Whole transcriptome analysis identifies differentially expressed mRNA, miRNA and lncRNA associated with male sterility in the silkworm, Bombyx mori.

Insect sterility technology is gradually being applied to the control of lepidoptera pests, and the target gene for male sterility is the core of this technology. JMS is a mutant silkworm that exhibits male sterility, and to elucidate its formation mechanism, this study conducted a full transcriptome analysis of the testes of JMS and its wild-type silkworms 48 h after pupation, identifying 205 DElncRNAs, 913 mRNAs, and 92 DEmiRNAs. The KEGG pathway enrichment analysis of the DEmRNAs revealed that they were involved in the biosynthesis of amino acids and ECM-receptor interactions. Combined with ceRNA regulatory network KEGG analysis suggests that pathways from amino acid biosynthesis to hydrolytic processes of protein synthesis may play a crucial role in the formation of JMS mutant variants. Our study deepens our understanding of the regulatory network of male sterility genes in silkworms; it also provides a new perspective for insect sterility technology.

Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori.

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.

CRISPR/Cas9-mediated Nap knockout affects female reproduction and egg shape in Bombyx mori.

Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies.

Evaluating bio-physicochemical properties of raw powder prepared from whole larvae containing liquid silk of the domestic silkworm.

The domestic silkworm, Bombyx mori, has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As "percentage yield" translates to "Budomari" in Japanese, this raw powder was named "B100rw." We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.

TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

Comparative analysis of testicular fusion in Spodoptera litura (cutworm) and Bombyx mori (silkworm): Histological and transcriptomic insights.

Spodoptera litura commonly known as the cutworm, is among the most destructive lepidopteran pests affecting over 120 plants species. The powerful destructive nature of this lepidopteran is attributable to its high reproductive capacity. The testicular fusion that occurs during metamorphosis from larvae to pupa in S.litura positively influences the reproductive success of the offspring. In contrast, Bombyx mori, the silkworm, retains separate testes throughout its life and does not undergo this fusion process. Microscopic examination reveals that during testicular fusion in S.litura, the peritoneal sheath becomes thinner and more translucent, whereas in B.mori, the analogous region thickens. The outer basement membrane in S.litura exhibits fractures, discontinuity, and uneven thickness accompanied by a significant presence of cellular secretions, large cell size, increased vesicles, liquid droplets, and a proliferation of rough endoplasmic reticulum and mitochondria. In contrast, the testicular peritoneal sheath of B.mori at comparable developmental stage exhibits minimal change. Comparative transcriptomic analysis of the testicular peritoneal sheath reveals a substantial difference in gene expression between the two species. The disparity in differential expressed genes (DEGs) is linked to an enrichment of numerous transcription factors, intracellular signaling pathways involving Ca2+ and GTPase, as well as intracellular protein transport and signaling pathways. Meanwhile, structural proteins including actin, chitin-binding proteins, membrane protein fractions, cell adhesion, extracellular matrix proteins are predominantly identified. Moreover, the study highlights the enrichment of endopeptidases, serine proteases, proteolytic enzymes and matrix metalloproteins, which may play a role in the degradation of the outer membrane. Five transcription factors-Slforkhead, Slproline, Slcyclic, Slsilk, and SlD-ETS were identified, and their expression pattern were confirmed by qRT-PCR. they are candidates for participating in the regulation of testicular fusion. Our findings underscore significant morphological and trancriptomic variation in the testicular peritoneal sheath of S.litura compared to the silkworm, with substantial changes at the transcriptomic level coinciding with testicular fusion. The research provides valuable clues for understanding the complex mechanisms underlying this unique phenomenon in insects.

Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori).

Mercury (Hg) contamination poses a global threat to the environment, given its elevated ecotoxicity. Herein, we employed the lepidopteran model insect, silkworm (Bombyx mori), to systematically investigate the toxic effects of Hg-stress across its growth and development, histomorphology, antioxidant enzyme activities, and transcriptome responses. High doses of Hg exposure induced evident poisoning symptoms, markedly impeding the growth of silkworm larvae and escalating mortality in a dose-dependent manner. Under Hg exposure, the histomorphology of both the midgut and fat body exhibited impairments. Carboxylesterase (CarE) activity was increased in both midgut and fat body tissues responding to Hg treatment. Conversely, glutathione S-transferase (GST) levels increased in the fat body but decreased in the midgut. The transcriptomic analysis revealed that the response induced by Hg stress involved multiple metabolism processes. Significantly differently expressed genes (DEGs) exhibited strong associations with oxidative phosphorylation, nutrient metabolisms, insect hormone biosynthesis, lysosome, ribosome biogenesis in eukaryotes, and ribosome pathways in the midgut or the fat body. The findings implied that exposure to Hg might induce the oxidative stress response, attempting to compensate for impaired metabolism. Concurrently, disruptions in nutrient metabolism and insect hormone activity might hinder growth and development, leading to immune dysfunction in silkworms. These insights significantly advance our theoretical understanding of the potential mechanisms underlying Hg toxicity in invertebrate organisms.

Transcriptomic analysis of Bombyx mori in its early larval stage (2nd instar) of development upon Nosema bombycis transovarial infection.

The infection caused by Nosema bombycis often known as pebrine, is a devastating sericulture disease. The infection can be transmitted to the next generation through eggs laid by infected female Bombyx mori moths (transovarial) as well as with N. bombycis contaminated food (horizontal). Most diagnoses were carried out in the advanced stages of infection until the time that infection might spread to other healthy insects. Hence, early diagnosis of pebrine is of utmost importance to quarantine infected larvae from uninfected silkworm batches and stop further spread of the infection. The findings of our study provide an insight into how the silkworm larval host defence system was activated against early N. bombycis transovarial infection. The results obtained from transcriptome analysis of infected 2nd instar larvae revealed significant (adjusted P-value < 0.05) expression of 1888 genes of which 801 genes were found to be upregulated and 1087 genes were downregulated when compared with the control. Pathway analysis indicated activation of the immune deficiency (IMD) pathway, which shows a potential immune defence response against pebrine infection as well as suppression of the melanin synthesis pathway due to lower expression of prophenoloxidase activating enzyme (PPAE). Liquid chromatography mass spectrometry (LC-MS/MS) analysis of haemolymph from infected larvae shows the secretion of serpin binding protein of N. bombycis which might be involved in the suppression of the melanization pathway. Moreover, among the differentially expressed genes, we found that LPMC-61, yellow-y, gasp and osiris 9 can be utilised as potential markers for early diagnosis of transovarial pebrine infection in B. mori. Physiological as well as biochemical roles and functions of many of the essential genes are yet to be established, and enlightened research will be required to characterize the products of these genes.

BmHSP19.9 targeting P6.9 and VLF-1 to mediate the formation of defective progeny viruses in the silkworm antiviral variety 871C.

The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains.

Identification of sulfakinin receptor regulating feeding behavior and hemolymph trehalose homeostasis in the silkworm, Bombyx mori.

Feeding behavior, the most fundamental physiological activity, is controlled by two opposing groups of factors, orexigenic and anorexigenic factors. The sulfakinin family, an insect analogue of the mammalian satiety factor cholecystokinin (CCK), has been shown to suppress food intake in various insects. Nevertheless, the mechanisms through which sulfakinin regulates feeding behavior remain a biological question. This study aimed to elucidate the signaling pathway mediated by the anorexigenic peptide sulfakinin in Bombyx mori. We identified the Bombyx mori neuropeptide G protein-coupled receptor A9 (BNGR-A9) as the receptor for sulfakinin through functional assays. Stimulation with sulfakinin triggered a swift increase in intracellular IP3, Ca2+, and a notable enhancement of ERK1/2 phosphorylation, in a manner sensitive to a Gαq-specific inhibitor. Treatment with synthetic sulfakinin resulted in decreased food consumption and average body weight. Additionally, administering synthetic sulfakinin to silkworms significantly elevated hemolymph trehalose levels, an effect markedly reduced by pre-treatment with BNGR-A9 dsRNA. Consequently, our findings establish the sulfakinin/BNGR-A9 signaling pathway as a critical regulator of feeding behavior and hemolymph trehalose homeostasis in Bombyx mori, highlighting its roles in the negative control of food intake and the positive regulation of energy balance.

Transcriptome Analysis Reveals Antioxidant Defense Mechanisms in the Silkworm Bombyx mori after Exposure to Lead.

Lead (Pb) is a major source of heavy metal contamination, and poses a threat to biodiversity and human health. Elevated levels of Pb can hinder insect growth and development, leading to apoptosis via mechanisms like oxidative damage. The midgut of silkworms is the main organ exposed to heavy metals. As an economically important lepidopteran model insect in China, heavy metal-induced stress on silkworms causes considerable losses in sericulture, thereby causing substantial economic damage. This study aimed to investigate Pb-induced detoxification-related genes in the midgut of silkworms using high-throughput sequencing methods to achieve a deeper comprehension of the genes' reactions to lead exposure. This study identified 11,567 unigenes and 14,978 transcripts. A total of 1265 differentially expressed genes (DEGs) were screened, comprising 907 upregulated and 358 downregulated genes. Subsequently, Gene Ontology (GO) classification analysis revealed that the 1265 DEGs were distributed across biological processes, cellular components, and molecular functions. This suggests that the silkworm midgut may affect various organelle functions and biological processes, providing crucial clues for further exploration of DEG function. Additionally, the expression levels of 12 selected detoxification-related DEGs were validated using qRT-PCR, which confirmed the reliability of the RNA-seq results. This study not only provides new insights into the detoxification defense mechanisms of silkworms after Pb exposure, but also establishes a valuable foundation for further investigation into the molecular detoxification mechanisms in silkworms.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

ABHD5 regulates midgut-specific lipid homeostasis in Bombyx mori.

Lipids are an important energy source and are utilized as substrates for various physiological processes in insects. Comparative gene identification 58 (CGI-58), also known as α/ß hydrolase domain-containing 5 (ABHD5), is a highly conserved and multifunctional gene involved in regulating lipid metabolism and cellular energy balance in many organisms. However, the biological functions of ABHD5 in insects are poorly understood. In the current study, we describe the identification and characterization of the ABHD5 gene in the lepidopteran model insect, Bombyx mori. The tissue expression profile investigated using quantitative reverse transcription polymerase chain reaction (RT-qPCR) reveals that BmABHD5 is widely expressed in all tissues, with particularly high levels found in the midgut and testis. A binary transgenic CRISPR/Cas9 system was employed to conduct a functional analysis of BmABHD5, with the mutation of BmABHD5 leading to the dysregulation of lipid metabolism and excessive lipid accumulation in the larval midgut. Histological and physiological analysis further reveals a significant accumulation of lipid droplets in the midgut of mutant larvae. RNA-seq and RT-qPCR analysis showed that genes related to metabolic pathways were significantly affected by the absence of BmABHD5. Altogether, our data prove that BmABHD5 plays an important role in regulating tissue-specific lipid metabolism in the silkworm midgut.

The Spatio-Temporal Expression Profiles of Silkworm Pseudogenes Provide Valuable Insights into Their Biological Roles.

Background: Pseudogenes are sequences that have lost the ability to transcribe RNA molecules or encode truncated but possibly functional proteins. While they were once considered to be meaningless remnants of evolution, recent researches have shown that pseudogenes play important roles in various biological processes. However, the studies of pseudogenes in the silkworm, an important model organism, are limited and have focused on single or only a few specific genes. Objective: To fill these gaps, we present a systematic genome-wide studies of pseudogenes in the silkworm. Methods: We identified the pseudogenes in the silkworm using the silkworm genome assemblies, transcriptome, protein sequences from silkworm and its related species. Then we used transcriptome datasets from 832 RNA-seq analyses to construct spatio-temporal expression profiles for these pseudogenes. Additionally, we identified tissue-specifically expressed and differentially expressed pseudogenes to further understand their characteristics. Finally, the functional roles of pseudogenes as lncRNAs were systematically analyzed. Results: We identified a total of 4410 pseudogenes, which were grouped into 4 groups, including duplications (DUPs), unitary pseudogenes (Unitary), processed pseudogenes (retropseudogenes, RETs), and fragments (FRAGs). The most of pseudogenes in the domestic silkworm were generated before the divergence of wild and domestic silkworm, however, the domestication may also involve in the accumulation of pseudogenes. These pseudogenes were clearly divided into 2 cluster, a highly expressed and a lowly expressed, and the posterior silk gland was the tissue with the most tissue-specific pseudogenes (199), implying these pseudogenes may be involved in the development and function of silkgland. We identified 3299 lncRNAs in these pseudogenes, and the target genes of these lncRNAs in silkworm pseudogenes were enriched in the egg formation and olfactory function. Conclusions: This study replenishes the genome annotations for silkworm, provide valuable insights into the biological roles of pseudogenes. It will also contribute to our understanding of the complex gene regulatory networks in the silkworm and will potentially have implications for other organisms as well.

A strategy for improving silk yield and organ size in silk-producing insects.

Insect silks possess excellent biodegradability, biocompatibility and mechanical properties, and have numerous applications in biomedicine and tissue engineering. However, the application of silk fiber is hindered by its limited supply, especially from non-domesticated insects. In the present study, the silk yield and organ size of Bombyx mori were significantly improved through genetic manipulation of the target of rapamycin complex 1 (TORC1) pathway components. Silk protein synthesis and silk gland size were decreased following rapamycin treatment, inhibiting the TORC1 signaling pathway both in vivo and ex vivo. The overexpression of posterior silk gland-specific Rheb and BmSLC7A5 improved silk protein synthesis and silk gland size by activating the TORC1 signaling pathway. Silk yield in BmSLC7A5-overexpression silkworms was significantly increased by approximately 25%. We have demonstrated that the TORC1 signaling pathway is involved in the transcription and translation of silk genes and transcriptional activators via phosphorylation of p70 S6 kinase 1 and 4E-binding protein 1. Our findings present a strategy for increasing silk yield and organ size in silk-producing insects.

GASZ is indispensable for gametogenesis in the silkworm, Bombyx mori.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Identification of long non-coding RNAs in response to microsporidia infection in Silkworm, Bombyx mori.

Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.

Effects of baculovirus infection on intestinal microflora of BmNPV resistant and susceptible strain silkworm.

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a serious pathogen causing huge economic losses to sericulture. There is growing evidence that the gut microbiota of silkworms plays a critical role in shaping host responses and interactions with viral infection. However, little is known about the differences in the composition and diversity of intestinal microflora, especially with respect to silkworm strain differences and BmNPV infection-induced changes. Here, we aim to explore the differences between BmNPV-resistant strain A35 and susceptible strain P50 silkworm and the impact of BmNPV infection on intestinal microflora in different strains. The 16S rDNA sequencing analysis revealed that the fecal microbial populations were distinct between A35 and P50 and were significantly changed post BmNPV infection in both strains. Further analysis showed that the BmNPV-resistant strain silkworm possessed higher bacterial diversity than the susceptible strain, and BmNPV infection reduced the diversity of intestinal flora assessed by feces in both silkworm strains. In response to BmNPV infection, the abundance of Muribaculaceae increased in P50 and decreased in A35, while the abundance of Enterobacteriaceae decreased in P50 and increased in A35. These results indicated that BmNPV infection had various effects on the abundance of fecal microflora in different silkworm strains. Our findings not only broadened the understanding of host-pathogen interactions but also provided theoretical help for the breeding of resistant strains and healthy rearing of silkworms based on symbiotic bacteria.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.