Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV) exhibit high genomic similarity but display significant differences in host range. The identification of key genes associated with this difference is of great importance for expanding its application in pest biocontrol. Fifteen genes specific to AcMNPV were identified. Among the constructed recombinant BmNPV, only ORF603 altered the virulence of BmNPV. Interestingly, our findings reveal a biological phenomenon that contradicts the prevailing theoretical framework. Current theory posits a positive correlation between viral virulence and replication efficiency; however, we observed that the recombinant virus vBmNPV-ORF603 exhibited high virulence despite significantly inhibited viral DNA replication. Furthermore, ORF603 exhibits entirely opposing roles during infections by AcMNPV and BmNPV. The underlying mechanism was determined to involve ORF603-induced G2/M phase cell cycle arrest in BmN cells, which consequently suppressed viral DNA replication. Meanwhile, the significant upregulation of genes associated with ribosome biogenesis and autophagy enhanced the transcription and translation efficiency of viral genes, thereby facilitating viral proliferation and intensifying the infection. The results demonstrate that the ORF603 of AcMNPV mediates a trade-off between viral virulence and transmission capacity in BmNPV. This functional dichotomy offers novel insights into the regulation of infection strategies in baculoviruses and highlights potential targets for engineering enhanced viral biopesticides.

Insect reproductive capacity is a key determinant of population fitness, though its regulatory mechanisms remain limited. Here, we identified an op50 silkworm mutant (derived from p50 strain) exhibiting female-specific reproductive defects, which provided an important model for studying insect reproductive regulation mechanism. We found that Bombyx mori xanthine dehydrogenase 1 (BmXDH1) in op50 contained a single-base insertion causing premature termination, deleting 154 C-terminal amino acids and losing catalytic activity. ΔBmXDH1 mutants recapitulated the op50 phenotype with 50 % reduced oviposition and 90 % lower hatchability, attributed to severe structural impairments of the eggshell surface and egg surface pore. In addition, female reproductive defects initiated at the pupal stage, characterized by oviduct malformation and impaired oviposition. This phenotype was associated with excessive accumulation of hypoxanthine, leading to ovarian and embryonic developmental disruption, including extracellular matrix remodeling, cellular signaling pathways, and oxidative stress regulation. The results uncovered how BmXDH1 regulates hypoxanthine metabolism to control silkworm reproduction, advancing understanding of insect reproductive networks and providing targets for pest control/economic insect breeding.

Cocoon silk quality is a critical determinant of the economic value in the sericulture industry for Bombyx mori L. (Lepidoptera: Bombycidae). Sur8 functions as a positive regulator of the RAS-MAPK signaling cascade, which plays an essential role in regulating cocoon silk production in Bombyx mori. However, the specific functions of BmSur8 in silkworms have not been fully elucidated. In this study, we demonstrate that BmSur8 is a highly conserved gene expressed across all life stages and various tissues at the third day of the fifth instar larvae (L5D3) stage in Bombyx mori. Knockout of the BmSur8 gene results in a 15.2% reduction in silk yield in females and a 13.4% reduction in males, with the cocoon layer rate in females decreasing by 3.8%. Through transcriptomic analysis of posterior silk glands (PSGs) at L5D3 stage from ΔBmSur8 mutants and Nistari controls, as well as mass spectrometry analysis of BmSur8-interacting proteins in BmN cells, we find that BmSur8 potentially interacts with BmEtfb and regulates the expression of BmCDA and BmjunD, thereby participating in material and energy metabolism. This study provides a comprehensive analysis of the function of BmSur8 in metabolic pathways and offers theoretical insights and experimental data for researching silkworm cocoon silk quality and yield, as well as for breed selection and breeding.

Autographa californica nucleopolyhedrovirus (AcMNPV) is a DNA virus with multiple host domains, and elucidating the mechanisms of its interactions with silkworms is crucial for its widespread use. Identifying key antiviral genes and analyzing their functions is an urgent task currently. Therefore, the identification and study of host genes associated with AcMNPV invasion is of great significance in solving the issue. Engulfment and cell motility (Elmo) is an identified viral infection-associated gene primarily involved in the regulation of cell motility and essential for phagocytosis and immune responses. However, its function in the silkworm response to viruses is still unclear. In this study, the sequence of BmElmo was analyzed first. It has a CED-12 functional domain that has been highly conserved among different species. Its expression peaks during the silkworm pupal stage, followed by the moth stage. Among various tissues, BmElmo expression is highest in the gonads, followed by the silk glands. BmElmo exhibits differential expression between resistant and susceptible strains. AcMNPV replication increased significantly after BmElmo knockdown in BmN cells, and decreased significantly after BmElmo overexpression. Furthermore, the expression of Janus kinase (JNK) pathway-related genes downstream of BmElmo showed altered expression that correlated positively with the expression of BmElmo. Hence, BmElmo may inhibit AcMNPV replication in the silkworm by activating the JNK pathway. The results of this study bridge the gap in understanding the role of Elmo genes in insect immunity and provides a theoretical reference for studying the interaction between insects and baculoviruses.

Ultrabithorax (Ubx) is a famous gene for insect body segment determination and c-myc plays a crucial role in cell fate and is well-known as proto-oncogene. As two pivotal transcription factors, whether there is a regulatory relationship between them has not yet been reported. In our previous research, we found that excess Ubx can strongly repress the expression of Myc in the posterior silk gland of Bombyx mori (B. mori). One unresolved question is whether Ubx inhibits the Myc expression directly or indirectly. In our current study, multiple Ubx binding sites were identified from the upstream sequence and 5'-UTR of Myc. Meanwhile, the expression profile data in silkDB 3.0 showed that Ubx and Myc were expressed simultaneously in multiple tissues and developmental stages of B. mori. Luciferase reporter assay results indicated that overexpressed Ubx down-regulated the activity of luciferase which was activated by the upstream sequence and 5'-UTR of Myc in BmN cells. Electrophoretic Mobility Shift Assay (EMSA) confirmed that Ubx bound to the 5'-UTR of Myc directly. Prediction results and ChIP-Seq data suggested that this interaction might be widespread in insects. In conclusion, our study uncovered the relationship between two famous gene, Ubx and Myc, which play critical regulatory roles in insect growth and development.

The molecular mechanism of baculovirus infection is the basis of baculovirus wide application. Identifying and elucidating the functional genes of virus replication is the focus of research. Eukaryotic initiation factor 4E (eIF4E) is a key component of the translation initiation process to synthesize proteins required for replication. Our pre-transcriptomics data suggest that BmeIF4E is associated with Autographacalifornica nucleopolyhedrovirus (AcMNPV) infection, but its exact role is unknown. The study first clarified, using bioinformatics, that the function of BmeIF4E2 is highly conserved, and expression profile analysis revealed that the high expression of BmeIF4E2 is mainly distributed in the egg stage, 4th instar molting stage, and pupal stage. To clarify the function of BmeIF4E2 in AcMNPV infestation, it was overexpressed and interfered in BmN cells. The increase of virus after overexpression and decrease of virus after interference indicated that BmeIF4E2 could promote AcMNPV infection. To analyze whether BmeIF4E2 is directly involved in promoting AcMNPV infection, His-tag pull down analysis was performed with prokaryotic expression of BmeIF4E2 protein as bait. It was found that BmeIF4E2 could interact directly with vAcMNPV-eGFP. Finally, we validated this in individual silkworms using the inhibitor 4E2RCat and the recombinant virus AcMNPV-BmeIF4E2, and the results were consistent with those obtained in cells. Thus, BmeIF4E2 is a key gene that promotes AcMNPV infection in silkworms via direct interaction. The results are valuable for pest biological control and improving the yield of baculovirus expression systems.

Baculovirus is a multipurpose virus with a wide range of applications, such as protein expression, disease therapy, biocontrol, and so forth. It is of great significance to elucidate the mechanisms of their application as biopesticides, since insect resistance to viruses limits their application. Autographa californica nucleopolyhedrovirus (AcMNPV) is a representative species of α-baculovirus, and the silkworm is an ideal model organism for Lepidoptera. Exploring the interactions between the two is an important reference for elucidating the mechanism. In this study, the transcriptome of the silkworm strain p50 hemolymph was examined after 36 h of AcMNPV infection, and the data obtained were assembled and annotated with messenger RNA and microRNA (miRNA), respectively. Three miRNAs related to AcMNPV infection were obtained, including miR-6497-5p, miR-6498-3p, and miR-6498-5p. Among them, miR-6497-5p was a key miRNA for AcMNPV inhibition, and its downstream target gene, organic cation transporter protein (BmOctp), was also confirmed. Furthermore, knockdown and overexpression of BmOctp revealed that it was beneficial for viral infection, and this function was found as early as 6 h after virus infection, which may be related to the early transmembrane transport of the virus. This study is the first screening of miRNAs related to AcMNPV in silkworms, and the results provide important theoretical references for elucidating the mechanism of baculovirus-host and its potential applications.

Bombyx mori ELAV-like-1 (BmEL-1) and B. mori ELAV-like-2 (BmEL-2) are 2 members of the ELAV-like family of RNA-binding proteins. Mutations in Bmel-1 and Bmel-2 resulted in 5.8% and 28.5% decreases in larval weight on the 3rd day of the 5th instar larva (L5D3), respectively. Triglycerides (TG) are the most important energy resource and are the main component of neutral fat (NF) in animals. To investigate the role of Bmelav-like genes in the synthesis and decomposition of TG, transcriptomic, and metabolic analyses were performed on the whole bodies on the 1st day of the 2nd instar larvae (L2D1) and on fat bodies on L5D3 of Bmel-1- and Bmel-2- mutants, respectively. As compared with the control silkworm, differentially expressed genes generated in both mutants were mainly enriched in lysine degradation, fatty acid (FA) metabolism, and unsaturated FAs biosynthesis. The diglyceride and phosphatide contents were significantly lower in Bmel-1- and Bmel-2- fat bodies than those of the control group. Consistently, the NF content of both mutants' fat bodies were reduced by 50% and 60%, respectively. BmEL-2 positively regulates BmAGPATγ (B. mori 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma, LOC101741736) and BmFaF2 (B. mori fatty acid synthetase-associated factor 2, LOC101739090) expression by binding to the specific regions of their 3' untranslated regions in BmN cells. This study suggests that BmEL-2 may be an important regulator of BmAGPATγ and BmFAF2 expression and thereby participates in TG metabolism in the silkworm fat body.

The study of functional genes involved in baculovirus infection is vital for its wide application in pest biocontrol. This study utilized the Autographa californica nucleopolyhedrovirus (AcMNPV) and silkworm as models to elucidate the role of BmRRS1, which has been found to exhibit notable differential expression between resistant and susceptible silkworm strains. The results showed that it was evolutionarily conserved in selected species. Among different tissues, it was expressed at the highest level in the gonads, followed by the hemolymph and silk glands; among the different developmental stages, it was the highest in the second instar, followed by the pupae and adults. Moreover, its vital role in suppressing AcMNPV infection was verified by the decreased expression of lef3 and vp39 protein after overexpression of BmRRS1 as well as by the increased expression of the viral gene lef3 and the viral protein vp39 after siRNA treatment against BmRRS1 expression in BmN cells. Additionally, the direct interaction between BmRRS1 and AcMNPV was detected by the GST pull-down assay. Finally, the homologue of BmRRS1 in Spodoptera frugiperda was found to be involved in larval resistance to AcMNPV. In a word, BmRRS1 plays a vital role in AcMNPV resistance in silkworms, and this might be related to the direct interaction with AcMNPV. The results of this study provide a potential target for protecting silkworm larvae from virus infection and controlling agricultural and forestry pests.

The silkworm ( Bombyx mori ) is an important model lepidopteran insect and can be used to identify pesticide resistance-related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post-feeding, indicating BmUGT 's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua : Its resistance to fenpropathrin decreased significantly after knocking down SeUGT . In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests.

Intestinal symbionts live in insects’ digestive systems and help with food digestion, nitrogen cycling, and energy use. Numerous symbionts, including bacteria, fungi, and protozoa, colonize the posterior gut of termites. Intestinal microbiota affects the Toll or Imd pathways during viral or entomopathogenic fungus infection in mosquitoes, but the role of intestinal symbionts in individual termite immunity is unclear. In the present research, Odontotermes formosanus and Metarhizium robertsii were selected to investigate the potential mechanism of the intestinal bacteria influencing termite innate immune while encountering an entomopathogen using metagenome sequencing and meta-transcriptome sequencing. We found that termite mortality declined within 48 hours under high spore infections and reached semi-lethal status within 72 hours. We sequenced the control and infestation termite and found that the dominant phyla changed below low-abundance flora during infestation. According to function annotation, the functions of spirochetes are significantly different from those of the rest of the phyla, which are mainly enriched in metabolism and cellular processes. There are 261 upregulated and 459 down-regulated DEGs derived from bacteria after infestation. We identified the glutamate-cysteine ligase gene, gshA, which is significantly upregulated in the infected group, and the GSH content in the regular infected group was higher than the 5% kanamycin-infected group. We speculated on a potential mechanism that one or more bacteria belonging to Spirochetes that upregulated gshA gene expression and increased GSH content in the hindgut to integrate with metarhizium toxins and then excrete out of the intestine.

Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, we constructed the Ubx overexpression (UbxOE) strain based on the Nistari strain of Bombyx mori. The UbxOE strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). In the current study, we focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three times in UbxOE, but the protein expression of Fib-H and Fib-L was not significantly different. We speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation between Bombyx mori ecdysone receptor B1 (BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics of BmEcR-B1 and its isoform, BmEcR-A, were initially analyzed. BmEcR-B1 was found to be more critical than BmEcR-A in silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showed BmEcR-B1 had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore, BmEcR-B1 was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection.

Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.

Bombyx mori, domesticated from wild silkworms, is an economic insect that feeds on mulberry leaves and produces silk. In the current study, we demonstrated the contribution of BmAbl1 in silk protein synthesis. The inhibition and knockout of BmAbl1 can reduce the larva weight and CSW. The effect on CSW of BmAbl1 is not on the transcriptional level, but on the translational level. RNA-sequencing data suggested that amino acid synthesis and the metabolism process had a great difference between the BmAbl1- and Control strain, particularly glutathione metabolism. An abnormality in glutathione metabolism led to the reduction of free glycine and serine content, which are the main components of fibroin protein. Finally, fibroin protein synthesis has been reduced, including fibroin-heavy chain, fibroin-light chain, and p25 protein. This finding brought to light the role of BmAbl1 in the silk protein synthesis process.

Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.

[目的]筛选RNA结合蛋白RBP9在黑腹果蝇Drosophila melanogaster头部中的互作蛋白.[方法]利用CRISPR/Cas9基因组编辑技术,分别将3 x Flag和V5标签编码序列插入到黑腹果蝇成虫头中RBP9和FNE基因起始密码子ATG的后面,构建黑腹果蝇转基因品系3×Flag-RBP 9/3 x Flag-RBP9(3FRBP9)和V5-FNE/V5-FNE(VFNE);利用免疫沉淀和质谱法鉴定黑腹果蝇3FRBP9和野生型品系成虫头部中RBP9的互作蛋白并进行生物信息学分析.利用免疫共沉淀方法检测RBP9与FNE蛋白之间的相互作用.[结果]质谱法从黑腹果蝇成虫头部共鉴定了 190个与RBP9相互作用的蛋白,其中包括ELAV和FNE.KEGG富集分析显示这些蛋白的基因主要参与核糖体、碳代谢、三羧酸循环、氨基酸生物合成等通路.免疫共沉淀实验结果验证了 RBP9与FNE蛋白之间存在相互作用.[结论]RNA结合蛋白ELAV家族成员在黑腹果蝇成虫头部中具有相互作用.本研究为RBP9在果蝇神经系统发育中的功能研究提供了重要实验依据.

Baculoviruses are enveloped rod-shaped viruses that with circular double-stranded and large DNA genome. Baculoviruses successfully invade by using the host factors, especially at the process of establishing early infection. In this study, we investigated the different resistant strains of silkworm in response to AcMNPV early infection by RNA-sequencing. Our data revealed that the genes involved in innate immunity and ribosomal proteins were suppressed and cytoskeleton were induced in susceptible strain p50, thus facilitating the viral replication. However, in resistant strain C108, the genes participated in endocytosis and RNA transport were down-regulated, while up-regulation of ribosomal proteins and energy metabolic to inhibit the infection of the virus. These data provide a new sight of the initial host transcriptional responses to viral infection in silkworm.

BACKGROUND Nucleopolyhedrovirus (NPV), one of the baculoviruses, is a promising biopesticide for pest control. Lepidopteran account for 70% of pests, therefore investigation on highly-conserved genes associated with viral infections in the lepidopteran model, the silkworm, will serve as a valuable reference for improving the effectiveness of pest management. BmE74A is a member of the erythroblast transformation-specific (ETS) family of transcription factors in Bombyx mori, which we previously found to be highly conserved and closely associated with BmNPV. This study aimed to elucidate the role of BmE74A in viral infection. RESULTS A significantly high expression of BmE74A in eggs indicated its important role in embryonic development, as did relatively high expressions in the hemolymph and midgut. Significant differences in BmE74A expression in different resistant strains after BmNPV infection suggested its involvement as a response to viral infection. Moreover, RNA interference (RNAi) and overexpression experiments confirmed the important role of BmE74A in promoting viral infection. BmNPV infection was significantly suppressed and enhanced by BmE74A knockdown and overexpression, respectively. Besides, BmE74A was found to regulate the expression of BmMdm2 and Bmp53. Furthermore, the binding of ETS, the functional domain of BmE74A, to occlusion-derived virus proteins was confirmed by far-western blotting, and four viral proteins that may interact with ETS proteins were identified by mass spectrometry. Similarly, a homolog of BmE74A in Spodoptera litura was also found to be involved in larval susceptibility to BmNPV. CONCLUSION BmE74A promotes BmNPV proliferation by directly interacting with the virus, which may be related to the suppression of the p53 pathway. This article is protected by copyright. All rights reserved.