The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2%. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

Single-domain von Willebrand factor type C proteins (SVWCs), primarily found in arthropods, responds to infections caused by various pathogens. Three SVWCs have been identified in the silkworm and BmSVWC2 might play a crucial role in the immune system. However, the regulatory mechanism of BmSVWC2 remains largely unknown. This study aimed to investigate the biochemical functions of BmSVWC2 in the immune system of B. mori comprehensively. Phylogenetic analysis revealed that BmSVWC1, BmSVWC3, and BmSVWC2 were distributed in diverse groups, suggesting distinct biochemical functions. The mRNA and protein levels of BmSVWC2 increased significantly in response to bacterial infection. BmSVWC2 exhibited clear binding activity to the polysaccharide pathogen-associated molecular patterns of bacteria and fungi, enhancing bacterial clearance in vivo but not in vitro. RNA-sequencing assays of the fat body and hemocytes showed that numerous immune genes were markedly up-regulated with higher level of BmSVWC2, primarily affecting recognition, signaling, and response production of the Toll and immune deficiency (IMD) signaling pathways. This led to the production of various antimicrobial peptides and significant antibacterial activities in the hemolymph. BmSVWC2 up-regulated phagocytosis-related genes in the fat body and hemocytes, and phagocytosis assays confirmed that BmSVWC2 improved the phagocytic ability of hemocytes against bacteria. Additionally, BmSVWC2 induced the expression of nitric oxide synthetase (NOS) in the fat body, and bioassays confirmed that BmSVWC2 increased NOS activity in the fat body and hemolymph, resulting in nitric oxide accumulation. However, BmSVWC2 did not affect phenoloxidase activity, despite it caused differential expression of a few serine proteases and serine protease inhibitors. Co-immunoprecipitation and mass spectrometry assays showed that BmSVWC2 interacted with 30 K proteins, such as 30 K protein 2, 30 K pBmHPC-19, 30 K 19G1-like, 30 K protein 8, 30 K protein 7, 30 K pBmHPC-23, and low molecular mass lipoprotein 4-like. Our study provides a comprehensive characterization of BmSVWC2 and elucidates the mechanism underlying its regulation of immune responses activation.

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.

Neem, a biopesticide, offers a safer alternative to the synthetic insecticides commonly used in mulberry cultivation, which can harm silkworms. This study aimed to investigate the effects of Thai neem seed extract on all instar larvae of the Thai polyvoltine hybrid silkworm, Bombyx mori L., Dok Bua strains, focusing on the mortality rate and the activities of esterase (EST) and glutathione S-transferases (GST) enzymes. Acute toxicity was assessed using the leaf-dipping method. Results showed that the mortality rate tended to be higher in younger instars than in older ones. The first instar larvae exhibited the highest mortality rate at 94%, whereas the LC50 was highest in the third instar at 5.23 mg L-1 at 72 h. This trend aligns with the activities of EST and GST, which were evaluated in the whole bodies of the first instar larvae and the midgut tissue of fifth instar larvae. As the extract concentration increased, EST activity decreased while GST activity increased in both the first and fifth instar larvae. These findings highlight that neem extract is toxic to all instar larvae, with GST playing a crucial role in detoxification, particularly in the whole body of the Thai polyvoltine hybrid silkworm.

The increasing prevalence of diabetic reproductive complications has prompted the development of innovative animal models. The use of the silkworm Bombyx mori as a model for diabetic reproductive damage shows potential as a valuable research tool. This study employed silkworms as a novel model to investigate diabetic reproductive damage. The silkworms were fed a high-glucose diet containing 10% glucose to induce a diabetic model. Subsequently, the study concentrated on assessing the influence of diabetes on the reproductive system of male silkworms. The results indicate that diabetes resulted in reduced luteinizing hormone (LH) and testosterone (T) levels, as well as elevated triglyceride (TG) levels in male silkworms. Moreover, diabetes mellitus was associated with pathological testicular damage in male silkworms, accompanied by decreased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels, along with increased malondialdehyde (MDA) levels in the testis. Additionally, diabetes mellitus reduced the expression of siwi1 and siwi2 genes in the testis of male silkworms. Overall, these results support using silkworms as a valuable model for studying diabetic reproductive damage.

Non-tuberculous mycobacteria (NTM) cause skin infections, respiratory diseases, and disseminated infections. Mycobacterium avium and Mycobacterium intracellulare, which are slow grown Mycobacterium, are main agents of those NTM diseases. A silkworm infection model with Mycobacterium abscessus, a rapidly growing Mycobacterium species, was established to quantitatively evaluate its virulence within a short period. However, a silkworm infection model to quantitatively evaluate the virulence of M. intracellulare has not yet been developed. In this study, we determined the virulence of M. intracellulare subspecies within 4 days using a silkworm infection model. The subspecies of M. intracellulare strains used in this study were estimated by phylogenetic tree analysis using core gene data. The median lethal dose (LD50) values, which are the dose of a pathogen required to kill half of the silkworms in a group, were determined 4 days after infection. The LD50 value of M. intracellulare subsp. chimaera DSM44623 was higher than that of M. intracellulare subsp. intracellulare ATCC13950. These results suggest that the virulence of M. intracellulare subspecies can be compared using a silkworm model within 4 days.

High temperature stress has long-term negative effects on the growth and development of silkworm (Bombyx mori). Different silkworm varieties show the different tolerance to high temperature. The induction of autophagy is linked to increased thermotolerance in diverse ectothermic organisms. However, the function of autophagy in the thermotolerant and thermosensitive silkworm strains under high-temperature conditions remains unclear. The thermotolerant Liangguang NO.2 and thermosensitive Jingsong × Haoyue strains were used to explore the role of autophagy in thermotolerance. Here, we first found that the larval body weight gain was increased in the thermosensitive Jingsong × Haoyue strain, but there was no difference in the thermotolerant Liangguang NO.2 strain under high temperature conditions. High temperature stress had a negative influence on the cocoon performance in both the Liangguang NO.2 and Jingsong × Haoyue strains. Additionally, the autophagy-related gene Atg5 mRNA expression in the Liangguang NO.2 strain was upregulated by high temperature, while the expression of Atg12 mRNA was reduced in the Jingsong × Haoyue strain. Titers of 20-Hydroxyecdysone and the ultraspiracle 1 mRNA expression in the Liangguang NO.2 strain were upregulated by high temperature, which might be associated with the induction of autophagy. These results demonstrate the potentially regulatory mechanism of autophagy in silkworms\' tolerance to high temperature, providing a theoretical basis for exploring the physiological mechanism of thermotolerance in insects.

The silkworm (Bombyx mori) is a model organism for lepidopteran insects. It is an oligophagous insect that primarily feeds on mulberry leaves and has industrial use for the production of raw silk. The development of artificial diets has provided an alternative nutrient source for silkworms; however, one significant issue is that the production of cocoons is lower in silkworms reared on artificial diets compared with those reared on mulberry leaves. The differences in the silk gland in the late-stage fifth instar silkworm larvae, when silk synthesis is most active, between those raised on artificial diets and mulberry leaves, are unknown. In this study, we identified differences in the transcriptomes of the middle and posterior silk glands of fifth instar day five silkworm larvae reared on artificial diets compared with those reared on mulberry leaves using three strains: Daizo, Nichi01, and J137 × C146. We found that the silk-related genes fibrohexamerin (fhx), fibroin-light-chain (fibL), and fibroin-heavy-chain (fibH) in the middle silk gland, and ser1 in the posterior silk gland, were differentially expressed in a strain-dependent manner. In silkworms reared on artificial diets, fhx, fibL, and fibH in the middle silk gland were upregulated in Nichi01 and downregulated in J137 × C146, whereas ser1 in the posterior silk gland was upregulated in J137 × C146 compared with silkworms reared on mulberry leaves. Our results demonstrate that the diet and strain of silkworm larvae affect the expression of genes related to silk production in their silk glands during the late fifth instar stage.

Insect guts offer unique habitats for microbial colonization, with gut bacteria potentially offering numerous benefits to their hosts. Although Enterococcus has emerged as one of the predominant gut commensal bacteria in insects, its establishment in various niches within the gut has not been characterized well. In this study, Enterococcus mundtii was inoculated into the silkworm (Bombyx mori L.) to investigate its biological functions. Genome-based analysis revealed that its successful colonization is related to adherence genes (ebpA, ebpC, efaA, srtC, and scm). This bacterium did not alter the activities of related metabolic enzymes or the intestinal barrier function. However, significant changes in the gene expressions levels of Att2, CecA, and Lys suggest potential adaptive mechanisms of host immunity to symbiotic E. mundtii. Moreover, 16S metagenomics analysis revealed a significant increase in the relative abundance of E. mundtii in the intestines of silkworms following inoculation. The intestinal microbiome displayed marked heterogeneity, an elevated gut microbiome health index, a reduced microbial dysbiosis index, and low potential pathogenicity in the treatment group. Additionally, E. mundtii enhanced the breakdown of carbohydrates in host intestines. Overall, E. mundtii serves as a beneficial microbe for insects, promoting intestinal homeostasis by providing competitive advantage. This characteristic helps E. mundtii dominate complex microbial environments and remain prevalent across Lepidoptera, likely fostering long-term symbiosis between the both parties. The present study contributes to clarifying the niche of E. mundtii in the intestine of lepidopteran insects and further reveals its potential roles in their insect hosts.

The utilization of nitenpyram for aphid and whitefly control may induce environmental contamination and negative repercussions on non-target organisms. Formerly, we found that nitenpyram would pollute the peripheral and sub-peripheral areas of the adjacent mulberry orchard. Under acute toxicity conditions, nitenpyram induced oxidative damage in silkworms, affected biological metabolism, synthesis, immunity, and signal transduction. Considering the impact of nitenpyram mist drift on mulberry leaves, we investigated the effects of low concentrations of nitenpyram on silkworms. The results showed that silkworms exposed to 0.17 mg/L, 0.35 mg/L and 0.70 mg/L of nitenpyram (1/40 LC50, 1/20 LC50 and 1/10 LC50) showed obvious poisoning symptoms. The cocoon weight and cocoon shell weight decreased gradually with increases in the concentration, and these decreases prolonged the growth and development time of silkworms and induced the detoxification enzymes carboxylesterase (CarE) and glutathione-S-transferase (GST) to cope with the stress damage caused by nitenpyram. Exposure to low concentrations of nitenpyram downregulates genes involved in the drug metabolism-other enzymes and peroxisome pathway in silkworms. Additionally, through injection of miRNA mimics and inhibitors, we discovered that detoxifying enzyme pathway genes are influenced by bmo-miR-3382-3P, bmo-miR-3213-5P and bmo-miR-133, regulating the immune response of silkworms. This study provides an overall view of the toxicity and detoxification metabolism of nitenpyram in silkworm, and provides a reference for environmental assessment.