Freezing Survival by Isolated Malpighian Tubules of the New Zealand Alpine Weta Hemideina Maori Available to Purchase

The ability of isolated Malpighian tubules from a freeze-tolerant insect, the New Zealand alpine weta (Hemideina maori), to withstand freezing was assessed by measuring post-freeze membrane potentials and rates of fluid secretion. The hemolymph of cold-acclimated Hemideina maori was found to contain relatively high concentrations of the cryoprotectants trehalose (>300 mmol l⁻¹) and proline (41 mmol l⁻¹). Survival of isolated Malpighian tubules was correspondingly high when a high concentration of trehalose was present in the bathing saline. Tubules allowed to recover for 20 min from a 1 h freeze to −5°C in saline containing 400 mmol l⁻¹ trehalose had a basolateral membrane potential of −53 mV compared with a potential of −63 mV in tubules not exposed to a freeze/thaw cycle. Fluid secretion in tubules that had experienced a freeze/thaw cycle in saline containing 400 mmol l⁻¹ trehalose was 9.9±2.6 nl h⁻¹ compared with 18.7±5.0 nl h⁻¹ (means ± S.E.M., N=18) in tubules that had not been frozen. Tubules frozen in saline containing a lower concentration of trehalose (200 mmol l⁻¹) or in glucose (400 mmol l⁻¹) showed a similar ability to survive freezing to −5°C. In contrast, freezing for 1 h at −5°C in saline containing 400 mmol l⁻¹ sucrose produced a 57% decrease in membrane potential and an 88% decrease in secretion rate. Tubules held in saline lacking high concentrations of sugars showed no survival after freezing to −5°C for 1 h. When frozen to −15°C, tubules appeared to survive best in saline with the highest trehalose concentration (400 mmol l⁻¹). Freezing damage was not simply the result of exposure to cold, since tubules chilled (unfrozen) to −5°C for 1 h were not compromised even when the bathing saline lacked a high sugar concentration. Exposure of tubules to a combination of low temperature and high osmolality mimicked damage caused by actual freezing: the membrane potential showed a 60% recovery when the test was performed in saline containing trehalose, but showed no recovery in saline containing sucrose.