transcriptional of related genes of silk gland,midgut,fat body and ovary respectively,as well as the activities of detoxification enzymes in silk gland,midgut and fat body were detected.The results showed that 0.001 mg/L pyriproxyfen could lead to silkworm larvae poisoning,affect the growth and development indicators,damage the silk gland,midgut,fat body and ovary.In addition,it can cause the damage of silk gland,midgut,fat body and ovary,change the transcription level of silk gland,midgut,fat body and ovary related genes and change the activity of detoxification enzyme.1.The symptoms of pyriproxyfen poisoning of silkworm larvae were slow mulberry eating,prolonged instar,shortened body,swollen and shiny,head up,soft and black body,and serious death;at the same time,the weight of larvae decreased,cocooning function was destroyed,resulting in the decrease of cocooning rate,the increase of dead cage rate,and the decrease of cocoon layer rate;after metamorphosis,mating and oviposition of the surviving larvae,the number of oviposition,hatching and hatching rate in the treatment group all decreased.2.The results of HE staining showed that the structure of silk gland was seriously damaged,the epithelial cell layer of gland became thinner,the content of particulate matter became higher,a large number of cells in connective tissue layer died,and the characteristics of vacuoles and rupture appeared;the expression levels of genes related to silk protein synthesis（Fib-H,Fib-L,P25,Ser-1,Ser-3,SGF-1）were down regulated,which indicated that the decrease of cocooning rate may be closely related to the down-regulation of silk protein synthesis gene expression.At the same time,the expression levels of the key genes（Pi3k,Akt,P70s6k,4e-bp,Cnc C,Keap1）of PI3K/Akt and Cnc C/Keap1 pathway were up-regulated,which indicated that these two signaling pathways were involved in the process of silk gland respond to pyriproxyfen.The results showed that the expression of detoxification enzyme genes（CYP9a19,CYP9a20,GSTd1,GSTs1,Car E8v1,Car E10）and the activity of detoxification enzymes（CYPs,GST and Car E）in silk gland could be increased by adding a small amount of pyriproxyfen.It is speculated that this is the main way for silk gland to resist the toxicity of pyriproxyfen.The results provide a theoretical basis for further study on the damage of silk gland and the effect of gene transcription induced by pyriproxyfen.3.Histopathological analysis of the midgut showed that the muscle layer of the midgut was destroyed,the cylindric cells and goblet cells were seriously damaged,and the peritrophic membrane was destroyed after feeding pyriproxyfen.The expression levels of digestion and absorption related genes（α-amylase,Trypsin-like protease,Lipase 1）,oxidative phosphorylation related genes（NDUFV 1,NDUFB 7,NDUFC 2,QCR 7,QCR 9,ATPe VOD）and antioxidant related genes（SOD,GST,GPX,CAT）in midgut of the treatment group were decreased to varying degrees.However,the expression of detoxification enzyme genes（CYP9a20,GSTe5,GSTo3,GSTs1,Car E10,Car E15）and the activities of detoxification enzymes（CYPs,GST and Car E）were up-regulated.According to the results can speculate that pyriproxyfen can inhibit the digestive absorption,energy metabolism and antioxidant enzyme system of the midgut in silkworm,but increasing the expression of detoxification enzyme genes and the activity of detoxification enzymes can be used to respond to the invasion of pyriproxyfen to the host.The results provide a certain reference value for the stress mechanism of the midgut to the toxicity of pyriproxyfen.4.The addition of pyriproxyfen cause that the cell membrane was destroyed,the cell contents were drained and the cytoplasm was vacuolated.At the same time,the key genes of PI3K/Akt and Cnc C/Keap1 pathway（Pi3k,Akt,PDK,P70s6k,Cnc C,Keap1）and detoxification enzyme genes（CYP9a19,CYP9a20,GSTd1,GSTo3,GSTs1,Car E10）were up-regulated,and the activities of detoxification enzymes（CYPs,GST and Car E）were up-regulated.It is suggested that the stress ability of fat body to pyriproxyfen can be improved through the above ways.5.Histopathological analysis showed that the number of oocytes and oogonia decreased and vacuoles appeared in the treatment group.At the same time,the expression of ovarian development related genes（Ovo,Otu）and shear regulation related genes（Sxl-S,Sxl-L）were decreased in varying degrees,and the expression of 20E receptor Ec R was also decreased,but the expression of JHBP2 responsible for JH binding and transport was increased.It is speculated that these gene transcription changes affect ovarian development and egg formation of silkworm ovary,which lead to the reproductive failure of silkworm.Therefore,this study revealed the response of silkworm reproduction to pyriproxyfen,and provided a certain reference value for the metabolic mechanism of ovary to pyriproxyfen.