Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Photoperiodism of Diapause Induction in the Silkworm, Bombyx mori.

The silkworm Bombyx mori exhibits a photoperiodic response (PR) for embryonic diapause induction. This article provides a comprehensive review of literature on the silkworm PR, starting from early works on population to recent studies uncovering the molecular mechanism. Makita Kogure (1933) conducted extensive research on the PR, presenting a pioneering paper on insect photoperiodism. In the 1970s and 80s, artificial diets were developed, and the influence of nutrition on PR was well documented. The photoperiodic photoreceptor has been investigated from organ to molecular level in the silkworm. Culture experiments demonstrated that the photoperiodic induction can be programmed in an isolated brain (Br)-subesophageal ganglion (SG) complex with corpora cardiaca (CC)-corpora allata (CA). The requirement of dietary vitamin A for PR suggests the involvement of opsin pigment in the photoperiodic reception, and a cDNA encoding an opsin (Boceropsin) was cloned from the brain. The effector system concerning the production and secretion of diapause hormone (DH) has also been extensively investigated in the silkworm. DH is produced in a pair of posterior cells of SG, transported to CC by nervi corporis cardiaci, and ultimately released into the hemolymph. Possible involvement of GABAergic and corazonin (Crz) signal pathways was suggested in the control of DH secretion. Knockout (KO) experiments of GABA transporter (GAT) and circadian clock genes demonstrated that GAT plays a crucial role in PR through circadian control. A model outlining the PR mechanism, from maternal photoperiodic light reception to DH secretion, has been proposed.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Lipid homeostasis is essential for oogenesis and embryogenesis in the silkworm, Bombyx mori.

Reproduction, a fundamental feature of all known life, closely correlates with energy homeostasis. The control of synthesizing and mobilizing lipids are dynamic and well-organized processes to distribute lipid resources across tissues or generations. However, how lipid homeostasis is precisely coordinated during insect reproductive development is poorly understood. Here we describe the relations between energy metabolism and reproduction in the silkworm, Bombyx mori, a lepidopteran model insect, by using CRISPR/Cas9-mediated mutation analysis and comprehensively functional investigation on two major lipid lipases of Brummer (BmBmm) and hormone-sensitive lipase (BmHsl), and the sterol regulatory element binding protein (BmSrebp). BmBmm is a crucial regulator of lipolysis to maintain female fecundity by regulating the triglyceride (TG) storage among the midgut, the fat body, and the ovary. Lipidomics analysis reveals that defective lipolysis of females influences the composition of TG and other membrane lipids in the BmBmm mutant embryos. In contrast, BmHsl mediates embryonic development by controlling sterol metabolism rather than TG metabolism. Transcriptome analysis unveils that BmBmm deficiency significantly improves the expression of lipid synthesis-related genes including BmSrebp in the fat body. Subsequently, we identify BmSrebp as a key regulator of lipid accumulation in oocytes, which promotes oogenesis and cooperates with BmBmm to support the metabolic requirements of oocyte production. In summary, lipid homeostasis plays a vital role in supporting female reproductive success in silkworms.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Domestication Gene Mlx and Its Partner Mondo Are Involved in Controlling the Larval Body Size and Cocoon Shell Weight of Bombyx mori.

Bombyx mori was domesticated from Bombyx mandarina. The long-term domestication of the silkworm has brought about many remarkable changes to its body size and cocoon shell weight. However, the molecular mechanism underlying the improvement in the economic characteristics of this species during domestication remains unclear. In this study, we found that a transposable element (TE)-Bm1-was present in the upstream regulatory region of the Mlx (Max-like protein X) gene in wild silkworms but not in all domesticated silkworms. The absence of Bm1 caused an increase in the promoter activity and mRNA content of Mlx. Mlx and its partner Mondo belong to the bHLHZ transcription factors family and regulate nutrient metabolism. RNAi of Mlx and Mondo decreased the expression and promoter activity of glucose metabolism-related genes (trehalose transport (Tret), phosphofructokinase (PFK), and pyruvate kinase (PK)), lipogenic genes (Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)), and glutamine synthesis gene (Glutamine synthase 2, (GS2)). Furthermore, the transgenic overexpression of Mlx and Mondo in the fat body of silkworms increased the larval body size, cocoon shell weight, and egg number, but the silencing of the two genes resulted in the opposite phenotypes. Our results reveal the molecular mechanism of Mlx selection during domestication and its successful use in the molecular breeding of Bombyx mori.

Injectable Bombyx mori (B. mori) silk fibroin/MXene conductive hydrogel for electrically stimulating neural stem cells into neurons for treating brain damage.

Brain damage is a common tissue damage caused by trauma or diseases, which can be life-threatening. Stem cell implantation is an emerging strategy treating brain damage. The stem cell is commonly embedded in a matrix material for implantation, which protects stem cell and induces cell differentiation. Cell differentiation induction by this material is decisive in the effectiveness of this treatment strategy. In this work, we present an injectable fibroin/MXene conductive hydrogel as stem cell carrier, which further enables in-vivo electrical stimulation upon stem cells implanted into damaged brain tissue. Cell differentiation characterization of stem cell showed high effectiveness of electrical stimulation in this system, which is comparable to pure conductive membrane. Axon growth density of the newly differentiated neurons increased by 290% and axon length by 320%. In addition, unfavored astrocyte differentiation is minimized. The therapeutic effect of this system is proved through traumatic brain injury model on rats. Combined with in vivo electrical stimulation, cavities formation is reduced after traumatic brain injury, and rat motor function recovery is significantly promoted.

The molecular basis of sugar detection by an insect taste receptor.

Animals crave sugars because of their energy potential and the pleasurable sensation of tasting sweetness. Yet all sugars are not metabolically equivalent, requiring mechanisms to detect and differentiate between chemically similar sweet substances. Insects use a family of ionotropic gustatory receptors to discriminate sugars1, each of which is selectively activated by specific sweet molecules2-6. Here, to gain insight into the molecular basis of sugar selectivity, we determined structures of Gr9, a gustatory receptor from the silkworm Bombyx mori (BmGr9), in the absence and presence of its sole activating ligand, D-fructose. These structures, along with structure-guided mutagenesis and functional assays, illustrate how D-fructose is enveloped by a ligand-binding pocket that precisely matches the overall shape and pattern of chemical groups in D-fructose. However, our computational docking and experimental binding assays revealed that other sugars also bind BmGr9, yet they are unable to activate the receptor. We determined the structure of BmGr9 in complex with one such non-activating sugar, L-sorbose. Although both sugars bind a similar position, only D-fructose is capable of engaging a bridge of two conserved aromatic residues that connects the pocket to the pore helix, inducing a conformational change that allows the ion-conducting pore to open. Thus, chemical specificity does not depend solely on the selectivity of the ligand-binding pocket, but it is an emergent property arising from a combination of receptor-ligand interactions and allosteric coupling. Our results support a model whereby coarse receptor tuning is derived from the size and chemical characteristics of the pocket, whereas fine-tuning of receptor activation is achieved through the selective engagement of an allosteric pathway that regulates ion conduction.

Bombyx mori triose-phosphate transporter protein inhibits Bombyx mori nucleopolyhedrovirus infection by reducing the cell glycolysis pathway.

Bombyx mori triose-phosphate transporter protein (BmTPT) is a member of the solute carrier (SLC) family. Its main function is to transport triose phosphate between intracellular and extracellular. In this study, BmTPT was cloned and characterised from the fat body of the silkworm Bombyx mori, resulting in an open reading frame (ORF) with a full length of 936 bp, which can encode 311 amino acid residues and has eight transmembrane structural domains. BmTPT was distributed throughout the cell and deposited the most in the nucleus, and is expressed in all tissues of Bombyx mori. Bombyx mori nucleopolyhedrovirus (BmNPV) infection significantly up-regulated BmTPT expression in immune tissue fat bodies. In addition, overexpression of BmTPT significantly inhibited BmNPV infection and markedly reduced the expression of enzymes related to the cellular glycolytic pathway; on the contrary, down-regulation of BmTPT expression by RNA interference resulted in robust replication of BmNPV and a significant increase in the expression of enzymes related to the cellular glycolytic pathway. This is the first report that BmTPT has antiviral effect in silkworm, and also could result in a lack of energy and raw materials for BmNPV replication and infection through down-regulation of the cellular glycolytic pathway.

Molecular basis of host plant recognition by silkworm larvae.

Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercetin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings have revealed that the two Grs, previously considered to be sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.

The ratio of fibroin to sericin in the middle silk gland of Bombyx mori and its correlation with the extensional behavior of the silk dope.

Understanding how native silk spinning occurs is crucial for designing artificial spinning systems. One often overlooked factor in Bombyx mori is the secretion of sericin proteins. Herein, we investigate the variation in amino acid content at different locations in the middle silk gland (MSG) of B. mori. This variation corresponds to an increase in sericin content when moving towards the anterior region of the MSG, while the posterior region predominantly contains fibroin. We estimate the mass ratio of sericin to fibroin to be ~25/75 wt% in the anterior MSG, depending on the fitting method. Then, we demonstrate that the improvement in the extensional behavior of the silk dope in the MSG correlates with the increase in sericin content. The addition of sericin may decrease the viscosity of the silk dope, a factor associated with an increase in the spinnability of silk. We further discuss whether this effect could also result from other known physicochemical changes within the MSG.

BmPGPR-L4 is a negative regulator of the humoral immune response in the silkworm Bombyx mori.

Toll, immune deficiency and prophenoloxidase cascade represent vital immune signaling pathways in insects. Peptidoglycan recognition proteins (PGRPs) are innate immune receptors that activate and regulate the immune signaling pathways. Previously, we reported that BmPGPR-L4 was induced in the silkworm Bombyx mori larvae by bacteria and peptidoglycan challenges. Here, we focused on the function of BmPGRP-L4 in regulating the expression of antimicrobial peptides (AMPs). The hemolymph from BmPGRP-L4-silenced larvae exhibited an enhanced inhibitory effect on the growth of Escherichia coli, either by growth curve or inhibitory zone experiments. Coincidentally, most of the AMP genes were upregulated after RNAi of BmPGRP-L4. Oral administration of heat-inactivated E. coli and Staphylococcus aureus after RNAi of BmPGRP-L4 resulted in the increased expression of BmPGRP-L4 in different tissues of the silkworm larvae, revealing an auto-regulatory mechanism. By contrast, the expression of most AMP genes was downregulated by oral bacterial administration after RNAi of BmPGRP-L4. The above results demonstrate that BmPGRP-L4 recognizes bacterial pathogen-associated molecular patterns and negatively regulates AMP expression to achieve immunological homeostasis. As a negative regulator, BmPGPR-L4 is proposed to be involved in the feedback regulation of the immune signaling pathways of the silkworm to prevent excessive activation of the immune response.

CRISPR/Cas9-Mediated Editing of BmEcKL1 Gene Sequence Affected Silk Gland Development of Silkworms (Bombyx mori).

The silkworm (Bombyx mori) has served humankind through silk protein production. However, traditional sericulture and the silk industry have encountered considerable bottlenecks and must rely on major technological breakthroughs to keep up with the current rapid developments. The adoption of gene editing technology has nevertheless brought new hope to traditional sericulture and the silk industry. The long period and low efficiency of traditional genetic breeding methods to obtain high silk-yielding silkworm strains have hindered the development of the sericulture industry; the use of gene editing technology to specifically control the expression of genes related to silk gland development or silk protein synthesis is beneficial for obtaining silkworm strains with excellent traits. In this study, BmEcKL1 was specifically knocked out in the middle (MSGs) and posterior (PSGs) silk glands using CRISPR/Cas9 technology, and ΔBmEcKL1-MSG and ΔBmEcKL1-PSG strains with improved MSGs and PSGs and increased silk production were obtained. This work identifies and proves that BmEcKL1 directly or indirectly participates in silk gland development and silk protein synthesis, providing new perspectives for investigating silk gland development and silk protein synthesis mechanisms in silkworms, which is of great significance for selecting and breeding high silk-yielding silkworm varieties.

Proteolytic Activity of Silkworm Thorn (Cudrania tricuspidata) Fruit for Enzymatic Hydrolysis of Food Proteins.

This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit (Cudrania tricuspidata) through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (v/v). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation (p < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

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.