Fa 45 24 11

The levels of volatile fatty acids present in the supernatant of both co-cultures and monocultures were measured using an Agilent1260 Infinity HPLC (Agilent) system. The samples were prepared by acidifying to 5 mM, using sulfuric acid, and then incubating at room temperature for 5 min. Samples were then centrifuged for 5 min at 21,000 g. The supernatant was syringe filtered into an HPLC vial (Eppendorf FA-45–24–11) using a 0.22-µm polyvinylidene difluoride (PVDF) filter. The samples were analyzed using an Agilent 1260 Infinity HPLC (Agilent) system equipped with an autosampler unit (1260 ALS). The separation of formate, acetate, glucose, and lactate was carried out using a Bio-Rad Aminex^® 87H Ion Exclusion Column for organic acids (Part No. 1250140; Bio-Rad Laboratories, Inc., Hercules, CA, USA), with a mobile phase of 5 mM sulfuric acid. In-house standards were prepared with MC-blank culture medium as a base and sodium formate (ACS Grade, Fisher Chemical S648500), sodium acetate (ACS Grade, Fisher Chemical S210500), and L-lactic acid sodium (99%, extra pure; Acros Organics, 439220100), and D-(+)-glucose (Sigma-Aldrich Cat. No. G8270) at concentrations of 0.1 g/L and 1 g/L.

Levels of volatile fatty acids present in the supernatant of both co-cultures and monocultures were measured using an Agilent1260 Infinity HPLC (Agilent). Samples were prepared by acidifying to 5 mM using sulfuric acid and subsequently incubating at room temperature for 5 min. Samples were then centrifuged for 5 min at 21,000 g. The supernatant was syringe filtered into an HPLC vial (Eppendorf FA-45-24-11) using a 0.22 µm PVDF filter. Samples were analyzed on an Agilent 1260 Infinity high-performance liquid chromatography system (HPLC, Agilent, Santa Clara, CA) equipped with an auto-sampler unit (1260 ALS). Separation of formate, acetate, and lactate was achieved with a Bio-Rad Aminex^® 87H Ion Exclusion Column for organic acids (Part No. 1250140, Bio-Rad, Hercules, CA) with a mobile phase of 5 mM sulfuric acid. In-house standards were prepared with MC- blank culture medium as a base and sodium formate (ACS Grade, Fisher Chemical S648500), sodium acetate (ACS Grade, Fisher Chemical S210500), and L-lactic acid sodium (99%, extra pure, Acros Organics 439,220,100) at VFA concentrations of 0.1 and 1 g/L.

Flies were homogenized using Cell Lysis Buffer (25 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1× liquid protease inhibitor [Gen DEPOT], 0.1 M Dithiothreitol (DTT)). The supernatant was collected after centrifugation at 13,000 rpm for 10 min at 4°C (Eppendorf 5424R with rotor Eppendorf FA-45-24-11). The supernatant was mixed with Laemmli Buffer containing β-mercaptoethanol and heated at 95°C for 10 min. Subsequently, the samples were loaded in 4–20% gradient polyacrylamide gels (BioRad Mini-PROTEAN TGX). Following electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon, Sigma). The membrane was blocked using skimmed milk and treated with the primary antibody for overnight. The following antibodies were used in the present study: rabbit anti-GFP (1:1000) (Thermo Fisher Scientific, #A-11122), mouse anti-Actin (1:5000) (EMD Millipore, #MAB1501). Horseradish peroxidase-conjugated secondary antibody was used to detect the respective primary antibody. Blots were imaged on a BioRad ChemiDocMP.

The fat body, hemolymph, midgut, muscle, and defensive glands were collected from B. rhynchopetera in Yunnan Province. One portion was stored in 4% paraformaldehyde (pH 7.4) for IHC analysis. The other portion was placed in radioimmunoprecipitation (RIPA) buffer with 1% protease inhibitors and 1% phosphatase inhibitors, ground with a grinder (Servicebio, Wuhan, China), placed in an ice bath for 1 h, and then centrifuged at low temperature for 30 min (Eppendorf, Germany; model FA-45-24-11, 4°C, 1,000 × g). Protein concentrations of the supernatant of different tissues were determined using the bicinchoninic acid (BCA) method. The samples were diluted to the same level for ELISA analysis.