Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection.Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori.

Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the Bmserpin2 gene which was identified from a transcriptome database in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Bmserpin2 was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of Bmserpin2 was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in Bmserpin2-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in Bmserpin2-depleted haemolymph. We conclude that Bmserpin2 is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of B. mori.

Rapid vascularization is vital for dermal regeneration, nutrient and nutrition transfer, metabolic waste removal, and prevention of infection. This study reports on a series of proangiogenic peptides designed to undergo self-assembly and promote angiogenesis and hence skin regeneration. The proangiogenic peptides comprised an angiogenic peptide segment, GEETEVTVEGLEPG, and a β-sheet structural peptide sequence. These peptides dissolved easily in ultrapure water and rapidly self-assembled into hydrogels in a pH-dependent manner, creating three-dimensional fibril network structures and nanofibers as revealed by a scanning microscope and a transmission electron microscope. In vitro experiments showed that the peptide hydrogels favored adhesion and proliferation of mouse fibroblasts (L929) and human umbilical vein endothelial cells (HUVECs). In particular, many connected tubes were formed in the HUVECs after 8 h of culture on the peptide hydrogels. In vivo experiments demonstrated that new blood vessels grew into the proangiogenic peptide hydrogels within 2 weeks after subcutaneous implantation in mice. Moreover, the proangiogenic-combined hydrogels exhibited faster repair cycles and better healing of skin defects. Collectively, the results indicate that the proangiogenic peptide hydrogels are a promising therapeutic option for skin regeneration.

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.

Previous studies have revealed that some proteins in Bombyx mori larvae digestive juice show antiviral activity. Here, based on the label-free proteomics data, BmLipase member H-A (BmLHA) was identified as being involved in the response to BmNPV infection in B. mori larvae digestive juice. In the present study, a gene encoding the BmLHA protein in B. mori was characterized. The protein has an open reading fragment of 999 bp, encoding a predicted 332 amino acid residue-protein with a molecular weight of approximately 35.9 kDa. The phylogenetic analysis revealed that BmLHA shares a close genetic distance with Papilio xuthus Lipase member H-A. BmLHA was highly expressed in the middle part of the B. mori gut, and the expression level increased with instar rising in larvae. There was higher expression of BmLHA in A35 than in P50 strains, and it was upregulated in both A35 and P50 strains, following BmNPV infection. The expression level of VP39 decreased significantly in appropriate recombinant-BmLHA-treated groups compared with the PBS-treated group in B. mori larvae and BmN cells. Meanwhile, overexpression of BmLHA significantly reduced the infectivity of BmNPV in BmN cells. These results indicated that BmLHA did not have digestive function but had anti-BmNPV activity. Taken together, our work provides valuable data for the clarification of the molecular characterization BmLHA and supplements research on proteins of anti-BmNPV activity in B. mori.