Effects Of 1-deoxyoxykomycin And Urease On Metabolism Of Silkworm,Bombyx Mori,Fed By Artificial Diet And Gene Expression Of Digestive Enzymes

Silkworm rearing with artificial diet is an important direction for the future development of the sericulture industry.After decades of research and exploration at domestic and foreign,the silkworm artificial diet has made great progress in terms of nutritional ratio and palatability.At present,the techniques of young silkworm rearing with artificial diet that has basically reached the requirement of practical production.However,the whole-age artificial diet sericulture still has many problems.Such as weak constitution,poor resistance,low enamel layer rate and so on.That is to say,the artificial diet with high mulberry leaf content still can not achieve the effect of mulberry leaf silkworm rearing.Existing studies have shown that silkworms fed by mulberry leaves can accumulate 1-deoxynojirimycin(DNJ),an alkaloid that is harmful to other insects,and there are regular changes of DNJ content in silkworms.Another study found that the silkworm fed by mulberry leaves can absorb urease in mulberry leaves,and can decompose the urea produced by silkworm metabolism in the blood,thereby increasing the nitrogen utilization.However,there is no urease in the blood of silkworm fed by artificial feed,resulting in high urea content in the body and excreta.In order to explore the physiological effects of DNJ and urease on silkworm,metabolomics was used to investigate the effects of exogenous DNJ and urease on the metabolism of silkworm reard by artificial diet in this article.And the expression of four digestive enzyme genes in the midgut of artificial diet silkworm was detected using real-time quantitative PCR.In order to provide reference for improving artificial feed formulation.The main results are as follows:1.We measured the DNJ content in the blood on the 5th instar 3rd day of silkworm which fed with DNJ 0.1% of dry pellet artificial diet of M10(Mulberry leaf powder content 10%)in whole instars.The result indicated that the DNJ content of the adding group was more than 6 times that of the control group.The addition of DNJ to the pellet artificial diet had an inhibitory effect on the body weight growth of the silkworm,and the cocoon weight,the cocoon shell weight and the cocoon shell rate were significantly increased.And the rate of dead cocoon decreased significantly,the rate of larva-pupa increased significantly.Adding DNJ to the midgut of the silkworm had different effects on sucrase activity,blood trehalase activity,blood trehalose content and glycogen content in fat body,but there was no significant difference from the control,indicating that DNJ does not affect the silkworm’s sugar absorption and blood glucose concentration.2.Gas chromatography-mass spectrometer(GC-MS)and liquid chromatography-mass spectrometer(LC-MS)were applied to analyze the metabolomics of the 5th instar 3rd day blood samples of silkworm,and a total of 130 substances were identified in the silkworm blood of artificial diet fed with DNJ and without DNJ.Among them,there were 23 kinds of differential metabolites between the the DNJ fed group and the control group,the majority were organic acids and amino acids.Further analysis on the differential metabolites and metabolic pathways,the results showed that the addition of DNJ had different degrees of influence on the sugar metabolism,energy metabolism and amino acid metabolism in the silkworm.Among them,the glycometabolites regulated by α-glycosidase were significantly reduced,and the ?-fructofuranoses metabolites were significantly elevated,indicating that silkworms can metabolize sugars through the fructofurose pathway to reduce the toxicity of DNJ.However,the physiological accumulation of periodical accumulation and excretion of DNJ in silkworm remains to be further studied.3.The addition of jack bean urease to pellet artificial diet can be absorbed by the silkworm and remain active in the blood.The urease-treated group had different degrees higher levels of the cocoon weight,the cocoon shell weight and the cocoon shell rate than the control group.In addition,when silkworms were matured,the content of uric acid in silkworm blood and silkworm feces fed with artificial diet without urease was significantly lower than that of silkworms added and injected urease,while the content of urea in silkworm excrement was extremely higher than that of the urease treatment group.4.The analysis of 29 kinds of amino acids and their metabolites in the blood by targeted metabolomics method showed that the relative content of ornithine in the blood of mature silkworm reared by artificial diet with urease was significantly higher than that of the control group.Compared with the artificial diet injected with urease and control silkworm at the age of 5th instar 5th day,the relative content of ornithine and spermidine was significantly higher,the relative content of histamine decreased significantly,and there was no significant difference in the relative content of other substances in the blood of the mature silkworm.Whether added or injected urease,it led to a significant increase in the content of ornithine.It was speculated that urease may decompose urea and promote the metabolism of arginine,which was resulted in an increase content of ornithine.5.It has been also found that there are significant differences in amino acids and their metabolites in the blood of the mulberry silkworm compared with artificial diet silkworm,whether it is a control group or a added or injected urease treatment group,during the results of the analysis of amino acids and their metabolites by target metabolomics.In general,the relative content of most free amino acids and their metabolites in the blood of artificial diet silkworm is higher than that of mulberry leaf breeding,and the difference in metabolites is greatest between the artificial diet control group and the mulberry leaf control group.This indicates that the utilization rate of amino acids in the artificial diet silkworm is lower than that of mulberry leaves,which leads to the accumulation of various amino acids and their metabolites in the blood during the mature silkworm period.This may be an important reason why the cocoon shell weight and the cocoon shell rate in artificial diet are lower than those in mulberry leaves.6.Fluorescence quantitative PCR technique was used to determine the changes in the expression of the 4 digestive enzyme genes such as α-amylase,maltase,lipase and tryptase in the midgut of the silkworm were determined in the 4th and 5th instars.It was found that the expression levels of various digestive enzyme genes in the artificial diet silkworms were significantly different from those of mulberry silkworms.In particular,the expression levels of lipase and tryptase genes in the 5th instar of artificial diet silkworms were significantly lower than those of mulberry silkworms which might be one of the reasons that affected the digestion and absorption of silkworm,the growth and development of silkworm body and disease resistance.