Adaptations to terrestrial overwintering of hatchling northern map turtles,<Emphasis Type="Italic"> Graptemys geographica</Emphasis>

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle (Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to –5.4 °C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g^–1 day^–1), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at –2.5 °C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to –14.8 °C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to –8 °C. Others, held at –6 °C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N₂ gas for 8 days, may promote survival during exposure to subfreezing temperatures.Abbreviations EWL evaporative water loss - FP_eq equilibrium freezing point - INA ice-nucleating agents - T_c temperature of crystallizationCommunicated by L.C.-H. Wang     

Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea

The effects of anoxia (N₂ atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1^-1 KCl (peak I) or 90–110 mmol·1^-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I ₅₀ value for l-alanine of 0.38 mmol·1^-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1^-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.Abbreviations AABS p-(p-aminophenylazo)benzene sulphonic acid - F2, 6P fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GP glycogen phosphorylase - GS glycogen synthase - I ₅₀ inhibitor concentration reducing enzyme velocity by 50% - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenopyruvate - PFK phosphofructokinase - PK pyruvate kinase - SW sea water - F _a air temperature - TCA trichloroacetic acid - UDPG uridine-diphosphate glucose - WW wet weight     

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.     

Anoxia tolerance and alcohol dehydrogenase activity in lettuce seedlings

In order to clarify the effect of protein synthesis inhibition on anoxiatolerance, lettuce (Lactuca sativa L.) seedlings weresubjected to anoxic stress in the presence of cycloheximide (CHM). Atconcentrations greater than 3 , CHM decreased thesurvivability of the roots and the survivability decreased with increasing CHMdoses. At 100 CHM, the survivability was 41% of thatof non-CHM-treated seedlings. Alcohol dehydrogenase (EC 1.1.1.1) activity andATP concentration in the roots of the seedlings were also decreased by CHM,which may be one of the causes of the reduced anoxia tolerance of the seedlings.     

Anoxia tolerance and ethanol sensitivity of rice and oat coleoptiles

Anoxia tolerance and ethanol sensitivity of rice (Oryza sativa L.) and oat (Avena sativa L.) seedlings were evaluated to clarify their growth habit in anoxia. Anoxic stress inhibited elongation and dry weight gain of coleoptiles of the oat and rice seedlings; however, the inhibition of the oat coleoptiles was much greater than that of the rice coleoptiles. Anoxic stress increased endogenous ethanol concentration and alcohol dehydrogenase activity in oat and rice coleoptiles and their increases in the rice coleoptiles were much greater than those in the oat coleoptiles. At concentrations greater than 30 mM and 300 mM, exogenously applied ethanol inhibited the elongation and weight gain for the oat and the rice coleoptiles, respectively, and the inhibition was increased with increasing ethanol concentrations with marked inhibition being achieved on the oat coleoptiles. These results suggest that anoxia tolerance and induction of ethanolic fermentation in anoxia may be greater in rice than oat, and ethanol sensitivity of rice may be lower than that of oat.     

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

Freeze tolerance in Aporrectodea caliginosa and other earthworms from Finland

Earthworms that live in subarctic and cold temperate areas must deal with frost even though winter temperatures in the soil are often more moderate than air temperatures. Most lumbricid earthworms can survive temperatures down to the melting point of their body fluids but only few species are freeze tolerant, i.e. tolerate internal ice formation. In the present study, earthworms from Finland were tested for freeze tolerance, and the glycogen reserves and glucose mobilization (as a cryoprotectant) was investigated. Freeze tolerance was observed in Aporrectodea caliginosa, Dendrobaena octaedra, and Dendrodrilus rubidus, but not in Lumbricus rubellus. A. caliginosa tolerated freezing at -5 degrees C with about 40% survival. Some individuals of D. octaedra tolerated freezing even at -20 degrees C. Glycogen storage was largest in D. octaedra where up to 13% of dry weight consisted of this carbohydrate, whereas the other species had only 3-4% glycogen of tissue dry weight. Also glucose accumulation was largest in D. octaedra which was the most freeze-tolerant species, but occurred in all four species upon freezing. It is discussed that freeze tolerance may be a more common phenomenon in earthworms than previously thought.     

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.     

Ethanolic fermentation and anoxia tolerance in four rice cultivars

The relationship between coleoptile elongation and ethanolic fermentation was investigated in rice (Oryza sativa L.) coleoptiles of four cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress; however, the elongation of cvs Yukihikari and Nipponbare was much greater than that of cvs Leulikelash and Asahimochi. The stress did not significantly increase lactate dehydrogenase (LDH) activity or lactate concentration, but increased alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities, as well as ethanol concentration in the coleoptiles of all cultivars. The elevated ADH and PDC activities and ethanol concentration in cvs Yukihikari and Nipponbare were much greater than those of cvs Leulikelash and Asahimochi, suggesting that ethanolic fermentation is likely more active in cvs Yukihikari and Nipponbare than in cvs Leulikelash and Asahimochi. ATP concentration in cvs Yukihikari and Nipponbare in anoxia was also greater than that in cvs Leulikelash and Asahimochi in anoxia. The ethanol concentration in the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation may be one of the determinants in anoxia tolerance of rice coleoptiles.     

Locomotion in hatchling leatherback turtles Dermochelys coriacea

Hatchling leatherback turtles can only swim forwards, and employ synchronized beating of the forelimbs whether swimming slowly or quickly. The hind limbs make no contribution to propulsion. Effectively, the hatchlings have two swimming speeds; subsurface and fast (30 cm s^-1) or surfaced and slow (8 cm s^-1). Intermediate velocities are transitory; the hatchlings were never seen to rest without movement, nor did they exhibit gliding of the type seen in green turtles. During fast ('vigorous') swimming, power is developed on both the upstroke and downstroke of the limb cycle. During slow swimming, power is only developed during the upstroke—a consequence of the orientation of the axis of limb beat which is opposite in direction to that of cheloniid sea turtles. Terrestrial locomotion is laboured and features an unstable gait which involves simultaneous movement of all four limbs and forward overbalancing during each limb cycle.     

中华花龟幼体热耐受性、体温昼夜变化和运动表现的热依赖性

为研究中华花龟(Ocadia sinensis)幼体的热耐受性和运动表现热依赖性，设计了具有和缺乏温度梯度两种热环境，研究幼龟体温的昼夜变化。高、低温耐受性分别用临界高温和临界低温表示，体温为泄殖腔温度，水温和气温分别是幼龟所处位置的水温和1cm高气温。临界高温和临界低温分别为41．9℃和1．8℃。在有温度梯度的热环境中，体温、水温和气温平均值有显著的昼夜差异，水温和体温的日平均值无显著差异，两者均大于气温的日平均值。在缺乏温度梯度的热环境中，体温、气温和水温平均值亦有显著的昼夜差异，但气温、水温和体温的日平均温度无显著差异。温度梯度是幼龟进行体温调节不可或缺的条件，选择体温有显著的昼夜变化，最大值和最小值分别为29．2℃和25．4℃。在02：00—06：00时间段内，幼龟选择体温明显较低，其它测定时刻的选择体温无显著差异。幼龟各测定时刻的平均体温与平均气温和水温均呈正相关。处于温度梯度中幼龟特定气温的体温比处于缺乏温度梯度中的幼龟高3．7℃，这种差异是前者利用温度梯度进行体温调节的结果；处于不同热环境中幼龟特定水温的体温无显著差异。体温显著影响幼龟的运动表现。18—39℃体温范围内，疾跑速随体温增加而增加，36℃和39℃体温的幼龟疾跑速最大；体温达到41℃时，疾跑速显著下降。体温较高的幼龟的最大持续运动距离大于体温较低的幼龟。偏相关分析显示，疾跑速与最大持续运动距离和停顿次数呈显著的正相关，停顿次数与最大持续运动距离呈负相关。     

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25 degrees C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20 degrees C with 0.25 M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75 M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35 degrees C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.     

Reptile freeze tolerance: metabolism and gene expression

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.     

高原低氧环境下牙周炎的发病机制

牙周炎的病理过程受全身和局部因素的综合调控,一些调查研究显示高原牙周炎患病率高于平原地区,显然高原特殊环境在牙周炎的发生和发展过程中起到一定的作用,因此本文就高原低氧环境下牙周病变组织的变化、可能的发病机制作一综述。     

MicroRNA Regulation in Extreme Environments:Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia

Several recent studies of vertebrate adaptation to environmental stress have suggested roles for microRNAs (miRNAs) in regulating glo- bal suppression of protein synthesis and/or restructuring protein expression patterns. The present study is the first to characterize stress-responsive alterations in the expression of miRNAs during natural freezing or anoxia exposures in an invertebrate species, the intertidal gastropod Littorina littorea. These snails are exposed to anoxia and freezing conditions as their environment constantly fluctuates on both a tidal and seasonal basis. The expression of selected miRNAs that are known to influence the cell cycle, cellular signaling pathways, carbohydrate metabolism and apoptosis was evaluated using RT-PCR. Compared to controls, significant changes in expression were observed for miR-1a-1, miR-34a and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-125b, miR-29b and miR-2a in foot muscle after freezing exposure at 6 °C for 24 h (P < 0.05). In addition, in response to anoxia stress for 24 h, significant changes in expression were also observed for miR-1a-1, miR-210 and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-29b and miR-2a in foot muscle (P < 0.05). Moreover, protein expression of Dicer, an enzyme responsible for mature microRNA processing, was increased in foot muscle during freezing and anoxia and in hepatopancreas during freezing. Alterations in expression of these miRNAs in L. littorea tissues may contribute to organismal survival under freezing and anoxia.     

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide.Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.