Methoxylamine hydrochloride

First grade methanol, hexane, water, D₂O [99.%, containing 0.05% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) as internal standard for NMR], methoxylamine hydrochloride, pyridine, L-ascorbic acid, 1,1-diphenyl-2-picrylhydrzyl (DPPH), Folin-Ciocalteu's phenol reagent, gallic acid, and sodium carbonate were purchased from Sigma (St. Louis, MO, USA). NaOD were obtained from Cortec (Paris, France). BSTFA [N,O-bis (trimethylsilyl) trifluoroacetamide containing 1% TMCS (trimethyl chlorosilane)] were purchased from Alfa Aesar (Ward Hill, MA, USA) and 2-Chloronaphtahalene as internal standard for GC-MS were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan).

Silkworms, strain Dazao (P50), were reared on fresh mulberry leaves at 26 ± 1 °C and 70% ± 10% relative humidity. Larval heads were dissected on the third day of the fifth instar and immediately stored at −80 °C. Each group (with 3 biological replicates) containing 30 heads was homogenized in a mortar with liquid nitrogen. Powdered samples (100 mg) from each group were suspended with a 500 μL methanol-water (v/v, 4:1) mixture and sonicated for 20 min, followed by incubation on ice for 40 min. Thereafter, the samples were centrifuged at 15,000× g for 30 min. The supernatant was transferred to a centrifuge tube and lyophilized for 5 h in a vacuum concentrator [10 (link)]. The lyophilized samples were then re-dissolved in 65 μL pyridine (containing 15 mg/mL methoxylamine hydrochloride; Sigma-Aldrich, St. Louis, MO, USA) for GC-MS analysis or in a 80 μL methanol-water (v/v, 4:1) mixture for LC-MS/MS analysis.

BBR, γ-oryzanol, pyridoxine (vitamin B₆), urease, pyridine, methoxylamine hydrochloride, L-phenylalanine-¹³C₉-¹⁵N, dulcitol, L-leucine-¹³C₆, L-isoleucine-¹³C₆-¹⁵N, L-valine-¹³C₅-¹⁵N, L-alanine-¹³C₃-¹⁵N and N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1 % trimethylchlorosflane (TMCS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pure water was obtained from Millipore Alpha-Q water system (Bedford, MA, USA). Methanol and ethanol for HPLC grade were purchased from Merck Chemicals (Darmstadt, Germany). Chloroform for analytical grade from Sinopharm Chemical Reagent Company (Shanghai, China). KAPA HiFi polymerase (KAPA Biosystems, USA), Agencourt AMPure XP beads (Beckman Coulter, USA), Qubit dsDNA HS assay kit (Invitrogen, CA), Agilent high sensitivity DNA Kit (Agilent, USA), Ion PGM template OT2 200 kit, Ion PGM sequencing 200 kit v2 and Ion 316 chip kit v2 (Life Technology, CA) and QIAamp^® Fast DNA stool mini kit (Qiagen, Germany) were obtain for fecal microbiota analysis.

N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) of analytical grade was from Regis (Morton Grove, IL, USA). Methanol and chloroform of analytical grade were purchased from CNW Technologies GmbH (Düsseldorf, Germany). Methoxylamine hydrochloride and anhydrous pyridine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Commercial kit used for determining adrenocorticotropic hormone (ACTH) was obtained from Phoenix Pharmaceuticals (Burlingame, CA, USA). Commercial kit used for determining thyroid-stimulating hormone (TSH) was purchased from R&D Systems (Minneapolis, USA). Commercial kit used for determining corticosterone (CORT) was obtained from Cayman Chemical Co. Ltd (MI, USA). Commercial kits used for determining, catecholamine (CA), testosterone, and serotonin were purchased from Cusabio Biotech Co. Ltd, (Wuhan, China). Total ginsenosides (GS, purity ≥ 80%, UV method) extracted from the roots of P. ginseng were purchased from Nanjing Zelang Biomedical Technology Co. Ltd. (Nanjing, China).

All the chemicals and reagents used in the study of LP toxicity to C. acutatum were of the highest commercially available grade. The extraction of the fungal metabolome for GC/EI/MS analyses was performed using ethyl acetate (EtOAc) and methanol (MeOH) (GC/MS grade, 99.9% purity, Carlo Erba Reagents, val de Reuil, France). For ¹H NMR metabolomics, metabolite extraction was performed using deuterium oxide (D₂O, 99.9%) containing 0.05 wt.% 3-(trimethylsilyl)propionic acid-2,2,3,3-d4-sodium salt (TSP, Sigma-Aldrich Ltd., Steinheim, Germany). Pyridine (99.8%, v/v), methoxylamine hydrochloride (98% w/w), and ribitol were purchased from Sigma-Aldrich Ltd. (Steinheim, Germany), and N-methyl-N-(trimethyl-silyl) trifluoroacetamide (MSTFA), used in sample preparation for GC/EI/MS metabolomics, was purchased from Macherey-Nagel (Düren, Germany).
The LP extract used in the study was obtained from liquid cultures of the isolate Bacillus sp. PTA13, as previously described [6 (link)]. Stock solutions of the LP extract (10,000 ppm) were prepared in methanol (MeOH) (GC/MS grade, 99.9% purity, Carlo Erba Reagents, val de Reuil, France) into 2 mL Eppendorf tubes, which were kept at −30 °C until further utilization.

CPT-11 (≥ 98%), L-(+)-lactic acid, D-sorbitol and CIF hydrochloride monohydrate were purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). Methoxylamine hydrochloride, 1,2-¹³C myristic acid and N,O-bis(trimethylsilyl)trifluoroacetamide were bought from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α, IL-1β, and IL-6 kits were purchased from Cell Signaling Technology (Beverly, MA, USA). The primers used for amplification were ordered in Sangon Biotech Co., Ltd. (Shanghai, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Jiangsu KeyGEN Bio-Tech Corp., Ltd (Nanjing, China). E.Z.N.A.® Stool DNA Kit was purchased from Omega Bio-Tek (Norcross, GA, USA). TransStart Fastpfu DNA Polymerase and AxyPrep DNA Gel Extraction Kits were purchased from Axygen Scientific Inc. (Silicon Valley, USA). QuantiFluor™-ST blue fluorescent quantitative system was purchased from Promega Biotech Co., Ltd. (Beijing, China). SDS-PAGE Gel Kit was purchased from Solarbio Life Sciences Co., Ltd. (Beijing, China).