| The domesticated silkworm (Bombyx mori) is a holometabolism insect, including four development stages with significant differences both in the configuration and physiological function (egg, larva, pupa and adult moth). The cocoon shell acts like a house for silkworm to avoid the wretched weather, natural enemy and germs. And it could provide a stable and safe environment for pupal metamorphosis. As the pupa eats nothing and could not adhere anywhere, the cocoon contributes to the pupal immobilization. On the other side, the pupa is also very momentous for silkworm reproduction. Silkworm is only fed during larval stages, and accumulates lots of nutrient for the development of reproductive system and imaginal organs at the pupal stage. So, the silk and pupa are essential for the silkworm life.Bombyx mandarina was domesticated 5000 years ago, during the domestication process, the silk production was greatly increased. Meanwhile, the size and weight of pupa were also raised. The cocoon shell ratio (cocoon shell weight/(cocoon shell weight+ pupa weight)* 100%) of current silkworm strains ranges from about 10% to 30%. The genetic correlation coefficient of the cocoon shell weight and pupa weight was high. The daily amount of utilization of dry matter, energy, and carbohydrates in Bombyx mori were approximate equivalently stored in the cocoon shell (silk) and pupa. So, we hypothesize that there is an accurate mechanism in regulating the nutrition allocation across the cocoon shell (silk) and pupa. The metabolomics analysis could reveal useful biological information, because the metabolites demonstrate biological end points. Metabolomics analysis of silkworm is urgently required to discover the pivotal metabolite for regulating the distribution of nutrients into silk and pupa. In this study, metabolomic analysis based on GC-MS was performed to obtain comprehensive and reliable hemolymph metabolic profiling of silkworm with different extent silk protein synthsis arrested, including following three experimental groups. The silk-gland-degenerated silkworm strain (Nd), Dazao and 21-872 as control (Nd group). Silk yield was artificially arrested by knocking out the Bmfib-H gene (FGKO group) or extirpating the silk glands (SGE group). Furthermore, the cocoon shell ratio and cocoon shell weight were increased by glycine-injection and ACPC-injection, screening for candicate gene glycine transporter gene (BmGT1) was analyzed by bioinformatics, RNAi and overexpression, and so on. The main results are as follows:1. Hemolymph metabolomics of naturally mutational silkworm with nutritional imbalance between silk and pupaThe cocoon shell ratio of seven silkworm strains, with significantly different larvae weights, ranges from 10% to 30%, except for the naked pupa Nd. The Nd strain has almost no ability to produce silk because the degeneration of its silk glands. Thus, the Nd was the good material for our study. However, we could not find the original silkworm strain as the control, so, we selected the usual scientific research srtain Dazao and the silk productive strain 21-872 as control (called Nd group). Firstly, the biochemical indicator of Nd group on the 3rd day of fifth instar was assayed, the results showed that the contents of trehalose, glycogen and ATP in Nd were higher than Dazao or 21-872, the contents of TG were lower.To obtain the accurate and comprehensive data for metabolic profiling, non-targeted metabolomics method was developed. And the method validations (reproducibility, intra-day and inter-day precision and stability of post-preparation) were performed, which showed that the metabolomics method is reliable. Firstly, the metabolomics method was used to analyze the metabolic profiling of hemolymph from the 3rd day of fifth instar to the 6th day of fifth instar between Nd group based on GC-MS. A total of 100 identified metabolites were observed,82 of them were verified by standard substances. Principal components analysis (PCA) score plot represented a clear differences between the Nd and its control (Dazao or 21-872), and the separation within one given strains at different developmental stages was also observed, suggesting that differential metabolites were exist in hemolymph between Nd and its control. Moreover, the absent separation between two silkworm strain with normal silk glands (Dazao and 21-872) could explain the reason that the separation between Nd and Dazao or 21-872 was resulted from the defect of the silk synthesis. Furthermore, hierarchical cluster analysis of significant differential metabolites between Nd vs. Dazao or Nd vs. 21-872 from the 3rd day to 6th day on the fifth instar was performed.Firstly, we focused on the significantly differential metabolites between Nd and Dazao on the 6th day of the fifth instar, the day was the anaphase of silk protein synthesis. A total of 46 identified differential metabolites were observed by GC-MS in Nd group. The levels of 15 identified differential metabolites were higher in Nd than Dazao, including uric acid, glycine, aminomalonic acid, serine, proline, lanthionine, glycolic acid, glutamic acid, and so on. Notably, the levels of uric acid, glycine were more abundant in Nd hemolymph during the developmental stages on the day 3rd to 6th day of fifth instar. In contrast, the decreased levels of 31 identified differential metabolites were also conspicuous discovered, mainly including carbohydrates (inositol, D-Lyxose, inositol,2-keto-L-Gluconic acid), intermediate metabolites of tricarboxylic acid cycle (citric acid, malic aicd, fumaric acid), free fatty acid (hexadecanoic acid, linolenic acid and octadecanoic acid), and amino acids (methionine, ornithine, isoleucine and hydroxyproline).2. Hemolymph metabolomics of artificially mutational silkworm nutributions imbalance between silk and pupaThe silk protein synthesis was artificially obstructed by knocking out the Bmfib-H gene (FGKO) or extirpating the silk gland (SGE) for in-depth study to find the regulatory factor of nutritional distribution in silk and pupa. The development stages of treated larvae were almost not affected, but as expected, the size and weight of pupal body were significantly elevated. The pupal body weight of FGKO or SGE was increased by 19.08% or 32.49% compared with their respective control, respectively. The cocoon shell ratio of FGKO was decreased by 67.2%. GC-MS analysis was applied to assay the relative metabolite levels in hemolymph samples of FGKO group or SGE group. Partial least squares-discriminate analysis (PLS-DA) score plot showed a clear distinction across the two experimental groups.60 or 66 identified differential metabolites in FGKO group or SGE group were detected, respectively. The levels of five or nine differential metabolites were increased in FGKO or SGE, respectively. Most of those differential metabolites change trends were consistent with those of Nd group, including glycine and its precursors, uric acid, free fatty acid, carbohydrate and some amino acids.Venn diagram displayed that there were at least 52 differential metabolites in random two experimental groups,16 differential metabolites detected in three groups. The pathway impact analysis exhibited that differential metabolites were concentrated on the pathway of aminoacyl-tRNA biosynthesis, glycine metabolism, nitrogen metabolism, carbohydrate metabolism, citrate cycle, fatty acid metabolism, and so on. The correlation network of cocoon shell ratio and differential metabolites in FGKO group was constructed using the Cytoscape software. All of them were divided into two separate cluster, the larger network was bridged by glycine. Cocoon shell ratio was closely relevant with glycine (0.892) or aminomalonic acid (0.74). Together all of above results, we speculate that glycine played the important role in regulating the nutritional balance between the silk and pupa.3. Proteomic analysis of fatbody in FGKO groupProteomics of fat body was analyzed to detect the protein changes in the FGKO fat body. A total of 1767 and 1705 protein groups were identified in FGKO and wide-type Dazao, respectively. All of identified proteins were classified into 20 categories based on their annotated molecular functions through BLAST2GO software, mainly including enzymes, transcription and translation, energy-related, cytoskeleton, protease and protease inhibitor, transferase, proteins termed as 30K proteins, SP proteins, heat shock proteins, and so on. Importantly, the enzymes functioning as the catalyst were the top abundance proteins,25.2% by relative molar abundance. The third profuse proteins were involved in transcription and translation processes, suggesting that the fat body is the important place for proteins synthesis as well. The abundances of enzymes, SP proteins, transferase, and protease inhibitor were down-regulated in FGKO, while transcription and translation proteins, energy-related proteins, and immune-related proteins were up-regulated. These results implied that the abundances of large-scale proteins in fat body were altered after knocking out a single gene of Bmfib-H in silk glands.To find differential proteins in the FGKO fat body, LFQ algorithm was performed. A total of 662 differential proteins with at least two fold-changes of LFQ intensities were obtained. The top two kinds of differential proteins were the enzymes (155), and transcription and translation related proteins (86). Interestingly, the numbers of down-regulated proteins, which belonged to enzymes, transferase, protease inhibitor, and SP proteins, were more than that of up-regulated proteins in FGKO fat body. In contrast, the numbers of up-regulated proteins involved in transcription and translation process were more than that of down-regulated proteins. The results of differential proteins demonstrated that the metabolism of fat body was greatly altered after knocking out Bmfib-H gene in silk gland.Furthermore, metabolic pathways that differential proteins participated were analyzed.153 differential proteins were located in metabolic pathways. The results of metabolic pathways analysis showed that both glycolysis/ glyconeogenesis and pentose phosphate pathway existed in silkworm fat body.10 of 12 differential proteins involving in glycolysis/glyconeogenesis, and 8 of 9 differential proteins taking part in pentose phosphate pathway were down-regulated in FGKO fat body, respectively.7 of 11 proteins participating in oxidative phosphorylation were up-regulated,8 of 10 differential expressed proteins in glycine, serine and threonine metabolism were significant decreased. These findings illustrated that many metabolic pathways were changed in fat body in respond to the Bmfib-H gene knocked out in silk gland. The contents of glycogen and proteins of dry weight fat body were robustly accumulated in FGKO fat body. The increasing contents of glycogen and proteins demonstrated that anabolism in fat body was activated after knocking out Bmfib-H gene.4. Functional analysis of silkworm glycine transporter geneConsidering that the level of glycine in hemolymph was significantly increased after breaking the nutritional balance between silk and pupa. And, the expression levels of the genes involved in glycine-serine synthetic pathway were decreased in FGKO fatbody. Hence, glycine or ACPC was injected into the hemolymph, the results showed that cocoon shell ratio was increased by approximately 3.5% or 3.89%, respectively. The level of glycine transporter gene (BmGT1) was depressed in the midgut of FGKO or SGE. The expression leves of BmPSGDH, BmPSAT, and BmPSPH involved in serine synthetic pathway were decreased in FGKO midgut. In contrast, the expression leves of BmSHMT, BmAGXT, BmGMT, and BmOAT involved in glycine synthetic pathway were increased in FGKO midgut.BmGT1 was a candicate gene that may be important for the domestication. It was similar to the growth disease gene of human, and the gene was specially and highly expressed in midgut. The three dimensional structure of BmGT1 is highly similar to the dopa transporters’. In addition, the expression level of BmGT1 was significantly decreased after supplying the extra glycine or ACPC into the cell culture. The expression levels of enzymes participating in glycine-serine synthetic pathway were changed after interfering or overexpressing the BmGTl. The cocoon shell ratio was increased after injecting the inhibitor of BmGT1 gene. These results suggested that BmGT1 may be involved in regulating the nutritional balance between silk and pupa.In this study, the integration of metabolomics and proteomics is helpful for us to understand the regulational mechanism of nutritional balance between silk and pupa in silkworm. Three different extent ways were used to arrest the silk synthesis for breaking the nutritional balance between silk and pupa. Those results showed that the level of glycine was notably increased, the levels of enzymes involved in glycine-serine synthetic pathway were significantly changed at protein and mRNA level. Taken together, these findings indicated that glycine plays an important role in regulating nutritional balance between silk and pupa. Furthermore, using biological technique, we found that BmGT1 gene may be taken part in regulating the levels of enzymes involved in glycine-serine synthetic pathway to control the level of glycine for maintaining the nutritional balance between silk and pupa in silkworm, Bombyx mori. |
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