Silkworm Molting Research Articles

Insights into the activation mechanism of Bm-CPA: Implications for insect molting regulation

Carboxypeptidase A has been found across various animal species, yet its activation mechanism during the insect molting process remains elusive. Our study specifically delved into the activation mechanism of carboxypeptidase A (Bm-CPA), identified in Bombyx mori's molting fluid during metamorphosis. Initially, western blotting identified two forms of Bm-CPA, 65 kDa and 54 kDa, in the epidermis of silkworms during the molting stage. Expressing the complete Bm-CPA sequence in Pichia pastoris allowed the identification, via mass spectrometry analysis, of a 75-amino-acid propeptide for the initial hydrolysis process. Subsequently, a 35 kDa form of Bm-CPA emerged in the molting fluid, confirmed as the active form through in vitro assays, demonstrating potent carboxypeptidase A activity and faint carboxypeptidase B activity. Four potential activation sites (including Lys158/Arg159 and Arg177/Arg178) were identified through mass spectrometry and amino acid mutation analysis. RNAi of Bm-CPA indicates its critical role in molting. Finally, the carboxypeptidase inhibitor (Bm-CPI) from silkworm molting fluid was expressed to explore its role in regulating Bm-CPA activity, demonstrating a direct interaction with the 35 kDa Bm-CPA. Our research implies Bm-CPA's potential involvement in the silkworm molting process, suggesting diverse regulatory roles. These findings highlight intricate protein regulation patterns during insect metamorphosis and development.

Molecular cloning, gene expression analysis, and in silico characterization of UDP-N-acetylglucosamine pyrophosphorylase from Bombyx mori.

The present study was aimed to explore the molecular and structural features of UDP-N-acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20-hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N-acetylglucosamine-1-phosphate, and UDP-N-acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.

Disruption of PTPS Gene Causing Pale Body Color and Lethal Phenotype in the Silkworm, Bombyx mori.

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene or by defects in the tetrahydrobiopterin (BH4) synthesis pathway. Here, by positional cloning, we report that the 6-pyruvoyl-tetrahydropterin synthase (PTPS) gene, encoding a key enzyme of BH4 biosynthesis, is responsible for the alc (albino C) mutation that displays pale body color, head shaking, and eventually lethality after the first molting in silkworm. Compared to wild type, the alc mutant produced more substrates (phenylalanine (Phe) and tyrosine (Tyr)) and generated less DOPA and dopamine. Application of 2,4-diamino-6-hydroxypyrimidine (DAHP) to block BH4 synthesis in the wild type effectively produced the alc-like phenotype, while BH4 supplementation rescued the defective body color and lethal phenotype in both alc and DAHP-treated individuals. The detection of gene expressions and metabolic substances after drugs treatments in alc and normal individuals imply that silkworms and humans have a high similarity in the drugs metabolic features and the gene pathway related to BH4 and the dopamine biosynthesis. We propose that the alc mutant could be used as an animal model for drug evaluation for BH4-deficient PKU.

Proteomic analysis of Bombyx mori molting fluid: Insights into the molting process

Molting is an essential biological process occurring multiple times throughout the life cycle of most Ecdysozoa. Molting fluids accumulate and function in the exuvial space during the molting process. In this study, we used liquid chromatography-tandem mass spectrometry to investigate the molting fluids to analyze the molecular mechanisms of molting in the silkworm, Bombyx mori. In total, 375 proteins were identified in molting fluids from the silkworm at 14–16h before pupation and eclosion, including 12 chitin metabolism-related enzymes, 35 serine proteases, 15 peptidases, and 38 protease inhibitors. Gene ontology analysis indicated that “catalytic” constitutes the most enriched function in the molting fluid. Gene expression patterns and bioinformatic analyses suggested that numerous enzymes are involved in the degradation of cuticle proteins and chitin. Protein-protein interaction network and activity analyses showed that protease inhibitors are involved in the regulation of multiple pathways in molting fluid. Additionally, many immune-related proteins may be involved in the immune defense during molting. These results provide a comprehensive proteomic insight into proteolytic enzymes and protease inhibitors in molting fluid, and will likely improve the current understanding of physiological processes in insect molting. Biological significanceInsect molting constitutes a dynamic physiological process. To better understand this process, we used LC-MS/MS to investigate the proteome of silkworm molting fluids and identified key proteins involved in silkworm molting. The biological processes of the old cuticle degradation pathway and immune defense response were analyzed in the proteome of silkworm molting fluid. We report that protease inhibitors serve as key factors in the regulation of the molting process. The proteomic results provide new insight into biological molting processes in insects.

Comparative proteomic analysis reveals that juvenile hormone binding protein and adenylate kinase may be involved in the molting process of silkworm, Bombyx mori.

The molting is an essential part of the silkworm metamorphosis development. Although previous studies have demonstrated that molting in silkworm is associated with prothoracicotropic hormone (PTTH), molting hormone (MH), and juvenile hormone (JH), the changes of proteins and genes during silkworm molting, as well as the molecular mechanism about its generating and maintaining remains unclear. In this paper, the proteomic approaches were employed to investigate this issue. Totally, 35 different proteins were successfully identified through mass spectrometry and database searches, among which 42 % proteins were involved in cell structure and 16 % proteins belonged to the metabolism group. Meanwhile, vacuolar ATP synthase, juvenile hormone binding protein precursor and adenylate kinase isoenzyme were found to be down-regulated at early, mid-molt stages, which were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Taken together, our data suggests that juvenile hormone binding protein (JHBP) and adenylate kinase (AK) play a critical role in the process of silkworm molting, which may participate in the regulation of silkworm molting.

Expression profile analysis of silkworm P450 family genes after phoxim induction

Silkworm (Bombyx mori) is an important economic insect and a model species for Lepidopteran. Each year, O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate (phoxim) pesticide poisoning in China results in huge economic losses in sericulture. Silkworm fat body is the main organ for nutrient storage, energy supply, intermediary metabolism, and detoxification. Microarray analysis of silkworm Cytochrome P450 detoxification enzyme genes revealed that all tested P450 4 (CYP4) family genes are expressed in the fat body. Quantitative Real-time PCR (QRT-PCR) was used to detect the expression of CYP4 family genes in silkworm fat body 0, 24, 48, and 72 h after phoxim exposure. The expression levels of silkworm molting hormone synthesis-related genes started to change 24 h after phoxim exposure, with those of CYP302A1, CYP306A1, and CYP314A1 being elevated by 1.38-, 1.33-, and 2.10-fold, respectively. The CYP18A1 gene that participates in steroid hormone inactivation and the CYP15C1 gene that participates in the epoxidation during the synthesis of juvenile hormone (JH) from methyl farnesoate (MF) were increased by 3.85- and 7.82-fold, respectively. Phylogenetic analysis indicated that these endogenous hormone metabolism-related genes belong to CYP mito clan and clan 2, and that phoxim exposure may affect silkworm development and metamorphosis. The CYP4, CYP6, and CYP9 families all showed some degrees of increases in gene expression; among them, CYP49A1, CYP4L6, CYP6AB4, CYP9G3, CYP9A19, and CYP9A22's transcription levels were significantly upregulated to 12.77-, 2.64-, 2.42-, 4.06-, 3.32-, and 2.98-fold, respectively, of the control levels. In the fat body, CYP49A1, CYP6AB4, CYP9A19, and CYP9A22 were constantly expressed at high levels after 24, 48, and 72 h of phoxim treatments; according to phylogenetic analysis, these genes belong to detoxification-related clan 3 and clan 4 CYP families. These genes may participate in the metabolism of phoxim in silkworm fat body. The results obtained in this study provide a basis for future in-depth investigations of insect P450 family genes in metabolic detoxification.

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.

Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein ( hsp) 23.7 promoter, with a neomycin-resistance gene ( neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene ( gfp) under the control of the B. mori actin 3 ( A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.

Simple Manipulation of Silkworm Molting by an Artificial Diet Containing Plant-Derived 20-Hydroxyecdysone

Simple Manipulation of Silkworm Molting by an Artificial Diet Containing Plant-Derived 20-Hydroxyecdysone