Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999     

Effect of hypertonicity and freezing on survival of unprotected synchronized mammalian cells

Survival of unprotected Chinese hamster cells frozen to −196 °C in tissue culture medium or exposed to a 6X NaCl solution is a function of the time in the cell cycle at which freezing or the exposure to hypertonicity takes place. In both cases, the cells are sensitive to exposure in M and G₂, and are resistant in late S. The survival curves are different in late G₁ or early S. This indicates that damage due to hypertonicity is not the only mode of injury present when cells are frozen to −196 °C.     

Survival of the first stage larva of the metastrongyloid nematode Elaphostrongylus rangiferi under various conditions of temperature and humidity

First-stage larvae of E. rangiferi kept in water at 50°C died within 80 minutes, while at 6° the last larvae died between day 180 and 210. The time it took to reach 1_x= 0.5 (half of the larvae dead) at various temperatures between 6° and 50° was well described by the exponential function y = 614.6e^−0.15x, giving a value of 615 days to reach 1_x= 0.5 at 0°C. There was no clear decrease in the survival of larvae frozen at −20° in faeces and in water, and at −80° in faeces after 360 days. When subjected to repealed freezing and thawing, all larvae died within 77 days. When kept in air at RH = 20% and 22°C, all larvae died within 11 days, while when frozen (−20°C) in air at RH approx. 0%, 1_x stayed at approx. 0.5 from day 5 to day 16.     

Ultrastructure-function correlative studies for cardiac cryopreservation. II. Hearts frozen to various temperatures without a cryoprotectant

Isolated rat hearts were perfused with balanced salt solution (BSS) for 20 min, sealed in a metal cannister, and cooled in a −20 °C acetone bath at a rate of 1 °C/min to one of four subzero core temperatures (−10, −12, −17, or −20 °C). Upon attainment of the desired temperature the hearts were rapidly thawed (40–50 ° C/min) and reperfused with BSS for an additional 20 min. Approximately half of the hearts cooled to −10 or −12 °C resumed spontaneous contractile activity after thawing. One of 16 hearts survived cooling to −17 °C, while no heart survived cooling to −20 °C. Nonfrozen controls gave a positive inotropic response to a standard test dose of ouabain; none of the thawed survivors did.     

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

Overwintering of benthic macroinvertebrates in ice and frozen sediment in a North Swedish river

In winter the water freezes into the substrate within considerable areas of unregulated northern rivers due to low temperature combined with a lowering of the water level. Living individuals of Nematoda, Gastropoda, Sphaeriidae, Oligochaeta, Hirudinea, Isopoda, Trichoptera and Chironomidae were found in samples of ice and frozen sediment from the bottom frozen hydrolittoral zone of the north Swedish river Vindelälven. All abundant species in the frozen substratum, except Asellus aquaticus , seemed to be well adapted to withstand overwintering in this special habitat free from predation. Generally, between 80 and l00% of enclosed animals survived thawing. Cysts or other kinds of resting stage constructions, similar to those found during drought, were common in several enclosed species. Specimens of Gyraulus acronicus, Pisidium ssp., Molanna albicans and Chironomidae survived exposure to −4°C for five month in a freezing experiment. Extracellular freezing of the invertebrates overwintering in the ice is probable, as the ambient temperature was below the true freezing point of most animals. The composition of the substratum may effect the survival of animals enclosed in ice.     

Ultrastructure-function correlative studies for cardiac cryopreservation. 3. Hearts frozen to--10 degrees and -17 degrees C with and without dimethyl sulfoxide (DMSO)

Isolated rat hearts were perfused with balanced salt solution (BSS), then with BSS containing DMSO in one of several concentrations (0.14, 0.70, 1.41, 2.11, or 2.82 m) at +30 °C, sealed in a metal cannister, and cooled slowly (1 °C/min) to a core temperature of −17 °C (total cooling time (TCT) = 37 min), thawed rapidly and reperfused with BSS. Groups of protected (0.70 m DMSO) or nonprotected hearts were cooled to −10 °C; of these, some were thawed immediately upon reaching −10 °C (TCT = 30 min), others were maintained at −10 °C for an additional 7 min and thawed (TCT = 37 min). Contractile activity was recorded during prefreeze and postthaw perfusion periods. Hearts which exhibited spontaneous A-V function after thawing were considered to be survivors.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Viability of cold-adapted L cells after freezing and storage in solid CO2

Mouse L cells adapted to low temperature by repeated exposures to 4 °C for 6–8 weeks were stored in solid CO₂ at −70 °C for 7 months simultaneously with unpretreated controls. The population of the cold-adapted subline LC2 contained more living cells after thawing than did the control L cells. The increased viability of the LC2 cells was expressed by a higher number of eosin-unstained cells immediately after thawing and by a higher increase in cell number after incubation at 36 °C. The difference between the two populations was more marked after longer storage.     

Evaluation of freezing injury and freeze-thaw injury to membranes of Saskatoon serviceberry twigs by measuring HCN release

The influence of thawing on freeze-injured Saskatoon serviceberry ( Amelanchier alnifolia Nutt.) twigs was evaluated by refreezing freeze-thawed twigs and comparing the HCN release at -5°C fro these twigs to the HCN release at -5°C from twigs that had not been thawed. An effect of thawing depended on the physiological state of the twigs, on the degree of freezing stress, or on both. Manifestation of membrane injury does not have an absolute dependence on thawing. Post-thaw temperature influences manifestation of injury, since twigs warmed to 30°C released more HCN than twigs warmed to 1°C when refrozen to -5°C. Although thawing and post-thaw conditions can influence the magnitude of membrane injury, the critical event leading to injury occurs while plants are frozen.     

Macromolecular synthesis by yeasts under frozen conditions

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8°C min⁻¹), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at −5°C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [³H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15°C and −15°C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at −5°C under frozen and liquid conditions revealed 2–3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of ≈ 1.4 mol l⁻¹ of NaCl at −5°C). The mesophile Saccharomyces cerevisae also incorporated [³H]leucine at −5°C and −15°C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.     

Rapid cold-hardening in a Karoo beetle, Afrinus sp.

Abstract.  In the insect rapid cold-hardening response, survival at subzero temperatures is greatly improved by a brief pre-exposure at a milder temperature. It is predicted that insects with minimal cold tolerance capabilities living in variable environments should use rapid cold-hardening to survive sudden cold snaps. This is tested in Afrinus sp., a beetle that lives in an exposed habitat on rock outcrops in the Karoo Desert, South Africa, where microclimate temperatures drop infrequently to below freezing. Afrinus sp. shows a significant rapid cold-hardening response: survival of a 2-h exposure to −6.5 °C is much improved after pre-exposure to −2 °C, to 0 °C with a 2-h return to the rearing temperature, and to 40 °C, but not after pre-exposure to 0 °C. Little is known about the mechanism of the rapid cold-hardening response, although the data suggest that rapid cold-hardening may be mediated via several different mechanisms.     

The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. Optimal conditions for fertilization including suitable medium and sperm:egg ratio were determined. Sperm was diluted in modified Kurokura's 'Extender 2'containing DMSO as cyroprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 2°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min⁻¹; −4°C–80°C: 11°C min⁻¹; from −80°C they were plunged directly into liquid nitrogen (− 196°C). Frozen samples were thawed in a water bath at 40°C. Fertilization rates achieved were much higher in water than in other solutions. Optimal ratios of frozen sperm:egg:water (1:20:20 in volume) and optimal number of frozen spermatozoa:egg (10⁵ spz: 1 egg) were determined. In such conditions, a strong positive correlation (c =+0.846) was found between the post-thaw motility and the fertility of frozen sperm securing high fertilization (99.6%, percent of control). No significant difference was found between fertilization and hatching rates achieved using frozen-thawed common carp sperm.     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

Field ecology of freeze tolerance: interannual variation in cooling rates, freeze-thaw and thermal stress in the microhabitat of the alpine cockroach Celatoblatta quinquemaculata

Microclimate recordings were made over four years on the Rock and Pillar Range, New Zealand. These are used in conjunction with mortality and freezing data derived in the laboratory to make inferences about the winter thermal ecology of the freeze-tolerant alpine cockroach Celatoblatta quinquemaculata (Dictyoptera: Blattidae). Threshold temperatures are identified through laboratory experiments at ecologically relevant cooling rates: 0°C; −3.1°C (FP₅: the temperature at which 5% of cockroaches are expected to be frozen); and −4.5°C (FP₅₀). The maximum cooling rate in the microhabitat across any of the thresholds was 0.06°C min⁻¹, considerably slower than the 1°C min⁻¹ normally used in laboratory studies. Freeze-thaw events occurred regularly in the field, and temperature minima occasionally fell to temperatures lethal to C. quinquemaculata . Variability in snow cover contributed to interannual variation in microclimate temperatures. Decreased snow cover is predicted with climate change scenarios, and this will probably result in more freeze-thaw cycles and more extreme minimum temperatures in this environment. It is concluded that the limited environmental tolerances of the animals living in this habitat make the few degrees of interannual variation ecologically relevant.     

Growth and survival of Azolla filiculoides in Britain I. Vegetative production

Azolla filiculoides Lam. causes serious weed problems in Britain, but its long-term survival might be limited by winter death. The aim of this study was to establish the low temperature responses and limitations of A. filiculoides sporophytes.
In the laboratory, normal vegetative growth was shown to continue at 5°C. Reddening of plants was a response to low temperature and high light conditions which could be prevented by shading. Adult plants died after short (18 h) exposure to −4°C but survived sub-zero temperatures >−4°C. Evidence was found of seasonal changes in chill tolerance, but not in freeze tolerance.
In outdoor culture, plants survived encasement in ice and air temperatures to −5°C. Additional evidence suggested that natural populations can readily survive air temperatures much lower than this. Microclimatic effects are likely to be responsible for this discrepancy between laboratory and outdoor culture results.
Three phenotyes were identified; survival, colonizing and mat forms.     

Potato cold hardiness development and abscisic acid. II. De novo synthesis of proteins is required for the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

During cold acclimation of potato plantlets ( Solanum commersonii Dun, PI 458317), there are two transitory increases in free ABA content corresponding to a three-fold increase on the 2nd day and a five-fold increase on the 6th day (Ryu and Li 1993). During this period, plantlets increased in cold hardiness from −5°C (killing temperature, control grown at 22/18°C, day/night) to −10°C by the 7th day of exposure to 4/2°C (day/night). This increase in free ABA was not found when cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis, was added to the culture medium 6 h before exposure to low temperatures. Plantlets treated with CHI did not acclimate to cold, maintaining a hardiness level (−5°C) similar to that of the 22/18°C-grown plantlets. When the CHI-treated plantlets were exposed to low temperatures for 3 days, transferred to CHI-free culture medium and grown at low temperatures, the plantlets showed a transitory increase in free ABA 2 days later. This increase was followed by the development of cold hardiness (−8°C). Application of CHI to the culture medium after 3 days of cold acclimation, when the first ABA peak and a partial development of cold hardiness (−8°C) had occurred, blocked the second transitory increase in free ABA and resulted in no further development of cold hardiness. These results suggest that de novo synthesis of proteins is required for these transitory increases in free ABA during cold acclimation of potato plantlets.     

Comparison of cold tolerance in eggs of two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, in relation to climate warming

The population of the cicada Cryptotympana facialis began to increase in Osaka, Japan, during the late 20th century. Climate warming is considered a major cause, although the relationship between temperature and the cicada population increase remains unclear. By examining cold tolerance in overwintering eggs of C. facialis in relation to another cicada, Graptopsaltria nigrofuscata , whose population has recently decreased in Osaka, we tested the hypothesis that warming has caused the population increase of C. facialis by decreasing egg mortality due to winter temperatures. A short-term (24 h) cold exposure experiment demonstrated that the half-lethal temperatures (LT50) of C. facialis and G. nigrofuscata were −23.3°C and −28.9°C, respectively, although these extreme low temperatures never occurred in Osaka during the 20th century. Prolonged exposure to −5°C for up to 30 days had no harmful effects on the hatching rate in either species. Overwintering mortality was also assessed under naturally fluctuating conditions by transferring eggs to cooler elevated sites that mimicked the environment prior to the current warming. Eggs of C. facialis that overwintered at the cooler site exhibited similar hatching rates to those maintained at the original site. The results of these experiments consistently indicated that overwintering eggs of C. facialis possess adequate tolerance to the low temperatures of the 20th century. Therefore, we rejected our initial hypothesis that recent increases in the C. facialis population have been caused by warming-related reductions in overwintering egg mortality.