Recrystallisation temperature (Trc) in an Antarctic springtail

Cryobiologists have traditionally assumed that the temperature of crystallisation (Tc) or supercooling point (SCP) of a chill-tolerant insect is not a stochastic event, i.e. that it is a biologically meaningful indicator of phenotypic characteristics, be they exogenous influences (e.g. acclimation/acclimatization) or endogenous factors (e.g. life history stage, moult state). Recent work by Wilson et al. (11) has suggested that SCPs--at least in non-biological samples--are more stochastic than previously thought. Here, this question is tested indirectly by the repetitive freezing of individuals of the Antarctic springtail, Cryptopygus antarcticus. The springtails were each supercooled ten times in succession to determine their re-crystallisation temperatures (Trc). SCPs were found to be deterministic i.e. related to their initial Tc. Despite the mortality of re-crystallised samples, 70 percent showed that less than 1 degree C difference between Tc and Trc1 and 95 percent showed less than 5 degree C difference. Tc and Trc1 were significantly correlated. Variability in re-crystallisation temperatures is hypothesised to be predominantly the result of differences in nucleator content and changes in body fluid osmolality during the experimental exposures. Factors affecting the relative variability of SCPs are discussed.     

Rapid cold hardening: a gut feeling

This study examined the rate of cold hardening of a field population of Antarctic springtails and the effect of eating food with particular levels of ice nucleating activity on the animal's whole body freezing point. The SCPs of samples of c. 20, freshly collected, Cryptopygus antarcticus were measured hourly over a 32 hour collection period using differential scanning calorimetry and related to habitat temperature. The mean SCP of the springtails increased from -24 to -10 degree C during which time the habitat temperature warmed slowly from -2.5 to +2.5 degree C. In laboratory experiments, previously starved, cold tolerant springtails were fed on selected species of algae with measured SCP's but there was no clear correlation between the SCP of food and that of the animals after feeding. Microscopic examination of faecal pellets and guts from springtails showed that algal cells were completely destroyed during digestion.     

Chill tolerance in the tropical beetle Stenotarsus rotundus

The tropical beetle Stenotarsus rotundus (Endomychidae) survived chilling at mildly low temperatures (above +5 degree C). With upper limit of cold injury zone (ULCIZ, the highest temperature that causes cold injury) well above freezing point, the supercooling ability (mean supercooling point - SCP; -11 degree C) has no cryoprotective importance. Mortality increases rapidly between -9 and +5 degree C, dependent on accumulated dose of chilling (sum of injurious temperatures - SIT; 2 degree-days below ULCIZ). The cold hardiness traits found in this species are by-products of deep diapause, and may serve as pre-adaptation for expansion into cooler regions.     

The tolerance of the field slug Deroceras reticulatum to freezing temperatures

Cold hardiness of ectotherms has been widely studied in arthropods, but there is a more limited literature on the survival of molluscs at low temperatures. A number of intertidal species have been examined in detail, but terrestrial molluscs have largely been overlooked until recently. This paper reports results of laboratory experiments to evaluate the cold hardiness of the terrestrial slug, Deroceras reticulatum. The mean supercooling point (SCP) rose from -4.2 degree C in summer to -3.6 degree C in winter. The SCP that caused 50 percent mortality (LSCP50) remained constant at -4.7 to -4.8 degree C in both seasons, but slugs were able to survive the frozen state for longer in winter (LD50 of 31.8 minutes compared with 17.0 minutes in summer). Slug survival at freezing temperatures was prolonged to at least five hours when placed on a moist, absorbent substrate. D. reticulatum exhibits partial freeze tolerance, with an increased survival in winter. The results are discussed in relation to the natural environment of slugs.     

Cold hardiness and supercooling capacity in the pea leafminer Liriomyza huidobrensis

Pupal SCP (supercooling point) of Liriomyza huidobrensis showed no variation with age, with an average of -20.9 degree C. Low temperature survival of different ages of pupae showed no correlation with their SCP. Nonlinear regression analysis found that the response of L. huidobrensis pupae to exposure time under different low temperature regimes above -5 degree C was best fitted by a logistic equation. Both low temperature and exposure time had significant effects on pupal mortality. Temperatures above 5 degree C do not prevent pupae from emergence. L. huidobrensis was shown to be a freeze susceptible, and at the same time, a chill tolerant insect. It can tolerate subzero temperatures by supercooling. Compared with L. sativae, another dominant leafminer in China, L. huidobrensis is more cold tolerant. Our results explain differences between the species in geographic distribution and phenology.     

The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance

The effects of fruit maturity, at the time of natural dispersal, on subsequent desiccation tolerance and sub-zero storage was investigated in three lots of Acer pseudoplatanus (sycamore) collected from northern to southern Europe. Fruits from the native plant distribution range in Italy had significantly higher desiccation tolerance (0.16 g water per g DW) than those from England (0.30) and Norway (0.50), confirming that the maximum potential desiccation tolerance in sycamore exceeds that of the recalcitrant type. In contrast, the unfrozen water content varied only slightly between seedlots, but systematically reduced with development (0.35 to 0.27 g water per g DW). Maximum survival (60 percent fruit germination) of seven days sub-zero temperature storage coincided with drying the Italian fruit lot to c. 0.2 g water per g DW followed by holding at -20 degree C, above the onset temperature for freezing, or at -196 degree C (liquid nitrogen). Fruit survival was much lower in the Italian fruits when held at this water content and -70 degree C, and in all other combinations of water content, temperature and fruit lot provenance. As the risk of nucleation in partially dried fruits held at -20 degree C is high, we recommend sycamore fruits are cryopreserved for long-term conservation.     

Survival of freezing by free-living Antarctic soil nematodes

Free-living microbivorous nematodes become numerically dominant in Antarctic terrestrial faunas as environmental conditions become more severe, while also reaching very high levels of abundance in moist, vegetated habitats. Nematodes have little resistance to freezing via exogenous ice nucleation, such as would occur as their microhabitat freezes. We report the results of experiments testing the ability of seven maritime Antarctic nematode taxa to survive freezing in small water droplets at high sub-zero temperatures. Isolated individuals of these species possessed supercooling characteristics similar to those previously reported (supercooling points -6 to -25 degree C). When frozen in water at -3 to -6 degree C, most showed high (> 70%) survival both (i) after rapid cooling (1 degree C/min) to c. -60 degree C followed by immediate rewarming, and (ii) when held for 7-12 h at either -10 or -30 degree C, although the proportions surviving varied between species. We propose that the ability to survive freezing while fully hydrated at high sub-zero temperatures is one of the most important aspects of these species' survival tactics.     

Survival at low temperatures in insects: what is the ecological significance of the supercooling point?

Many freezing-intolerant insects may die during long or even brief exposures to temperatures above their supercooling point (SCP). Consequently, the real ecological value of the SCP remains ambiguous, particularly for tropical species that never experienced cold exposures. The bimodal distribution of SCP is discussed in the light of sexual dimorphism. The importance of sex in insect cold hardiness has been regularly neglected and although we admit that in some species sex may be uneasy to determine, it should be taken into account in further studies. We suggest that supercooling ability may be, at least partially, a result of adaptations to other functions unrelated to cold, including the desiccation resistance. The potential causes of insect death at low temperatures during survival experiments have also been examined. Prolonged exposures at lethal low temperatures can produce deleterious effects (including death) even if the insect does not freeze; during long-term exposure to low temperatures the organisms may finally die from the exhaustion of energy reserves.     

Cold resistance in the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

We have investigated cold resistance, measured by the supercooling point (SCP) temperature, in life stages of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), collected in Brittany poultry houses. Mean SCP values drastically increased during the insect ontogeny: egg (-26.1 C), first instar larvae (-21.6 C), last instar larvae (-15.5 C), pupae (-11.6 C), teneral adults (-12.0 C) and mature adults (-13.1 C). Nymphal metamorphosis and adult maturation did not promote substantial decrease of freezing resistance. The SCP values reflect the physiological states of the developmental stages especially the absence of ice nucleating agents: (i) lower SCP values in egg and unfed newly-emerged larvae I (i.e. -25.1 C), (ii) higher SCP values in fed larvae (i.e. -14.7 C), pupae and adults most likely due to the presence of ice nucleation sites in the gut. A tropical species, A. diaperinus, seems not to use its potential cold hardiness even in winter to remain in this warm habitat in temperate regions.     

Snow white and the seven dwarfs: a multivariate approach to classification of cold tolerance

Two main cold hardiness strategies of insects - freeze tolerance in some species, and overwintering in a supercooled state without tolerance of freezing in many others - were recently reclassified. However, I present several problems with the current systems. My suggested classification is based on clearer definitions of the causes of cold injury. I recognize three main mortality factors: freezing of body liquids, cold shock, and cumulative chill injury. Presence or absence of each of these factors produce eight combinations. I have named the eight classes after Snow White and the Seven Dwarfs to avoid nomenclatural confusion. Some of these classes are probably not used as tactics against cold injury by any insect species. Other classes contain so many species that they might be reclassified in more detail, using values of supercooling point and other quantitative parameters. However, widely comparable parameters, like the upper limit of cold injury zone and the sum of injurious temperatures are still rarely published, thus we still lack comprehensive data for multivariate analyses. Every cold hardiness strategy should be characterized by a meaningful class or subclass together with the physiological, biochemical, and behavioural mechanisms employed by the insects. I also point out the existence of strategies that combine two tactics - either a switching strategy (during preparation for winter, population "chooses" which tactic will be used), or a dual strategy (individuals are ready to use one of the tactics depending on the prevailing environmental conditions).     

Cryopreservation of Salix species using sections from winter vegetative scions

Twigs of Salix species are candidates for cryopreservation procedures because they become tolerant of freezing temperatures during mid-winter. We examined several variables in developing a two-step cryopreservation procedure for sections from these twigs. Samples of Salix triandra cooled to -30 or -35 degree C and then transferred to the vapor phase over liquid nitrogen gave the greatest percent shoot formation. Cooling rate to -35 degree C had a major influence on shoot formation. Samples cooled at rates greater than 10 degree C/hour showed no shoot formation. The highest percent of shoot formation was achieved by cooling at 0.21 degree C/hour. Cooling rate from -35 degree C to liquid nitrogen did not influence shoot formation. Warming procedures affected shoot formation. Transferring samples from -160 degree C to either a +2 degree C cold room or to -3 degree C methanol gave similar levels of shoot formation. No shoot formation occurred either with warming in +40 degree C water or very slowly in a Styrofoam box. Eight of eleven Salix taxa tested using the established protocol had significant levels of shoot formation after cryogenic treatment.     

Insect cold hardiness: a Canadian perspective

The cold climates and diverse environments of Canada have allowed key studies of insect cold hardiness that developed and widened the understanding of this subject. For example, freezing tolerance, chilling tolerance, freezing resistance, supercooling, cryoprotectants and other features can be combined in many different ways, reflecting a wide range of adaptations. Many other factors interact with and influence cold hardiness, such as habitats and their selection, and water and energy balances. These findings suggest several topics that would be especially fruitful for further study in northern Canada.     

A simple method to adjust cooling rates for supercooling point determination

A simple method to obtain predetermined constant cooling rates for insect supercooling point (SCP) determination is described. A transient heat transfer equation was used to design polystyrene cubes of different sizes to yield constant rates of cooling at their centers when held at a constant surrounding temperature. Cubes of 0.29 x 0.29 x 0.29 m and 0.19 x 0.19 x 0.19 m were found to produce cooling rates of about -0.5 and -1 degree C per min, respectively, from 0 to -40 degree C. The observed temperature variations at the geometrical center of the cubes were similar to those predicted by the equation. Temperature plots showed a nearly constant rate of cooling. Supercooling points of Tribolium castaneum (Herbst) at different stages of development were recorded using polystyrene cubes. These SCPs compared favorably with published values. This method of obtaining cooling rates is economical, flexible, and allows for multiple simultaneous SCP measurements.     

壁面温度对疏水表面上水滴冻结的影响

本文研究了冷壁面温度对疏水表面(θ=108.8°)上单个水滴冻结过程的影响,比较了水滴的冻结时间及冻结前后的形态变化。结果表明,水滴冻结所需要的时间随冷表面温度的降低而减小,冷表面温度越低,水滴冻结后其顶端越容易出现树枝状霜晶生长。最后,根据能量守恒原理建立了冷表面上水滴冻结的数学模型,给出了相应的表达式,进而分析了壁面温度对水滴冻结时长的影响,计算结果与实验结果一致。     

Supercooling capacity and cold hardiness of the adults of the sycamore lace bug, corythucha ciliata (Hemiptera:Tingidae)

Supercooling point (SCP) of female adults of Corythucha ciliata was significantly lower than that of male adults, with an average being -11.49 degrees C and -9.54 degrees C, respectively. Low temperature survival of adults of different ages indicated that there were differences in cold survival ability among age groups of adults. Nonlinear regression analysis found that the response of C. ciliata adults to exposure time under different low temperature regimes (above -5 degrees C) was best fitted by a logistic equation. Both low temperature and exposure time had significant effects on mortality of adults. Temperatures above 5 degrees C did not prevent C. ciliata adults from surviving. C. ciliata was shown to be a freeze-intolerant but chill-tolerant insect. C. ciliata could tolerate subzero temperatures by supercooling. Temperature around -8 degres C is a critical point for successful overwintering of C. ciliata adults, which can establish in the whole areas where Platanus trees are planted in China.     

Seasonal changes in antifreeze protein gene transcription and water content of beetle Microdera punctipennis (Coleoptera, Tenebrionidae) from Gurbantonggut desert in Central Asia

Desert beetle Microdera punctipennis (Coleoptera: Tenebriondae) is a special species in Gurbantonggut Desert in Central Asia. To investigate the possible strategy it employs for cold survival, seasonal changes in supercooling point (SCP), body water content, haemolymph osmolality and antifreeze protein gene (Mpafp) expression were measured over 13 months. Our results show SCPs in M. punctipennis adults changed from -8.0°C in summer to -18.7°C in winter. During winter, adults endured modest water loss; total water decreased from 65.4 percent in summer to 55.9% in winter. Mpafp mRNAs level increased by 13.1 fold from summer to early winter, and haemolymph osmolality increased accordingly from 550 mOsm to 1486 mOsm. Correlation coefficient of Mpafp mRNAs level and SCP indicates that Mpafp mRNA explained 65.3 percent of the variation in SCPs. The correlation between Mpafp mRNA level and total water reflected an indirect influence of antifreeze protein on water content via reducing SCP.     

The effect of cold acclimation on the water relations and freezing tolerance of Hordeum vulgare L

During a 5 degree C and a 5/-1 degree C cold acclimation (CA) regime there was a significant decline in the water potential of winter barley, and a concurrent decline in tissue water content of the 5/-1 degree C CA plants. Results of carbohydrate analysis illustrated a significant (P < 0.001) accumulation of sucrose, fructose and glucose in the 5/-1 degree C CA plants, which was inversely correlated to water potential. Using an infrared imaging radiometer during a convection frost test the water release time (WRT) of 5/-1 degree C CA was demonstrated to be significantly (P < 0.001) longer than that observed in non-cold acclimated plants. This observation is consistent with visual analysis of exotherm curves where the rate of cellular water release to extracellular ice is reduced in the 5/-1 degree C CA plants, compared to the non-cold acclimated plants. These biochemical and physiological changes were correlated to increased plant health following a non-lethal freezing test to -5 degree C, where non-cold acclimated plants produced 2.3 +/- 0.3 tillers and 5 degree C and 5/-1 degree C CA plants produced 2.4 +/- 0.3 and 4.7 +/- 0.7 tillers, respectively. Results from this study imply that cold acclimation leads to changes in the physical state of water that result in a less osmotically responsive cellular environment and subsequently significantly less damage to meristematic tissue.     

Solute accumulation in heat seedlings during cold acclimation: contribution to increased freezing tolerance

Accumulation of sugars, amino acids and glycinebetaine in leaf tissues during cold acclimation was simultaneously monitored and compared in three wheat cultivars that have different freezing tolerance. Freezing tolerance was the decreasing order of cv. Norstar (NO) > cv. Chihokukomugi (CH) > or = cv. Haruyutaka (HA). During cold acclimation, there was a significant increase in osmotic concentration in the three cultivars. The increase was largely due to the increase in soluble sugars and the extent of the increase was the greatest in NO and the least in HA. While there was a considerable increase in glucose, fructose and sucrose during the first week of cold acclimation, the increase in raffinose occurred only after the second week. The total sugar content was the order of NO > CH > HA after 4 weeks of cold acclimation. Proline increased in all cultivars after 1 week of cold acclimation but a prolonged cold acclimation resulted in different profiles: no further increase occurred in HA while an additional increase occurred in other two cultivars. In all three cultivars, a noticeable increase of glycinebetaine occurred only after the second week of cold acclimation with the amount being the order of NO > CH > HA. It is concluded that a substantial part of the increase in osmotic concentration during cold acclimation was due to the increase in sugars, but the extent of the contribution of each compatible solute is cultivar-specific and can be associated with the degree of the maximum freezing tolerance attainable.     

Cold tolerance and overwintering of an introduced New Zealand frog, the brown tree frog (Litoria ewingii)

The overwintering strategy of Litoria ewingii in Otago, New Zealand, was studied under laboratory and field conditions. Microhabitat temperature measurements showed that the frogs were often exposed to subzero temperatures. In the laboratory, Litoria ewingii tolerated freezing for up to 6 hrs at -1 degrees C, and after the completion of the freezing event (about 1 hr) at -2 degrees C. Frogs frozen with insulation survived freezing for 12 hrs at -1 degrees C. Frogs supercooled to -1.2 +/- 0.1 degrees C and -1.7 +/- 0.3 degrees C on wet and dry substrates respectively. L. Ewingii tolerated up to 47.5% of its body water frozen. Plasma glucose levels and osmolality were not increased during freezing. It is concluded that l. Ewingii cannot avoid freezing and is sufficiently freeze tolerant to survive the subzero temperatures encountered during winter in Otago.     

过冷水滴碰撞结冰的实验与模拟研究

以宏观结冰/霜过程中过冷水滴的碰撞结冰现象为背景,实验对比了亲水和超疏水表面上常温水滴碰撞、常温水滴碰撞结冰和过冷水滴碰撞结冰的过程,建立了过冷水滴碰撞结冰过程的数值模型,研究了We数和接触角对碰撞结冰的影响。结果表明:相比于常温水滴的碰撞及其碰撞结冰过程,过冷水滴碰撞结冰过程的稳态铺展系数更大;随着过冷度和We数的增大以及接触角的减小,过冷水滴的碰撞结冰与常温水滴的碰撞在水滴形态和铺展系数上的差异逐渐增大。