Aminoguanidine

We obtained grubs of the Japanese horned beetle, Trypoxylus dichotomus septentrionalis, at the third instar larval stage, from a local insect shop. Hemolymph from these grubs was obtained as previously described [22] (link), [23] (link) with minor modification. Briefly, phosphate buffer saline (PBS, 100 μL) was injected into the body of grubs, and the grubs were reared in leaf mold for 5 h. After surface sterilization and anesthesia on an ice pack, bleeding was done through the amputated prologs. Hemolymph was collected into tubes on ice and immediately heated at 95 °C for 5 min. The hemolymph fluids were subsequently centrifuged at 20,000×g for 10 min at 4 °C. The supernatant from this step was the source of the hemolymph samples used in the study. The hemolymph samples were stored at −80 °C, after addition of 20 μg/mL aprotinin (Roche, Penzberg, Germany), until use.
Aminoguanidine, glucose, amino acids, and bovine serum albumin (BSA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Maillard reaction products of amino acids or BSA were obtained by incubating 25 mg/mL amino acid or BSA with 125 mg/mL glucose in water at 70 °C for 17 h. Inhibition of the Maillard reaction was performed by the addition of 230 mM Aminoguanidine into the reaction solution.