Experimental studies on supercooling in two Antarctic micro-arthropods

Samples of mites and Collembola which had been acclimated at 5°C and provided with natural foods were cooled at four constant cooling rates: 1, $\text{1}\text{2}$, $\text{1}\text{4}$, $\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$ and ca 20 deg min^?1, and their individual supercooling points measured. Frequency distributions of supercooling points comprised not less than 84 (Alaskozetes antarcticus) and 96 (Cryptopygus antarcticus) individuals in each case. Two modal groups were displayed in these distributions, which were widely separated in temperature and termed low group and high group. In Alaskozetes a trough between ?3 and ?4°C was present in the high-group distribution, which may be due to a lack of a certain class of nucleators. The highest temperatures at which animals froze occurred at the slowest cooling rate ($\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$), whereas rapid cooling removed the trough to form a single high-group peak. In Cryptopygus, the high groups were narrow and peaked (<2 deg wide) at all cooling rates, with a downward shift of ca 1 deg between the rates $\text{1}\text{8}$ and $\text{1}\text{2}\text{deg min}{}^{\mathrm{?1}}$. Both species showed a trend towards a lower mean low-group supercooling point at faster rates of cooling, but these were not significant. Regressions of cooling rate on individual low-group supercooling points (≥?20°C) for both species showed a significant negative correlation, which did not differ between species. The distribution of the deviations about each rate-defined mean in the low group for each species was skewed to the right, with 88% occurring between ±2 deg of the means. It is suggested that minor deviations (e.g. halving or doubling of the cooling rate) do not affect the resultant supercooling points at non-constant cooling rates, but a rate of 1 deg min^?1 is to be preferred.     

Comparison of the cold hardiness of two larval Lepidoptera (Noctuidae)

Abstract Diapause larvae of the European corn borer (Ostrinia nubilalis (Hubn.)) and the related Mediterranean noctuid Sesamia cretica Led. possess sufficient supercooling ability to avoid freezing over their normal environmental temperature ranges. In progressive chilling experiments (10 days acclimation at each 5° step in the temperature range from 15 to ?5°C), mean supercooling points (measured at a cooling rate of 0.1°C min^?1) were lowered from ?20.4°C at 15°C to ?24.0°C at 5°C (lower lethal temperatures: c.?28°C) in O.nubilalis, compared with ?15.0 to ?17.2°C (lower lethal temperatures: ?15 to ?17°C respectively) in S.cretica. Concentrations of glycerol and trehalose determined by gas chromatography of whole body extracts were consistently higher in the former than in the latter species at both 15 and 5°C, and may be responsible for the deeper supercooling in O.nubilalis larvae. Acclimation to 5°C increased glycerol levels in O. nubilalis extracts compared with 15°C, and this was enhanced in larvae exposed for a further 10 days at each of 0 and ?5°C (glycerol being 438μmol ml^?1 body water). Haemolymph glycerol concentrations showed a similar pattern to whole body extracts in this species. Fat body glycogen was reduced during low temperature acclimation in both species. Body water contents did not change with acclimation in O.nubilalis, whilst S.cretica, containing significantly more water, lost c.7% during acclimation from 15 to 5°C. Haemolymph osmolalities increased during acclimation, especially in Ostrinia larvae, probably as a result of the accumulation of cryoprotectants. The majority of O.nubilalis larvae survived freezing under the conditions of the cooling experiments, whilst larvae of S.cretica did not, thereby confirming an element of freezing tolerance in the former.     

Survival of sub-zero temperatures by two South Georgian beetles (Coleoptera,Perimylopidae)

Summary The ability of adults and larvae of two species of perimylopid beetles (Hydromedion sparsutum, Perimylops antarcticus) to survive sub-zero temperatures was studied at Husvik, South Georgia in summer during October–December 1990. Experiments determined their survival at constant sub-zero temperatures, their lower lethal temperatures and individual supercooling points. The effects of cooling rates (0.015°, 0.5° and 2.0°C min^–1) and starvation on survival were also assessed. Mean supercooling points of field-collected individuals of both species were in the range -3.0° to -5.4°C with Perimylops having a deeper capacity (ca. 1.5°C) for supercooling relative to Hydromedion. The former species also survived freezing temperatures significantly better than the latter and its mean lower lethal temperature was 2.5°C lower. At a constant temperature of -8.5°C, the median survival times for Perimylops adults and larvae were 19 and 26 h respectively, whilst both stages of Hydromedion died within 3 h. The three cooling rates resulted in significantly different median survival temperatures for adult Hydromedion with 0.5°C min^–1 producing maximum survival. Prior starvation did not have a significant influence on the survival of either species at sub-zero temperatures although both adults survived less well. The results support field observations on the habitats and distribution of these insects, and suggest differing degrees of freezing tolerance.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intracellular freezing: effect of extracellular supercooling.

Human erythrocytes were frozen on the stage of a cryomicroscope at accurately controlled constant-cooling rates with varying degrees of extracellular supercooling. The formation of intracellular ice was detected by direct observation of the frozen cells through the microscope. A significant coupling effect was determined between the minimum cooling rate necessary to produce intracellular freezing and the extent of supercooling. Increased degrees of extracellular supercooling reduced the range of cooling rates for which water would freeze within the cell. Specific data points were obtained at ΔT_SC = 0, ?5, and ?12 °C for which the corresponding transition cooling rates were respectively ?845, ?800, and ?11 °C/min.An explanation for the occurrence of this phenomenon is presented based on the physiochemical processes that govern the freezing of a cell suspension.     

Effects of low-temperature acclimation on the survival and cold tolerance of an antarctic mite

The effects of long-term exposures to constant temperatures (+4, 0, −5, −10, −15 and −20°C) on the survival of a cryptostigmatid mite, Alaskozetes antarcticus, were studied during 1983–1984 on Signy Island, South Orkney Islands, in the maritime Antarctic. Field-fresh samples collected during the austral summer showed very large (e.g. about 60 percentage points) variations in survival when placed at constant temperatures, as a result of collection-date effects. Pretreatment acclimations (10-day) at +4 and 0°C (especially) reduced this variation. Short-term modulations in cold-hardiness levels were related to ambient temperature fluctuations. However, samples collected on the same occasion, from microhabitats 20 m apart, also showed significant cold-hardiness variation. For twelve summer samples, survival after 24 h at −15°C was highly correlated with supercooling capacity. Winter samples showed little variation in survival, interms of collection-date. Percentage survival remained greater than 85% at −5, −10 and −15°C, for exposures up to 100 days. Samples with median supercooling points of about −30°C, showed 52% survival after 250 days at −15°C, and 73% survival after 100 days at −20°C. At −15°C, supercooling capacity was used up at an estimated rate of 0.06 deg day⁻¹, as a result of a time-temperature interaction effect on the probability of heterogeneous nucleation. Adult mites showed 78% survival after 21 days encasement in distilled water ice, at −15°C. Survival differences between post-larval stages were not detected. In conclusion, survival ability under controlled laboratory conditions appeared to exceed the requirements of average winter-habitat temperatures, but the effects of fluctuating and extreme temperatures require investigation. Supercooling points are considered to be accurate indicators of low-temperature survival capability in this species.     

Low temperature mortality of the peach-potato aphid Myzus persicae

ABSTRACT.

• 1 The mean supercooling points of first instar and adult Myzus persicae (Sulzer) maintained at 20°C and cooled at 1°C min^?1 were ?26.6 and ?25.0°C respectively.
• 2 The LT₅₀ (temperature) of the same age groups drawn from the same population and cooled at the same rate were ?8.1 and ?6.9°C, indicating extensive pre-freeze mortality in M.persicae under laboratory conditions.
• 3 Acclimation at 10 and 5°C did not affect supercooling but depressed the LT₅₀ of both first instars and adult aphids.
• 4 Freezing of leaves during feeding did not increase mortality above that expected from the direct effects of low temperature.
• 5 The level of cold in different winters can be expressed in terms of the total number of frost days, and the frequency of abnormally cold days. Winter temperatures differ markedly in a vertical profile from the soil to the soil or grass surface, and then to the air (and foliage) above.
• 6 The time of the first record of M.persicae in suction trap samples is correlated with January and February temperatures except in the west of England and Wales. Further north December and January temperatures are relatively more important.
• 7 Winter temperatures and the resultant aphid mortality is a primary determinant of the timing of the spring migration.

     

Seasonal changes of glycogen/trehalose contents,supercooling points and survival rate in mature larvae of the overwintering soybean pod borer Leguminivora glycinivorella

The mature larvae of the soybean pod borer Leguminivora glycinivorella, spend over 9 months (October-next August) in the inactive state until pupation down to 3 cm below the surface in soil. Trehalose content of inactive larvae increases in early winter, attaining a maximum (ca 30 mg/g), and decreases in spring, with a concomitant decrease and increase of glycogen. The median supercooling points seasonally change from ?19.8°C (October) to ?25.0°C (February), and to ?17.0°C (June). The lower supercooling points in winter are in part due to the absence of unusually high values (> ?18°C). The increase in trehalose does not seem to be effective in depressing the supercooling points. The larvae are freeze-intolerant, but ambient temperatures in outdoor conditions are always above the supercooling points. The survival rates are very high throughout the inactive period.     

Cold hardiness of Lymantria monacha and L. dispar (Lepidoptera: Erebidae) eggs to extreme winter temperatures: implications for predicting climate change impacts

1. It has been predicted that temperature increases of 3.6–5.8 °C would shift the northern distribution limit of Lymantria monacha (Linnaeus) and Lymantria dispar (Linnaeus) by 500–700 km, but these predictions ignore the effects of minimum winter temperatures. It was hypothesised that winter cold can limit range expansion due to high egg mortality in cold temperatures. 2. The present study determined the supercooling points of overwintering eggs of these forest pests, and compared these with recent minimum winter temperatures in the areas of origin of three populations. Eggs from one L. monacha and one L. dispar population from the species' core distribution area in Germany were included, as well as L. monacha eggs from Finland, near the northern border of the species' distribution. 3. The median supercooling points of both species were more than 10 °C lower than the median minimum winter temperatures of their areas of origin, and the median supercooling points of Finnish and German L. monacha eggs did not differ significantly. The median supercooling point of German L. monacha eggs differed from that of German L. dispar eggs. 4. Previous literature on the topic is referenced, and translations of the old German and Russian sources are given. Based on these results, it is argued that the frequent claim that L. monacha eggs can survive cold down to ?40 °C is unsupported, with a value near ?30 °C being a more likely limit. 5. Winter cold alone can limit the predicted range shifts of these species to 200–300 km under 3.6–5.8 °C increase scenarios, which is less than half the value of earlier estimates.     

Cold tolerance of the invasive larch casebearer and implications for invasion success

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Within‐generation variation of critical thermal limits in adult Mediterranean and Natal fruit flies Ceratitis capitata and Ceratitis rosa: thermal history affects short‐term responses to temperature

Insect thermal tolerance shows a range of responses to thermal history depending on the duration and severity of exposure. However, few studies have investigated these effects under relatively modest temperature variation or the interactions between short‐ and longer‐term exposures. In the present study, using a full‐factorial design, 1 week‐long acclimation responses of critical thermal minimum (CT_min) and critical thermal maximum (CT_max) to temperatures of 20, 25 and 30 °C are investigated, as well as their interactions with short‐term (2 h) sub‐lethal temperature exposures to these same conditions (20, 25 and 30 °C), in two fruit fly species Ceratitis capitata (Wiedemann) and Ceratitis rosa Karsch from South Africa. Flies generally improve heat tolerance with high temperature acclimation and resist low temperatures better after acclimation to cooler conditions. However, in several cases, significant interaction effects are evident for CT_max and CT_min between short‐ and long‐term temperature treatments. Furthermore, to better comprehend the flies' responses to natural microclimate conditions, the effects of variation in heating and cooling rates on CT_max and CT_min are explored. Slower heating rates result in higher CT_max, whereas slower cooling rates elicit lower CT_min, although more variation is detected in CT_min than in CT_max (approximately 1.2 versus 0.5 °C). Critical thermal limits estimated under conditions that most closely approximate natural diurnal temperature fluctuations (rate: 0.06 °C min^?1) indicate a CT_max of approximately 42 °C and a CT_min of approximately 6 °C for these species in the wild, although some variation between these species has been found previously in CT_max. In conclusion, the results suggest critical thermal limits of adult fruit flies are moderated by temperature variation at both short and long time scales and may comprise both reversible and irreversible components.     

Winter survival of an adult bark beetle Ips acuminatus Gyll

Freezing-susceptible adult Ips acuminatus hibernate underneath bark of Scots pine. The beetles lower their supercooling points from ?20 to ?34°C due to accumulation of low molecular weight antifreezes. The capability of specimens to supercool to about ?20°C in the absence of cryoprotective solutes during winter, seemed to be at least partially attributable to the presence of a thermal hysteresis factor at 3–4°C.Using a GC-MS-COM technique, a unique combination of accumulated solutes present only in specimens demonstrating supercooling points below ?20°C was identified as ethylene glycol, mannitol, sorbitol and dulcitol. Not previously found in nature, ethylene glycol was the major solute (90%) synthesized at sub-zero temperatures. Exposure to ?10°C was an effective cue to accumulation of ethylene glycol and nearly 5 times as effective in promoting sorbitol synthesis than was ?5°C. When low molecular weight substances were lost at high temperatures, they were not re-synthesized in beetles re-exposed to sub-zero temperature. The supercooling point was closely related to both the concentration of ethylene glycol and to the haemolymph melting point. Attempts to correlate changes in sorbitol concentrations to changes in supercooling points were not conclusive.Proliferation of thermal hysteresis was observed in the beginning of November. A melting-hysteresis freezing point differential of about 3.6°C was demonstrated in the haemolymph of beetles during December. No thermal hysteresis was demonstrated in the haemolymph of positive phototactic beetles or in the outdoor beetles in May. The combination of high temperature and long photoperiod appeared to be a more effective cue to the final loss of thermal hysteresis than was high temperature alone.     

Critical temperature for skin necrosis in experimental cryosurgery

To investigate the minimal lethal freezing temperature required to produce skin necrosis in dogs, multiple skin sites were frozen with cryosurgical equipment. Tissue temperatures were recorded from thermocouple sites placed at diverse distances, usually 5 mm from the edge of the freezing probe. In single freezing cycles of about 3 min duration, tissue temperatures in the range of 0 to ?60 °C were produced. Punch biopsies of the skin at the thermocouple sites 3 days after freezing injury provided tissues for estimation of viability by histologic examination.The histologic findings permitted classification of the biopsy tissue into three groups, that is, viable, borderline, or necrotic. When classified as borderline, the division between the necrotic and viable tissue was evident on the histologic slide. The viable specimens were scattered through the 0 to ?35 °C range. All specimens frozen to ?10 °C or warmer were viable. In biopsies classified as borderline, the range of viability extended from ?11 ° to ?50 °C. The necrotic biopsies covered a range of ?14 ° to ?50 °C. Cell death was certain at temperatures colder than ?50 °C. The data showed cryosurgical freezing conditions produced a range of temperatures in which viability or death of tissue may occur and that the ranges of viability and necrosis overlapped to a great extent.The wide range of temperatures at which cells were viable shows the need to achieve tissue temperatures in the range of ?50 °C in the cryosurgical treatment of cancer.     

Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis

Two populations of the gall fly Eurosta solidaginsis utilize different strategies to endure seasonal exposure to temperatures below freezing. Both populations are freezing tolerant. In north temperate populations, supercooling points rise from ?10.2°C to ?6.2°C following exposures to temperatures below freezing. This level is maintained throughout winter and ensures frequent and prolonged periods of tissue freezing. South temperate populations depress the supercooling point to ?14.2°C during autumn and early winter, and this depression precludes extracellular ice formation during periods of supra-optimal temperature fluctuations. During mid-winter, supercooling points rise to the same level as in northern groups.Both populations accumulate three principal cryoprotective agents following first frost exposures (glycerol, sorbitol and trehalose). Cryoprotectants levels do not peak in northern populations until 4–6 weeks after first frost. In southern populations the accumulation profile is characterized by a high initial rate of synthesis, a protective overshoot and pronounced seasonal fluctuations. The relative survival advantages of each strategy are discussed.     

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min⁻¹. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.     

Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration

Eggs of the stonefly, Arcynopteryx compacta, that overwinter in the alpine region of Norwegian mountains, increase their cold-hardiness by dehydration. Eggs enclosed in ice at −22°C survive the loss of about two-thirds of their total water content by shrinkage due to passive diffusion of body water along the concentration gradient. Fully hydrated eggs are killed by freezing at their supercooling point of −26°C, and by direct cooling to −30°C. Dehydrated eggs have a mean supercooling point of −31°C, and survive exposure at −27 and −29°C in ice. Judged from their melting points the eggs do not accumulate low-molecular-weight cryoprotective substances. The difference between freezing and melting points corresponds to a thermal hysteresis of up to 1.8°C. The presence of thermal hysteresis antifreezes may stabilize their supercooled state when enclosed by ice during overwintering. The eggs enter diapause in the autumn, and diapause completion is enhanced both by temperature and time during enclosure in ice.     

Ultrastructural appearance of freeze-substituted schistosomula of Schistosoma mansoni frozen by a two-step cooling schedule

Schistosomula of the parasitic helminth Schistosoma mansoni were frozen by two-step cooling, then examined for ultrastructural changes by the freeze-substitution method. Samples were cooled at 1 °C min^?1 to ?20, ?25, ?28, and ?38 °C before being cooled at 10,000 °C min^?1 to ?196 °C. The results showed that progressive partial dehydration of the parasites occurred during slow cooling. Numerous cavities, indicating the presence of intracellular ice crystals, were observed in organisms which did not become shrunken. The sizes of the ice cavities varied between organisms and also within the same cell type in individual organisms indicating that intracellular ice nucleation may occur at any time during the slow cooling step. Some organisms cooled first to ?28 or ?38 °C contained no evidence of ice crystal formation. When correlated with previously reported infectivity studies, the results indicated that successful cryopreservation of schistosomula requires slow cooling to approximately ?30 °C to induce cryodehydration, followed by rapid cooling to ?196 °C to prevent ice nucleation or crystal growth.     

A method for quantitative determination of ice nucleating agents in insect hemolymph

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 10³, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph.This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.     

Freezing avoidance in Andean giant rosette plants

Abstract Frost avoidance mechanisms were studied in Espeletia spicata and Espeletia timotensis, two Andean giant rosette species. The daily courses of soil, air and tissue temperatures were measured at a site at circa 4000 m. Only the leaves were exposed to subzero temperatures; the apical bud and stem pith tissues were insulated by surrounding tissues. The leaf tissues avoided freezing by supercooling rather than by undergoing active osmotic changes. The temperatures at which ice formed in the tissues (the supercooling points) coincided with injury temperatures indicating that Espeletia tissue does not tolerate any kind of ice formation. For insulated tissue (apical bud, stem pith, roots) the supercooling point was around - 5°C coinciding with the injury temperature. Supercooling points of about –13 to - 16°C were observed for leaves. These results contrast with those reported for Afroalpine giant rosettes which tolerate extracellular freezing. The significance of different adaptive responses of giant rosettes to similar cold tropical environments is discussed.