Linc20486 promotes BmCPV replication through inhibiting the transcription of AGO2 and Dicers.

The silkworm holds pivotal economic importance, serving not only as a primary source of silk but also as a prominent model organism in scientific research. Nonetheless, silkworm farming remains vulnerable to diverse factors, with viral infections posing the gravest threat to the sericulture industry. Among these, the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a member of the Reoviridae family and the cytoplasmic polyhedrosis virus genus, emerges as a significant pathogen in silkworm production. BmCPV infection primarily induces midgut sepsis in silkworms, spreads rapidly, and can inflict substantial economic losses on sericulture production. Presently, effective strategies for preventing and treating BmCPV infections are lacking. Long non-coding RNA (lncRNA) constitutes a class of RNA molecules with transcripts exceeding 200 nt, playing a crucial role in mediating the interplay between pathogens and host cells. Investigation through high-throughput technology has unveiled that BmCPV infection markedly upregulates the expression of Linc20486. This observation suggests potential involvement of Linc20486 in regulating virus replication. Indeed, as anticipated, knockdown of Linc20486 in cells profoundly impedes BmCPV replication, whereas overexpression significantly enhances virus propagation. To probe into the mechanism underlying Linc20486's impact on virus replication, its effects on autophagy, innate immunity, and RNAi-related pathways were scrutinized. The findings revealed that Linc20486 exerts significant influence on the expression of RNAi pathway-related genes, such as Dicer1, Dicer2 and AGO2. This discovery holds promise for unveiling novel avenues to comprehend and combat BmCPV infections in silkworms.

Multi-omics reveals the ecological and biological functions of Enterococcus mundtii in the intestine of lepidopteran insects.

Insect guts offer unique habitats for microbial colonization, with gut bacteria potentially offering numerous benefits to their hosts. Although Enterococcus has emerged as one of the predominant gut commensal bacteria in insects, its establishment in various niches within the gut has not been characterized well. In this study, Enterococcus mundtii was inoculated into the silkworm (Bombyx mori L.) to investigate its biological functions. Genome-based analysis revealed that its successful colonization is related to adherence genes (ebpA, ebpC, efaA, srtC, and scm). This bacterium did not alter the activities of related metabolic enzymes or the intestinal barrier function. However, significant changes in the gene expressions levels of Att2, CecA, and Lys suggest potential adaptive mechanisms of host immunity to symbiotic E. mundtii. Moreover, 16S metagenomics analysis revealed a significant increase in the relative abundance of E. mundtii in the intestines of silkworms following inoculation. The intestinal microbiome displayed marked heterogeneity, an elevated gut microbiome health index, a reduced microbial dysbiosis index, and low potential pathogenicity in the treatment group. Additionally, E. mundtii enhanced the breakdown of carbohydrates in host intestines. Overall, E. mundtii serves as a beneficial microbe for insects, promoting intestinal homeostasis by providing competitive advantage. This characteristic helps E. mundtii dominate complex microbial environments and remain prevalent across Lepidoptera, likely fostering long-term symbiosis between the both parties. The present study contributes to clarifying the niche of E. mundtii in the intestine of lepidopteran insects and further reveals its potential roles in their insect hosts.

Effects of high temperature on the growth performance and midgut autophagy of thermotolerant and thermosensitive silkworm strains.

High temperature stress has long-term negative effects on the growth and development of silkworm (Bombyx mori). Different silkworm varieties show the different tolerance to high temperature. The induction of autophagy is linked to increased thermotolerance in diverse ectothermic organisms. However, the function of autophagy in the thermotolerant and thermosensitive silkworm strains under high-temperature conditions remains unclear. The thermotolerant Liangguang NO.2 and thermosensitive Jingsong × Haoyue strains were used to explore the role of autophagy in thermotolerance. Here, we first found that the larval body weight gain was increased in the thermosensitive Jingsong × Haoyue strain, but there was no difference in the thermotolerant Liangguang NO.2 strain under high temperature conditions. High temperature stress had a negative influence on the cocoon performance in both the Liangguang NO.2 and Jingsong × Haoyue strains. Additionally, the autophagy-related gene Atg5 mRNA expression in the Liangguang NO.2 strain was upregulated by high temperature, while the expression of Atg12 mRNA was reduced in the Jingsong × Haoyue strain. Titers of 20-Hydroxyecdysone and the ultraspiracle 1 mRNA expression in the Liangguang NO.2 strain were upregulated by high temperature, which might be associated with the induction of autophagy. These results demonstrate the potentially regulatory mechanism of autophagy in silkworms' tolerance to high temperature, providing a theoretical basis for exploring the physiological mechanism of thermotolerance in insects.

Compensatory effects of other olfactory genes after CRISPR/cas9 editing of BmOR56 in silkworm, Bombyx mori.

Bombyx mori is an oligophagous economic insect. Cis-Jasmone is one of the main substances in mulberry leaf that attract silkworm for feeding and BmOR56 is its receptor. Potential interaction ways between BmOR56 and cis-Jasmone were explored, which included some crucial amino acids such as Gln172, Val173, Ser176, Lys182, His322, and Arg345. BmOR56 was edited using CRISPR/cas9 for Qiufeng, and a homozygous knockout strain QiufengM was obtained. Compared with Qiufeng, the feeding ability of QiufengM on mulberry leaf did not change significantly, but on artificial diet decreased significantly. QiufengM also showed a dependence on the concentration of mulberry leaf powder. The result indicated that other olfactory genes had a compensatory effect on the attractance of mulberry leaf after the loss of BmOR56. Transcriptome analysis of antennae showed that many genes differentially expressed between Qiufeng and QiufengM, which involved in olfactory system, glucose metabolism, protein metabolism, amino acid metabolism, and insect hormone biosynthesis. Particularly, BmIR21, BmOR53 and BmOR27 were significantly up-regulated, which may have a compensatory effect on BmOR56 loss. In addition, detoxification mechanism was activated and may cause the passivation of feeling external signals in silkworm.

The Bombyx mori G protein ß subunit 1 (BmGNß1) gene inhibits BmNPV infection.

G protein ß subunit 1 (GNß1) has several functions, including cell growth regulation, the control of second messenger levels, and ion channel switching. Previous transcriptome analyses in our laboratory have shown that BmGNß1 transcription is reduced following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), but it is unknown what role this gene may have in the host response to BmNPV infection. In this study, the BmGNß1 gene was cloned using the RACE method. After BmNPV infection, BmGNß1 was downregulated in Baiyu strains in tissues such as the hemolymph and midgut. Indirect immunofluorescence showed that BmGNß1 was localized to the cytoplasm. We further constructed a BmGNß1-pIZ/V5-His-mCherry overexpression plasmid and designed siRNA to evaluate the role of BmGNß1 in host response to infection. The results showed that BmGNß1 overexpression inhibited BmNPV proliferation, while knockdown of BmGNß1 was correlated with increased BmNPV proliferation. The siRNA-mediated reduction of BmGNß1 was correlated with an increase in BmNPV infection of BmN cells, increased BmNPV vp39 transcription, and reduced survival time of BmNPV-infected B. mori. Overexpression of BmGNß1 in BmN cells was also correlated with apoptosis and a modification in transcript levels of genes involved in host response to BmNPV infection (PI3K, AKT, Bmp53, BmFOXO, Caspase-1, Bmp21, BmPKN and BmCREB), suggesting that BmGNß1 may influence the apoptotic host response of infected B. mori through the PI3K-AKT pathway. This study provides potential targets and theoretical support for breeding BmNPV-resistant silkworm varieties.

Knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy.

It has been demonstrated that angiopoietin-like protein 4 (ANGPTL4) plays an important regulatory role in lipid metabolism and backfat deposition appears to vary in different pig breeds. However, the correlation between ANGPTL4 and backfat deposition have not been well characterized and the role of ANGPTL4 in regulating adipogenesis remains unclear. Therefore, this study aimed to investigate correlation between ANGPTL4 and backfat deposition and to explore the effects of ANGPTL4 on preadipocyte differentiation and the underlying mechanism. Our results showed that the backfat thickness and the ANGPTL4 gene expression of Laiwu pigs were significantly higher than those in DLY pigs and the ANGPTL4 gene expression was positively correlated with backfat thickness both in DLY pigs and Laiwu pigs. Moreover, an increase in ANGPTL4 expression and activation of autophagy were observed during the differentiation of stromal vascular fraction cells. In addition, knockdown of ANGPTL4 inhibited the differentiation of 3T3-L1 cells with decreased expression of LC3-II and ATG5 and increased expression of SQSTM1, suggesting the involvement of autophagy in ANGPTL4-mediated adipogenesis. In conclusion, these results suggested that ANGPTL4 is positively correlated with backfat deposition in pigs and knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy, providing new insights into the regulation of fat deposition and to improve the carcass quality and meat quality of porcine.

Identification of long noncoding RNAs of silkworm at the early stage of Bombyx mori bidensovirus infection.

Bombyx mori bidensovirus (BmBDV) is one of the most important pathogens of silkworm. It mainly infects midgut cells of silkworm and causes losses to the sericulture industry. Long noncoding RNAs (lncRNAs) have been reported to play an important role in the regulation of antiviral immune response in silkworm. To explore whether lncRNAs are involved in BmBDV infection and immune response of silkworm, we performed a comparative transcriptome analysis to identify the lncRNAs and mRNAs between the BmBDV infected and noninfected silkworm larvae at the early stage. A total of 16,069 genes and 974 candidate lncRNAs were identified, among which 142 messenger RNA (mRNAs) and four lncRNAs were differentially expressed (DE). Target gene prediction revealed that 142 DEmRNAs were coexpressed with four DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans-regulation activities. A regulatory network of DElncRNAs and DEmRNAs was constructed, showing that many genes targeted by different DElncRNAs are involved in metabolism and immunity, which implies that these genes and lncRNAs play an important role in the replication of BmBDV. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmBDV infection, providing a new perspective for further exploring the interaction between host and BmBDV.

Ebony plays an important role in egg hatching and 30k protein expression of silkworm (Bombyx mori).

QiufengN is a silkworm strain. During the feeding process of QiufengN, a mutant of black pupal cuticle QiufengNBP was found. Some silkworm pupae of the mutant were unable to easily molt during pupation, and some silkworm eggs produced by developed normally but larvae were unable to break out of the eggshells. These phenomena had not been observed in other black pupa mutants. Genetic analysis showed that the melanization trait of QiufengNBP is controlled by a recessive gene located on the autosome and follows Mendelian inheritance. Results of positional cloning and qRT-PCR showed that the occurrence of black pupae was caused by the mutation of the ebony gene on chromosome 26. 2-DE analysis of the pupal cuticle of QiufengN and QiufengNBP found that the 30K protein, the main storage protein for the growth and development of silkworms and an important energy substance for embryonic development, has changed significantly. In addition, the expression level of Bombyx mori hatching enzyme (BmHEL), which can soften the eggshell during the hatching process of silkworm, was significantly higher in the eggs of black pupae before and after hatching than in normal eggs. The mutation of ebony makes hatching difficult for silkworms, and increases in BmHEL is needed to soften the eggshell. This study showed that ebony may have important effects on the formation of silkworm pigment and egg hatching, and its formation mechanism is complex and deserves further study.

Proteomics Analysis to Explore the Resistance Genes of Silkworm to Bombyx mori Nuclear Polyhedrosis Virus.

The resistance of silkworms to Bombyx mori nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified genes, it is difficult to determine the main gene by positional cloning. In this study, the main anti-BmNPV gene of BmNPV-resistant silkworm variety N was introduced into the susceptible variety Su to breed the near-isogenic line SuN with BmNPV resistance. The infection process of BmNPV in the hemolymph of Su and SuN was analyzed using the cell analysis system TissueFAXS PLUS. According to the law of infection and proliferation, hemolymph was extracted every 6 h for two-dimensional electrophoresis (2-DE) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Seven DEPs were found in comparisons between Su and SuN by 2-DE analysis. Among them, acid phosphatase, storage protein, and phenoloxidase can prevent pathogen invasion, which may play a role against BmNPV. Polyamine oxidase plays an important role in energy metabolism, which may be indirectly involved in the process of resisting BmNPV. Most of the transcriptional expression profiles of the seven DEPs were consistent with the 2-DE results. This study can provide a reference for the identification of anti-BmNPV genes and the breeding of BmNPV-resistant silkworm varieties.

The Bmtret1 Gene Family and Its Potential Role in Response to BmNPV Stress in Bombyx mori.

Trehalose is a non-reducing disaccharide and participates in physiological activities such as organ formation, energy metabolism, and stress resistance in insects. The Bmtret1 gene family is mainly involved in in the sugar metabolism of silkworm. In the present study, phylogenetic analysis divided 21 Bmtret1 orthologs into three clades. These genes are equally distributed on the nine chromosomes. The cis-elements in the promoter regions of Bmtret1s indicated the possible function of Bmtret1s in response to hormones and environmental stimulus. The qPCR analysis showed the significantly different expression levels of Bmtret1s in different tissues and organs, indicating possible functional divergence. In addition, most Bmtret1s showed disturbed expression levels in response to silkworm nuclear polyhedrosis virus (BmNPV) stresses. Our results provide a clue for further functional dissection of the Tret1s in Bombyx mori and implicate them as potential regulators of antiviral responses.

Parasitism and Suitability of Trichogramma chilonis on Large Eggs of Two Factitious Hosts: Samia cynthia ricini and Antheraea pernyi.

Trichogramma, an effective biological control agent, demonstrates promise in environmentally sustainable pest management through its parasitic action toward insect eggs. This study evaluates the parasitism fitness and ability of T. chilonis with regard to two factitious host eggs, aiming to develop a cost-effective biological control program. While T. chilonis demonstrated the ability to parasitize both host eggs, the results indicate a preference for ES eggs over COS eggs. The parasitism and emergence rates of T. chilonis regarding ES eggs (parasitism: 89.3%; emergence: 82.6%) surpassed those for COS eggs (parasitism: 74.7%; emergence: 68.8%), with a notable increase in the number of emergence holes observed in the ES eggs compared to the COS eggs. Moreover, the developmental time of T. chilonis for ES eggs (10.8 days) was shorter than that for COS eggs (12.5 days), resulting in a lower number of dead wasps produced. Notably, no significant difference was observed in the female ratios between the two species. A comprehensive analysis was conducted, comparing the size and shell thickness of the two factitious hosts. The ES eggs exhibited smaller dimensions (length: 1721.5 µm; width: 1178.9 µm) in comparison to the COS eggs (length: 2908.8 µm; width: 2574.4 µm), with the ES eggshells being thinner (33.8 µm) compared to the COS eggshells (47.3 µm). The different host species had an effect on the body length of the reared parasitoids, with T. chilonis reared on COS hosts exhibiting a larger body length (female: 626.9 µm; male: 556.7 µm) than those reared on ES hosts (female: 578.8 µm; male: 438.4 µm). Conclusively, the results indicate that ES eggs present a viable alternative to COS eggs for the mass production of Trichogramma species in biological control programs.

Identification of Key Genes Involved in Resistance to Early Stage of BmNPV Infection in Silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens restricting the sustainable development of the sericulture industry. Currently, there is no effective treatment for BmNPV infection in silkworms, and the mechanism underlying BmNPV resistance in silkworms is also not clear. In this study, comparative transcriptome analyses were carried out in midguts of two silkworm varieties, namely BaiyuN, which is a resistance variety, and Baiyu, which is a susceptible variety, at five different time points (i.e., 0, 1, 3, 6, and 9 h) post-BmNPV infection to detect the early-stage transcriptional changes in these silkworms. In total, 1911 and 1577 differentially expressed genes (DEGs) were identified in the Baiyu and BaiyuN varieties, respectively, involving a total of 48 metabolic pathways. Of these pathways, eight were shared by the Baiyu and BaiyuN varieties in response to BmNPV infection. Notably, four genes (i.e., BGIBMGA08815, BGIBMGA003935, BGIBMGA003571, BGIBMGA010059) were upregulated in the Baiyu variety while downregulated in the BaiyuN variety. The inhibited expression of these four genes in the resistant variety highlighted their potential roles in the resistance of early-stage viral replication. Thus, our study provided a new avenue for the further study of the mechanism underlying BmNPV infection in silkworms and the potential treatment of BmNPV infection.

High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation.

The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

Metabolic characterization of hemolymph in Bombyx mori varieties after Bombyx mori nucleopolyhedrovirus infection by GC-MS-based metabolite profiling.

The "Huakang 2" silkworm variety, bred by the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, is highly resistant to Bombyx mori nucleopolyhedrovirus (BmNPV) and effectively solves the issue of frequent Bombyx mori nuclear polyhedrosis in sericultural production. The molecular mechanism of its resistance to BmNPV, however, is still unknown. The purpose of the present study was therefore to identify these anti-BmNPV mechanisms by using metabolomics in combination with transcriptomics after subcutaneous injection of budded virus (BV) with high concentrations of BmNPV from specimens of the Baiyu N variety (which is highly resistant to BmNPV) and the Baiyu variety (which is sensitive to BmNPV). A total of 375 differential metabolites were identified, which mainly included sugars, acids, amines, alcohols, glycosides, and other small molecules. KEGG enrichment analysis and functional clustering of differential metabolites identified possible metabolic pathways, including tyrosine metabolism, oxidative phosphorylation, and alanine, aspartate, and glutamate metabolism. The differentially expressed genes (DEGs) identified by transcriptome analysis were annotated in KEGG. Association analysis showed that the metabolic pathways of different silkworm varieties are affected differently by BmNPV infection, triggering a series of complex physiological and biochemical changes in the organism. In particular, oxidative phosphorylation might be an essential pathway involved in regulation of disease resistance.

Integration of Transcriptomic and Proteomic Analyses Reveals New Insights into the Regulation of Immune Pathways in Midgut of Samia ricini upon SariNPV Infection.

Samia ricini nucleopolyhedrovirus (SariNPV) is one of the main pathogens of S. ricini sericulture and its infection causes severe impacts on economic sericulture development. To explore and reveal the molecular mechanisms of S. ricini in response to SariNPV infection, we employed RNA sequencing (RNA-seq), adopting isobaric tags for relative and absolute quantitation (iTRAQ), and carried out combination analysis of the obtained differentially expressed genes (DEGs) and proteins (DEPs). Through transcriptome sequencing, a total of 2535 DEGs were detected, and with iTRAQ, 434 DEPs with significant expression difference were identified. Through correlation analysis, we found that the expression trends of 116 differentially expressed proteins were the same as those of differentially expressed genes (including 106 up-regulated and 10 down-regulated). Twenty-five key differentially expressed genes (proteins) involved in several signaling and immune related pathways (mainly involving Toll, Imd, Jak-STAT and Wnt signaling pathways, as well as other immune related pathways) were screened through real-time quantitative PCR. Our results, not only provide insights into the pathogenic mechanism of SariNPV infection in ricin silkworm and the immune response mechanism within the host, but also provide a significant contribution for identifying and preventing diseases caused by SariNPV.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori.

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKß), via binding to the target mRNA 3'-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

Pathogenicity Detection and Genome Analysis of Two Different Geographic Strains of BmNPV.

The pathogenicity of different concentrations of Bombyx mori nuclear polyhedrosis virus- Zhenjiang strain (BmNPV ZJ) and Yunnan strain (BmNPV YN) was assessed in Baiyu larvae. The structures of the two viral strains were observed by negative-staining electron microscopy, and their proliferation was examined by quantitative polymerase chain reaction (qPCR). The genomic sequences of these two viruses were obtained to investigate the differences in their pathogenicity. The lethal concentration 50 (LC50) of BmNPV ZJ against Baiyu larvae was higher than that of BmNPV YN, indicating a relatively more robust pathogenicity in BmNPV YN. Electron microscopic images showed that the edges of BmNPV YN were clearer than those of BmNPV ZJ. The qPCR analysis demonstrated significantly higher relative expressions of immediately early 1 gene (ie-1), p143, vp39, and polyhedrin genes (polh) in BmNPV ZJ than in BmNPV YN at 12-96 h. The complete genomes of BmNPV ZJ and BmNPV YN were, respectively, 135,895 bp and 143,180 bp long, with 141 and 145 coding sequences and 40.93% and 39.71% GC content. Considering the BmNPV ZJ genome as a reference, 893 SNP loci and 132 InDel mutations were observed in the BmNPV YN genome, resulting in 106 differential gene sequences. Among these differential genes, 76 (including 22 hub genes and 35 non-hub genes) possessed amino acid mutations. Thirty genes may have been related to viral genome replication and transcription and five genes may have been associated with the viral oral infection. These results can help in understanding the mechanisms of pathogenicity of different strains of BmNPV in silkworms.

Two Putative Cypovirus-Encoded miRNAs Co-regulate the Host Gene of GTP-Binding Nuclear Protein Ran and Facilitate Virus Replication.

microRNA (miRNA) plays important roles in regulating various biological processes, including host-pathogen interaction. Recent studies have demonstrated that virus-encoded miRNAs can manipulate host gene expression to ensure viral effective multiplication. Bombyx mori cypovirus (BmCPV), a double-stranded RNA virus with a segmented genome, is one of the important pathogens for the economically important insect silkworm. Our present study indicated that two putative miRNAs encoded by BmCPV could promote viral replication by inhibiting the gene expression of B. mori GTP-binding nuclear protein Ran (BmRan), an essential component of the exportin-5-mediated nucleocytoplasmic transport of small RNAs. BmCPV-miR-1 and BmCPV-miR-3 are two of the BmCPV-encoded miRNAs identified in our previous studies. BmRan is a common target gene of them with binding sites all located in the 3'-untranslated region (3'-UTR) of its mRNA. The expression levels of the two miRNAs in the midgut of larvae infected with BmCPV gradually increased with the advance of infection, while the expression of the target gene BmRan decreased gradually. The miRNAs and the recombinant target gene consisting of reporter gene mCherry and 3'-UTR of BmRan mRNA were expressed in HEK293T cells for validating the interaction between the miRNAs and the target gene. qRT-PCR results revealed that BmCPV-miR-1 and BmCPV-miR-3 negatively regulate target gene expression not only separately but also cooperatively by binding to the 3'-UTR of BmRan mRNA. By transfecting miRNA mimics into BmN cells and injecting the mimics into the body of silkworm larvae, it was indicated that both BmCPV-miR-1 and BmCPV-miR-3 could repress the expression of BmRan in BmN cells and in the silkworm, and the cooperative action of the two miRNAs could enhance the repression of BmRan expression. Furthermore, the repression of BmRan could facilitate the replication of BmCPV genomic RNAs. It is speculated that BmCPV-miR-1 and BmCPV-miR-3 might reduce the generation of host miRNAs by inhibiting expression of BmRan, thus creating a favorable intracellular environment for virus replication. Our results are helpful to better understand the pathogenic mechanism of BmCPV to the silkworm, and provide insights into one of the evasion strategies used by viruses to counter the host defense for their effective multiplication.

Effect of pyriproxyfen exposure on cocooning and gene expression in the silk gland of Bombyx mori （Linnaeus, 1758）.

Bombyx mori（Linnaeus, 1758） is an important economical insect, and the sericulture is a flourishing industry in many developing countries. Pyriproxyfen, a juvenile hormone pesticide, is often applied to cultivations widely in the world, and its exposure often resulted in silk yield reduction and non-cocooning. However, the effect of pyriproxyfen exposure on cocooning and gene expression level in the silk gland of B. mori has not been studied yet, and this study focused on the above issues. The result indicated that pyriproxyfen exposure can lead to silk gland injury, reduction of silk yield and cocooning rate. Furthermore, the expression levels of silk protein synthesis related genes were down regulated significantly. The same change trends were shown between PI3K/Akt and CncC/Keap1 pathway, which is the expressions of key genes can be elevated by pyriproxyfen exposure. In addition, the activity of detoxification enzymes (P450, GST and CarE) and the expression levels of detoxification genes were elevated after pyriproxyfen exposure, suggesting that detoxification enzymes may play an important role in detoxification of pyriproxyfen in silk gland. These results provided possible clues to the silk gland injury and gene transcriptional level changes in silkworm after pyriproxyfen exposure.

Metabolic Characterisation of the Midgut of Bombyx mori Varieties after BmNPV Infection Using GC-MS-Based Metabolite Profiling.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a silkworm disease that is especially harmful to cocoon production and seriously restricts sericultural development. Our laboratory successfully cultivated a new highly BmNPV-resistant silkworm variety, Huakang 2; however, its mechanism of BmNPV resistance remains unclear. To understand its resistance mechanism, we conducted a metabolomic and transcriptomic study of the midgut of silkworm varieties, Baiyu N and Baiyu after BmNPV infection. We identified 451 differential metabolites, which were mostly comprised of small molecules, such as saccharides, acids, amines, alcohols, and glycosides. We found that the primary differences in disease resistance between the silkworm varieties are metabolic-pathways, tryptophan metabolism, oxidative phosphorylation, ABC-transporters, beta-alanine metabolism, and phenylalanine metabolism. Combined analysis with transcriptomic data suggested that tryptophan metabolism and oxidative phosphorylation are closely related to the silkworms' BmNPV resistance. We hypothesize that the roles of the two metabolic pathways in the BmNPV resistance mechanism might be the following: Oxidative phosphorylation generates a large amount of adenosine triphosphate (ATP) in response to BmNPV infection to provide silkworms the energy required for establishing BmNPV resistance. Tryptophan metabolism then activates the aryl hydrocarbon receptor (AhR) through the exogenous virus BmNPV, which activates the silkworm's immune system to defeat BmNPV infections.