Freezing survival by isolated Malpighian tubules of the New Zealand alpine weta Hemideina maori

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 degrees 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 degrees C. In contrast, freezing for 1 h at -5 degrees 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 degrees C for 1 h. When frozen to -15 degrees 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 degrees 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.