Jon P. Costanzo

Cold Hardiness and Overwintering Strategies of Hatchlings in an Assemblage of Northern Turtles

Field and laboratory studies were conducted during 1989-1994 to investigate the overwintering strategies of hatchling turtles representing four families native to western Nebraska. Whereas hatchling snapping turtles (Chelydra serpentine) and spiny soft-shelled turtles (Apalone spinifera) overwinter in aquatic habitats, yellow mud turtles (Kinosternon flavescens) and ornate box turtles (Terrapene ornata) burrow below the natal nest and hibernate in sandy soil. Painted turtles (Chrysemys picta) overwinter within their shallow natal nests, but this species, and T. ornata, tolerate extensive tissue freezing. Overwintering behaviors of these species are consistent with indices of physiological cold hardiness and patterns of geographic distribution. Frost commonly penetrated and persisted below 10 cm, the soil depth at which hatchling C. picta routinely hibernate. Field and laboratory data suggested that hatchling C. picta survive either by remaining supercooled (unfrozen) or by tolerating tissue freezing, the strategy employed depending on prevailing physiological and microenvironmental conditions. Whereas relatively lower temperatures can be survived in the supercooled state, supercooling capacity may be limited via the inoculation of body fluids by environmental ice. Alternatively, whereas freeze tolerance fortuitously is promoted by ice inoculation, this strategy may be viable only at relatively high subzero temperatures. A cold-hardiness strategy based on both survival mechanisms may promote winter survival in hatchling C. picta by conferring protection under dynamic physiological and microen- vironmental conditions. Physiological cold hardiness and behavior are integrated deter- minants of the northern distributions of temperate region turtles.

Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms

Abstract In this paper we present the results of physiological responses to winter acclimation and tissue freezing in a freeze-tolerant Siberian earthworm, Eisenia nordenskioeldi, and two freeze-intolerant, temperate earthworm species, Lumbricus rubellus and Aporrectodea caliginosa. By analysing the physiological responses to freezing of both types we sought to identify some key factors promoting freeze tolerance in earthworms. Winter acclimation was followed by a significant increase in osmolality of body fluids in E. nordenskioeldi, from 197 mosmol kg−1 in 10 °C-acclimated animals to 365 mosmol kg−1 in animals acclimated to 0 °C. Cold acclimation did not cause any change in body fluid osmolality in the two freeze-intolerant species. As a response to ice formation in the body, the freeze-intolerant species produced copious amounts of slime and expulsion of coelomic fluids, and thereby lost 10–30% of their total water content. Contrary to this, the freeze-tolerant species did not lose water upon freezing. At temperatures down to −6.5 °C, the ice content in the freeze-tolerant E. nordenskioeldi was significantly lower than in L. rubellus. At lower temperatures there were no differences in ice content between the two species. Cold acclimated, but unfrozen, specimens of all three species had low levels of ammonia, urea, lactate, glycerol and glucose. As a response to ice formation, glucose levels significantly increased within the first 24 h of freezing. This was most pronounced in E. nordenskioeldi where a 153-fold increase of glucose was seen (94 mmol · l−1). In L. rubellus and A. caliginosa a 19-fold and 17-fold increase in glucose was seen. This is the first study on physiological mechanisms promoting freeze tolerance in E. nordenskioeldi, or any other oligochaete. Our results suggest that the cryoprotective system of this species more closely resembles that of freeze-tolerant anurans, which synthesize cryoprotectants only after tissues begin to freeze, than that of cold-hardy invertebrates which exhibit a preparatory accumulation of cryoprotectants during seasonal exposure to low temperature.

Physiological ecology of overwintering in hatchling turtles

Temperate species of turtles hatch from eggs in late summer. The hatchlings of some species leave their natal nest to hibernate elsewhere on land or under water, whereas others usually remain inside the nest until spring; thus, post-hatching behavior strongly influences the hibernation ecology and physiology of this age class. Little is known about the habitats of and environmental conditions affecting aquatic hibernators, although laboratory studies suggest that chronically hypoxic sites are inhospitable to hatchlings. Field biologists have long been intrigued by the environmental conditions survived by hatchlings using terrestrial hibernacula, especially nests that ultimately serve as winter refugia. Hatchlings are unable to feed, although as metabolism is greatly reduced in hibernation, they are not at risk of starvation. Dehydration and injury from cold are more formidable challenges. Differential tolerances to these stressors may explain variation in hatchling overwintering habits among turtle taxa. Much study has been devoted to the cold-hardiness adaptations exhibited by terrestrial hibernators. All tolerate a degree of chilling, but survival of frost exposure depends on either freeze avoidance through supercooling or freeze tolerance. Freeze avoidance is promoted by behavioral, anatomical, and physiological features that minimize risk of inoculation by ice and ice-nucleating agents. Freeze tolerance is promoted by a complex suite of molecular, biochemical, and physiological responses enabling certain organisms to survive the freezing and thawing of extracellular fluids. Some species apparently can switch between freeze avoidance or freeze tolerance, the mode utilized in a particular instance of chilling depending on prevailing physiological and environmental conditions.

Dynamics of body water during freezing and thawing in a freeze-tolerant frog (Rana sylvatica)

Abstract 1. 1.|Calorimetric analyses showed that wood frogs ( Rana sylvatica ) frozen to −2.5°C contained 7.8 g ice, as 65.4% of the body water had frozen. 2. 2.|During 24 h of freezing, water content decreased in liver (58.9%), intestine (58.6%) and skeletal muscle (22–36%). Complete rehydration during thawing at 3.5°C required from 3 to >;48 h, depending on the organ. 3. 3.|Because organs dehydrate, increases in tissue metabolite concentrations associated with freezing, if calculated on a per wet-weight basis, may be greatly exaggerated. 4. 4.|Reversible organ dehydration during freezing may enhance freeze tolerance of R. sylvatica by concentrating cryoprotectant and reducing cryoinjury to tissues.

Avoidance and tolerance of freezing in ectothermic vertebrates.

Summary Ectothermic vertebrates have colonized regions that are seasonally or perpetually cold, and some species, particularly terrestrial hibernators, must cope with temperatures that fall substantially below 0°C. Survival of such excursions depends on either freeze avoidance through supercooling or freeze tolerance. Supercooling, a metastable state in which body fluids remain liquid below the equilibrium freezing/melting point, is promoted by physiological responses that protect against chilling injury and by anatomical and behavioral traits that limit risk of inoculative freezing by environmental ice and ice-nucleating agents. Freeze tolerance evolved from responses to fundamental stresses to permit survival of the freezing of a substantial amount of body water under thermal and temporal conditions of ecological relevance. Survival of freezing is promoted by a complex suite of molecular, biochemical and physiological responses that limit cell death from excessive shrinkage, damage to macromolecules and membranes, metabolic perturbation and oxidative stress. Although freeze avoidance and freeze tolerance generally are mutually exclusive strategies, a few species can switch between them, the mode used in a particular instance of chilling depending on prevailing physiological and environmental conditions.

Influence of soil hydric parameters on the winter cold hardiness of a burrowing beetle, Leptinotarsa decemlineata (Say)

This investigation examined the influence of soil moisture and associated parameters on the cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a temperate-zone species that overwinters in terrestrial burrows. The body mass and water content of adult beetles kept in sand at 4 °C varied over a 16-week period of diapause according to the substratums moisture content. Changes in body water content, in turn, influenced the crystallization temperature (range −3.3 to −18.4 °C; n = 417), indicating that environmental moisture indirectly determined supercooling capacity, a measure of physiological cold hardiness. Beetles held in dry sand readily tolerated a 24-h exposure to temperatures ranging from 0° to −5 °C, but those chilled in sand containing as little as 1.7% water (dry mass) had elevated mortality. Thus, burrowing in dry soils not only promotes supercooling via its effect on water balance, but may also inhibit inoculative freezing. Mortality of beetles exposed to −5 °C for 24 h was lower in substrates composed of sand, clay and/or peat (36–52%) than in pure silica sand (78%) having an identical water content (17.0% dry mass). In addition to moisture, the texture, structure, water potential, and other physico-chemical attributes of soil may strongly influence the cold hardiness and overwintering survival of burrowing insects.

Survival mechanisms of vertebrate ectotherms at subfreezing temperatures: applications in cryomedicine.

Various marine fishes, amphibians, and reptiles survive at temperatures several degrees below the freezing point of their body fluids by virtue of adaptive mechanisms that promote freeze avoidance or freeze tolerance. Freezing is avoided by a colligative depression of the blood freezing point, supercooling of the body fluids, or the biosynthesis of unique antifreeze proteins that inhibit the propagation of ice within body fluids. Conversely, freeze tolerance is an adaptation for the survival of tissue freezing under ecologically relevant thermal and temporal conditions that is conferred by the biosynthesis of permeating carbohydrate cryoprotectants and an extensive dehydration of tissues and organs. Such cryoprotective responses, invoked by the onset of freezing, mitigate the osmotic stress associated with freeze‐concentration of cytoplasm, attendant metabolic perturbations, and physical damage. Cryomedical research has historically relied on mammalian models for experimentation even though endotherms do not naturally experience subfreezing temperatures. Some vertebrate ectotherms have “solved” not only the problem of freezing individual tissues and organs, but also that of simultaneously freezing all organ systems. An emerging paradigm in cryomedicine is the application of principles governing natural cold hardiness to the development of protocols for the cryopreservation of mammalian tissues and organs.—Costanzo, J. P., Lee, R. E., Jr., DeVries, A. L., Wang, T., Layne, J. R., Jr. Survival mechanisms of vertebrate ectotherms at subfreezing temperatures: applications in cryomedicine. FASEB J. 9, 351–352 (1995)

Supercooling, ice inoculation and freeze tolerance in the European common lizard, Lacerta vivipara

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter subzero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5°C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0°C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (≤0.1°C·h-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.

Physiological Ecology of Overwintering in the Hatchling Painted Turtle: Multiple‐Scale Variation in Response to Environmental Stress

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°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.

A simple moder for estimating the ice content of freezing ectotherms

Abstract 1. 1.Although body ice content is an important variable affecting freeze tolerance, present calorimetric methods for its measurement necessarily require the termination of a freezing protocol. 2. 2.A simple iterative model, based on the colligative properties of solutions and requiring precise measurements of only equilibrium freezing point (of the unfrozen organism) and of core body temperature, allows estimation of the percentage of body water frozen at any time during a freezing episode. 3. 3.This model can also predict the lethal temperature for a freezing ectotherm, assuming that death occurs due to osmotic dehydration when 67% (of any other known lethal fraction) of the body water is frozen. 4. 4.The basic model is easily extended to evaluate the effects of variables such as: body mass, initial body water content, initial osmotic concentration, and test chamber microenvironment. 5. 5.This model is not intended to supplant existing more exact biophysical models of freezing kinetics. Rather it is proposed as a first approximation which is generally supported by published data and which should be of significant practical value for investigators of freeze tolerant organisms.