Optimization Of Artificial Diet For Silkworm Based On Metabonomics

Up to now,turning dry mulberry leaves powders into diet is the prerequisite for reducing sericulture labor and large-scale breeding in the future.It is not only an important symbol of the transformation of sericulture from a labor-intensive industry to a technology-intensive industry,but also an important direction for the development of sericulture agriculture in the future.In view of this,under the premise of clarifying the difference between mulberry leaf and artificial diet for silkworm rearing performance,this paper compared the composition differences between mulberry leaf and artificial diet,and further utilised metabolomics to compare the differences of hemolymph and silkworm sand between mulberry leaves and artificial diet for feeding silkworms.Based on this,different metabolites were added into the artificial diet to raise silkworm and the possibility of further improving the quality of artificial diet was analyzed.The main results are as follows:1.Artificial diet formulation optimizationIn order to explore the effects of different artificial diets and mulberry leaves on the growth and development of silkworms,This part compares the feeding results of Japanese artificial diet,domestic artificial diet and basic artificial diet with that of mulberry leaf.The results showed that the cocoon shell rate of silkworm fed with different artificial diets was significantly lower than that of mulberry leaf group(P <0.05).Compared with the artificial diet group,the whole cocoon weight,cocoon shell weight and cocoon shell rate of the silkworm fed with mulberry leaves at the 5th instar were significantly increased(P < 0.05).In order to seek for suitable artificial diet formulations,the chemical composition differences between mulberry leaves and artificial diets were compared based on gas chromatography-mass spectrometry(GC-MS).The results showed that a total of 129 metabolites were annotated(matching value > 0).These metabolites include a variety of substances,such as sugars,amino acids,organic acids,fatty acids,vitamins and nucleosides.There were significant differences in 56 metabolites between mulberry leaf(M)and artificial diet(A)(P < 0.05).Among them,compared with mulberry leaves,40 substances were up-regulated and 16 substances were down regulated in artificial diet.The abundance in mulberry leaves was significantly higher than that in artificial diet,and the top 10 metabolites were 9,12,15-octadecatrienoic acid 、L-ascorbic acid 、 glucofuranoside 、 glyceryl-glycoside TMS ether 、13-methyltetradec-9-enoic acid trimethylsilyl ester、aminomalonic acid、urea、sucrose、L-(+)-threose 、 tyrosine.The abundance of metabolites in artificial diet was significantly higher than that in mulberry leaves,and the top 10 metabolites were1,8-naphthyridine、citric acid、D-(-)-tagatose、sorbic acid、ethanedioic acid、dulcitol、L-alanine、glutamic acid、acetic acid、serine.Based on the analysis of the results,it is recommended to seek for the lipids,proteins,inositol,citric acid and vitamins in the diet.Soybean oil and β-sitosterol were used in the experiment,and they were combined according to their different contents.The single addition of soybean oil had no significant effect on the moulter body weight of silkworms than the group without soybean oil(P > 0.05).Adding different amounts of soybean oil to diet containing 0.1%β-sitosterol had no significant effect on the moulter body weight of silkworms(P >0.05).Six kinds of artificial diets containing soybean meal in 0%,5%,10%,15%,20%and 30% groups were prepared respectively.The results showed that the level of soybean meal in artificial diet had a significant effect on the weight of silkworm(P <0.05).Compared with each treatment group,the weight of molter body of silkworm in30% group was the highest.Formulated with 5 kinds of artificial diet with myo-inositol content in the 0%,0.05%,0.1%,0.15%,0.2% groups,the weight of molter body of 1st instar in the inositol group was significantly higher than that of the 0% group(P <0.05).The weight of molter body of 2nd instar was significantly higher in the 0.05%group and 0.1% group than the other treatment groups(P < 0.05).Four kinds of artificial diets containing citric acid in 0%,0.5%,1% and 2% groups were prepared respectively.The weight of molter body of 1st instar in 2% group was significantly lower than that in other groups(P < 0.05),and the weight of molter body of 2nd instar in 2% group was significantly higher than that in other groups(P < 0.05).Four kinds of artificial diets containing vitamin B mixture in 0%,0.5%,1% and 1.5% groups were prepared respectively.The addition of vitamin B mixture had no significant effect on the molter body weight of the 1st instar(P > 0.05),and the molter body weight of the2 nd instar in 1.5% group was significantly higher than that in other groups(P > 0.05).Four kinds of artificial diets containing vitamin C in 0%,1%,2% and 3% groups were prepared respectively.The weight of molter body of 1st instar in 0% group was significantly lower than that in each vitamin C group(P < 0.05),and there was no significant difference in the molter body weight of the 2nd instar between the 2%group and the 3% group(P > 0.05).Based on the results of the previous diet formula optimization,we adjusted the artificial diet formula.The main adjustments are as follows: soybean meal content is 30%,inositol is 0.05%,citric acid is 2%,vitamin B mixture is 1.5%,and vitamin C is 2%,other components are shown in Table 3.3.After comparing the feeding effects of the optimized artificial diet and the basic diet,the molter body weight of the 1st instar was not significantly different(P > 0.05),and the molter body weight of the 2nd instar in the optimized artificial diet breeding group was significantly higher than that of the basic diet breeding group(P < 0.05).The growth and development of the Daizo and Heigao varieties fed with the optimized artificial diet is better than that of the basic artificial diet.The feeding performance is compared based on the optimized artificial diet and mulberry leaves.The feeding time of silkworm rearing with artificial diet was 25.5days,which was 10.9% longer than that of silkworm rearing with mulberry leaves(～23 days).For the artificial diet group,the maximum body weight of silkworm larvae reached 2.79 g on the 5th day of the 5th instar.For the mulberry leaf group,the maximum body weight of 3.48 g was reached on the 6th day of the 5th instar.The total cocoon weight,cocoon shell weight and cocoon shell rate in mulberry leaf group were significantly higher than those in artificial diet group(P < 0.05).The cocoon layer rate of mulberry leaf group was 20.32%,which was 12% higher than that of artificial diet group(18.13%).The contents and abundances of seven main silk proteins in silk were compared.The results showed that there was no significant change in the contents of fibroin light chain(Fib L),Fibroin Heavy Chain(Fib H),fibroin P25,sericin 1,sericin3,seroin 1 and seroin 2 in mulberry leaves group compared with artificial diet group(P > 0.05).The main component content of silk protein was not changed by artificial diet.The reelability,non-broken cocoon filament and neatness of artificial diet group were higher than those of mulberry leaf group.The degumming rate in 0.5% sodium carbonate solution in artificial diet breeding group was higher than that in mulberry leaf breeding group,and the degumming rate of silk in aqueous solution in artificial diet breeding group was higher than that in mulberry leaf breeding group.We also measured the silk long axis diameter of mulberry leaf group and artificial feed group.The results showed that the silk long axis diameter of mulberry leaf group was 10.1 μm and was significantly less than 12.4 μm in artificial diet group(P < 0.05).The results showed that the strength and elastic modulus of mulberry leaf group were significantly higher than those of artificial diet group(P < 0.05),while the toughness and elongation had no significant change(P > 0.05).Finally,the secondary structures of different silk were characterized by Attenuated total reflection Fourier Transform Infrared Spectroscopy(ATR-FTIR).The results showed that there was no significant difference in β-sheet content in silk from artificial diet group and the mulberry leaf group(P >0.05).2.Comparative fecal/hemolymph metabolomes of silkworms being fed mulberry leaf and artificial dietIn order to systematically evaluating the growth and metabolism of the artificial diet-bred silkworm,based on GC-MS metabolomics,the metabolic differences of the hemolymph of the artificial diet and the mulberry leaf-bred silkworm were compared.The results showed that a total of 38 different metabolites were identified.Among them,compared with the hemolymph of the artificial diet group,24 metabolites were up-regulated and 16 were down regulated in the hemolymph of the mulberry leaf group.Differential metabolites are mainly concentrated in the following eight different pathways: Aminoacyl-t RNA biosynthesis,Valine,leucine and isoleucine biosynthesis,Pantothenate and COA biosynthesis,Alanine,aspartate and glutamate metabolism,Glyoxylate and dicarboxylate metabolism,Galactose metabolism,Glycine,serine and threonine metabolism,Valine,leucine and isoleucine degradation.Metabonomic analysis was carried out on the feces of mulberry leaf breeding and artificial diet breeding of silkworm.The results showed that there were 57 silkworm fecal metabolites(P < 0.05)with significant difference between mulberry leaves group and artificial diet group.Compared with artificial diet group,there were 39up-regulated metabolites and 18 down regulated metabolites in mulberry leaves group.The relative content of metabolites in mulberry leaf group was significantly higher than that in artificial diet group,and the top 10 metabolites were L-methionine,D-allose,L-Alanine,9,12,15-octadecatrienoic acid,2-keto-L-gluconic acid,benzoic acid and L-leucine,L-valine,galactinol and L-threonic acid.The relative content of metabolites were significantly higher in artificial diet group than in mulberry leaf group,and the top 10 metabolites were D-Pinitol,3-hydroxyanthranilic acid,D-(+)-cellobiose,sorbic acid and butanoic acid,N-acetylglucosamine,urea,citric acid,D-gluconic acid and propionic acid.Further comprehensive analysis showed that the differential metabolites in the two groups of biomaterials were mainly concentrated in amino acid metabolic pathway,valine and glycine are the targets for subsequent optimization of diet formulations.3.Optimization of artificial diet for silkworm based on metabonomicsIn order to optimize the diet formula through metabolomics,this section conducts experiments on target differential metabolites.We selected valine for feeding experiment and prepared four kinds of artificial diets with valine content of 0%,0.5%,2% and 4%,The results showed that the addition of valine had no significant effect on the digestibility of silkworm at the 5th instar(P > 0.05).The food intake of 2% and 4%groups was significantly lower than that of 0% group(P < 0.05).The whole cocoon production efficiency was significantly improved in the 2% group and 4% group(P <0.05),which were 11.3% and 25.1% higher than that in the 0% group,respectively.The cocoon shell production efficiency of 2% group and 4% group was significantly lower than that of 0% group,decreased by 7.7% and 13.9% respectively.The wondering time of 0.5% and 2% groups was shorter than that of 0% group.Then we conducted a feeding experiment on glycine,succinic acid and D-mannitol.Every metabolites was added at four concentrations of 0%,0.05%,0.1%and 0.15% respectively.The results are as follows: Succinic acid and D-mannitol had no significant effect on cocoon weight,cocoon shell weight,pupa weight and cocoon shell rate(P > 0.05).The cocoon shell weight and cocoon shell rate of 0.15% glycine group were significantly higher than those of other groups(P < 0.05).The cocoon shell weight and cocoon shell rate of 0.15% glycine group were 13.04% and 5.4% higher than those of 0% glycine group,respectively.