For positive controls, salivary glands were dissected from a laboratory colony of Reticulitermes speratus (Blattodea: Rhinotermitidae). The insects were dissected and their fore- and midguts were retained for proteomics analysis. Gut volumes of walking sticks and termites were measured as per Fujita et al. (2010). Midguts were divided into even thirds to analyze the different sections separately. Genetic analysis was performed on salivary glands from E. calcarata and from

adults of fresh E. okinawaensis, lab-reared on Rubus sp. at the National Institute of Agrobiological Sciences (Tsukuba, Ibaraki, selleckchem Japan). Fresh or rehydrated (for acetone-preserved specimens) Dasatinib supplier fore- and midguts with contents were homogenized on ice in 50 mM sodium acetate buffer (pH 5.5) with a single proteinase inhibitor cocktail tablet (Complete Mini, EDTA-free, Rosche Diagnosis GmbH, Nannheim, Germany) and 1% Triton X-100, then centrifuged at 20,000×g for 10 min. Samples that were not immediately used were stored in a 50 mM sodium acetate, 1 M NaCl, and 20% glycerine buffer solution and frozen. For hydrophobic interaction chromatography, the supernatant of the homogenate was precipitated with four volume of cold acetone and pelleted by centrifugation at 10,000×g for 10 min. The pellet was dried and rehydrated with

1 M ammonium sulfate with 20 mM Tris–HCl buffer (pH 7.6) (loading buffer), then applied to a HiTrap Glutamate dehydrogenase Phenyl FF (high sub) column

(GE Healthcare Life Sciences®) equilibrated with loading buffer. Ten microliters of diluted sample (1/100) were mixed with 100 μL of 1% CMC (Aldrich Chemical Company) in 100 mM sodium acetate buffer (pH 5.5), vortexed, and incubated at 37 °C for 13 minutes. To stop the reaction, 0.8 mL of tetrazolium blue (TZB) reagent was added and the mixture was boiled for 5 min (Jue and Lipke, 1985). Controls without enzyme, without substrate, and of just MilliQ water and 0.5 mM glucose were used (Calderón-Cortés et al., 2010). Absorbance at 660 nm was measured using a Pharmacia Biotec® Ultrospec 2000 spectrophotometer and reducing sugar concentration was calculated by comparison with the glucose solution. EG activities of the termite midguts were calculated from the body weights of workers and previously reported values (Tokuda et al., 2004 and Tokuda et al., 2005). For EG purification, a HiTrap Phenyl FF high-sub column was employed. After the protein-loaded column was washed with 10 mL of loading buffer, proteins absorbed on the column were eluted by stepwise concentrations of ammonium sulfate (0.35 M for 20 mL and 0 M for 24 mL) in 20 mM Tris–HCl buffer (pH 7.5). Chromatography was conducted with a BioLogic DuoFlow™ chromatography system (Bio-Rad®).