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Microbial degradation of chloramphenicol (CAP) has become important for reducing the adverse impact of environmental pollution with antibiotics. Although several pathways for CAP degradation have been identified in various bacteria, multiple metabolic pathways and their respective intermediate metabolites within a single strain are rarely reported. Here, Raoultella ornithinolytica CT3 was first isolated from silkworm excrement using CAP as the sole carbon source, and 100 mg/L CAP was almost completely degraded within 48 h. The biodegradation type of CAP followed first-order kinetics. Twenty-two CAP biotransformation products were identified using high-performance liquid chromatography and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. The CAP biotransformation pathways were predicted mainly in the acetylation and auxiliary pathways of propionylation and butyrylation. The toxicity of CAP biotransformation products was evaluated using the ecological structure-activity relationship (ECOSAR) model and biological indicators. The results showed that the toxicity of the intermediate metabolites changed slightly, but the final metabolite was harmless to the environment. Genomic analysis predicted that genes encoding acetyltransferase, amido-linkage hydrolase, nitroreductase, haloacetate dehalogenase, and protocatechuate 3,4-dioxygenase were associated with CAP biodegradation. This study provides new insights into the microbial degradation pathway of CAP and constitutes an ecological safety assessment for CAP-contaminated environments., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hao LI reports financial support was provided by National Natural Science Foundation of China. Zhongzheng GUI reports financial support was provided by the Graduate Research and Innovation Projects of Jiangsu Province. Zhongzheng GUI reports a relationship with National Natural Science Foundation of China that includes: employment. Zhongzheng GUI has patent pending to ZL202111325904.8. No If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 LC 50 , 1/20 LC 50 and 1/10 LC 50 ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms., Competing Interests: Declaration of competing interest The authors that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The permeation of cryoprotectants into insect embryos is critical for successful cryopreservation. However, the permeability of silkworm embryos to cryoprotectants and the effects of cryopreservation remain poorly studied. In this study, we evaluated the permeability and toxicity of four cryoprotective agents (CPAs) as well as the vitrification effect on the viability of silkworm embryos. Among the four CPAs, propylene glycol (PG) showed the best permeability. Ethylene glycol (EG) and PG were the least toxic CPAs, but glycerol (GLY) and dimethyl sulfoxide (DMSO) were more toxic. Moreover, we examined several factors including the kind and the concentration of CPAs, exposure time, embryonic stage, and silkworm strains. Embryos at the earlier phases of stage 25 were more tolerant to vitrification using EG. We found that over 21% of embryos treated with EG at the early 2 phase of stage 25: 163 h after egg laying (AEL) developed and progressed to serosa ingestion after vitrification and rewarming. The result was the same in other strains as well. Our results are valuable for the development of new cryopreservation protocols of silkworm embryos.

This study investigates the toxic effects of the insecticide spinetoram on the model organism Bombyx mori (Linnaeus) and explores the potential ameliorative properties of O-Vanillin. Sub-lethal concentrations of spinetoram were given to silkworm larvae via oral feed, resulting in reduced body weight, larval length, and impaired cocoon characteristics. A study of the enzymatic and non-enzymatic antioxidants revealed oxidative stress in the gut, fat body, and silk gland tissues, characterized by decreased antioxidants and increased lipid peroxidation. However, post-treatment with O-Vanillin effectively mitigated these toxic effects, preserving antioxidant capacities and preventing lipid peroxidation. Additionally, O-Vanillin prevented the loss of body weight and improved cocoon characteristics. At the histological level, spinetoram exposure caused mild histological damage in the gut, fat body, and silk gland. However, O-Vanillin post-treatment had ameliorative effects and mitigated the histological damages. To delve deeper into the mechanism of amelioration of O-Vanillin, in silico studies were used to study the interaction between an important xenobiotic metabolism protein of the Bombyx mori, i.e., Cytochrome p450, specifically CYP9A19, and O-Vanillin. We performed blind molecular docking followed by molecular dynamic simulation, and the results demonstrated stable binding interactions between O-Vanillin and CYP9A19, a cytochrome P450 protein in silkworm, belonging to the subfamily CYP9A, suggesting a potential role for O-vanillin in modulating xenobiotic metabolism., (© 2024. Sociedade Entomológica do Brasil.)

Background/objectives: The utilization of methyl-thiophanate (MT) in vegetables and fruits is widespread due to its broad efficiency, yet its potential impact on silkworm growth remains uncertain. This study aims to examine the effects of MT on the growth of silkworms. Specifically, we assessed the weights of fifth-instar larvae that were fed mulberry leaves saturated with three concentrations (2.5, 5, and 10 mg/mL) of MT, as well as the weights of a control group., Methods: TEM was used to show the status of the silkworm midgut after MT supplementation. Oxidative stress was evaluated in the presence of MT. Furthermore, a transcriptomic sequencing experiment was conducted to investigate the mechanism through which the development of silkworms is induced by MT., Results: Our findings indicate that the supplementation of MT hindered larval growth compared to the control group, suggesting a toxic effect of MT on silkworms. The transmission electron microscopy (TEM) results show that MT supplementation induced autophagy in the silkworm midgut. MT was also found to induce oxidative stress in silkworms through the activation of reactive oxygen (ROS), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Subsequent transcriptomic analysis revealed 1265 significantly differentially expressed genes (DEGs) in response to MT. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these DEGs were associated with antioxidant defense, detoxification processes, lysosome biogenesis, and metabolic pathways., Conclusions: These findings suggest that MT toxicity in silkworm larvae is mediated through the induction of oxidative stress and alterations in metabolism. This study contributes to our understanding of the impacts of MT exposure on silkworms and provides insights into potential pesticides for use in mulberry gardens.

Bombyx mori is an insect species of great economic importance, and its silk gland is a vital organ for the synthesis and secretion of silk protein. However, long-term artificial domestication of B. mori has resulted in high sensitivity to chemical toxins, especially insecticides. Cyantraniliprole (Cya), a second-generation ryanodine receptor modulator insecticide, is widely utilized in agriculture for pest control. In this study, the impact of Cya toxicity on the development of silk glands in the 5th instar larvae of B. mori was assessed using Cya LC 5 , LC 10 and LC 20 , as well as a starvation treatment group for comparison. Short-term exposure (24 h) to different concentrations of Cya resulted in delayed development of silk glands in B. mori. Meanwhile, the body weight, silk gland weight, silk gland index and cocoon quality were significantly reduced in a concentration-dependent manner, except for the Cya LC 5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC 10 induced disruption of the nuclear membrane and endoplasmic reticulum in the posterior silk gland (PSG) cells, leading to the formation of intracellular vacuoles. Transcriptome sequencing of PSGs identified 2152 genes that were differentially expressed after exposure to Cya LC 10 , with 1153 down-regulated genes and 999 up-regulated genes. All differentially expressed genes were subjected to functional annotation using gene ontology and Kyoto encyclopedia of genes and genomes database, and it was found that protein synthesis-related pathways were significantly enriched, with the majority of genes being down-regulated. Furthermore, the transcription levels of genes involved in "protein processing in endoplasmic reticulum", "protein export", "proteasome" and "DNA replication" were quantified using qRT-PCR. Our findings suggested that short-term exposure to Cya LC 10 resulted in disruption of DNA replication, as well as protein transport, processing and hydrolysis in the PSG cells of B. mori. The results of this study provide a theoretical foundation for the safe utilization of Cya in sericulture production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Our previous research reported the influence of 50 μM selenium (Se) on the cytosolization (endocytosis) pathway, which in turn stimulates the growth and development of Bombyx mori. Lately, dynamin is recognized as one of the key proteins in endocytosis. To explore the underlying mechanisms of Se impact, the dynamin gene was knocked down by injecting siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3). This was followed by an analysis of the target gene and levels of silk protein genes, as well as growth and developmental indices, Se-enrichment capacity, degree of oxidative damage, and antioxidant capacity of B. mori. Our findings showed a considerable decrease in the relative expression of the dynamin gene in all tissues 24 h after the interference and a dramatic decrease in the silkworm body after 48 h. RNAi dynamin gene decreased the silkworm body weight, cocoon shell weight, and the ratio of cocoon. In the meantime, malondialdehyde level increased and glutathione level and superoxide dismutase/catalase activities decreased. 50 μM Se markedly ameliorated these growth and physiological deficits as well as decreases in dynamin gene expression. On the other hand, there were no significant effects on fertility (including produced eggs and laid eggs) between the interference and Se treatments. Additionally, the Se content in the B. mori increased after the dynamin gene interference. The dynamin gene was highly expressed in the silk gland and declined significantly after interference. Among the three siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3), the dynamin-2 displayed the highest interference effects to target gene expression. Our results demonstrated that 50 μM Se was effective to prevent any adverse effects caused by dynamin knockdown in silkworms. This provides practical implications for B. mori breeding industry., (© 2024 Wiley Periodicals LLC.)

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 % (LC 50 ). 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 LC 50 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., Competing Interests: Declaration of Competing Interest There is no conflict of interest in the submission of this manuscript, which is approved by all my co-authors for publication., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shengxiang Zhang reports financial support was provided by China Agriculture Research System of MOF and MARA. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Until recently, chemical pesticides were one of the most effective means of controlling agricultural pests; therefore, the search for insecticide targets for agricultural pests has been an ongoing problem. Estrogen-related receptors (ERRs) are transcription factors that regulate cellular metabolism and energy homeostasis in animals. Silkworms are highly sensitive to chemical pesticides, making them ideal models for pesticide screening and evaluation. In this study, we detected ERR expression in key organs involved in pesticide metabolism in silkworms (Bombyx mori), including the fat body and midgut. Using ChIP-seq technology, many estrogen- related response elements were identified in the 2000-bp promoter region upstream of metabolism-related genes, almost all of which were potential ERR target genes. The ERR inhibitor, XCT-790, and the endocrine disruptor, bisphenol A, significantly inhibited expression of the ERR target genes, BmTreh-1, BmTret-1, BmPK, BmPFK, and BmHK, in the fat bodies of silkworms, resulting in pupation difficulties in silkworm larvae that ultimately lead to death. In addition, based on the clarification that the ERR can bind to XCT-790, as observed through biofilm interferometry, its three-dimensional spatial structure was predicted, and using molecular docking techniques, small-molecule compounds with a stronger affinity for the ERR were identified. In summary, utilizing the powerful metabolic regulatory function of ERR in Lepidoptera pests, the developed small molecule inhibitors of ERR can be used for future control of Lepidoptera pests., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microplastics (MPs) existing extensively in various ecosystems can be ingested by marine organisms and enter the food chain, resulting the health risks from the presence of MPs in aquatic and terrestrial ecosystems. In the present study, an ideal model for Lepidoptera, the silkworm, Bombyx mori, was exposed to environmental concentrations (0.125 μg, 0.25 μg or 0.5 μg/diet) of MPs for 5 days, and the global changes in gut microbes and metabolites were subsequently examined via 16S rDNA sequencing and GC‒MS-based metabolomics. The results showed that MPs exposure did not seriously threaten survival but may regulate signaling pathways involved in development and cocoon production. MPs exposure induced gut microbiota perturbation according to the indices of α-diversity and β-diversity, and the functional prediction of the altered microbiome and associated metabolites demonstrated the potential roles of the altered microbiome following MPs exposure in the metabolic and physiological states of silkworm. The metabolites markedly altered following MPs exposure may play vital biological roles in energy metabolism, lipid metabolism, xenobiotic detoxification and the immune system by directly or indirectly affecting the physiological state of silkworms. These findings contribute to assessing the health risks of MPs exposure in model insects and provide novel insight into the toxicity mechanism of MPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The commercial value of silkworms has been widely explored and the effects of fluoride exposure on silkworms' breeding and silk production cannot be ignored. Bombyx mori is a commonly used model to explore the mechanisms of fluorosis. In the present study, we analyzed the differences in physiological and biochemical indicators after exposing larva to NaF, then evaluated differential genes and proteins. Compared to control, larvae exposed to 600 mg L -1 NaF presented decreased bodyweight, damaged midgut tissue, and were accompanied by oxidative stress. The RNA-seq showed 1493 differentially expressed genes (574 upregulated and 919 downregulated). Meanwhile, the TMT detected 189 differentially expressed proteins (133 upregulated and 56 downregulated). The integrative analysis led to 4 upregulated and 9 downregulated genes and proteins. Finally, we hypothesized that fluoride exposure might affect the intestinal digestion of silkworms, inhibit the gene expression of detoxification enzymes and stimulate cellular immune responses. Our current findings provided new insights into insect fluorosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 μg/cm 2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 μg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Flavonoids are secondary metabolites that help plants resist insect attack. It can resist insect attack by inhibiting insect immune defense, and pathogens can also inhibit insect immune defense. It is speculated that the combination of flavonoids and pathogens may inhibit the immune defense and have stronger toxicity to silkworm. In this study, the combined treatment of quercetin with Bombyx mori nuclear polyhedrosis virus (BmNPV) had significant negative effects on the growth and survival of silkworm compared with BmNPV group. The detoxifying enzyme activity of BmNPV group was significantly increased at 96 h, while the activity of the combined treatment group was significantly decreased with the increase of quercetin exposure time (72 or 96 h). The activity of antioxidant enzymes also showed a similar trend, that was, the activity of antioxidant enzymes in the combined treatment group also decreased significantly with the increase of quercetin exposure time, which led to the increase of reactive oxygen species content. The silkworm cells would produce lipid peroxidation, malondialdehyde content was significantly increased, so that the expression of immune-related genes (the antimicrobial peptide, Toll pathway, IMD pathway, JAK-STAT pathway, and melanin genes) were decreased, leading to the damage of the immune system of silkworm. These results indicated that quercetin combined with BmNPV could inhibit the activities of protective enzymes and lead to oxidative damage to silkworm. It can also affect the immune response of the silkworm, and thus resulting in abnormal growth. This study provides the novel conclusion that quercetin accumulation will increase the susceptibility of silkworm to pathogens., (© 2021 Wiley Periodicals LLC.)

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca 2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca 2+ . The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca 2+ binding assay indicated that rCNB shows affinity for Ca 2+ . The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca 2+ or efflux Ca 2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes great economic losses in sericulture. Many genes play a role in viral infection of silkworms, but silkworm metabolism in response to BmNPV infection is unknown. We studied BmE cells infected with BmNPV. We performed liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based non-targeted metabolomics analysis of the cytosolic extract and identified 36, 76, 138, 101, 189, and 166 different molecules at 3, 6, 12, 24, 48, and 72 h post BmNPV infection (hpi) compared with 0 hpi. Compounds representing different areas of metabolism were increased in cells post BmNPV infection. These areas included purine metabolism, aminoacyl-tRNA biosynthesis, and ABC transporters. Glycerophosphocholine (GPC), 2-hydroxyadenine (2-OH-Ade), gamma-glutamylcysteine (γ-Glu-Cys), hydroxytolbutamide, and 5-pyridoxolactone glycerophosphocholine were continuously upregulated in BmE cells post BmNPV infection by heat map analysis. Only 5-pyridoxolactone was found to strongly inhibit the proliferation of BmNPV when it was used to treat BmE cells. Fewer infected cells were detected and the level of BmNPV DNA decreased with increasing 5-pyridoxolactone in a dose-dependent manner. The expression of BmNPV genes ie1, helicase, GP64, and VP39 in BmE cells treated with 5-pyridoxolactone were strongly inhibited in the BmNPV infection stage. This suggested that 5-pyridoxolactone may suppress the entry of BmNPV. The data in this study characterize the metabolism changes in BmNPV-infected cells. Further analysis of 5-pyridoxolactone, which is a robust antiviral molecule, may increase our understanding of antiviral immunity.

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain., (© 2021 Wiley Periodicals LLC.)

Nocardia is a ubiquitous environmental microbe that causes nocardiosis against immunosuppressed and immunocompromised hosts. The assay system for the quantitative evaluation of virulence of Nocardia sp. or therapeutic effectiveness of antimicrobials for treatment of nocardiosis is not established so far. In this study, we established an infection model of Nocardia sp. using silkworm as an alternative animal model. We found that all tested Nocardia sp. such as Nocardia asiatica, Nocardia elegans, Nocardia exalbida, Nocardia farcinica, and Nocardia nova killed silkworm and their killing ability were different by species. N. farcinica showed higher pathogenicity among tested strain, similar to the mouse model as previously reported. In addition, we found that antimicrobials such as amikacin and minocycline showed therapeutic effectiveness in silkworms infected with N. farcinica, and we could determine effective doses 50 (ED₅₀) values. These results suggest that silkworm is a useful alternative animal to evaluate the pathogenicity of Nocardia pathogen and the therapeutic effects of antimicrobials against Nocardia sp. in a quantitative manner.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-β-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

This study aimed to explore the toxicity of environmental residues of graphene oxide nanoparticles (GONPs) to reproduction of Lepidopteron insects using both ovary cell line (BmN) and individual female Bombyx mori as the research subjects. The results showed that GONPs dose dependently affect BmN cells. At higher concentrations (>25 mg/L), GONPs led to oxidative stress, ROS accumulation and DNA damage in BmN cells and significantly reduced their survival rate (p ≤ 0.05). Moreover, feeding female B. mori larvae with mulberry leaves treated with 25 mg/L GONPs significantly decreased their gonadosomatic index (GSI) by 40.84%, and increased oxidation levels and antioxidant enzyme activity in silkworm ovary tissues. Pathological analysis found that exposure to GONPs decreased the numbers of both oogonia and oocytes in ovarian tissues, increased the formation of peroxisome and vacuoles in follicle cells, reduced the transcription of genes (Vg, Ovo, Sxl-s, Sxl-l, and Otu) related to ovarian development in B. mori by 0.61, 0.65, 0.75, 0.72, and 0.42-fold, respectively, and lowered the amount of spawning by 52.25%. Overall, these results revealed that GONPs exposure is toxic to the reproduction of B. mori. The underlying mechanism is that oxidative stress due to GONPs causes oxidative damage to DNA, damages ovarian tissues, as well as hinders B. mori development and spawning. Thus, this study provides important experimental data for safety evaluation of reproductive toxicity due to GONPs exposure., (Copyright © 2021. Published by Elsevier Inc.)

Five new tremulane sesquiterpenoids were isolated from co-culture of endophyte Irpex lacteus, phytopathogen Nigrospora oryzae, and entomopathogen Beauveria bassiana. All compounds showed obvious antifeedant activities against silkworm with inhibition percentages of 73-99%, at concentrations of 50 μg/cm 2 . Compound 11 indicated notable antifeedant activity with inhibition percentage of 93% at concentration of 6.25 μg/cm 2 among them. Compounds 2, 3, 4, 8, 9, 15 and 16 indicated anti-fungal activities against I. lacteus with MIC values ≤8 μg/mL, compounds 11, 12, 16-18 showed significant anti-fungal activity against N. oryzae with MICs ≤ 4 μg/mL, and compounds 2, 5, 12 and 18 indicated significant anti-fungal activity against B. bassiana with MICs ≤ 8 μg/mL. In addition, the I. lacteus should unite B. bassiana to inhibit the production of phytotoxins from N. oryzae in the ternary culture., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Quercetin is a flavonoid produced as a defense by plants. The effects of 1% quercetin on the growth and development of Bombyx mori were studied. The activities of the enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), carboxy-lesterase (CarEs), and glutathione S-transferase (GST) were all measured at 24, 48, 72, and 96 h after quercetin exposure. The results show that quercetin induces the activities of antioxidant and detoxification enzymes. With longer exposure times, enzyme activity first increased and then decreased. The relative expressions of AMP (defensin, CecA), the Toll pathway (cactus, Spatzle, and Rel), the IMD pathway (Imd, Fadd, and Dorsal), the JAK-STAT pathway (STAT, HOP, and Pi3k60), and the Melanization gene (DDC and PAH) were analyzed using quantitative polymerase chain reaction (qPCR). The results indicated that long-term exposure to quercetin could inhibit the expression of immune-related pathway genes in silkworms. This suggests that it can inhibit the activities of antioxidant and detoxifying enzymes, thus inhibiting the immune system and affecting the growth and development, resulting in an increase in the death rate in silkworm. This study provides the novel conclusion that quercetin accumulation inhibits the immune system of silkworm and increases its death rate, a result that may promote the development and utilization of better biopesticides that avoid environmental pollution., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Fenoxycarb 0-ethyl N-(2-(4-pheoxyphenoxy)-ethyl) carbamate is the most potent juvenile hormone analogue against a variety of insect species including the silkworm Bombyx mori. In this study, the effects of fenoxycarb on silkworm Bombyx mori brain neurosecretory cells in 5th instar were investigated. Fenoxycarb (1 ng/10 µl) was applied topically along the dorsa-medial line to the animals in the spinning behavior on day 1 of the experimental group. Brains removed by dissection were histologically examined by hematoxylin eosin (hem&eosin) and paraldehyde fuchsin staining. Three types of neurosecretory cells (NSCs) were identified, NSC-1, NSC-2 and NSC-3. It was determined that cell secretions were in different density on different days. It was shown that the secretion density of cells on different days was not the same as the experimental and control groups. The fenoxycarb was found to suppress the bombyxin (insulin-like peptides) secretion of cells in the spinning behavior on day 2. Also, it stimulated the division of NSCs on the spinning behavior on day 5.

Bombyx mori（Linnaeus, 1758） is an important economical insect, and the sericulture is a flourishing industry in many developing countries. Pyriproxyfen, a juvenile hormone pesticide, is often applied to cultivations widely in the world, and its exposure often resulted in silk yield reduction and non-cocooning. However, the effect of pyriproxyfen exposure on cocooning and gene expression level in the silk gland of B. mori has not been studied yet, and this study focused on the above issues. The result indicated that pyriproxyfen exposure can lead to silk gland injury, reduction of silk yield and cocooning rate. Furthermore, the expression levels of silk protein synthesis related genes were down regulated significantly. The same change trends were shown between PI3K/Akt and CncC/Keap1 pathway, which is the expressions of key genes can be elevated by pyriproxyfen exposure. In addition, the activity of detoxification enzymes (P450, GST and CarE) and the expression levels of detoxification genes were elevated after pyriproxyfen exposure, suggesting that detoxification enzymes may play an important role in detoxification of pyriproxyfen in silk gland. These results provided possible clues to the silk gland injury and gene transcriptional level changes in silkworm after pyriproxyfen exposure., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Information on the occurrence and effects of nanoplastics in ecosystems worldwide currently represent one of the main challenges from the ecotoxicological point of view. This is particularly true for terrestrial environments, in which nanoplastics are released directly by human activities or derive from the fragmentation of larger plastic items incorrectly disposed. Since insects can represent a target for these emerging contaminants in land-based community, the aim of this study was the evaluation of ingestion of 0.5 μm polystyrene nanoplastics and their effects in silkworm (Bombyx mori) larvae, a useful and well-studied insect model. The ingestion of nanoplastics, the possible infiltration in the tissues and organ accumulation were checked by confocal microscopy, while we evaluated the effects due to the administered nanoplastics through a multi-tier approach based on insect development and behaviour assessment, as endpoints at organism level, and the measurements of some biochemical responses associated with the imbalance of the redox status (superoxide dismutase, catalase, glutathione s-transferase, reactive oxygen species evaluation, lipid peroxidation) to investigate the cellular and molecular effects. We observed the presence of microplastics in the intestinal lumen, but also inside the larvae, specifically into the midgut epithelium, the Malpighian tubules and in the haemocytes. The behavioural observations revealed a significant (p < 0.05) increase of erratic movements and chemotaxis defects, potentially reflecting negative indirect effects on B. mori survival and fitness, while neither effect on insect development nor redox status imbalance were measured, with the exception of the significant (p < 0.05) inhibition of superoxide dismutase activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A series of linear hydrophilic cationic polymers with different charge density and molecular weights were synthesized by one-step polymerization process. The effect of the hydrophobicity and molecular weights on the antifungal activity against Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4) was assessed. The biotoxicity of the cationic polymers were evaluated based on their median lethal concentration (LC 50 ) for zebrafish and silkworm and median lethal dose (LD 50 ) for Kunming mice. The results indicated that the balance between antifungal activity and biotoxicity could be well tuned by controlling the hydrophobic-hydrophilic balance. The minimum inhibitory concentration (MIC) of PEPB10 and PEPB25 against R. solani were 160 μg/mL and 80 μg/mL, respectively. And the LD 50 for Kunming mice of PEPB10 and PEPB25 were more than 5000 mg/kg, which mean that PEPB10 and PEPB25 with high hydrophilicity show low toxicity and better selectivity for R. solani. The cationic polymers can kill the R. solani by damaging their membranes and exchanging the Ca 2+ or/and Mg 2+ cations of their membranes or cell wall. These results help to understand the antifungal mechanism of low-toxic polymeric quaternary ammonium salts and highlight their potential application as highly selective fungicidal agents for controlling plant diseases., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a serious disease harmful to silk industry, which is one of the major sources of financial support for farmers in many developing countries. So far, there is still no good way to prevent or treat this disease. In this study, titanium dioxide nanoparticles (TiO 2 NPs) were used to pretreat silkworm larvae, and good results were achieved in improving silkworm immunity and alleviating the damage of cytoplasmic polyhedrosis virus. The results showed that nano-titanium dioxide pretreatment could inhibit the proliferation of BmCPV in the midgut of silkworm, activate JAK/STAT and PI3K-AKT immune signaling pathways, and upregulate the expression of key immune genes, so as to improve the immunity of silkworm and enhance the resistance of silkworm to BmCPV.

Ecdysteroids are widely found in terrestrial organisms, including insects, crustaceans, fungi, and plants. The function of ecdysteroids has been extensively studied in insects for decades because ecdysteroids regulate metamorphosis. In plants, in contrast, ecdysteroids (called phytoecdysteroids) do not show apparent hormonal activity and their function remains unclear. However, it has been proposed that phytoecdysteroids have an antifeedant function. Ecdysteroid ingestion disrupts insect development and alters behavior to deter insect feeding, resulting in reduced plant damage by the insect. These points of view are generally accepted, but the function of phytoecdysteroids in specific contexts has not been unveiled. In the present study, we used larvae of the silkworm, Bombyx mori, to investigate the biological significance of phytoecdysteroids. To mimic the situation where larvae consume plant leaves that contain phytoecdysteroids, 26 or 30 larvae were fed the diet containing ecdysteroid or the control diet. We show that ecdysteroid ingestion dramatically suppressed carbohydrate processing in the larval midgut to reduce the nutritional value of the ingested diet. Based on the present results, we propose a new explanation of phytoecdysteroid function: ingested ecdysteroids may lead to the erroneous perception that the plant is poor in nutrients and consequently result in cessation of feeding.

Silkworm (Bombyx mori L.) has a clear genetic background, parts of which are highly homologous to certain genes related to human hereditary diseases. Thus, the species presents an excellent interspecies model for drug screening and microbe-host interaction studies. Chloramphenicol (CAM) and vancomycin (VCM) are antibiotics commonly used to treat specific bacterial infections in medical care, animal husbandry, and agriculture. However, inappropriate dosages and prolonged therapy increase their risk of toxicity. In this work, we investigated the physiological and toxicological responses of silkworm to combined oral administration of CAM and VCM. Results showed that antibiotics promote the feeding behavior of silkworm and significantly reduce (P < 0.05) intestinal cultivable bacterial counts. Moreover, antibiotics decreased the antioxidant enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, and thioredoxin reductase and caused oxidative damage to the silkworm intestine; the degree of damage was confirmed by histopathology analysis. The gene expression levels of antimicrobial peptides (attacin, lysozyme, and cecropins) were also perturbed by antibiotics. After antibiotic exposure, 16S rRNA metagenomic sequencing revealed increases in the relative abundance of Sphingobium, Burkholderia, Barnesiella, Bacteroides, Bradyrhizobium, Acinetobacter, Phenylobacterium, Plesiomonas, Escherichia/Shigella, and unclassified bacteria, as well as a reduction of Enterococcus. The metabolic and functional profiles of intestinal microbiota, particularly metabolic processes, such as energy, cofactors and vitamins, lipid, amino acid, and carbohydrate metabolisms, changed after antibiotic exposure. In conclusion, our findings reveal that antibiotics exert substantial effects on silkworm. The present study may promote the applications of silkworm as an interspecies model in the medical and pharmaceutical fields., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nanosilver is an environment-friendly, harmless alternative of traditional disinfectants which can be potentially applied in the sericulture industry. However, the effects of nanosilver on the intestinal bacterial community of the silkworms (Bombyx mori L.) are unclear. In this study, Illumina MiSeq high-throughput sequencing technology was used to assess the intestinal bacterial community in both male and female silkworms while treated with different concentrations of nanosilver. We found that nanosilver significantly influenced the composition of silkworm intestinal bacterial community on the different taxonomic levels. Most conspicuously, the abundance of Firmicutes was increased by the treatment of 20 mg L-1 nanosilver but decreased by that of 100 mg L-1 nanosilver at the phylum level. The same trend was observed in Bacilli at the class level and in Enterococcus at the genus level. In some extreme cases, application of nanosilver eliminated the bacterium, e.g., Brevibacillus, but increased the population of several other bacteria in the host intestine, such as Blautia, Terrisporobacter, Faecalibacterium, and some bacteria could only be found in nanosilver treatment groups, e.g., Dialister. In addition, although nanosilver generally showed negative effects on the cocooning rate in a dose-dependent manner, we found that 20 mg L-1 nanosilver treatment significantly increased the body weight of silkworms and did not show negative effects on the survival rate. These results indicated that the intestinal bacteria community of silkworm larvae was significantly changed after nanosilver treatment which might consequently influence host growth and development.

Hydrogen sulfide (H2S) is a highly poisonous gas with an unpleasant smell of rotten eggs. Previous studies of H2S have primarily focused on its effects on mammalian nervous and respiratory systems. In this study, silkworm developmental parameters and changes in metabolites in response to H2S exposure were investigated using a hemolymph metabolomic approach, based on liquid chromatography-mass spectrometry (LC-MS). The developmental parameters, body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio, were noticeably increased following H2S exposure, with the greatest effects observed at 7.5-μM H2S. Metabolites upregulated under H2S exposure (7.5 μM) were related to inflammation, and included (6Z, 9Z, 12Z)-octadecatrienoic acid, choline phosphate, and malic acid, while hexadecanoic acid was downregulated. Identified metabolites were involved in biological processes, including pyrimidine, purine, and fatty acid metabolism, which are likely to affect silk gland function. These results demonstrate that H2S is beneficial to silkworm development and alters metabolic pathways related to spinning function and inflammation. The present study provides new information regarding the potential functions of H2S in insects and metabolic pathways related to this phenomenon., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Destruxin A (DA), a cyclodepsipeptidic mycotoxin produced by entomopathogenic fungus Metarhizium anisopliae , has good insecticidal activity and potential to be a new pesticide. However, the mechanism of action is still obscure. Our previous experiments showed that DA was involved in regulation of transcription and protein synthesis and suggested that silkworms' arginine tRNA synthetase (BmArgRS), Lamin-C Proteins (BmLamin-C) and ATP-dependent RNA helicase PRP1 (BmPRP1) were candidates of DA-binding proteins. In this study, we employed bio-layer interferometry (BLI), circular dichroism (CD), cellular thermal shift assay (CETSA), and other technologies to verify the interaction of DA with above three proteins in vitro and in vivo. The results of BLI indicated that BmArgRS and BmLamin-C were binding-protein of DA with K D value 5.53 × 10 -5 and 8.64 × 10 -5 M, but not BmPRP1. These interactions were also verified by CD and CETSA tests. In addition, docking model and mutants assay in vitro showed that BmArgRS interacts with DA at the pocket including Lys228, His231, Asp434 and Gln437 in its enzyme active catalysis region, while BmLamin-C binds to DA at His524 and Lys528 in the tail domain. This study might provide new insight and evidence in illustrating molecular mechanism of DA in breaking insect., Competing Interests: The authors declare no conflict of interest.

: Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins., Competing Interests: We declare no conflict of interest, and the funders had no role in the design of the study.

Organophosphate pesticides are widely applied worldwide for agricultural purposes, and their exposures often result in adverse effects on Bombyx mori. The insect gut is a complicated ecosystem inhabited by a large number of microbes that play important roles in insect physiology and behavior. Recent studies have reported that alteration of their microbiota due to stressful conditions or environmental changes has been linked to a compromised health status and a susceptibility to diseases. In the present study, we aimed to assess the effects of phoxim exposure on intestinal microbes in silkworms. The results showed that phoxim exposure increased the bacterial community evenness and altered the structure of gut microbiota in silkworm larvae. The abundances of several genera, such as Methylobacterium and Aurantimonadaceae, in phoxim-treated larval guts were significantly reduced compared with the H 2 O-treated group, whereas the abundances of non-dominant bacteria, such as Staphylococcus, were significantly increased. Moreover, phoxim inhibited the expressions of antimicrobial peptides (AMPs) at the mRNA level and enhanced the pathogenesis of Enterobacter cloacae (E. cloacae) against silkworm larvae, suggesting that the immune system was inhibited after phoxim exposure. Therefore, the gut microbial community shifts were apparent after phoxim exposure. The compositional and structural changes of intestinal microbes caused by phoxim exposure might affect the normal function of the intestinal tract of silkworm. These results highlighted the importance of the gut bacterial community when investigating the mechanisms of midgut injury after pesticide exposure in Bombyx mori., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin-III (BmApoLp-III). First, the expression of BmApoLp-III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp-III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp-III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp-III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp-III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition., (© 2019 The Royal Entomological Society.)

Biological modifications of the silk fibroin (silk) material have broad applications in textiles, biomedical materials and other industrial materials. It is economical to incorporate nanoparticles to the biosynthesis of silk fibroin by adding them to silkworm larval diets. This strategy may result in the rapid stable production of modified silk. Glucose-coated silver nanoparticles (AgNPs) were used to improve the AgNPs' biocompatibility, and the AgNPs were efficiently incorporated into silk by feeding. Larvae fed with AgNPs produced silk with significantly improved antibacterial properties and altered silk secondary structures. Both positive and negative effects on the growth and synthesis of silk proteins were observed after different AgNPs doses. Larvae feeding with low concentration of 0.02% and medium 0.20% AgNPs have greater transfer efficiencies of AgNPs to silk compared with feeding high concentration of 2.00% AgNPs. In addition, the elongation and tensile strength of the produced silk fibers were also significantly increased, with greater mammalian cell compatibility. The appropriate AgNPs concentration in the diet of silkworms can promote the synthesis of silk proteins, enhance their mechanical properties, improve their antibacterial property and inhibit the presence of Gram-negative bacteria.

Cells under stress generate reactive oxygen species (ROS) in excess, which causes mitochondrial dysfunction and stimulates the apoptotic cascade. However, mild stress or pre-conditioning lead to the evasion of apoptosis by activating mitogenic signaling, including the signaling of inhibitors of apoptosis proteins (IAPs), or by inactivating certain apoptotic molecules. The silkworm (Bombyx mori) is an important economic insect which serves as a model organism in biological research. Bombyx mori apoptotic protease inducing factor (BmApaf1), a death-related ced-3/Nedd2-like protein (BmDredd), and BmSurvivin-2 (BmSvv2) are known to play significant roles in metamorphosis. Azaserine is an analogue of glutamine and irreversibly inhibits glutamine-utilizing enzymes and cysteine-glutamate transporter genes EAAT2. In the present study, we experimentally demonstrated stress induced by azaserine along with the capacity of antioxidants to modulate apoptotic/anti-apoptotic gene expression in determining the fate of the larvae. We observed higher larval survival with higher azaserine dosages and attributed this to the quantum of ROS generated and AOEs response, which favoured the BmSvv2 expression. Meanwhile higher levels of ROS with concomitant changes in AOEs were found to be responsible for BmApaf1 and BmDredd expression, which reflected a higher mortality rate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study investigates the effects of the insect growth regulator Novaluron on the silk gland (SG) and silk cocoon production in a nontarget insect, the silkworm Bombyx mori, which is a model research insect among Lepidoptera and of great economic importance for the commercial production of silk threads. Larvae were segregated into experimental groups: the control group (CG) and the treatment group (TG), which was exposed to a Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed the cytotoxic effects on the epithelial cells of the anterior, middle and posterior regions of the SG of B. mori larvae in the 3rd, 4th, and 5th instars, as well as the quality of the cocoons from larvae in the 5th instar. Cytotoxic effects were observed in the TG, such as the dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum, intracellular and intercellular spaces, spacing between the epithelial cells and the basal lamina and detachment of some cells towards the lumen of the SG, and decreased protein in the lumen, with faults in its composition. In addition, we verified ultrastructural changes in the production of fibers and silk cocoons, including a reduction in the weight of the cocoons constructed by both males and females in the TG and the construction of defective cocoons. Novaluron exposure impairs the SG and may affect the physiological functions of this organ; additionally, it compromises the quality of silk cocoons, potentially causing serious damage to sericulture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Traditional Indian medicine has utilized Aeglemarmelos (L.) Corr. commonly called as bael in several indigenous systems against various diseases. Bioactive components isolated from various plant parts of A. marmelos were used in ethno-medicine. More precisely they are known for its antiviral property against various human and animal viruses., Aim of the Study: The study was conducted to investigate the antiviral activity of A.marmelos against Bombyx mori nucleopolyhedrovirus (BmNPV)., Materials and Methods: Among the various crude extracts tested, hexane extracts of leaves of A. marmelos with promising anti-BmNPV activity was subjected to bioactivity guided fractionation based on column chromatography. Out of 40 fractions obtained from the fractionation, fractions showing similar TLC profiles were pooled into 14 fractions. A fraction with potential activity was used to purify a molecule with anti-BmNPV activity. This molecule was characterized through structural and functional analyses., Results: The functionally and structurally characterized molecule in the fraction with prospective anti-BmNPV activity revealed a single crystal compound 'seselin' (8, 8-dimethyl pyrido oxazine-2-one)., Conclusion: It is therefore understood that this seselin compound could be used as a natural medicine for the management of NPV infection in the silkworm larvae under commercial conditions after suitable field evaluations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To investigate the biological processes affected by long-term iron supplementation, newly hatched silkworms were exposed to high iron mulberry diet (10 and 100 ppm) and its effect on silkworm transcriptom was determined. The results showed that the silkworm was responsive to iron by increasing iron concentration and ferritin levels in the hemolymph and by regulating the expression of many other genes. A total of 523 and 326 differentially expressed genes were identified in 10 and 100 ppm Fe group compared to the control, respectively. Of these genes, 249 were shared between in both the 10 ppm and 100 ppm Fe group, including 152 up-regulated and 97 down-regulated genes. These shared genes included 19 known Fe regulated, 24 immune-related, 12 serine proteases and serine proteases homologs, 41 cuticular and cuticle genes. Ten genes (carboxypeptidases A, serine protease homologs 85, fibrohexamerin/P25, transferrin, sex-specific storage-protein 2, fungal protease inhibitor F, insect intestinal mucin, peptidoglycan recognition protein B, cuticle protein CPH45, unknown gene) were involved in the regulation of iron overload responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Silkworm (Bombyx mori) is one of the most important economic insects in the world, while pesticides impact its economic benefits. Tebuconazole is a fungicide that has been frequently detected in agriculture systems at concentrations that affect endocrine function in organisms. In the present study, silkworm larvae at different instar stages were exposed to tebuconazole, respectively. Cocoon weight, cocoon shell weight and cocoon shell rate were significantly decreased by 6.8%, 11.8% and 4.4% respectively, after exposure to 0.40 mg/L tebuconazole at 2nd -3rd instar stage. Vacuolization was found in the exposure silkworm under histopathological study at all stages exposures, indicating potential damage to silk gland. Downregulation of genes transcription (Fibh, Fibl, P25, Ser2, Ser3) involved with protein synthesis in the silk gland were further observed, and the results showed significant decreasing in mRNA expression among the tebuconazole treatments. Ecdysteroid levels in silkworm were changed with pronounced decreases after exposed to tebuconazole. In contrast, exposure to tebuconazole significantly increased juvenile hormone 1 concentrations and the maximum increasing fold of juvenile hormone 1 was up to 3.73 which was observed at stage I exposure. In addition, co-exposure to 2 and 10 mg/L forskolin able to mitigate tebuconazole-induced downregulate of mRNA expression of Sgf1 in the present study, indicating the potential mechanism of tebuconazole-induced chronic toxicity in silkworm may relative to PI3K/AKT/TORC1/Sgf1 pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05-0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Silkworm (Bombyx mori) is an economic insect of the Lepidoptera. Chlorantraniliprole (CAP) exposure results in reduced growth and development of B. mori and failure in cocooning, seriously affecting the development of sericulture. To study the mechanisms underlying the damage to silkworm caused by sublethal doses of CAP, we examined the oxidative damage, the activities of digestive enzymes in midgut, and the expressions of midgut-related genes at the mRNA level. We found that CAP exposure inhibited the growth of silkworm, decreased the body mass and caused the accumulation of reactive oxygen species (ROS) [the levels of O 2 - , H 2 O 2 and lipid peroxidation (MDA) were increased by 1.62-, 1.87- and 1.46-fold, respectively]. Moreover, we also found that the midgut cells were disintegrated, microvilli disappeared, the stroma became thinner, and the chromatin of nucleus became aggregated after CAP exposure by the analysis of transmission electron microscopy (TEM). In addition, the activities of digestive enzymes were dysregulated in midgut (the activities of α-amylase and trypsin were decreased 0.69- and 0.20-fold, respectively). Furthermore, digital gene expression (DGE) profiling analysis revealed that the expressions of oxidative phosphorylation pathway and antioxidant defense system related genes in midgut were decreased, indicating that it was the oxidative damage in midgut caused by CAP that mainly affected the growth of silkworm, rather than the toxicological effects of CAP. Collectively, this study provided valuable insights into the toxic effects of CAP on insects.

Background: Neonicotinoid insecticides are widely used in the prevention and control of pests in agriculture and forestry, but they can also affect the development of nontarget economic insects. In order to determine the effects of trace acetamiprid on the development of reproductive system of silkworm, we studied the gonad development and endogenous hormone metabolism in silkworms exposed to trace acetamiprid., Result: The silkworm showed mild poisoning symptoms after being exposed to trace acetamiprid (0.01 mg L -1 ). After 96 h of acetamiprid exposure, the body weight of silkworm was decreased by 7.67% and the ovary and fallopian tube were abnormally developed. The egg production in adults was decreased by 197 ± 29, and the egg weight was decreased by 0.52 ± 0.01 mg. Quantitative RT-PCR analysis showed that the relative expression levels of ovarian development-related genes Vg, Ovo, Otu, Sxl-S and Sxl-L were downregulated by 0.71, 0.77, 0.47, 0.67 and 0.88-fold, respectively. The transcriptional expression of ecdysone metabolism-related gene (EcR) in the ovary was downregulated by 0.46-fold while the expression of juvenile hormone-related gene (JHBP2) was upregulated by 1.36-fold. In hemolymph of acetamiprid exposed larvae, the content of ecdysone was reduced whereas the content of juvenile hormone was increased., Conclusion: Trace acetamiprid may cause reproductive dysfunction in silkworms through regulating the metabolism of endogenous hormones. Our study provides a reference for elucidating the mechanism of acetamiprid-induced reproductive disorders in insects. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Acetamiprid is widely used for agricultural pest control. However, it remains poorly understood whether the environmental residues of acetamiprid have the potential effects on economic insect. In this study, we evaluated the effects of acetamiprid on silkworm growth and development. The exposure to trace amounts of acetamiprid significantly decreased body weight, viability, and spinning ability. In addition, the activity of trypsin in the midgut was decreased after exposure. DGE and KEGG pathway enrichment analysis revealed that the significantly differentially expressed genes were mainly involved in nutrient metabolism, stress responses, and inflammation pathways. These results, in combination with hematoxylin-eosin staining and transmission electron microscopy, indicated that acetamiprid could cause oxidative damage to midgut, lead to inflammatory responses, and affect the activities of midgut digestive enzymes, thus resulting in abnormal growth and development. Our findings greatly contributed to the evaluation of the effects of acetamiprid residues on other nontarget beneficial insect., (© 2019 Wiley Periodicals, Inc.)