Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens causing severe economic losses in sericulture. However, the molecular mechanism of silkworm resistance to BmNPV remains largely unknown. Here, the recurrent parent P50 (susceptible strain) and the near-isogenic line BC9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with BmNPV. A total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (DEGs) were identified after comparing the four transcriptomes. Many DEGs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to BmNPV infection. Moreover, some immunity related genes were also altered following BmNPV infection. Specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing BmNPV infection. These genes were related to transport, virus replication, intracellular innate immune, and apoptosis. Our study provided an overview of the molecular mechanism of silkworm resistance to BmNPV infection and laid a foundation for controlling BmNPV in the future.

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.

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 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, reversetranscription 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.

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.