How Do Butterflies Use Silk to Attach their Pupae to Trees?

Butterflies constitute approximately 10% of lepidopteran insects, and along with silkworms, they can produce silk; however, this feature is often ignored. In the present study, we observed two primary methods used by butterflies to hang pupae on trees using silk: pupa adheraena (Danaus chrysippus) and pupa contigua (Papilio polytes). Anchoring the abdominal ends of pupae with a silk pad was the most common method used in both cases, whereas wrapping silk around the body using a silk girdle was a method unique to pupa contigua. The connection between the cremaster and silk pad was observed to be similar to that between the hook and loop of a Velcro fastener, except that the cremaster hook is anchor-shaped rather than being a single hook. Such a connection will remain secure, ensuring the safety of the pupae during exposure to wind and rain. Through determining the mechanical properties of silk, the performance of butterfly silk was found to be weaker than that of silkworm silk. Therefore, the P. polytes silk girdle adopts the strategy of merging a dozen silk threads to improve its strength and toughness, thereby making it difficult to break. In addition, we explained how the protein sequence and structure of butterfly silk impact its performance. In conclusion, we discovered that butterfly pupae develop unique body features to establish secure bonds with silk. This enables them to effectively undergo metamorphosis and endure harsh weather conditions and surroundings.

Silkworm Hemolymph and Cocoon Metabolomics Reveals Valine Improves Feed Efficiency of Silkworm Artificial Diet.

Artificial silkworm diets significantly impact farm profitability. Sustainable cocoon production depends on the continuous improvement of feed efficiency to reduce costs and nutrient losses in the feed. This study used metabolomics to explore the differences in silkworm cocoons and hemolymph under two modes of rearing: an artificial diet and a mulberry-leaf diet. Nine metabolites of silkworm cocoons and hemolymph in the mulberry-leaf group were higher than those in the artificial-diet group. Enrichment analysis of the KEGG pathways for these metabolites revealed that they were mainly enriched in the valine, leucine, and isoleucine biosynthesis and degradation pathways. Hence, the artificial silkworm diet was supplemented various concentrations of valine were supplemented to with the aim of examining the impact of valine on their feeding and digestion of the artificial diet. The results indicated that valine addition had no significant effect on feed digestibility in the fifth-instar silkworm. Food intake in the 2% and 4% valine groups was significantly lower than that in the 0% valine group. However, the 2% and 4% valine groups showed significantly improved cocoon-production efficiency, at 11.3% and 25.1% higher, respectively. However, the cocoon-layer-production efficiencies of the 2% and 4% valine groups decreased by 7.7% and 13.9%, respectively. The research confirmed that valine is an effective substance for enhancing the feed efficiency of silkworms.

BmSPP is a virus resistance gene in Bombyx mori.

Introduction: Signal peptide peptidase (SPP) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, Bombyx mori , may be helpful in breeding virus-resistant silkworms. Methods: First, we performed RT-qPCR to analyze the expression pattern of BmSPP. Subsequently, we inhibited BmSPP using the SPP inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)2-ketone) and downregulated the expression of BmSPP using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV). Results: We observed a decreased in the expression of BmSPP during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of BmSPP in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, BmSPPKO silkworms exhibited a 12.4% reduction in mortality rate. Discussion: Collectively, this work demonstrates that BmSPP plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that BmSPP servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.

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.

Targeted DNA N6-methyladenine editing by dCas9 fused to METTL4 in the lepidopteran model insect Bombyx mori.

We have established a novel CRISPR-dCas9-METTL4 epigenome editing tool that can methylate target regions to achieve site-specific DNA 6mA methylation in both hypermethylated and hypomethylated genes. Targeted methylation on genes by dCas9-METTL4 results in misexpression, allowing for the functional investigation of target genes of interest in silkworm.

Ecdysteroid UDP-Glucosyltransferase Expression in Beauveria bassiana Increases Its Pathogenicity against Early Instar Silkworm Larvae.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500µg/mL G418 sulfate solution through the protoplast transformation method. This enzyme has the function of 20E (20-hydroxyecdysone) inactivation, thus increasing the mortality of the early instar larvae infected with B. bassiana. In this study, we transformed B. bassiana with the egt gene, which deactivates 20-hydroxyecdysone, a key hormone in insect development. The results showed that transgenic B. bassiana killed more silkworms of the 2nd instar larvae than the wild-type with a shorter LT50 time, which was reduced by approximately 20% (day 1 of the 2nd instar silkworm infection of B. bassiana) and 26.4% (day 2 of the 2nd instar silkworm infection of B. bassiana) compared to the wild-type, and also showed a higher mortality number before molting. The transgenic B. bassiana had a higher coverage of the body surface of silkworms compared to the wild type on the 3rd instar. In summary, improving entomopathogenic fungi using biological methods such as genetic engineering is feasible.

A Study on the Effect of Energy on the Development of Silkworm Embryos Using an Estrogen-Related Receptor.

Energy metabolism is a fundamental process in all organisms. During silkworm (Bombyx mori) embryonic development, there is a high demand for energy due to continuous cell proliferation and differentiation. Estrogen-related receptors (ERRs) are transcriptional regulatory factors that play crucial roles in mammalian energy storage and expenditure. Although most insects have one ERR gene, it also participates in the regulation of energy metabolism, including carbohydrate metabolism in Drosophila, Aphid, and Silkworm. However, no study has reported the direct impact of energy metabolism on embryonic development in silkworms. In this study, we used transgenic technology to increase silkworm (B. mori; Bm) BmERR expression during embryonic development and explored the impact of energy on embryonic development. We found no significant change in the quality of silkworm eggs compared to that of wild-type silkworms. However, there was an increase in the consumption of vitellin, a major nutrient in embryos. This resulted in a decrease in glucose content and a significant increase in ATP content. These findings provide evidence that the acceleration of energy metabolism promotes embryonic development and enhances the motility of hatched silkworms. In addition, these results provide a novel perspective on the relationship between energy metabolism and embryonic development in other insects.

Vitellogenin receptor transports the 30K protein LP1 without cell-penetrating peptide, into the oocytes of the silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30 kDa lipoproteins (30 K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori.

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

Effects of Microbial Transfer during Food-Gut-Feces Circulation on the Health of Bombyx mori.

Change in habitual diet may negatively affect health. The domestic silkworm (Bombyx mori) is an economically important oligophagous insect that feeds on mulberry leaves. The growth, development, and immune-disease resistance of silkworms have declined under artificial dietary conditions. In this study, we used B. mori as a model insect to explore the relationship between changes in diet and balance of intestinal microbes due to its simpler guts compared with those of mammals. We found that artificial diets reduced the intestinal bacterial diversity in silkworms and resulted in a simple intestinal microbial structure. By analyzing the correlations among food, gut, and fecal microbial diversity, we found that an artificial diet was more easily fermented and enriched the lactic acid bacteria in the gut of the silkworms. This diet caused intestinal acidification and microbial imbalance (dysbiosis). When combined with the artificial diet, Enterococcus mundtii, a colonizing opportunistic pathogen, caused dysbiosis and allowed the frequent outbreak of bacterial diseases in the silkworms. This study provides further systematic indicators and technical references for future investigations of the relationship between diet-based environmental changes and intestinal microbial balance. IMPORTANCE The body often appears unwell after habitual dietary changes. The domestic silkworm (Bombyx mori) raised on artificial diets is a good model to explore the relationship between dietary changes and the balance of intestinal microbes. In this study, the food-gut-feces microbial model was established, and some potential key genera that could regulate the balance of intestinal microbiota were screened out. Our findings will provide a reference for future research to further our understanding of healthy silkworm development and may even be useful for similar research on other animals.

Transcriptional Activation of Ecdysone-Responsive Genes Requires H3K27 Acetylation at Enhancers.

The steroid hormone ecdysone regulates insect development via its nuclear receptor (the EcR protein), which functions as a ligand-dependent transcription factor. The EcR regulates target gene expression by binding to ecdysone response elements (EcREs) in their promoter or enhancer regions. Its role in epigenetic regulation and, particularly, in histone acetylation remains to be clarified. Here, we analyzed the dynamics of histone acetylation and demonstrated that the acetylation of histone H3 on lysine 27 (H3K27) at enhancers was required for the transcriptional activation of ecdysone-responsive genes. Western blotting and ChIP-qPCR revealed that ecdysone altered the acetylation of H3K27. For E75B and Hr4, ecdysone-responsive genes, enhancer activity, and transcription required the histone acetyltransferase activity of the CBP. EcR binding was critical in inducing enhancer activity and H3K27 acetylation. The CREB-binding protein (CBP) HAT domain catalyzed H3K27 acetylation and CBP coactivation with EcR, independent of the presence of ecdysone. Increased H3K27 acetylation promoted chromatin accessibility, with the EcR and CBP mediating a local chromatin opening in response to ecdysone. Hence, epigenetic mechanisms, including the modification of acetylation and chromatin accessibility, controlled ecdysone-dependent gene transcription.

CRISPR-Cas-mediated diagnostics.

An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.

SUMOylation of Translationally Regulated Tumor Protein Modulates Its Immune Function.

Translationally controlled tumor protein (TCTP) is a highly conserved protein possessing numerous biological functions and molecular interactions, ranging from cell growth to immune responses. However, the molecular mechanism by which TCTP regulates immune function is largely unknown. Here, we found that knockdown of Bombyx mori translationally controlled tumor protein (BmTCTP) led to the increased susceptibility of silkworm cells to virus infection, whereas overexpression of BmTCTP significantly decreased the virus replication. We further demonstrated that BmTCTP could be modified by SUMOylation molecular BmSMT3 at the lysine 164 via the conjugating enzyme BmUBC9, and the stable SUMOylation of BmTCTP by expressing BmTCTP-BmSMT3 fusion protein exhibited strong antiviral activity, which confirmed that the SUMOylation of BmTCTP would contribute to its immune responses. Further work indicated that BmTCTP is able to physically interact with interleukin enhancer binding factor (ILF), one immune molecular, involved in antivirus, and also induce the expression of BmILF in response to virus infection, which in turn enhanced antiviral activity of BmTCTP. Altogether, our present study has provided a novel insight into defending against virus via BmTCTP SUMOylation signaling pathway and interacting with key immune molecular in silkworm.

Type III CRISPR-based RNA editing for programmable control of SARS-CoV-2 and human coronaviruses.

Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.

Bioengineered silkworm model for expressing human neurotrophin-4 with potential biomedical application.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays important roles in maintaining nerve cell survival, regulating neuronal differentiation and apoptosis, and promoting nerve injury repair. However, the source of sufficient NT-4 protein and efficient delivery of NT-4 remain a challenge. This study aims to express an activated human NT-4 protein in a large scale by genetically engineering silk gland bioreactor of silkworm as a host. We showed that the expression of human NT-4-functionalized silk material could promote proliferation of mouse HT22 cells when compared to the natural silk protein, and no obvious cytotoxicity was observed under the conditions of different silk materials. Importantly, this functional silk material was able to induce the potential differentiation of HT22 cells, promote peripheral neural cell migration and neurite outgrowth of chicken embryo dorsal root ganglion (DRG). All these results demonstrated a high bioactivity of human NT-4 protein produced in silk gland. Therefore, based on the silkworm model, the further fabrication of different silk materials-carrying active NT-4 protein with good mechanical properties and great biocompatibility will give promising applications in tissue engineering and neurons regeneration.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Function of Polyamines in Regulating Cell Cycle Progression of Cultured Silkworm Cells.

BACKGROUND: Putrescine, spermidine, and spermine are polyamines that are ubiquitously distributed in prokaryotic and eukaryotic cells, which play important roles in cell proliferation and differentiation. METHODS: We investigated the expression profiles of polyamine pathway genes by qRT-PCR in different tissues of the lepidopteran silkworm. The polyamine levels in cultured silkworm cells were measured by HPLC. Spermidine and polyamine biosynthetic inhibitors were used for treating the cultured silkworm cells in order to clarify their effects on cell cycle progression. RESULTS: We identified the anabolic and catabolic enzymes that are involved in the polyamine biosynthetic pathway in silkworm. Transcriptional expression showed at least seven genes that were expressed in different silkworm tissues. Treatments of the cultured silkworm cells with spermidine or inhibitor mixtures of DFMO and MGBG induced or inhibited the expression of cell cycle-related genes, respectively, and thus led to changed progression of the cell cycle. CONCLUSIONS: The present study is the first to identify the polyamine pathway genes and to demonstrate the roles of polyamines on cell cycle progression via regulation of the expression of cell cycle genes in silkworm.

Estrogen-Related Receptor Influences the Hemolymph Glucose Content by Regulating Midgut Trehalase Gene Expression in the Last Instar Larvae of Bombyx mori.

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism.

The Response of the Estrogen-Related Receptor to 20-Hydroxyecdysone in Bombyx mori: Insight Into the Function of Estrogen-Related Receptor in Insect 20-Hydroxyecdysone Signaling Pathway.

Estrogen-related receptor (ERR) is an orphan nuclear receptor that was first discovered in animals, and play an important role in metabolism, development, and reproduction. Despite extensive research on the function of ERR, its transcriptional regulation mechanism remains unclear. In this study, we obtained the upstream region of Bombyx mori ERR (BmERR) and confirmed the promoter activity of this region. Interestingly, we found that 10 and 50 nM 20-hydroxyecdysone (20E) up-regulated the transcriptional activity of BmERR promoter. In addition, eight putative ecdysone response elements (EcREs) were predicted in the upstream sequence of BmERR. Based on their positions, the upstream sequence of BmERR was truncated into different fragments. Finally, an EcRE-like sequence (5'-AGTGCAGTAAACTGT-3') was identified. Electrophoretic mobility shift assay (EMSA) and cell transfection experiments confirmed that this motif specifically binds to the complex formed between ecdysone receptor (BmEcR) and the ultraspiracle (BmUSP), a key complex in the 20E signaling pathway. Interference of BmERR or BmEcR mRNA in the embryonic cells of Bombyx mori significantly affected the expression of BmEcR and BmUSP. Overall, these results suggested that an EcRE element was identified from BmERR, and this will help understanding the detailed regulatory mechanism of ERR in insects.