5 CryoLetters 23, 5-10 (2002)  CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK FREEZING RATE AFFECTS THE SURVIVAL OF A SHORT-TERM FREEZING STRESS IN Panagrolaimus davidi, AN ANTARCTIC NEMATODE THAT SURVIVES INTRACELLULAR FREEZING D.A. Wharton 1* , G. Goodall 1,2 and C.J. Marshall 2 Departments of Zoology 1 and Biochemistry 2 , University of Otago, P.O. Box 56, Dunedin, New Zealand. *Corresponding author, Email: david.wharton@stonebow.otago.ac.nz Abstract The ability of the Antarctic nematode Panagrolaimus davidi to survive a short-term freezing stress depended upon the rate of freezing of its surroundings, measured as the duration of the sample exotherm. The freezing rate increased as the sample volume and freezing temperature decreased and resulted in fewer nematodes surviving. This appears to be due to the greater risk of physical damage by ice crystal growth at high freezing rates. Once frozen the nematodes will then survive exposure to lower temperatures. The environment of the nematode is likely to produce the slow rate of freezing of its surroundings that is necessary for its survival. Keywords: Freeze tolerance, cooling rate, freezing rate, exotherm duration, intracellular freezing INTRODUCTION Panagrolaimus davidi is a small (up to 1 mm long), free-living Antarctic nematode that survives extensive intracellular freezing (17). Inoculative freezing occurs via body openings and the ice spreads rapidly to all parts of the body. The freezing tolerance of this nematode has two components (21). Nematodes grown at 20°C will survive a short-term, but not a long- term, freezing stress whilst those acclimated at lower temperatures will survive a long-term stress. Survival of a long-term freezing stress may be related to the production of a protein that inhibits recrystallisation (11). In this paper we investigate the effect of freezing rate on the nematode’s ability to survive a short-term freezing stress. Baust & Rojas (2), in their review of insect cold hardiness, suggested that cooling rate is an important factor determining whether or not an insect survives freezing. However, most studies of insect cold tolerance have used a standard cooling rate of 1°C min –1 , following the suggestion of Salt (12), and there are relatively few studies that have investigated the effects of cooling rate on the freezing survival of animals. A cooling rate of 1°C min –1 is much faster than animals are likely to experience in nature. Sincla ir (14) measured a maximum cooling rate of 0.06°C min –1 in the alpine habitat of the freezing tolerant cockroach, Celatoblatta quinquemaculata as the temperature crossed biologically-relevant thresholds (0°C, and the temperatures at which 5% and 50% of the cockroaches would be expected to be frozen). Those studies that have investigated the effect of cooling rate on freezing tolerance (1, 8-10,