Carbopacma1

Hemolymph was collected after cutting the first abdominal prolegs of mutant L5 larvae. A few phenylthiourea (Sigma-Aldrich Korea, Seoul, South Korea) granules were added to the hemolymph sample to prevent coagulation. After centrifugation at 400 × g for 3 min, the supernatant plasma was diluted 20× with distilled water. The diluted plasma sample was cleaned with a Sep-Pak C18 cartridge (Walters Associates, Milford, MA, United States). Trehalose was identified and quantified using high-performance liquid chromatography (HPLC) (BioLC, Dionex, Sunnyvale, CA, United States) with the main column (CarboPacMA1, 4 × 250 mm, Dionex) and a guard column (CarboPacMA1, 4 × 50 mm, Dionex) following the method described by Park and Kim (2013) (link). The injection volume of the sample was 25 μL. NaOH (400 mM) was used as an elution buffer at a constant rate of.4 mL/min. Pulse amperometry mode of an electrochemical detector (ED40, Dionex) was used for detecting trehalose and other sugars.

Glycerol, glucose, fructose, mannose, myo‐inositol, and ribose were quantified in culture supernatant using an ion chromatograph ICS‐3000 system with a Dionex CarboPac MA1 (250 × 4.0 mm) column. Injection volume was 5 μL; eluents used were 100 mM NaOH and 5 mM NaOH. Lactate, acetate, formate and pyruvate were determined using the same HPLC system but fitted with a Dionex IonPac AS‐11‐HC column (250 × 4.0 mm) with a 5 μL injection volume and three eluents: 100 mM NaOH, 5 mM NaOH, and purified water. Each fermentation sample was measured once per method.

Alditols contents were determined by HPAEC. CCE powder (0.025 g) was dissolved in 100 mL distilled water and the solution was filtered through a 0.45-μm microporous membrane before ion chromatography determination using a CarboPac MA1 anion exchange column (4 mm × 250 mm, Dionex, USA). The injection volume was 25 μL, the mobile phase was 48-mmol/L NaOH, the flow rate was 0.4 mL/min, and the column temperature was 30°C.