Sucrose

To morphologically confirm the mitochondria of E. tenella, the sporozoites prepared as described above were fixed with 2% (w/v) glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% (w/v) paraformaldehyde (PFA, TAAB laboratories Equipment, Berks, UK) in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 2 h and kept at 4 °C overnight. The cells were washed with 0.1 M PB (pH 7.4) containing 0.1 M sucrose (Wako, Osaka, Japan), post-fixed with 1% (w/v) OsO4 in 0.1 M PB containing 0.1 M sucrose at 4 °C for 2 h, dehydrated in an ethanol series, and embedded in Epon resin (TAAB Laboratories Equipment). Ultra-thin sections were stained with saturated uranyl acetate (TAAB Laboratories Equipment) in distilled water for 20 min and Reynold’s lead citrate for 3 min and examined with an electron microscope (H-7500, Hitachi, Tokyo, Japan).

The natural sugars maltose (Wako, Osaka, Japan) and sucrose (Wako) were used for sweet taste solutions. Each compound was dissolved in deionized water to make 1, 3, 10, 30, and 100 mM solutions for the behavioral tests. To prevent potential osmotic effects, we used a maximum concentration of 100 mM. For the in vitro functional analysis, the compounds were dissolved in assay buffer (10 mM HEPES, 130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl₂, 1.2 mM MgCl₂, pH 7.4).

Cultured cells were scraped in ice-cold T20 buffer (20 mM Tris-HCl [pH7.4, Wako Pure Chemical Industries, Ltd., Osaka, Japan], 300 mM sucrose [Wako], and a proteinase inhibitor mixture, Complete [Roche Diagnostics K.K., Tokyo, Japan]) and sonicated using a probe sonicator (Ohtake Works, Tokyo, Japan). Frozen tissues (1–100 mg) were homogenized in ice-cold T100 buffer (100 mM Tris-HCl (pH7.4), 300 mM sucrose, and Complete) using a Physcotron homogenizer (Microtec Co. Ltd., Chiba, Japan). The homogenate was centrifuged at 800×g for 10 min. The supernatant was used for western blot analysis of MTP and PDI. The same supernatant was centrifuged at 100,000×g for 1 hr to obtain membrane fractions. The pellet was resuspended in TSE buffer (20 mM Tris-HCl [pH7.4], 300 mM sucrose, and 1 mM EDTA [Dojindo Laboratories, Kumamoto, Japan]). This membrane fraction was used for western blot analysis of LPCAT3 and the enzymatic assays. Protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA). Protein samples were snap frozen in liquid nitrogen and stored at −80°C until use.

Oocytes were fixed to observe cytochrome C oxidase (CCO) activity, as described in a previous report^{47 (link)}. Briefly, they were fixed in 2% (v/v) glutaraldehyde (TAAB Laboratories, Aldermaston, UK) and 0.15 M sucrose (Wako Chemicals) in 0.05 M PBS for 15 min at 4 °C. After rinsing in the same buffer, the oocytes were incubated in 1.4 mM 3,3′-diaminobenzidine (DAB) tetrahydrochloride (Merck Millipore Co.), 0.1% (w/v) cytochrome C type II (Sigma), 0.23 M sucrose, and 0.0002% catalase (w/v) (Merck Millipore Co.) in 0.05 M PBS for 3 h at 37 °C. Oocytes were then washed in PBS for 1 h. Subsequently, they were post-fixed in 1% (v/v) osmium tetroxide (TAAB Laboratories) for 2 h at 4 °C. Next, they were dehydrated in increasing concentrations of ethanol and embedded in epoxy resins. Ultrathin sections were stained with uranyl acetate and examined using TEM (JEM-1011; JEOL, Tokyo, Japan). The content of neutral lipids was estimated by calculating the surface ratio of neutral lipids per surface area of the oocyte excluding the surface area of the nucleus. The surface areas of each of neutral lipids, oocyte, and nucleus were measured using Digital Micrograph version 3.7.4 (Gatan, Pleasanton, CA, USA)^{48 (link)}. In this study, a total of five oocytes were examined in each treatment.

Pectinex Ultra SP-L, a commercial pectinolytic and cellulolytic enzyme derived from Aspergillus aculeatus, was purchased from Novozymes (Bagsværd, Denmark). Commercial brown sugar and white sugar derived from sugar canes were used as substrates in this study. 3,5-dinitrosalicylic acid (DNS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard carbohydrates including 1-kestose, nystose, 1^F-β-fructofuranosylnystose, sucrose, glucose and fructose were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Deionized water was purified using an Ultrapure Water System RFU424CA (Advantec, Tokyo, Japan). LC-MS grade of methanol, acetonitrile, formic acid, dehydrated pyridine, sucrose, and acarbose were purchased from Fujifilm Wako Pure Chemical Corporation, Osaka, Japan. Methoxyamine hydrochloride was obtained from Aldrich (St Louis, Mo., USA). N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) + 1% trimethylchlorosilane (TMCS) was purchased from Thermo Scientific, Rockford, IL, USA. C₈–C₂₀ alkane analytical standard solution containing ~40 mg/L of each C₈–C₂₀ alkane in hexane was obtained from Fluka (Menlo Park, CA, USA). Triterpenoid standards of ganoderic acid A, ganoderic acid B, ganoderenic acid C, ganoderic acid C1, ganoderic acid C2, ganoderic acid C6, ganoderenic acid D, ganoderic acid I, and ganoderic acid K were purchased from Chem Faces Co., Ltd. (Wuhan, China). Ultrol grade HEPES was purchased from Calbiochem (San Diego, CA, USA). α-Glucosidase derived from yeast was obtained from Oriental Yeast Co. Ltd. (Tokyo, Japan).

Female mosquitoes Aedes albopictus were reared at 28 °C, 70% relative humidity with a photoperiod of 12 h light:12 h dark (lights on at 8 AM). Eggs of Aedes albopictus were purchased from Sumika Technoservice Corporation and allowed to hatch in deoxygenated, deionized water. Larvae were fed TetraMin Baby (Tetra). Pupae were placed in a small cup of deionized water and moved to a 30 cm × 30 cm × 30 cm-insect cage (BugDorm-1, Mega View Science Co., Ltd), and allowed to eclose. Adult mosquitoes were mated in the insect cage for at least 1 week. Mosquitoes were provided with unlimited access to 10 wt% sucrose (Wako chemicals) solution. Experiments were performed during 10 am–5 pm using mosquitoes 14–21 days after eclosion.