Environmental concentrations of microplastic-induced gut microbiota and metabolite disruption in silkworm, Bombyx mori.

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 ß-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.

Proteotranscriptomic Integration analyses reveals new mechanistic insights regarding Bombyx mori fluorosis.

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.

Antifeedant and Antifungal Activities of Metabolites Isolated from the Coculture of Endophytic Fungus Aspergillus tubingensis S1120 with Red Ginseng.

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/cm2 . 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.

Molecular Characterization and Tissue Distribution of Calcineurin Regulatory B Subunit during the Prevention of Diapause in the Silkworm, Bombyx mori.

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 Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. 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 Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. 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 Ca2+ or efflux Ca2+ 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.

The effects of quercetin combined with nucleopolyhedrovirus on the growth and immune response in the silkworm (Bombyx mori).

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.

The plant-derived triterpenoid, cucurbitacin B, but not cucurbitacin E, inhibits the developmental transition associated with ecdysone biosynthesis in Drosophila melanogaster.

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.

The Antiviral Molecule 5-Pyridoxolactone Identified Post BmNPV Infection of the Silkworm, Bombyx mori.

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.

Transcriptome reveal the response to Cry1Ac toxin in susceptible Bombyx mori.

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.

Evaluation of pathogenicity and therapeutic effectiveness of antibiotics using silkworm Nocardia infection model.

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.

Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

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-ß-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.

An assessment of the reproductive toxicity of GONPs exposure to Bombyx mori.

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.

Antifeedant and antiphytopathogenic metabolites from co-culture of endophyte Irpex lacteus, phytopathogen Nigrospora oryzae, and entomopathogen Beauveria bassiana.

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/cm2. Compound 11 indicated notable antifeedant activity with inhibition percentage of 93% at concentration of 6.25 µg/cm2 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.

Effects of Quercetin on the Growth and Expression of Immune-Pathway-Related Genes in Silkworm (Lepidoptera: Bombycidae).

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.

Histological investigation of the effects of fenoxycarb on neurosecretory cells in the silkworm, Bombyx mori brain.

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.

Effect of pyriproxyfen exposure on cocooning and gene expression in the silk gland of Bombyx mori （Linnaeus, 1758）.

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.

Synthesis and antifungal activities of hydrophilic cationic polymers against Rhizoctonia solani.

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 (LC50) for zebrafish and silkworm and median lethal dose (LD50) 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 LD50 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 Ca2+ or/and Mg2+ 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.

Ingestion and effects of polystyrene nanoparticles in the silkworm Bombyx mori.

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.

Ecdysteroid ingestion suppresses carbohydrate hydrolysis in larvae of the silkworm Bombyx mori.

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.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

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.

Evaluation of the Metabolic Effects of Hydrogen Sulfide on the Development of Bombyx mori (Lepidoptera: Bombycidae), Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

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.