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Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs inBombyxand that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier genebroad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level ofbroad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentifiedbroad-inducing factor, i.e., a competence factor.

Bombyx mori , the domesticated silkworm, is of great importance as a silk producer and as a powerful experimental model for the basic and applied research. Similar to other animals, abundant microorganisms live inside the silkworm gut; however, surprisingly, the microbiota of this model insect has not been well characterized to date. Here, we comprehensively characterized the gut microbiota of the domesticated silkworm and its wild relatives. Comparative analyses with the mulberry-feeding moths Acronicta major and Diaphania pyloalis revealed a highly diverse but distinctive silkworm gut microbiota despite thousands of years of domestication, and stage-specific signatures in both total (DNA-based) and active (RNA-based) bacterial populations, dominated by the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Most fungal sequences were assigned to the phyla Ascomycota and Basidiomycota. Environmental factors, including diet and human manipulation (egg production), likely influence the silkworm gut composition. Despite a lack of spatial variation along the gut, microbial community shifts were apparent between early instars and late instars, in concert with host developmental changes. Our results demonstrate that the gut microbiota of silkworms assembles into increasingly identical community throughout development, which differs greatly from those of other mulberry-feeding lepidopterans from the same niche, highlighting host-specific effects on microbial associations and the potential roles these communities play in host biology.

Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval–pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93. Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.

Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules. Silk fibres currently used in biotechnology are chemically reconstituted silk fibroins (RSF), which are more stable than native silk fibroin (NSF) but possess different biophysical properties. Here, the authors use microfluidic droplets to encapsulate and store NSF, preserving their native structure.

Herein, we report the synthesis of silver nanoparticles (AgNPs) by a green route using the aqueous leaf extract of Morus indica L. V1. The synthesized AgNPs exhibited maximum UV-Vis absorbance at 460 nm due to surface plasmon resonance. The average diameter (~54 nm) of AgNPs was measured from HR-TEM analysis. EDX spectra also supported the formation of AgNPs, and negative zeta potential value (−14 mV) suggested its stability. Moreover, a shift in the carbonyl stretching (from 1639 cm −1 to 1630 cm −1 ) was noted in the FT-IR spectra of leaf extract after AgNPs synthesis which confirm the role of natural products present in leaves for the conversion of silver ions to AgNPs. The four bright circular rings (111), (200), (220) and (311) observed in the selected area electron diffraction pattern are the characteristic reflections of face centered cubic crystalline silver. LC-MS/MS study revealed the presence of phytochemicals in the leaf extract which is responsible for the reduction of silver ions. MTT assay was performed to investigate the cytotoxicity of AgNPs against two human cell lines, namely HepG2 and WRL-68. The antibacterial study revealed that MIC value of the synthesized AgNPs was 80 µg/ml against Escherichia coli K12 and Staphylococcus aureus (MTCC 96). Finally, the synthesized AgNPs at 10 µg/ml dosages showed beneficial effects on the survivability, body weights of the Bombyx mori L. larvae, pupae, cocoons and shells weights via enhancing the feed efficacy.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

The domesticated silkworm, Bombyx mori , is crucial for global silk production, which is a significant economic activity supporting millions of livelihoods worldwide. Beyond traditional silk production, the growing demand for insect larvae in cosmetics, biomedical products, and animal feed underscores the need to enhance B . mori productivity. This study investigates the role of the circadian clock gene Clock in B . mori using CRISPR/Cas9-mediated mutagenesis to establish the Clk Δ29 knock-out mutant strain. Dysregulation of the circadian clock in Clk Δ29 was demonstrated by altered temporal transcriptional profiles of core circadian clock genes in adult heads and disrupted circadian-controlled behaviors, including adult eclosion and egg hatching rhythms under constant darkness. By analysing larval development timing, as well as the weights of late instar larvae, pupae, and cocoon components in Clk Δ29 mutants and in Clk Δ1922 silkworms (carrying an independently generated Clk - null allele), we showed that CLK contributes to physiological processes regulating B . mori development and growth. Importantly, Clk Δ29 mutants reared on a standard sericulture diet exhibited significant increases in key economic traits, with silk production increasing by up to 7%, and pupal weight increasing by up to 25% compared to wild-type controls. This study highlights the potential of circadian clock gene manipulation to significantly enhance sericultural productivity. Future research should focus on elucidating the molecular mechanisms driving these phenotypes and determining whether they result from circadian clock functions or pleiotropic effects of B . mori Clk . These findings provide a foundation for advancing sustainable sericulture and developing new commercial applications for silkworm-derived products.

Background Florfenicol (FF) is widely used in animal husbandry due to its broad-spectrum bactericidal activity, while there is little research focused on its toxic effects on the silkworm( Bombyx mori L.), a model organism. Result It was demonstrated in this paper that high-concentration florfenicol exposure significantly inhibited the activities of digestive and antioxidant enzymes, reduced the body weight and cocooning ability of silkworms, prolonged the instar duration, and simultaneously led to organelle swelling and vacuolization in the midgut, a large number of autophagosomes, and nuclear condensation. Meanwhile, it was found that exposure to FF reduced the α diversity and richness of the midgut flora, leading to a severe loss of core microbiota. The results of this study also indicated that Bacillus velezensis LY5, as a functional bacterium, effectively mitigated the toxic effects of FF on silkworm larvae. This strain significantly improved the body weight and cocoon quality of silkworms exposed to FF, promoted the repair of midgut tissue damage, and restored the structure of the midgut core flora. Furthermore, B. velezensis LY5 treatment increased the relative abundance of potentially beneficial bacteria while suppressing the abnormal proliferation of opportunistic pathogens. Conclusion This study reveals the toxicological mechanism by which high concentrations of antibiotics (FF) disrupt the midgut microbiota balance in silkworms, induce tissue damage, and subsequently impede their growth and development. It also demonstrates for the first time that functional probiotics Bacillus velezensis LY5 effectively reverse this damage by restoring the silkworm midgut microbiota and intestinal tissue.

As a representative species of Lepidoptera, the silkworm ( Bombyx mori ) serves as both a model organism and an insect of economic importance. The abundant genetic mutation resources of silkworms provide a critical foundation for related studies. Globally, exposure to various pesticides often leads to failure in cocooning or direct mortality in silkworms. However, systematic investigations into the effects of novaluron exposure on silkworm growth, development, cocooning, and gene expressions are still lacking. This study revealed that novaluron exposure induced significant signs of intoxication in silkworms, accompanied by reductions in body weight and cocoon quality. Furthermore, novaluron exposure caused histopathological damage to the silk gland, reduced the cocooning rate, and significantly downregulated the expression of genes related to silk protein synthesis. Concurrent alterations were observed in the transcriptional levels of genes associated with the PI3K/Akt and CncC/Keap1 signaling pathways. In addition, novaluron exposure upregulated the relative expression levels of detoxification enzyme genes and enhanced corresponding enzymatic activities. These findings provided further insights into the response mechanisms of silkworms to novaluron exposure.

Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.