Bing Li

The silkworm (Bombyx mori) is a key economic insect in sericulture, and silk production relies on nutrition and posterior silk gland (PSG) development. Nutritional deficiency impairs silk protein synthesis and secretion in the silkworm PSG, potentially affecting silk yield and quality. In this study, we investigated the cellular and molecular changes associated with starvation-induced injury in the PSG. Starvation impaired PSG development and altered NADPH levels, accompanied by increased dihydroethidium (DHE) fluorescence, elevated glutathione S-transferase (GST) activity, increased superoxide dismutase (SOD) activity at 24 and 48 h, and increased BmCAT transcript abundance. Transmission electron microscopy (TEM) revealed that short-term starvation was associated with the accumulation of autophagic structures, including double-membrane autophagosomes and autolysosomes. By contrast, prolonged starvation resulted in severe ultrastructural damage, characterized by nuclear envelope disruption and chromatin condensation. Furthermore, reverse transcription quantitative PCR (RT-qPCR) and Western blotting revealed increased expression of autophagy-related markers and apoptosis-associated changes, including cleaved caspase-3 accumulation after 72 h of starvation. Notably, prolonged starvation was accompanied by increased intracellular Ca²⁺ levels and elevated NtATG5 abundance, suggesting that altered Ca²⁺ homeostasis may be associated with the apoptosis-associated responses observed in the PSG. Together, these findings identify coordinated changes in redox status, Ca²⁺ homeostasis, and autophagy- and apoptosis-associated markers during starvation-induced PSG injury, providing a basis for further mechanistic studies and evaluation of food deprivation in sericulture.

The silkworm ( Bombyx mori ), a classical lepidopteran model, is evolving into a highly tractable synthetic biology chassis. In modern sericulture, genetic sexing circuits provide a rational design framework for efficient all‐male rearing, effectively overcoming the gene dosage disruptions and fitness costs associated with traditional radiation‐induced chromosomal translocations. This review systematically summarizes the construction strategies and industrial applications of these circuits. We outline the optimization of sex determination elements and highlight the advantages of far‐red optical markers in penetrating host physical barriers and reducing cytotoxicity. Furthermore, we trace the evolution of genomic manipulation tools from random transposon insertions to targeted CRISPR/Cas9 integration into genomic safe harbors (GSHs). To address circuit instability during multigenerational rearing, protective molecular strategies—including boundary insulators, conditional expression, and automatic excision networks—are evaluated. We also explore the industrial interface that integrates standardized biological optical reporters with artificial intelligence and high‐throughput optoelectronic sorting hardware. Ultimately, engineering principles derived from the silkworm chassis can provide a molecular framework for the sterile insect technique in global pest management, with future directions pointing toward footprint‐free genome editing and dynamic conditional regulation.

The widespread application of cyantraniliprole (Cya) and λ-cyhalothrin (λ-cyh) in agriculture has led to increasing environmental residues, posing potential risks to non-target beneficial insects. However, their reproductive toxicity at sublethal concentrations remains largely unexplored. In this study, the silkworm (Bombyx mori), a representative lepidopteran model organism and economically important insect, was exposed to sublethal concentrations of Cya (0.16 mg/L) and λ-cyh (0.21 mg/L) for 24 h during the 5th instar larval stage to evaluate the effects of the two insecticides on ovarian development and fecundity. Sublethal exposure to either compound significantly impaired larval growth and gonadal development, and reduced ovariole development and fecundity, with Cya exhibiting stronger effects. Crossing experiments further revealed that the reproductive impairment predominantly originated from direct toxicity to females, uncovering a sex-specific susceptibility. Histopathological and ultrastructural analyses showed that both insecticides induced ovarian structural damage, and disrupted mitochondrial and endoplasmic reticulum integrity. Transcriptomic and molecular analyses demonstrated that Cya and λ-cyh suppressed ribosome-related pathways, interfered with mitochondrial respiratory chain function, induced excessive reactive oxygen species (ROS) accumulation and antioxidant defense imbalance, and consequently activated both caspase-dependent and -independent apoptotic pathways. Overall, these findings indicate that sublethal Cya and λ-cyh exposure impairs silkworm reproductive fitness through mitochondrial dysfunction-driven oxidative injury and apoptosis, with preferential targeting of the female reproductive system. This study extends the risk assessment of diamide and pyrethroid insecticides from acute toxicity to sublethal reproductive toxicity, providing a scientific basis for ecological risk assessment and pesticide management in sericulture regions.

Immediate-early protein IE1 is an essential protein of baculoviruses that is involved in transcription and replication. In the present study, we provide several lines of evidence for how ancient IE1 evolved into its current version. Using progressive truncations and site-directed mutations coupled with fluorescence microscopy, surprisingly, we showed that the previously identified nuclear localization sequence (NLS), basic domain II, was essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and demonstrated that, BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. The non-canonical NLS alone was sufficient to launch infection. We also found that N-terminally truncated IE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, residues 58 to 151 of BmIE1 were shown to be dispensable, and residue 152 was found to be critical for launching a productive infection. To gain insight into how ancient IE1 acquired its multi-functionality, we reorganized the N-terminal 23 aa and 132 aa of BmIE1 with EGFP in a variety of manners and found that both fragments are separable and transferable. Notably, we observed that the nuclear levels of BmIE1 should reach certain thresholds to initiate infection. Additionally, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes. Collectively, these findings suggest a hypothesis where ancient IE1 might have evolved the two additional NLSs and acquired the N-terminal 132 aa through gene fusion so as to reach infection-initiating thresholds at a faster pace. Author Summary: Basic domain II was previously shown to be the NLS of AcMNPV IE1 protein. In the present study, we demonstrated that it is essential for sequence non-specific DNA binding, but not required for IE1 nuclear import, and found that BmNPV IE1 (BmIE1) possesses not only a unique bipartite NLS that contains a monopartite NLS but also a cryptic non-canonical NLS that is correlated with sequence non-specific DNA binding. We also found that N-terminally truncated BmIE1 can enter the nucleus through its sequence non-specific binding ability. Remarkably, the monopartite NLS, when fused to the N-terminus of EGFP, can form a novel NLS that is also functional in a mammalian cell line. Moreover, the domains of BmIE1 was shown to be separable and transferable. We also demonstrated that higher expression of functionally impaired BmIE1 achieved by higher MOIs or higher transfection efficiency can partially complement its compromised functions. Consistently, we found that BmNPV could launch infection more efficiently in a BmN cell line over another BmN cell line by increasing transcription levels of immediate early genes.

Bacillus thuringiensis (Bt) is widely used for the control of agricultural insect pests; however, trace residues of Bt may induce sublethal effects in insects. In this study, Bombyx mori was used as a model insect to investigate the mechanisms underlying midgut injury and tissue apoptosis induced by continuous exposure to a sublethal concentration of Bt. The results showed that prolonged exposure to a sublethal concentration of Bt progressively aggravated structural damage to midgut epithelial cells, accompanied by chromatin condensation. Moreover, the apoptotic rate of midgut cells was significantly higher in Bt-treated silkworms than in the control group (P < 0.05). Transcriptomic analysis indicated that the mitochondria-mediated intrinsic apoptotic pathway was activated earlier at 48 h, whereas activation of the death receptor-mediated extrinsic apoptotic pathway was delayed until 72 h. Consistent with these transcriptional changes, The protein expression level of Caspase-9 showed a sustained increase from 48 h to 96 h, while that of Caspase-8 began to increase at 72 h. Injection of the pan-caspase inhibitor Z-VAD-FMK into Bt-treated silkworms significantly reduced Caspase-3 protein expression and enzymatic activity, and markedly alleviated Bt-induced abnormal developmental phenotypes. These findings provide a basis for evaluating the biosafety risks associated with sublethal environmental residues.

Chlorantraniliprole (CAP), a novel anthranilic diamide insecticide, has been extensively used in agricultural pest management. Given their environmental persistence, low‐concentration CAP residues warrant a systematic assessment of their safety risks. The silkworm ( Bombyx mori ) is the lepidopteran model insect. This study was conducted to characterize the changes in Ca ²⁺ levels and the expression of the molting‐related gene Ftz‐f1 in the midgut of fourth‐instar B. mori after exposure to low‐concentration CAP at different time intervals (CAP 0–24 h, CAP 24–48 h, and CAP 48–72 h). Low‐concentration CAP exposure significantly reduced the molting rate of fourth‐instar silkworms in the CAP‐exposed groups to 97%, 92%, and 80% of the control group, respectively ( p < 0.001), and the percentage proportion of newly molted fifth instar in the CAP‐treated groups to 92%, 82%, and 67% of the control group, respectively ( p < 0.001). CAP exposure disrupts intracellular Ca ²⁺ homeostasis and induces dysregulation in the transcription of Ca ²⁺ ‐regulating genes, consequently suppressing both transcription and protein expression of the molting target gene Ftz‐f1 , which ultimately leads to molting defects in silkworms. The present study reveals the molecular mechanism of the impaired molting of fourth‐instar molting defects caused by low‐concentration CAP exposure, which provides critical evidence for the safety evaluation of low‐concentration CAP residues and the sublethal toxicity to the lepidopteran insect.

Silk glands are crucial for silk protein synthesis and secretion. However, the degeneration process takes place at pupation, as mediated by programmed cell death (PCD). The differences of PCD in the anterior silk gland (ASG) and middle silk gland (MSG) with different physiological functions remain unexplored. In this study, we investigated the occurrence of autophagy and apoptosis in the MSG and the ASG of silkworms from the mature silkworm to the 1st d of pupation (P1) based on Ca²⁺ homeostasis. The results showed high autophagy but low apoptosis at the early stage. After Ca²⁺ levels peak, calpain‐mediated autophagy‐related protein 5 (ATG5) cleavage drove the autophagy‐to‐apoptosis conversion. The ASG exhibited significant apoptosis at the P1 stage (P < 0.001), showing a 24 h delay compared to MSG. The peak of Ca²⁺ levels coincided with the onset of significant apoptosis. Further investigation revealed that delayed inositol‐1,4,5‐trisphosphate receptor (IP3R) protein expression in ASG, resulting in delayed Ca²⁺ release from the endoplasmic reticulum and lagging ATG5 cleavage, is responsible for the delayed onset of apoptosis in the ASG. The rescue of 20‐hydroxyecdysone‐induced apoptosis by IP3R inhibitors further confirmed this mechanism. The results indicate that delayed expression of IP3R in the ASG regulates Ca²⁺ release from the endoplasmic reticulum and calpain‐mediated ATG5 cleavage, leading to asynchronous PCD in different parts of the silk gland. This finding not only reveals the differences in the regulation of PCD between the secretory region (MSG) and non‐secretory region (ASG) but also provides new insights into the tissue‐specific degradation mechanisms in insect metamorphosis.

Baculovirus bacmids have been widely used in over-expression and gene deletion. Traditionally, baculovirus bacmids are developed by inserting an 8.6 kbp bacterial DNA cassette into baculovirus genomes either through homologous recombination in cultured cells or via in vitro cloning. In this study, by introducing Bsu36i-attached egfp to the 8.6 kbp bacterial DNA cassette, we develop a novel method for generating baculovirus bacmids. An 11.6 kbp bacterial DNA cassette containing the introduced egfp was used to generate an intermediate bacmid. With the EGFP reporter, purification was performed in cultured cells, increasing the proportions of recombinants. The intermediate bacmid containing the 11.6 kbp bacterial DNA cassette was obtained by transforming DH10B competent cells with viral DNA after 3 rounds of purification. The intermediate bacmid DNA was linearized by digestion with Bsu36i and then was co-transfected with the PCR-amplified 8.6 kbp bacterial cassette into BmN cells, where homologous recombination occurred between them. The final BmNPV bacmid was obtained by transforming DH10B competent cells with viral DNA. Capable of increasing the proportions of recombinants via purification and linearization, this method has great potential to be used for bacmid generation for baculoviruses, especially those that are not capable of producing high titers of viruses.

Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) plays a critical role in maintaining intracellular Ca²⁺ homeostasis by transporting excess Ca²⁺ into the endoplasmic reticulum (ER) calcium reservoir. Calcium is essential for regulating key biological processes in Bombyx mori, including development, metamorphosis, and reproduction. To explore the role of BmSERCA in the proliferation of the B. mori ovary cell line (BmN), intracellular Ca²⁺ levels were measured following BmSERCA interference. At 48 h post‐interference, intracellular Ca²⁺ levels increased significantly by 1.38 times. Moreover, transcript levels of genes involved in cell proliferation and division exhibited notable changes. Phenotypic assessments further demonstrated that BmSERCA interference significantly accelerated the proliferation and migration of BmN cells, with a marked increase in EdU‐positive cells. Additionally, flow cytometry analysis revealed that BmSERCA interference notably affected the cell cycle by inducing G2/M phase accumulation and elevating the expression of G2/M phase regulators, including BmCyclinB and BmCDK1. Collectively, these findings suggested that BmSERCA interference disrupted intracellular Ca²⁺ homeostasis, thereby promoting BmN cell proliferation. This study provided a valuable foundation for understanding the role of BmSERCA in BmN cell proliferation.

Cyantraniliprole (Cya), a diamide insecticide, is widely utilised for the management of Lepidoptera pests owing to its potent insecticidal efficacy and broad spectrum of activity. The extensive use and prolonged environmental persistence of this insecticide pose a significant threat to the sustainable development of sericulture. This study firstly assessed the lethal toxicity of cyantraniliprole to the 5th instar larvae of Bombyx mori . Exposure to cyantraniliprole (LC ₅ , LC ₁₀ and LC ₂₀ ) resulted in a concentration‐dependent reduction in larval weight, pupal weight and survival rate and a prolongation of larval development time. Moreover, cyantraniliprole LC ₁₀ resulted in substantial structural damage to the epithelial cells, suppressed the mRNA levels of oxidative phosphorylation genes, perturbed ATP synthesis and led to an imbalance of intracellular reactive oxygen species. Meanwhile, the starvation treatment suggested that the impacts of cyantraniliprole on silkworms cannot be solely ascribed to nutritional deficiencies. Additionally, the results revealed that cytochrome P450s might serve as a pivotal factor in the detoxification metabolism of cyantraniliprole in the midgut of silkworms. The findings of this study offer evidence for the ecological risk posed by environmental residues of cyantraniliprole to non‐target organisms and are also of great significance for sericulture production.