We integrated field and laboratory studies in an investigation of water balance, energy use, and mechanisms of cold-hardiness in hatchling painted turtles (Chrysemys picta) indigenous to west-central Nebraska (Chrysemys picta bellii) and northern Indiana (Chrysemys picta marginata) during the winters of 1999-2000 and 2000-2001. We examined 184 nests, 80 of which provided the hatchlings (n=580) and/or samples of soil used in laboratory analyses. Whereas winter 1999-2000 was relatively dry and mild, the following winter was wet and cold; serendipitously, the contrast illuminated a marked plasticity in physiological response to environmental stress. Physiological and cold-hardiness responses of turtles also varied between study locales, largely owing to differences in precipitation and edaphics and the lower prevailing and minimum nest temperatures (to -13.2 degrees C) encountered by Nebraska turtles. In Nebraska, winter mortality occurred within 12.5% (1999-2000) and 42.3% (2000-2001) of the sampled nests; no turtles died in the Indiana nests. Laboratory studies of the mechanisms of cold-hardiness used by hatchling C. picta showed that resistance to inoculative freezing and capacity for freeze tolerance increased as winter approached. However, the level of inoculation resistance strongly depended on the physical characteristics of nest soil, as well as its moisture content, which varied seasonally. Risk of inoculative freezing (and mortality) was greatest in midwinter when nest temperatures were lowest and soil moisture and activity of constituent organic ice nuclei were highest. Water balance in overwintering hatchlings was closely linked to dynamics of precipitation and soil moisture, whereas energy use and the size of the energy reserve available to hatchlings in spring depended on the winter thermal regime. Acute chilling resulted in hyperglycemia and hyperlactemia, which persisted throughout winter; this response may be cryoprotective. Some physiological characteristics and cold-hardiness attributes varied between years, between study sites, among nests at the same site, and among siblings sharing nests. Such variation may reflect adaptive phenotypic plasticity, maternal or paternal influence on an individual's response to environmental challenge, or a combination of these factors. Some evidence suggests that life-history traits, such as clutch size and body size, have been shaped by constraints imposed by the harsh winter environment.

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Freezing survival in hatchling turtles may be limited by ischemic anoxia in frozen tissues and the associated accumulation of lactate and reactive oxygen species (ROS). To determine whether mechanisms for coping with anoxia are also important in freeze tolerance, we examined the association between capacities for freezing survival and anoxia tolerance in hatchlings of seven species of turtles. Tolerance to freezing (-2.5 degrees C) was high in Emydoidea blandingii, Chrysemys picta, Terrapene ornata, and Malaclemys terrapin and low in Graptemys geographica, Chelydra serpentina, and Trachemys scripta. Hatchlings survived in a N(2) atmosphere at 4 degrees C for periods ranging from 17 d (M. terrapin) to 50 d (G. geographica), but survival time was not associated with freeze tolerance. Lactate accumulated during both stresses, but plasma levels in frozen/thawed turtles were well below those found in anoxia-exposed animals. Activity of the antioxidant enzyme catalase in liver increased markedly with anoxia exposure in most species, but increased with freezing/thawing only in species with low freeze tolerance. Our results suggest that whereas oxygen deprivation occurs during somatic freezing, freeze tolerance is not limited by anoxia tolerance in hatchling turtles.