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.

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.

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.

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.

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.

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.

A novel system for separation and purification of egg pigments from the nondiapause red-egg mutant Re-nd in the silkworm, Bombyx mori.

Re-nd, which was induced from the wild-type C108 by the chemical mutagen N-methane-N-methylnitrourea, is a nondiapause red-egg mutant of silkworm Bombyx mori. The special significance of the Re-nd mutant is that it is an independent dominant mutant. The aim of this study was to establish the type of pigment responsible for the red coloration in the Re-nd mutant eggs in silkworm. We compared the eggs of Re-nd mutants with those of the other B. mori egg color strains and confirmed that the Re-nd mutant is the only strain with red color and red pigment granules in nondiapause, showing this mutant belongs to the pigmentation in the serosa. We speculated that the red substance, which contributed to the bright red pigmentation for nondiapause eggs of the Re-nd mutant, could potentially be a novel pigment according to its solubility, optimum absorption peak, and oxidation-reduction reaction. Moreover, we have successfully constituted the system for enrichment, extraction, and purification of the red substance responsible for the Re-nd mutant, providing a new method for the separation and purification of other known and unknown pigments or substances.

Identification and Characterization of Aldehyde Oxidase 5 in the Pheromone Gland of the Silkworm (Lepidoptera: Bombycidae).

Aldehyde oxidases (AOXs) are a subfamily of cytosolic molybdo-flavoenzymes that play critical roles in the detoxification and degradation of chemicals. Active AOXs, such as AOX1 and AOX2, have been identified and functionally analyzed in insect antennae but are rarely reported in other tissues. This is the first study to isolate and characterize the cDNA that encodes aldehyde oxidase 5 (BmAOX5) in the pheromone gland (PG) of the silkworm, Bombyx mori. The size of BmAOX5 cDNA is 3,741 nucleotides and includes an open reading frame, which encodes a protein of 1,246 amino acid residues. The theoretical molecular weight and isoelectric point of BmAOX5 are approximately 138 kDa and 5.58, respectively. BmAOX5 shares a similar primary structure with BmAOX1 and BmAOX2, containing two [2Fe-2S] redox centers, a FAD-binding domain, and a molybdenum cofactor (MoCo)-binding domain. RT-PCR revealed BmAOX5 to be particularly highly expressed in the PG (including ovipositor) of the female silkworm moth, and the expression was further confirmed by in situ hybridization, AOX activity staining, and anti-BmAOX5 western blotting. Further, BmAOX5 was shown to metabolize aromatic aldehydes, such as benzaldehyde, salicylaldehyde, and vanillic aldehyde, and fatty aldehydes, such as heptaldehyde and propionaldehyde. The maximum reaction rate (Vmax) of benzaldehyde as substrate was 21 mU and Km was 1.745 mmol/liter. These results suggested that BmAOX5 in the PG could metabolize aldehydes in the cytoplasm for detoxification or participate in the degradation of aldehyde pheromone substances and odorant compounds to identify mating partners and locate suitable spawning sites.

Effects of Certain cis-Regulatory Elements on Stage-Specific vitellogenin Expression in the Bombyx mori (Lepidoptera: Bombycidae).

Bombyx mori vitellogenin (BmVg) is highly upregulated during pupation, and the 20-hydroxyecdysone and amino acids may regulate stage-specific BmVg expression. However, previous studies showed that other factors may also affect stage-specific BmVg expression. Here, we characterized effective BmVg transcription factors by identifying the corresponding cis-regulatory elements (CREs). We prepared transgenic B. mori, in which DsRed was driven by various lengths of BmVg promoter. qRT-PCR analysis showed that DsRed expression driven by a 1.0-kb BmVg promoter (VgP1.0K) was consistent with endogenous BmVg. VgP1.0K specificity was closer to the endogenous BmVg promoter than that of VgP0.8K. These results suggest that CREs affecting stage-specific BmVg expression were localized to the 1.0-kb BmVg promoter. We investigated the effects of certain CREs that could influence the stage specificity of BmVg promoter on BmVg expression in transgenic B. mori. The relative DsRed expression was significantly reduced in transgenic female B. mori and the peak in DsRed expression was delayed after E-box CRE mutation. These results demonstrate that the E-box element enhanced BmVg expression and also affected stage-specific BmVg expression. Moreover, the relative DsRed expression was significantly increased in transgenic female of B. mori after 3×BD CRE mutation in BmVg promoter. However, the stage specificity of the mutated promoter was consistent with that of the endogenous BmVg promoter. The 3×BD element downregulated BmVg but had no effect on stage-specific BmVg expression. The present study promoted the process of elucidating the regulatory network for stage-specific BmVg expression and furnished a theoretical basis for the application of BmVg promoter.

Heat Shock Cognate 70 Functions as A Chaperone for the Stability of Kinetochore Protein CENP-N in Holocentric Insect Silkworms.

The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin-proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle.

Quality Formation Mechanism of Stiff Silkworm, Bombyx batryticatus Using UPLC-Q-TOF-MS-Based Metabolomics.

Bombyx batryticatus is a well-known animal in traditional Chinese medicine. The aim of the research was to reveal the quality formation mechanism of B. batryticatus and to screen out the characteristic component used for the quality control. The anticonvulsant effects of B. batryticatus with a stiff time of one, five, and nine days (D1, D5 and D9, respectively) and healthy silkworm of the same developmental stage (SW) were determined by animal experiment. The dynamic changes in chemical composition were analyzed using UPLC-Q-TOF-MS-based metabolomics. D5 and D9 B. batryticatus exhibited significant anticonvulsant effects (p < 0.05 and p < 0.01, respectively). Accordingly, principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA) indicated that the chemical composition of D5 and D9 B. batryticatus changed significantly. The different metabolites mainly consisted of primary metabolites such as lipids and amino acids and secondary metabolites such as flavonoids, beauvericin, and glycolipids. Interestingly, the relative abundance of quercetin-7-O-ß-d-4-O-methylglucoside, the characteristic component of B. batryticatus, increased with stiff time and was promised to be used as an index component of quality control. The results expand our understanding of the quality formation mechanism of B. batryticatus. In addition, it highlights the potential of UPLC-Q-TOF-MS-based metabolomics for the quality control purpose of TCMs.

Vitellogenin receptor selectively endocytoses female-specific and highly-expressed hemolymph proteins in the silkworm, Bombyx mori.

VgR, a member of the LDLR family, functions to transport vitellogenin into the ovaries to protome ovarian growth and embryonic development. In insects, the only widely accepted ligand of VgR is Vg. Recently, BmVgR has been shown to interact with BmSP1 in vitro. Therefore, in this study, we evaluated whether BmVgR could transport BmSP1 into certain cells. Although BmVgR could combine with BmVg and BmSP1, BmVgR did not affect the amount of BmSP1 taken up by Sf9 cells. Parallel immunofluorescence showed that most BmVg and BmVgR were localized in the inner oocyte membrane, showing tissue localization similar to that of BmVg labeled with pHrodo Red absorbed by the ovaries on day 2 of pupation. Although BmSP1 showed localization similar to BmVgR during the same phase, little BmSP1 was present in the ovary. Additionally, BmSP1 did not exist in ovaries when the ovaries contained BmVgR on day 5 of pupation, suggesting that BmSP1 in the ovaries was not endocytosed by BmVgR. In summary, BmVgR could facilitate uptake of BmVg by developing oocytes, but did not modulate in the transport of BmSP1.

The Broad Complex isoform 2 (BrC-Z2) transcriptional factor plays a critical role in vitellogenin transcription in the silkworm Bombyx mori.

BACKGROUND: Vitellogenin (Vg) is synthesized in the fat body of the female silkworm Bombyx mori and transported to the oocyte as a source of nutrition for embryo development. It is well known that ecdysone regulates physiological, developmental and behavioral events in silkworm. However, it is still not clear how the ecdysone regulates B. mori Vg (BmVg) transcription. METHODS: Electrophoretic mobility shift assay (EMSA) and cell transfection assay were used to reveal whether BmBrC-Z2 is involved in regulating BmVg transcription. RNAi was employed to illustrate the function of BmBrC-Z2 in the silkworm egg formation and development. RESULTS: (1) The transcription of BmVg can be induced by ecdysone in the female fat body. (2) Three putative BrC-Z2 cis-response elements were mapped to regions flanking the BmVg gene. (3) BmBrC-Z2 required direct binding to the cis-response elements on the BmVg promoter. (4) Over-expression of three BmBrC isoforms in the cell line showed that only BmBrC-Z2 could induce the BmVg promoter activity. (5) RNA interference (RNAi) of BmBrC-Z2 in female remarkably reduced BmVg synthesis and led to destructive affection on egg formation. The dsRNA of BmBrC-Z2 treated moths laid fewer and whiter eggs compared to the control. CONCLUSIONS: BmBrC-Z2 transported the ecdysone signal then regulated BmVg transcription directly to control vitellogenesis and egg formation in the silkworm. GENERAL SIGNIFICANCE: The results of this study revealed that BmBrC-Z2 as a key factor to mediate ecdysone regulates reproduction in the silkworm.

Vertebrate estrogen regulates the development of female characteristics in silkworm, Bombyx mori.

The vertebrate estrogens include 17-ß-estradiol (E2), which has an analog in silkworm ovaries. In this study, the Bombyx mori vitellogenin gene (BmVg) was used as a biomarker to analyze the function of the E2 in silkworm. In most oviparous animals, Vg has female-specific expression. However, BmVg expression was also detected in B. mori males. Stage specific fluctuation of BmVg expression was similar in males and females, but expression levels in males were lower than in females. E2 treatment by injection or feeding of male larvae in the final instar stage induced and stimulated male BmVg transcription and protein synthesis. When silkworm ovary primordia were transplanted into males, BmVg was induced in male fat bodies. Transplanted ovaries primordia were also able to develop into ovaries and produce mature eggs. When females were treated with E2 promoted BmVg/BmVn protein accumulation in hemolymph, ovaries and eggs. However, BmVg transcription was decreased in female fat bodies. An E2 analog was identified in the hemolymph of day 3 wandering silkworms using high-performance liquid chromatography. Estradiol titers from fifth late-instar larvae to pupal stage were determined by enzyme-linked immunosorbent assay. The results suggested that silkworms synthesized a vertebrate E2 analog. This study found that E2 promoted the synthesis of BmVg, a female typical protein in silkworms.

Female qualities in males: vitellogenin synthesis induced by ovary transplants into the male silkworm, Bombyx mori.

Female qualities in males are common in vertebrates but have not been extensively reported in insects. Vitellogenin (Vg) is highly expressed in the female fat body and is generally required for the formation of yolk proteins in the insect egg. Vg upregulation is generally regarded as a female quality in female oviparous animals. In this study, we found that Bombyx mori Vg (BmVg) is especially highly expressed in the female pupa. Downregulation of the BmVg gene in the female pupa by RNA interference (RNAi) interfered with egg formation and embryonic development, showing the importance of BmVg in these processes. So, we used BmVg as a biomarker for female qualities in the silkworm. Hematoxylin-eosin staining and immunofluorescence histochemistry showed that ovary transplants induced BmVg synthesis in the male pupa fat body. Ovaries transplanted into male silkworms produced only a few eggs with deformed yolk granules. These results suggested that the amount of BmVg in the male silkworm was insufficient for eggs to undergo complete embryonic development. After 17-beta-estradiol was used to treat male pupae and male pupal fat bodies, BmVg was upregulated in vivo and in vitro. These findings indicated that the male silkworm has innate female qualities that were induced by a transplanted ovary and 17ß-estradiol. However, in silkworms, female qualities in males are not as complete as in females.

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.

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.

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.

Transgenic silkworm expressing bioactive human ciliary neurotrophic factor for biomedical application.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in neuronal survival and regeneration, and can also induce the differentiation of several stem cells into neurons, which highlights the broad application of CNTF in biomedicine. However, large-scale production of bioactive recombinant human CNTF protein remains to be explored. Herein, this study aims to express a bioactive human CNTF protein on a large scale by genetically engineering a silk gland bioreactor of silkworm. Our results showed that CNTF protein was successfully expressed in the middle silk gland (MSG) of silkworm, which can be secreted into the silks with the amount of 3.2 mg/g cocoons. The fabrication of human CNTF-functionalized silk material was able to promote proliferation and migration of neural cells when compared to the natural silk protein. Importantly, this functional silk material could also facilitate neurite outgrowth of mouse retinal ganglion cell (RGC-5) cells. All these data demonstrated a high bioactivity of the recombinant human CNTF protein expressed in the MSG of silkworm. The further fabrication of different silk materials with CNTF bioactivity will give biomedical applications in tissue engineering and neuroregeneration.

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.