Bmo-miR-6498-5p suppresses Bombyx mori nucleopolyhedrovirus infection by down-regulating BmPLPP2 to modulate pyridoxal phosphate content in B. mori.

The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR-6498-5p was down-regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR-6498-5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR-6498-5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4-deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR-6498-5p has a potential anti-BmNPV role by down-regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR-6498-5p down-regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori-BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture.

Bombyx mori serpin 3 is involved in innate immunity by interacting with serine protease 7 to regulate prophenoloxidase activation.

A subfamily of conserved proteins called serpins plays crucial roles in various physiological functions, particularly in the activation pathway of the serine protease cascade, an essential component of insect innate immunity. Here, we found Bombyx mori serpin 3 (BmSerpin3) was most highly expressed in the fat body, and was up-regulated after exposure to bacteria, fungus and virus. Further, the expression of BmSerpin3 in the hemocytes, fat body, midgut of silkworm larvae, and BmN cells was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Through Bac-to-Bac expression system, we obtained the active protein of BmSerpin3, and the enzyme activity assay showed that BmSerpin3 significantly inhibited the activity of both subtilisin and trypsin. In addition, BmSerpin3 could inhibit the activation of prophenoloxidase (PPO) in larvae. The knockdown of BmSerpin3 showed increased phenoloxidase (PO) activity compared to control after BmNPV infection. Ultimately, we confirmed that BmSerpin3 interacts with B. mori Serine Protease 7 (BmSP7). Hence, we hypothesize that BmSerpin3 is involved in innate immunity by interacting with BmSP7 to regulate the PPO activation cascade. Taken together, these results showed that BmSerpin3 play a role in silkworm innate immunity and lay a foundation for studying its functions.

RNA methyltransferase BmMettl3 and BmMettl14 in silkworm (Bombyx mori) and the regulation of silkworm embryonic development.

N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in regulating many biological processes, especially embryonic development. However, regulation of m6A methylation during silkworm embryonic development and diapause remains to be investigated. In this study, we analyzed the phylogeny of subunits of methyltransferases BmMettl3 and BmMettl14, and detected the expression patterns of BmMettl3 and BmMettl14 in different tissues and at different developmental stages in silkworm. To investigate the function of m6A on the development of silkworm embryo, we analyzed the m6A/A ratio in diapause and diapause termination eggs. The results showed that BmMettl3 and BmMettl14 were highly expressed in gonads and eggs. Moreover, the expression of BmMettl3 and BmMettl14 and the m6A/A ratio were significantly increased in diapause termination eggs compared with diapause eggs in the early stage of silkworm embryonic development. Furthermore, in BmN cell cycle experiments, the percentage of cells in the S phase increased when lacking BmMettl3 or BmMettl14. This work contributes to understanding the role of m6A methylation during insect embryogenesis and gametogenesis. It also provides a research orientation to further analyze the role of m6A methylation in diapause initiation and termination during insect embryonic development.

Identification of FerLCH, isolation of ferritin and functional analysis related to interaction with pathogens in Eri-silkworm, Samia cynthia ricini.

Ferritin is a ubiquitous and conserved iron storage protein that plays a significant role in host detoxification, iron storage, and immune response. Although ferritin has been studied in many species, little is known about its role in the Eri-silkworm (Samia cynthia ricini). In this study, the ferritin light-chain subunit gene, named ScFerLCH, was identified from S. c. ricini. The full-length gene, ScFerLCH, was 1,155 bp and encoded a protein consisting of 231 amino acids with a deduced molecular weight of 26.38 kDa. Higher ScFerLCH expression levels were found in the midgut, silk gland, and fat body by quantitative reverse-transcription polymerase chain reaction and western blot analysis. Injection of Staphylococcus aureus and Pseudomonas aeruginosa could induce upregulation of ScFerLCH in the hemolymph, fat body, and midgut, indicating that ScFerLCH may contribute to the host defense against invading pathogens. In addition, the native ferritin protein was isolated from S. c. ricini by native polyacrylamide gel electrophoresis and its two subunits, ferritin heavy-chain subunit (ScFerHCH) and ferritin light-chain subunit (ScFerLCH), were identified by mass spectrometry. Specifically, we found that recombinant ferritin subunits could self-assemble into a protein complex in vitro; moreover, both recombinant subunits and the protein complex were found to bind different bacteria, including Escherichia coli, P. aeruginosa, S. aureus, and Bacillus subtilis. However, bactericidal tests showed that the protein complex could not inhibit the growth of bacteria directly. Taken together, our results suggest that ScFerritin might play an important role in mediating molecular interaction with pathogens.

DNA Methylation Is Correlated with Gene Expression during Diapause Termination of Early Embryonic Development in the Silkworm (Bombyx mori).

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

DNA methyltransferase BmDnmt1 and BmDnmt2 in silkworm (Bombyx mori) and the regulation of silkworm embryonic development.

DNA methylation is one of the most widespread epigenetic marks and has been linked to insect development, especially influencing embryonic development. However, the regulation of DNA methylation in silkworm embryonic development and diapause remain to investigate. In this study, reverse-transcription quantitative polymerase chain reaction was performed to identify the expression level of Bombyx mori DNA methyltransferases (BmDNMTs) 1 and 2 ( BmDnmt1 and BmDnmt2) in different tissues, different embryonic developmental stages, and different strains of the silkworm. The results showed that BmDNMTs were the most highly expressed during embryonic development, especially at early embryonic stages. In particular, the expression of BmDNMTs was significantly upregulated in diapause-terminated eggs by HCl treatment. Moreover, tissue distribution showed that BmDnmt2 was highly expressed in testis and ovary, and BmDnmt1 was highly expressed in testis. This study contributes to understanding the correlation of DNA methylation occurs with embryogenesis and gametogenesis in insect, meanwhile, it provides a research orientation to further analyze the role of DNA methylation in diapause initiation and termination in insect embryonic development.

The regulation of crecropin-A and gloverin 2 by the silkworm Toll-like gene 18 wheeler in immune response.

The innate immune system is conserved among different insect species in its response to microorganism infection. The transmembrane receptors of the Toll superfamily play an important role in activating immune response, however, the function of silkworm Toll family member 18 Wheeler (18 W) remained unclear. Here, the 18w gene in silkworm was characterized. A relatively high transcription level of Bm18w mRNA was found in Malpighian tubules, and in eggs, larvae pre-molt to fourth instar, pupae and adults. When silkworm larvae were infected with E. coli or S. aureus, Bm18w showed a significant response, especially to E. coli, but did not have antibacterial activity. To further identify the downstream antimicrobial peptide genes of Bm18w, expression of Bm18w was knocked down with siRNA in vitro, resulting in significant decreases of cecropin-A, gloverin 2, and moricin B3. The overexpression of Bm18w was carried out using pIZT/V5-His-mCherry insect vector in BmN cells and significant upregulation of cecropin-A and gloverin 2 was detected, as well as upregulation of attacin and defensin. Based on the results, we concluded that Bm18w is involved in response to bacterial infection by selectively inducing the expression of antimicrobial peptide genes, especially cecropin-A and gloverin 2. This study provides valuable data to supplement understanding of the immune pathway of the silkworm.

Sanguinarine caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori.

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.

Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection.

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.

Molecular characterisation of Apolipophorin-III gene in Samia cynthia ricini and its roles in response to bacterial infection.

Apolipophorin-III (ApoLp-III) is an abundant hemolymph protein mainly involved in lipid transport and innate immunity in insects. In the present study, the gene Samia cynthia ricini ApoLp-III (ScApoLp-III) was identified from a transcriptome database, and contained 790 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that ScApoLp-III had significant homology with ApoLp-III protein from Antheraea pernyi. Higher ScApoLp-III expression levels were found in the fat body and silk gland by reverse transcription quantitative PCR (RT-qPCR). Injection of Staphylococcus aureus induced up-regulation of ScApoLp-III in the midgut, fat body and hemocytes. However, ScApoLp-III was down-regulated in the midgut and fat body after Pseudomonas aeruginosa injection, indicating that ScApoLp-III may contribute to the host's defense against invading pathogens. Additionally, recombinant ScApoLp-III was found to bind different bacteria, including E. coli, P. aeruginosa, S. aureus and B. subtilis. Bactericidal tests showed that recombinant ScApoLp-III strongly inhibited Gram-negative bacteria, including Escherichia coli and P. aeruginosa. However, it had no obvious influence on Gram-positive bacteria. Taken together, our results suggest that the ScApoLp-III might play an important role in the innate immunity of S. c. ricini.

A toxicological, metabonomic and transcriptional analysis to investigate the property of mulberry 1-deoxynojirimycin against the growth of Samia cynthia ricini.

1-Deoxynojirimycin (DNJ) is a natural d-glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1-5, and mass deaths happened in DNJ groups on day 3-5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.

Identification of Four ATP-Binding Cassette Transporter Genes in Cnaphalocrocis medinalis and Their Expression in Response to Insecticide Treatment.

The ATP-binding cassette (ABC) transporters belong to a superfamily of genes involved in the transport of specific molecules across lipid membranes, as well as insecticide resistance, present in all living organisms. In this study, we combined the Cnaphalocrocis medinals transcriptome database with a bioinformatics approach to identify four C. medinals ABCs (CmABCs), including CmABCG1, CmABCG4, CmABCC2 and CmABCC3. Tissue expression analysis showed that these genes had a tissue-specific expression pattern. CmABCG1 had significantly higher expression in the haemolymph and head compared to the other tissues. The expression of CmABCG4, CmABCC2 and CmABCC3 was highest in the midgut, followed by expression in the fat body. The developmental stage expression analysis showed that CmABCG1, CmABCG4, CmABCC2 and CmABCC3 were mainly expressed in adults. The transcription of CmABCG1, CmABCG4 and CmABCC2 was significantly induced by chlorpyrifos. Taken together, the results of our study provided useful information for understanding of the detoxification system of C. medinalis.

Molecular Characterization and Functional Analysis of a Ferritin Heavy Chain Subunit from the Eri-Silkworm, Samia cynthia ricini.

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host's defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis.

Screening weight related genes of velvet antlers by whole genome re-sequencing.

The velvet antler is a special organ that has important biological significance for deer, and its growth is a complicated biological metabolism process. Growing evidence suggests that genetics factors play essential roles in the weight of velvet antlers. In this study, we investigated five sika deer (Cervus nippon) populations under the same feeding condition, and screened genetic variations in the 100 samples (including 50 heavy and 50 light velvet antler weight samples) by whole genome re-sequencing. The results showed that 94 genetic variations were related to the velvet antler weight, among which two single nucleotide polymorphism (SNP) sites were located on the exon regions of OAS2 and ALYREF/THOC4, respectively. Furthermore, ALYREF/THOC4 is highly expressed in the velvet antler. The biological functions of these genetic variations were highly related to the growth and development of deer velvet antlers. Collectively, we screened genes related to the velvet antler weight in sika deer populations by whole genome re-sequencing and identified 94 sites as candidate genetic variations related to the velvet antler weight. We hope that it will contribute to further mechanistic studies of velvet antler development and weight variations.

Identification of Genes Putatively Involved in Chitin Metabolism and Insecticide Detoxification in the Rice Leaf Folder (Cnaphalocrocis medinalis) Larvae through Transcriptomic Analysis.

The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest.

Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori.

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a highly pathogenic virus in the sericultural industry, often causing severe damage leading to large economic losses. The immune mechanisms of B. mori against this virus remain obscure. Previous studies had demonstrated Bmlipase-1, BmNox and Bmserine protease-2 showing antiviral activity in vitro, but data on the transcription levels of these proteins in different resistant strains were not reported. In order to determine the resistance level of the four different strains (P50, A35, A40, A53) and gain a better understanding of the mechanism of resistance to BmNPV in B. mori, the relative expression level of the genes coding the three antiviral proteins in larval haemolymph and midgut of different B. mori strains resistant to BmNPV was determined. The results showed that these genes expressed significantly higher in the resistant strains compared to the susceptible strain, and the differential expression levels were consistent with the LC50 values in different strains. The transcription level of the target genes almost all up-regulated in the larvae midgut and down-regulated in the haemolymph. The results indicate the correlation of these genes to BmNPV resistance in B. mori.

Connections between body composition and dysregulation of islet α- and ß-cells in type 2 diabetes.

BACKGROUND: Accompanying islet α- and ß-cell dysregulation in type 2 diabetes (T2D) at the microscopic scale, alterations in body composition at the macroscopic scale may affect the pathogenesis of T2D. However, the connections between body composition and islet α-cell and ß-cell functions in T2D have not been thoroughly explored. METHODS: For this cross-sectional study, we recruited a total of 729 Chinese Han patients with T2D in a consecutive manner. Dual-energy X-ray absorptiometry (DXA) was used to measure body composition, which included total bone-free mass, total fat and lean mass, trunk fat and lean mass and limb fat and lean mass. Every patient underwent an oral glucose tolerance test to simultaneously detect glucose, C-peptide and glucagon. The indices of islet α-cell function included fasting glucagon levels and the area under the curve of glucagon after a challenge (AUCglucagon), while the indices of ß-cell function included the insulin sensitivity index derived from C-peptide (ISIC-peptide) and the area under the curve of C-peptide after a challenge (AUCC-peptide). RESULTS: Among all patients, fat mass, especially trunk fat mass, was significantly correlated with ISIC-peptide and AUCC-peptide levels (r = - 0.330 and 0.317, respectively, p < 0.001), while lean mass, especially limb lean mass, was significantly correlated with fasting glucagon and AUCglucagon levels (r = - 0.196 and - 0.214, respectively, p < 0.001). Moreover, after adjusting for other relevant variables via multivariate linear regression analysis, increased trunk fat mass was independently associated with decreased ISIC-peptide (ß = - 0.247, t = - 3.628, p < 0.001, partial R2 = 10.9%) and increased AUCC-peptide (ß = 0.229, t = 3.581, p < 0.001, partial R2 = 8.2%), while decreased limb lean mass was independently associated with increased fasting glucagon (ß = - 0.226, t = - 2.127, p = 0.034, partial R2 = 3.8%) and increased AUCglucagon (ß = - 0.218, t = - 2.050, p = 0.041, partial R2 = 2.3%). Additionally, when separate analyses were performed with the same concept for both sexes, we found that increased trunk fat mass was still independently associated with decreased ISIC-peptide and increased AUCC-peptide, while decreased limb lean mass was still independently associated with increased fasting glucagon and AUCglucagon. CONCLUSIONS: Increased trunk fat mass may partly account for decreased insulin sensitivity and increased insulin secretion, while decreased limb lean mass may be connected to increased fasting glucagon and postprandial glucagon secretion.

Effects of baculovirus infection on intestinal microflora of BmNPV resistant and susceptible strain silkworm.

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a serious pathogen causing huge economic losses to sericulture. There is growing evidence that the gut microbiota of silkworms plays a critical role in shaping host responses and interactions with viral infection. However, little is known about the differences in the composition and diversity of intestinal microflora, especially with respect to silkworm strain differences and BmNPV infection-induced changes. Here, we aim to explore the differences between BmNPV-resistant strain A35 and susceptible strain P50 silkworm and the impact of BmNPV infection on intestinal microflora in different strains. The 16S rDNA sequencing analysis revealed that the fecal microbial populations were distinct between A35 and P50 and were significantly changed post BmNPV infection in both strains. Further analysis showed that the BmNPV-resistant strain silkworm possessed higher bacterial diversity than the susceptible strain, and BmNPV infection reduced the diversity of intestinal flora assessed by feces in both silkworm strains. In response to BmNPV infection, the abundance of Muribaculaceae increased in P50 and decreased in A35, while the abundance of Enterobacteriaceae decreased in P50 and increased in A35. These results indicated that BmNPV infection had various effects on the abundance of fecal microflora in different silkworm strains. Our findings not only broadened the understanding of host-pathogen interactions but also provided theoretical help for the breeding of resistant strains and healthy rearing of silkworms based on symbiotic bacteria.

Bmo-miR-3351 modulates glutathione content and inhibits BmNPV proliferation by targeting BmGSTe6 in Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles in the host response to invading pathogens. Among these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of substantial economic losses in sericulture, and there are relatively few studies on the specific functions of miRNAs in the B. mori-BmNPV interaction. Therefore, we conducted transcriptome sequencing to identify differentially expressed (DE) messenger RNAs (mRNAs) and miRNAs in the midgut of 2 B. mori strains (BmNPV-susceptible strain P50 and BmNPV-resistant strain A35) after BmNPV infection. Through correlation analysis of the miRNA and mRNA data, we identified a comprehensive set of 21 miRNAs and 37 predicted target mRNAs. Notably, miR-3351, which has high expression in A35, exhibited remarkable efficacy in suppressing BmNPV proliferation. Additionally, we confirmed that miR-3351 binds to the 3' untranslated region (3' UTR) of B. mori glutathione S-transferase epsilon 6 (BmGSTe6), resulting in its downregulation. Conversely, BmGSTe6 displayed an opposite expression pattern to miR-3351, effectively promoting BmNPV proliferation. Notably, BmGSTe6 levels were positively correlated with glutathione S-transferase activity, consequently influencing intracellular glutathione content in the infected samples. Furthermore, our investigation revealed the protective role of glutathione against BmNPV infection in BmN cells. In summary, miR-3351 modulates glutathione content by downregulating BmGSTe6 to inhibit BmNPV proliferation in B. mori. Our findings enriched the research on the role of B. mori miRNAs in the defense against BmNPV infection, and suggests that the antiviral molecule, glutathione, offers a novel perspective on preventing viral infection in sericulture.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex.

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.