Transgenic overexpression of bmo-miR-6498-5p increases resistance to Nosema bombycis in the silkworm, Bombyx mori.

Microsporidia are unfriendly microorganisms, and their infections cause considerable damage to economically or environmentally important insects like silkworms and honeybees. Thus, the identification of measures to improve host resistance to microsporidia infections is critically needed. Here, an overexpressed miR-6498-5p transgenic silkworm line was constructed. Importantly, the survival rates and median lethal doses of the transgenic line were clearly higher after infection with Nosema bombycis. H&E staining and RT-qPCR analyses revealed an inhibitory effect on the proliferation of N. bombycis in the transgenic larvae. Metabolomics analysis further revealed the presence of 56 differential metabolites between the two lines. KEGG analysis of these 56 metabolites found that they were involved in various amino acid and vitamin metabolism pathways. Notably, VB6 metabolism was enriched among the metabolites, and the pathway was well known for its involvement in the synthesis, interconversion, and degradation of amino acids. These suggest that miR-6498-5p modifies parasitic environments to inhibit the proliferation of N. bombycis by affecting the host amino acid metabolism. These results demonstrate the potential of microRNAs as biomolecules that can promote resistance to microsporidia and provide new insights and a new approach to generate microsporidia-resistant biological materials.IMPORTANCEMicrosporidia have an extremely wide host range and are capable of infecting a wide variety of insects and vertebrates, including humans, and their lethality to multiple species often poses significant environmental management challenge. Here, we successfully constructed a microsporidium-resistant line in the silkworm, based on the overexpression of miR-6498-5p. Our results strongly support the hypothesis that miR-6498-5p efficiently suppresses the proliferation of Nosema bombycis by regulating the host VB6 metabolism, a key pathway for enzymes involved in amino acid transport and protein metabolism. Our study provides new insights for understanding host anti-pathogen defenses toward microsporidia.

Identification of genes associated with the silk gland size using multi-omics in silkworm (Bombyx mori).

Silk gland size in silkworms (Bombyx mori) affects silk output. However, the molecular mechanisms by which genes regulate silk gland size remain unclear. In this study, silk glands from three pure silkworm strains (A798, A306 and XH) with different silk gland weight phenotypes were compared using transcriptomics and proteomics to identify differentially expressed genes (DEGs) and proteins (DEPs). When comparing A798 to A306 and A798 to XH, 830 and 469 DEGs were up-regulated, respectively. These genes were related to the gene ontology terms, metabolic process, transport activity and biosynthesis process. In addition, 372 and 302 up-regulated differentially expressed proteins were detected in A798 to A306 and A798 to XH, respectively, related to the gene ontology terms, ribosome and protein export, ribosome and polypeptide biosynthesis processes. Moreover, combined transcriptomics, proteomics and weighted correlation network analyses showed that five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711 and BGIBMGA010889) were significantly associated with the silk gland weight. Reverse Transcription-quantitative real-time Polymerase Chain Reaction (RT-qPCR) and Enzyme linked immunosorbent assay (ELISA) were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The results showed that four genes have higher expression levels in heavier silk glands. These genes are associated with glycogen metabolism, fatty acid synthesis and branched chain amino acid metabolism, thus potentially promoting growth and silk protein synthesis. These findings provide valuable insights into the molecular mechanisms underlying the relationship between silk gland weight and silk yield in silkworms.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.

Recent advances in the biosensors application for reviving infectious disease management in silkworm model: a new way to combat microbial pathogens.

Silkworms are an essential economic insect but are susceptible to diseases during rearing, leading to yearly losses in cocoon production. While chemical control is currently the primary method to reduce disease incidences, its frequent use can result in loss of susceptibility to pathogens and, ultimately, antibiotic resistance. To effectively prevent or control disease, growers must accurately, sensitively, and quickly detect causal pathogens to determine the best management strategies. Accurate recognition of diseased silkworms can prevent pathogen transmission and reduce cocoon loss. Different pathogen detection methods have been developed to achieve this objective, but they need more precision, specificity, consistency, and promptness and are generally unsuitable for in-situ analysis. Therefore, detecting silkworm diseases under rearing conditions is still an unsolved problem. As a consequence of this, there is an enormous interest in the development of biosensing systems for the early and precise identification of pathogens. There is also significant room for improvement in translating novel biosensor techniques to identify silkworm pathogens. This study explores the types of silkworm diseases, their symptoms, and their causal microorganisms. Moreover, we compare the traditional approaches used in silkworm disease diagnostics along with the latest sensing technologies, with a precise emphasis on lateral flow assay-based biosensors that can detect and manage silkworm pathogens.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Alteration of ß-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence.

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as ß-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of ß-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface ß-glucan while low pH triggers an increase in ß-glucan. There is no inverse pattern between exposure levels of ß-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in ß-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in ß-glucan triggers an increase in the virulence of C. auris. This study demonstrates that ß-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Nosema bombycis: A remarkable unicellular parasite infecting insects.

Microsporidia are opportunistic fungal-like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host-parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens.

Therapeutic potential of silkworm sericin in wound healing applications.

Chronic wounds are characterised by an imbalance between pro and anti-inflammatory signals, which result in permanent inflammation and delayed re-epithelialization, consequently hindering wound healing. They are associated with bacterial infections, tissue hypoxia, local ischemia, reduced vascularization, and MMP-9 upregulation. The global prevalence of chronic wounds has been estimated at 40 million in the adult population, with an alarming annual growth rate of 6.6%, making it an increasingly significant clinical problem. Sericin is a natural hydrophilic protein obtained from the silkworm cocoon. Due to its biocompatibility, biodegradability, non-immunogenicity, and oxidation resistance, coupled with its excellent affinity for target biomolecules, it holds great potential in wound healing applications. The silk industry discards 50,000 tonnes of sericin annually, making it a readily available material. Sericin increases cell union sites and promotes cell proliferation in fibroblasts and keratinocytes, thanks to its cytoprotective and mitogenic effects. Additionally, it stimulates macrophages to release more therapeutic cytokines, thus improving vascularization. This review focuses on the biological properties of sericin that contribute towards enhanced wound healing process and its mechanism of interaction with important biological targets involved in wound healing. Emphasis is placed on diverse wound dressing products that are sericin based and the utilisation of nanotechnology to design sericin nanoparticles that aid in chronic wound management.

Contrasting gut bacteriomes unveiled between wild Antheraea assamensis Helfer (Lepidoptera: Saturniidae) and domesticated Bombyx mori L. (Lepidoptera: Bombycidae) silkworms.

BACKGROUND: Insect gut microbiomes play a fundamental role in various aspects of insect physiology, including digestion, nutrient metabolism, detoxification, immunity, growth and development. The wild Muga silkworm, Antheraea assamensis Helfer holds significant economic importance, as it produces golden silk. METHODS AND RESULTS: In the current investigation, we deciphered its intricate gut bacteriome through high-throughput 16S rRNA amplicon sequencing. Further, to understand bacterial community dynamics among silkworms raised under outdoor environmental conditions, we compared its gut bacteriomes with those of the domesticated mulberry silkworm, Bombyx mori L. Most abundant bacterial phyla identified in the gut of A. assamensis were Proteobacteria (78.1%), Bacteroidetes (8.0%) and Firmicutes (6.6%), whereas the most-abundant phyla in B. mori were Firmicutes (49-86%) and Actinobacteria (10-36%). Further, Gammaproteobacteria (57.1%), Alphaproteobacteria (10.47%) and Betaproteobacteria (8.28%) were the dominant bacterial classes found in the gut of A. assamensis. The predominant bacterial families in A. assamensis gut were Enterobacteriaceae (27.7%), Comamonadaceae (9.13%), Pseudomonadaceae (9.08%) Flavobacteriaceae (7.59%) Moraxellaceae (7.38%) Alteromonadaceae (6.8%) and Enterococcaceae (4.46%). In B. mori, the most-abundant bacterial families were Peptostreptococcaceae, Enterococcaceae, Lactobacillaceae and Bifidobacteriaceae, though all showed great variability among the samples. The core gut bacteriome of A. assamensis consisted of Pseudomonas, Acinetobacter, Variovorax, Myroides, Alteromonas, Enterobacter, Enterococcus, Sphingomonas, Brevundimonas, Oleispira, Comamonas, Oleibacter Vagococcus, Aminobacter, Marinobacter, Cupriavidus, Aeromonas, and Bacillus. Comparative gut bacteriome analysis revealed a more complex gut bacterial diversity in wild A. assamensis silkworms than in domesticated B. mori silkworms, which contained a relatively simple gut bacteriome as estimated by OTU richness. Predictive functional profiling of the gut bacteriome suggested that gut bacteria in A. assamensis were associated with a wide range of physiological, nutritional, and metabolic functions, including biodegradation of xenobiotics, lipid, amino acid, carbohydrate metabolism, and biosynthesis of secondary metabolites and amino acids. CONCLUSIONS: These results showed great differences in the composition and diversity of gut bacteria between the two silkworm species. Both insect species harbored core bacterial taxa commonly found in insects, but the relative abundance and composition of these taxa varied markedly.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Effects of high temperature on the growth performance and midgut autophagy of thermotolerant and thermosensitive silkworm strains.

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.

Establishment of diabetes mellitus model using Bombyx mori silkworms in a low-temperature environment.

Due to the high prevalence of diabetes mellitus, researchers have conducted numerous experimental animal studies. However, the mammalian diabetes model is cumbersome and expensive to operate, while the cheap and simple common silkworm diabetes model has the disadvantage of a short cycle time. Since the growth of silkworms is greatly affected by environmental factors, we extended the five-age cycle of silkworms by lowering the ambient temperature to establish a novel low-temperature silkworm diabetes model. Our goal was to determine whether the low-temperature feeding of a high-sugar diet to silkworms could serve as an effective animal model for diabetes. Also, we aimed to resolve certain issues concerning the normal temperature silkworm diabetes model, such as the short time frame for experiments and erratic fluctuations in blood sugar levels. Silkworms weighing between 0.9 and 1.0 g at the beginning of the fifth instar were selected, and we created diabetic silkworms by feeding mulberry leaves containing 4% glucose daily in a 16-20°C environment. When the silkworms were kept at a cooler temperature, the fifth instar stage lasted for an additional 9-11 days. In the model group, 83.3% of the silkworms had blood glucose levels greater than 7.8 mmol/L, while the total prevalence of diabetic silkworms was 89.8%. Moreover, JNK phosphorylation expression rose in the model group, while PI3K expression fell. Additionally, the JNK and PI3K signaling pathway expressions matched diabetic signals. Therefore, using silkworms to create a diabetes model in a cool environment is a straightforward and cost-effective approach to studying diabetes in animals.

Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Distribution and genetic diversity of Bombyx mori nucleopolyhedrovirus in mass-reared silkworms in Thailand.

Silk is an economically and culturally important product of Thailand that is threatened by the outbreaks of the Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworm rearings. BmNPV infects the larval stages of the silkworm Bombyx mori, causing fatal viral infection and preventing the larvae from reaching the pupal stage. Numerous BmNPV isolates have been described from silk producing countries from all over the world. In this study, the geographic distribution of BmNPV in Thailand was analyzed by collecting 18 samples from different regions. The BmNPV samples were analyzed by PCR and whole genome sequencing was performed for eight specimens. Their genome size ranged from 125,888 bp to 126,783 bp, comprising 138 open reading frames. Although the sequenced BmNPV genomes could be phylogenetically differentiated, a correlation between geographic and genetic distance was not observed indicating a close relationship between the BmNPV from Thailand.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure.

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

A comprehensive prediction system for silkworm acute toxicity assessment of environmental and in-silico pesticides.

The excessive application and loss of pesticides poses a great risk to the ecosystem, and the environmental safety assessment of pesticides is time-consuming and expensive using traditional animal toxicity tests. In this work, a pesticide acute toxicity dataset was created for silkworm integrating extensive experiments and various common pesticide formulations considering the sensitivity of silkworm to adverse environment, its economic value in China, and a gap in machine learning (ML) research on the toxicity prediction of this species, which addressed the previous limitation of only being able to predict toxicity classification without specific toxicity values. A new comprehensive voting model (CVR) was developed based on ML, combined with three regression algorithms, namely, Bayesian Ridge (BR), K Neighbors Regressor (KNN), Random Forest Regressor (RF) to accurately calculate lethal concentration 50 % (LC50). Three conformal models were successfully constructed, marking the first combination of conformal models with confidence intervals to predict silkworm toxicity. Further, the mechanism by analyzing structural alerts was summarized, and identified 25 warning structures, 24 positive compounds and 14 negative compounds. Importantly, a novel comprehensive prediction system was constructed that can provide LC50 and confidence intervals, structural alerts analysis, lipid-water partition coefficient (LogP) and similarity analysis, which can comprehensively evaluate the ecological toxicity risk of substances to make up for the incomplete toxicity data of new pesticides. The validity and generalization of the CVR model were verified by an external validation set. In addition, five new, low-toxic and green pesticide alternatives were designed through 50,000 cycles. Moreover, our software and ST Profiler can provide low-cost information access to accelerate environmental risk assessment, which can predict not only a single chemical, but also batches of chemicals, simply by inputting the SMILES / CAS / (Chinese / English) name of chemicals.

Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.