Purification and partial characterization of thermal hysteresis proteins from overwintering larvae of pine needle gall midge,Thecodiplosis japonensis (Diptera: cecidomiidae)

The pine needle gall midge of Thecodiplosis japonensis is a serious forest pest and overwinters as a 3rd instar larva at soil surface in Korea. The time necessary for killing 50% of larvae at -15 degree C is 160 min. During overwintering period, T. japonensis larvae accumulate relatively high content of trehalose as the main cryoprotectant. In this paper, the proteinaceous cryoprotectants were identified. Two thermal hysteresis proteins (THP-1S and 2S) were purified from overwintering larvae by ethanol fractionation, trichloroacetic acid precipitation, ion-exchange chromatography (DEAE-Sephadex A-25) and gel permeation chromatography (Sephadex G-100). Their molecular weights are 34.9 and 37.8 kD respectively. T. japonensis THPs cannot be stained by periodic acid-Schiffs' reagent, suggesting no carbohydrate in them. The thermal hysteresis activity of THP-2 at the concentration of 50 mg/ml is 11.02 +/- 0.08 degree C (mean +/- SD, n=10), perhaps the highest active insect THP. It is the first report of purified T. japonensis THPs in Diptera.