Lee, Richard

http://hdl.handle.net/2374.MIA/5808

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Cryoprotectants and extreme freeze tolerance in a subarctic population of the wood frog.
Costanzo, Jon P.; Reynolds, Alice M.; do Amaral, M. Clara F.; Rosendale, Andrew J.; Lee, Richard Lee
Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.
Compact genome of the Antarctic midge is likely an adaptation to an extreme environment
Kelley, Joanna L.; Peyton, Justin T.; Fiston-Lavier, Anna-Sophie; Teets, Nicholas M.; Muh-Ching, Yee; Johnston, J. Spencer; Bustamante, Carlos D.; Lee, Richard E.; Denlinger, David L.
The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment.
Seasonality of Freeze Tolerance in a Subarctic Population of the Wood Frog, Rana sylvatica
Costanzo, Jon P.; do Amaral, M. Clara F.; Rosendale, Andrew J.; Lee, Richard E.
We compared physiological characteristics and responses to experimental freezing and thawing in winter and spring samples of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA. Whereas winter frogs can survive freezing at temperatures at least as low as −16°C, the lower limit of tolerance for spring frogs was between −2.5°C and −5°C. Spring frogs had comparatively low levels of the urea in blood plasma, liver, heart, brain, and skeletal muscle, as well as a smaller hepatic reserve of glycogen, which is converted to glucose after freezing begins. Consequently, following freezing (−2.5°C, 48 h) tissue concentrations of these cryoprotective osmolytes were 44–88% lower than those measured in winter frogs. Spring frogs formed much more ice and incurred extensive cryohemolysis and lactate accrual, indicating that they had suffered marked cell damage and hypoxic stress during freezing. Multiple, interactive stresses, in addition to diminished cryoprotectant levels, contribute to the reduced capacity for freeze tolerance in posthibernal frogs.
Identification and expression of a putative facilitative urea transporter in three species of true frogs (Ranidae): implications for terrestrial adaptation.
Rosendale, Andrew J.; Costanzo, Jon P.; Kiss, Andor J.; Lee, Richard E.
Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrial R. sylvatica, intermediate in the semiaquatic R. pipiens, and quite low in the aquatic R. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments.
Enzymatic regulation of glycogenolysis in a subarctic population of the wood frog: implications for extreme freeze tolerance
do Amaral, M. Clara F.; Lee, Richard E.; Costanzo, Jon P.
The wood frog, Rana sylvatica, from Interior Alaska survives freezing at –16°C, a temperature 10–13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region (southern Ohio, USA). In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold) increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype’s exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.
Seasonal variation in the hepatoproteome of the dehydration- and freeze-tolerant wood frog, Rana sylvatica
Kiss, Andor J.; Muir, Tim J.; Lee, Richard E.; Costanzo, Jon P.
Winter’s advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation.
Brief chilling to subzero temperature increases cold hardiness in the hatchling painted turtle (Chrysemys picta)
Muir, Tim J.; Costanzo, Jon P.; Lee, Richard E.
Although many studies of ectothermic vertebrates have documented compensatory changes in cold hardiness associated with changes of season, much less attention has been paid to adjustment of physiological functions and survival limits following more acute exposure to cold. We investigated the ability of hatchling painted turtles (Chrysemys picta) to increase cold hardiness in response to brief exposure to a subzero temperature. Winter‐acclimated turtles were “cold conditioned” by chilling them in the supercooled (unfrozen) state to −7°C over a few days before returning them to 4°C. These turtles fared no better than control animals in resisting freezing when cooled in the presence or absence of ice and exogenous ice nuclei. Survival following tests of freeze tolerance (freezing for about 70 h; minimum body temperature, −3.75°C) was nominally higher in cold‐conditioned turtles than in controls (36% vs. 13%, respectively), although the difference was not statistically significant. Of the survivors, cold‐conditioned turtles apparently recovered sooner. Turtles subjected to cold shock (supercooling to −13°C for 24 h, followed by rewarming to 0°C) were strongly affected by cold conditioning: all controls died, but 50% of cold‐conditioned turtles survived. We investigated potential mechanisms underlying the response to cold conditioning by measuring changes in levels of putative cryoprotectants. Plasma levels of glucose and lactate, but not urea, were higher in cold‐conditioned turtles than in controls, although the combined increase in these solutes was only 23 mmol L−1. Cold conditioning attenuated cold‐shock injury to brain cells, as assessed using a vital‐dye assay, suggesting a link between protection of the nervous system and cold hardiness at the organismal level.
Carrion - It's what's for dinner: Wolves reduce the impact of climate change.
Constible, Juanita M.; Sandro, Luke H.; Lee, Richard E.
Humans have viewed wolves as competitors, threats to personal safety, and symbols of evil throughout history. By the early part of the 20th century, grey wolves (Canis lupus) had been eradicated from 42% of their historic range in North America (Laliberte & Ripple, 2004). In Yellowstone National Park, grey wolves were hunted to local extinction by 1926, but were reintroduced in 1995 after a decades-long process involving biologists, politicians, ranchers and the general public. By the end of 2006, the wolf population in the park was at least 136 wolves in 13 packs (Smith et al., 2007). In this activity, high school students use mathematical models to explore how the presence of wolves buffers other carnivores and scavengers from the effects of climate change. By the end of the lesson, students should be able to: Define and give examples of keystone species, Demonstrate, using mathematical models, that ecosystems are more resilient to environmental change, when they contain a full complement of species, including top carnivores, Recognize that math is a vital tool in scientific investigations.
Winter biology & freeze tolerance in the goldenrod gall fly
Sandro, Luke H.; Lee, Richard E.
Birds migrate. Bears hibernate. Turtles and frogs retreat to the bottom of lakes. Most animals must avoid harsh winter conditions; few can survive freezing. Larvae of the goldenrod gall fly (Eurosta solidaginis), can survive freezing to -40°C or below. The study of survival at low temperature is called cryobiology. This article provides an introduction to the winter biology of this widely distributed and unusual species, and suggests classroom activities that illuminate principles of cryobiology through insect overwintering. A variety of opportunities for educational activities are found in the complex, yet easy-to-manipulate, trophic relationships between goldenrod plants, insects that induce gall formation, and the natural enemies of these gallmakers. Gall collection, measurement, and observation (exit holes, larval response, temperature, etc.) can help students develop scientific process skills including observation, classification, measurement, inference, prediction, control of experimental variables, and material manipulation (Peard, 1994). Galls can also be studied to learn about insect ovipositing behavior and plant responses to three types of gallmakers—each with its own distinct gall type (Newell, 1994). Likewise, classroom activities can focus on the collection and study of galls to discover principles of ecology and insect life cycles (Kahn, 1997). One aspect of goldenrod gallmakers that has received little attention in the science education literature is the winter biology of these unusual insects. In autumn, the overwintering larva enters a state of dormancy, called diapause, and gradually acquires the capacity to survive freezing to temperatures of -40°C and below (Baust & Lee, 1981). In contrast, a beetle larva and two parasitic wasps that also overwinter in goldenrod galls are intolerant of freezing and must avoid internal ice formation.
Survival and physiological responses of hatchling Blanding's turtles (Emydoidea blandingii) to submergence in normoxic and hypoxic water under simulated winter conditions.
Dinkelacker, Stephen A.; Costanzo, Jon P.; Iverson, John B.; Lee, Richard E.
Abstract Overwintering habits of hatchling Blanding’s turtles (Emydoidea blandingii) are unknown. To determine whether these turtles are able to survive winter in aquatic habitats, we submerged hatchlings in normoxic (155 mmHg Po2) and hypoxic (6 mmHg Po2) water at 4°C, recording survival times and measuring changes in key physiological variables. For comparison, we simultaneously studied hatchling softshell (Apalone spinifera) and snapping (Chelydra serpentina) turtles, which are known to overwinter in aquatic habitats. In normoxic water, C. serpentina and A. spinifera survived to the termination of the experiment (76 and 77 d, respectively). Approximately one‐third of the E. blandingii died during 75 d of normoxic submergence, but the cause of mortality was unclear. In hypoxic water, average survival times were 6 d for A. spinifera, 13 d for E. blandingii, and 19 d for C. serpentina. Mortality during hypoxic submergence was probably caused by metabolic acidosis, which resulted from accumulated lactate. Unlike the case with adult turtles, our hatchlings did not increase plasma calcium and magnesium, nor did they sequester lactate within the shell. Our results suggest that hatchling E. blandingii are not particularly well suited to hibernation in hypoxic aquatic habitats.
Physiological Ecology of Overwintering in the Hatchling Painted Turtle: Multiple-Scale Variation in response to Environmental Stress
Costanzo, Jon P.; Dinkelacker, Stephen A.; Iverson, John B.; Lee, Richard E.
We integrates 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 analysis. 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.
Postfreeze reduction of locomotor endurance in the freeze-tolerant wood frog, Rana sylvatica.
Irwin, Jason T.; Costanzo, Jon P.; Lee, Richard E.
Considerable study has focused on the physiological adaptations for freeze tolerance in the wood frog, Rana sylvatica, a northern species that overwinters within the frost zone, but little attention has been paid to the associated costs to organismal performance. Here we report that freezing causes transient impairment of locomotor endurance and adverse changes in exercise physiology that persist for at least 96 h. Wood frogs frozen at −2°C for 36 h exhibited normal behaviors and hydro‐osmotic status and near‐normal metabolite (glycogen, glucose, and lactate) levels within 24 h after thawing began. However, when exercised to exhaustion on a treadmill, these frogs showed a 40% reduction in endurance as compared to sham‐treated (unfrozen) controls, a reduction that persisted for at least 96 h. Previously frozen frogs exhibited higher rates of lactate accumulation during exercise than controls, suggesting that prior freezing forces greater reliance on the glycolytic pathways of energy production to support exercise. Given that this species breeds in late winter, when subzero temperatures are common, freezing may result in reduced fitness by hampering their ability to reach the pond, avoid predators, and successfully obtain mates.
Cold-hardiness and evaporative water loss in hatchling turtles.
Costanzo, Jon P.; Litzgus, Jacqueline D.; Iverson, John B.; Lee, Richard E.
North American turtles hatch in late summer and spend their first winter either on land or underwater. Adaptations for terrestrial overwintering of hatchlings in northern regions, where winter thermal and hydric regimes are harsh, have not been systematically investigated in many species. We measured intrinsic supercooling capacity, resistance to inoculative freezing, and desiccation resistance in hatchlings of terrestrial and aquatic turtles collected from northern (Terrapene ornata, Chrysemys picta bellii, Kinosternon flavescens, Chelydra serpentina) and southern (Chrysemys picta dorsalis, Trachemys scripta, Sternotherus odoratus, Sternotherus carinatus) locales. Supercooling capacity was estimated from the crystallization temperature of turtles cooled in the absence of external ice nuclei. Mean values ranged from −8.1° to −15.5°C and tended to be lower in terrestrial hibernators. Inoculation resistance was estimated from the crystallization temperature of turtles cooled in a matrix of frozen soil. These values (range of means: −0.8° to −13.6°C) also tended to be lower in the terrestrial hibernators, especially C. picta bellii. Mean rates of evaporative water loss varied markedly among the species (0.9–11.4 mg g−1 d−1) and were lowest in the terrestrial hibernators. Most species tolerated the loss of a modest amount of body water, although half of the sample of S. carinatus died from desiccation. In general, turtles did not regain lost body water from wet soil, and immersion in free water was required for rehydration. Therefore, desiccation resistance may be an important adaptation to terrestrial hibernation. Resistances to inoculative freezing and desiccation were directly correlated, perhaps because they are governed by the same morphological characteristics.
Identification of ice nucleating active Pseudomonas fluorescens strains for biological control of overwintering Colorado potato beetles (Coleoptera: Chrysomelidae).
Castrillo, Louela A.; Lee, Richard E.; Lee, Marcia R.; Rutherford, Steven T.
Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to elevate the supercooling point, the temperature at which freezing is initiated in body fluids, of Colorado potato beetles, Leptinotarsa decemlineata (Say), and to persist in their gut. Adult beetles fed ice-nucleating active strains of Pseudomonas fluorescens, P. putida, or P. syringae at 10(6) or 10(3) bacterial cells per beetle had significantly elevated supercooling points, from -4.5 to -5.7 degrees C and from -5.2 to -6.6 degrees C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was -9.2 degrees C. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88-335 still had significantly elevated supercooling points, indicating that these strains of bacteria were retained. Furthermore, beetle supercooling points were comparable to those observed immediately after ingestion, suggesting that beetle gut conditions were favorable not only for colonization but also for expression of ice-nucleating activity by these two strains. The results obtained from exposure to a single, low dose of either bacterial strain also show that a minimum amount of inoculum is sufficient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also confirmed using a polymerase chain reaction technique that detected ice-nucleating active bacteria by virtue of their ina genes. Application of these ice-nucleating active bacteria to elevate the supercooling point of this freeze-intolerant insect pest could significantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.
Ice nuclei in soil compromise cold hardiness of hatchling painted turtles, Chrysemys picta.
Costanzo, Jon P.; Litzgus, Jacqueline D.; Iverson, John B.; Lee, Richard E.
Hatchling painted turtles (Chrysemys picta) commonly overwinter within their natal nests and survive exposure to temperatures as low as -12 degrees C by supercooling. We report that the supercooling capacity of hatchling C. picta is reduced by direct contact with nest soil which, in samples from northwestern and north-central Nebraska, Indiana, and Ontario, contained potent ice nuclei active in the range of -3.5 degrees to -5 degrees C. These nuclei were sensitive to autoclaving and extractable in water. The supercooling capacity of C. picta hatched in native nest soil, or hatched in sterilized vermiculite (which lacks water-extractable nuclei), and subsequently exposed to nest soil, was reduced by ∼10 degrees C relative to control turtles that were hatched and reared in sterilized vermiculite. The effect of these nuclei was potentiated by the presence of environmental moisture, although even transient exposure to dry nest soil markedly reduced supercooling capacity in ∼ 50% of the turtles. Unlike turtle species that hibernate underwater (Sternotherus odoratus, Chelydra serpentina, Apalone spinifera), hatchlings of C. picta exhibited an extraordinary capacity for supercooling (temperature of crystallization, -16 degrees to -20 degrees C) when cooled in isolation from external ice nuclei. However, hatchlings of these four species were equally susceptible to inoculation by suspensions of the ice-nucleating bacterium, Pseudomonas syringae. Indirect evidence suggests that the soil nuclei are associated with such microbes. Nucleating activity was higher in soil collected within nests than in soil collected at the same depth, adjacent to these nests. Differences in the activities of ice nuclei in nesting soils may account for geographic and local variation in winter survival of hatchling C. picta. Our finding that similar agents occur in various other terrestrial habitats in central North America suggests that such nuclei may pose a formidable challenge to the overwintering survival of ectothermic animals that rely on supercooling to withstand frost exposure.
Freezing impairment of male reproductive behaviors of the freeze-tolerant wood frog, Rana sylvatica.
Costanzo, John P.; Irwin, Jason T.; Lee, Richard E.
The wood frog (Rana sylvatica), a temperate-zone anuran that overwinters within the frost zone, is adapted to tolerate the freezing and thawing of its tissues. Because the effects of freezing on complex neurobehavioral function are unknown and because R. sylvatica encounters subfreezing temperatures during its late-winter breeding season, we investigated the reproductive behaviors and physiology of male frogs after freezing (minimum body temperature, -2°C) and postthaw recovery (4°C). In tests simulating conditions at the breeding pool, these frogs, which otherwise behaved normally, exhibited reduced mate-searching effort and fewer assaults on mates and did not amplex females until 16-24 h after thawing. Although amplectic ability was ultimately restored in most frogs, they competed poorly for mates against never frozen controls. Further study suggested that the level of behavioral impairment depends on the severity of the freezing exposure. During freezing, tissues accumulated large quantities of the cryoprotectant glucose and desiccated extensively, responses that promote freezing survival. Freezing also caused marked hydroosmotic and metabolic perturbations, which may have impaired neurobehavioral function, perhaps by interfering with the processing of audio, visual, and tactile stimuli. Individuals that encounter subfreezing temperatures shortly before arriving at the breeding pools may incur reduced reproductive success.
Inoculative freezing and the problem of winter survival for freshwater macroinvertebrates
Frisbie, Malcolm P.; Lee, Richard E.
Due to the thermal buffering of their environment, aquatic invertebrates are less likely than their terrestrial counterparts to face temperatures substantially below 0°C. Aquatic invertebrates may not be able to avoid internal freezing by supercooling (remaining unfrozen at temperatures below the freezing point of their body fluids), however, because when their body temperatures reach the freezing point of body fluids, these organisms will likely be in contact with external ice, which may induce formation of internal ice (i.e., inoculative freezing). In this study, a variety of winter-collected, aquatic invertebrates (a clam, Sphaerium sp.; an isopod, Lirceus fontinalis; a mayfly, Stenomena femoratum; a belostomatid, Belostoma flumineum; 2 dytiscids, Ilybius oblitus and Agabus disintegratus) and, for comparison, a terrestrial beetle (Hippodamia convergens) were studied with respect to their low temperature tolerance. No species appeared to lower its freezing point appreciably by accumulating colligatively active solutes in body fluids, and all aquatic species supercooled moderately (-5 to -7°C), but significantly less than the terrestrial beetle (-16°C), before freezing when chilled in a dry environment. However, when chilled in contact with external ice, all animals froze at their melting points or just below (as low as ∼-2°C for the beetles), showing that they are susceptible to inoculative freezing. All aquatic species readily survived exposures to sub-zero temperatures when supercooled, but succumbed to the same conditions when inoculated by external ice. Survival of low temperatures by temperate zone aquatic invertebrates appears to depend upon thermal buffering provided by the aquatic environment, slow progress of ice formation in bodies of water at high sub-zero temperatures, avoidance of contact with external ice, and tolerance of high sub-zero temperatures when freezing does not occur in the animals themselves.
Cold-hardiness of a laboratory colony of lone star ticks.
Needham, Glen R.; Jaworski, Deborah C.; Chen-Ping, Chen; Lee, Richard E.
The cold-hardiness of a lone star tick, Ambylomma americium (L.) laboratory colony was characterized. Fed and unfed larvae, fed and unfed nymphs, and unfed adults did not survive exposure to -17C for 7 d. After an 8-d exposure to -10C, adults tolerated cold better than immatures and unfed specimens fared better than fed ticks. Exposing unfed 6-wk old (post mold) adult males and females to -15C for increasing intervals up to 2 hrs suggests that males were more tolerant to cold then were females. Half of all adults were alive 3 d after the 2-h low-temperature treatment. Males may have survived because of a significantly higher hemolymph osmotic pressure, although the solute concentration increased for both sexes after a 2-h exposure to 0C. Acclimation to 5C for 7 d had no influence on supercooling points for unfed males and females, engorged nymphs and larvae, and eggs. None of the life stages survived supercooling, which strongly suggests that this species is freeze-intolerant. Intolerance of immature stages to chilling may be a limiting factor in the northern distribution of lone star ticks in North America.
Anatomic site of application of ice-nucleating active bacteria affects supercooling in the Colorado potato beetle (Coleoptera: Chrysomelidae)
Lee, Richard E.; Steigerwald, Kira A.; Wymen, Jeffrey A.; Costanzo, Jon P.; Lee, Marcia R.
Most overwintering insects do not survive internal freezing and must avoid low temperatures or enhance the capacity of their body fluids to supercool to survive low temperature exposure. Recent reports have demonstrated that the application of ice-nucleating active microorganisms markedly diminishes supercooling. Topical application of as little as 20 ppm of a suspension of a freeze-dried preparation of the ice-nucleating active bacterium Pseudomonas syringae van Hall was sufficient to elevate the mean supercooling point of the overwintering adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say), from −8.7 to −4.7°C. Previous reports have demonstrated that topical application of these biological ice nucleators to insects whose mouths have been sealed still reduces supercooling capacity; however, the anatomical route by which these agents make contact with body water is unclear. Application of the P. syringae suspension to the ventral abdomen did not significantly increase the supercooling point (−5.5°C) compared with beetles treated with the non-ice-nucleating active (control) bacterium Escherichia coli (Migula). However, application of the ice-nucleating agent to the thoracic spiracle, ventral cervix, or abdominal spiracle elevated supercooling point values above those of beetles treated on the ventral abdomen. These data are instructive in the development of methods for the use of ice-nucleating active microorganisms for the biological control of overwintering pests.
Cold hardiness and overwintering strategies of hatchlings in an assemblage of northern turtles.
Constanzo, Jon P.; Iverson, John B.; Wright, Michael F.; Lee, Richard E.
Field and laboratory studies were conducted during 1989-1994 to investigate the overwintering strategies of hatching turtles representing four families native to western Nebraska. Whereas hatchling snapping turtles (Chelydra serpentina) 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 as which hatchling C. picta routinely hibernate. Field and laboratory data suggested that hatchling C. picta survive either by remaining super-cooled (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, wheras freeze tolerence fortuitously is promoted by ice inoculation, this strategy may be viable only at relatively high subzero temperatures. A cold-hardiness stragefy based on both survival mechanisms may promote winter survival in hatchling C. picta by conferring protection under dynamic physiological and microenvironmental conditions. Physiological cold hardiness and behavior are integrated determinants of the northern distributions of temperature region turtles.

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