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.     

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.     

Some attributes of cold hardiness of the gregarious ectoparasitoid Colpoclypeus florus (Hymenoptera: Eulophidae)

Cold hardiness of diapause and non-diapause larvae of the parasitoid Colpoclypeus florus Walker (Hymenoptera: Eulophidae) was examined in the laboratory. Mean supercooling point (SCP) for diapausing larvae was -26.7 degree C and for non-diapausing larvae immediately after their larval development, was -16.2 degree C. Mean SCP for non-diapause larvae at the prepupa stage was -19.1 degree C. A short period of acclimation (1 week at 5 degree C) had no influence on the mean SCPs of both diapause and non-diapause larvae. Pre-freeze mortality for diapause and non-diapause larvae was also studied. A constant exposure of diapause larvae to -6 degree C resulted in high mortality (70.7 percent) after a period of 40 days. In contrast, 6 days at -6 degree C were sufficient to cause the same level of mortality in non-diapause larvae. After exposure of 15 days at -9 degree C, mortality for non-diapause larvae was 70 percent, whereas after 20 days at the same temperature mortality of diapause larvae was 25 percent. The importance of these findings for the cryobiology of C. florus is discussed.     

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.     

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.     

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.     

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.     

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.     

Refining the risk of freezing mortality for antarctic terrestrial microarthropods

In studies of three common, freezing susceptible, Antarctic microarthropods, the springtail Cryptopygus antarcticus and the mites Alaskozetes antarcticus and Halozetes belgicae, we report (i) the consequences on cold tolerance of cooling in contact with water, and (ii) the risk of freezing when held at temperatures above the typical freezing point (measured using standard techniques) for up to 12 h. The springtail showed no change in SCP distribution when in contact with freezing water while, in contrast, the mites showed clear shifts towards decreased cold tolerance, in addition to death of c. 33% of individuals during the freezing of the water. The springtail showed a bimodal SCP distribution, with the population divided into "high"(typically -8 to -12 degree C) and "low" (typically below -20 degree C) groups. Some animals held at temperatures above these values froze, over a timescale between minutes and several hours. These results highlight the danger of equating standard cold tolerance measures with mortality risk under more realistic water and thermal regimes.     

Cold tolerance of field-collected and laboratory reared larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae)

Sesamia nonagrioides Lefébvre (Lepidoptera: Noctuidae) is considered one of the most destructive pests of corn in the Mediterranean region. The purpose of the present study was to investigate some aspects of the cold tolerance of non-diapausing and diapausing laboratory reared larvae of S. nonagrioides, as well as of field-collected larvae, taking into consideration various parameters, such as supercooling ability, mean lethal temperature and accumulation of cryoprotectant substances, in relation to diapause. Our results provide evidence that S. nonagrioides has limited cold tolerance as it displays a low ability of supercooling. This is strongly supported by the fact that mortality of the individuals occurred after extended exposure to subzero temperatures, equivalent or slightly lower to their mean supercooling point. However, lethal temperatures of diapausing larvae were significantly lower in relation to that of non-diapausing larvae, indicating the existence of a direct link between diapause and cold tolerance. Regarding the role of cryoprotectant substances, accumulation of glycerol seems to be closely related to diapause, in contrast to accumulation of trehalose, which is more related to exposure to low temperatures slightly higher than 0 degree C. Finally, non-diapausing larvae of different instars displayed a similar ability of supercooling and tolerance to low temperatures as well as accumulation of cryoprotectant substances. The ecological significance of our findings on cold tolerance of this species is being discussed with particular reference to the microclimate observed in northern Greece.     

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.     

Suppression of ice nucleation in supercooled water under temperature gradients

Understanding the behaviours of ice nucleation in non-isothermal conditions is of great importance for the preparation and retention of supercooled water. Here ice nucleation in supercooled water under temperature gradients is analyzed thermodynamically based on classical nucleation theory (CNT). Given that the free energy barrier for nucleation is dependent on temperature, different from a uniform temperature usually used in CNT, an assumption of linear temperature distribution in the ice nucleus was made and taken into consideration in analysis. The critical radius of the ice nucleus for nucleation and the corresponding nucleation model in the presence of a temperature gradient were obtained. It is observed that the critical radius is determined not only by the degree of supercooling, the only dependence in CNT, but also by the temperature gradient and even the Young's contact angle. Effects of temperature gradient on the change in free energy, critical radius, nucleation barrier and nucleation rate with different contact angles and degrees of supercooling are illustrated successively. The results show that a temperature gradient will increase the nucleation barrier and decrease the nucleation rate, particularly in the cases of large contact angle and low degree of supercooling. In addition, there is a critical temperature gradient for a given degree of supercooling and contact angle, at the higher of which the nucleation can be suppressed completely.     

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.     

Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species

Differential thermal analysis (DTA) has indicated that xylem ray parenchyma cells (XRPCs) of hardwood species adapt to freezing of apoplastic water either by deep supercooling or by extracellular freezing, depending upon the species. DTA studies indicated that moderately cold hardy hardwood species exhibiting deep supercooling in the XRPCs were limited in latitudinal distribution within the −40°C isotherm, while very hardy hardwood species exhibiting extracellular freezing could distribute in colder areas beyond the −40°C isotherm. Predictions based on the results of DTA, however, indicate that XRPCs exhibiting extracellular freezing may appear not only in very hardy woody species native to cold areas beyond the −40°C isotherm but also in less hardy hardwood species native to tropical and subtropical zones as well as in a small number of moderately hardy hardwood species native to warm temperate zones. Cryo-scanning electron microscopic (cryo-SEM) studies on the freezing behavior of XRPCs have revealed some errors in DTA. These errors are originated mainly due to the overlap between exotherms produced by freezing of water in apoplastic spaces (high temperature exotherms, HTEs) and exotherms produced by freezing of intracellular water of XRPCs by breakdown of deep supercooling (low temperature exotherms, LTEs), as well as to the shortage of LTEs produced by intracellular freezing of XRPCs. In addition, DTA results are significantly affected by cooling rates employed. Further, cryo-SEM observations, which revealed the true freezing behavior of XRPCs, changed the previous knowledge of freezing behavior of XRPCs that had been obtained by freeze-substitution and transmission electron microscopic studies. Cryo-SEM results, in association with results obtained from DTA that were reconfirmed or changed by observation using a cryo-SEM, revealed a clear tendency of the freezing behavior of XRPCs in hardwood species to change with changes in the temperature in the growing conditions, including both latitudinal and seasonal temperature changes. In latitudinal temperature changes, XRPCs in less hardy hardwood species native to tropical and subtropical zones exhibited deep supercooling to −10°C, XRPCs in moderately hardy hardwood species native to temperate zones exhibited a gradual increase in the supercooling ability to −40°C from warm toward cool temperate zones, and XRPCs in very hardy hardwood species native to boreal forests exhibited extracellular freezing via an intermediate form of freezing behavior between deep supercooling and extracellular freezing. In seasonal temperature changes, XRPCs in hardwood species native to temperate zones changed their supercooling ability from a relatively low degree in summer to a high degree in winter. XRPCs in hardwood species native to boreal forests changed their freezing behavior from deep supercooling to −10°C in summer to extracellular freezing in winter. These results indicate that the freezing behavior of XRPCs in hardwood species tends to shift gradually from supercooling of −10°C, to a gradual increase in the deep supercooling ability to −40°C or less, and finally to extracellular freezing as a result of cold acclimation in response to both latitudinal and seasonal temperature changes. It is thought that these temperature-dependent changes in the freezing behavior of XRPCs in hardwood species are mainly controlled by changes in cell wall properties, although no distinct changes were detected by electron microscopic observations in cell wall organization between hardwood species or between seasons. Evidence of temperature-dependent changes in the freezing behavior of XRPCs in hardwood species provided by the results of studies using a cryo-SEM has indicated the need for further investigation to clarify cold acclimation-induced cell wall changes at the sub-electron microscopic level in order to understand the mechanisms of freezing adaptation.     

醇类低温保护剂水溶液过冷和水合性质研究

利用差示扫描量热仪研究了乙二醇、丙三醇、1,3丙二醇、1,3丁二醇和2,3丁二醇水溶液的过冷行为和水合性质,得出了这些性质与线性多元醇溶液浓度的关系.研究发现,在低浓度过冷度与浓度之间没有明显关系;而在中高浓度则具有相似的变化规律,即随着溶液浓度增大而增大.各低温保护剂在水合性质上所表现出的差异性,体现了保护剂的官能团(羟基、甲基)所起到的重要作用.     

Investigation of the interaction of oxygen with an Al(100) surface by work function methods

The change in work function (Δφ) of an Al(100) surface due to oxygen adsorption at different pressures (10⁻⁶– 10⁻⁵ Torr) and different crystal temperatures (115–600 K) has been investigated. The data show an initially positive Δφ at low temperatures and negative Δφ values for temperatures above 160 K. The results are pressure independent during the first 500 L oxygen exposure but become pressure dependent at higher exposures. Measurements of Δφ in vacuum after exposure to 1040 L O₂ show a nearly exponential decrease of Δφ with time. The time constants of this exponential behaviour are temperature dependent and vary between 1.5 min for 370 K and 33 min at 115 K. These time dependent effects are believed to be related to the movement of adsorbed oxygen to sites below the aluminum surface.     

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.     

Rapid cold hardening in young hoppers of the migratory locust Locusta migratoria L. (Orthoptera: Acridiidae)

This paper describes a rapid cold hardening process for first instar hoppers of the migratory locust Locusta migratoria L. First instar hoppers of this species are often subjected to subzero temperatures or frosts in early April or May after their emergence from the soil. The mean supercooling point of hoppers is -13.0 +/- 1.4 degrees C; the fact that none could survive this temperature suggests they are freezing intolerant. When young hoppers were transferred directly from 30 degrees C to -7 degrees C for 2 h, there was only 35.8% survival. However, exposure to 0 degrees C for 2 h prior to transfer to -7 degrees C increased the apparent survival to 75%. A similar rapid cold hardening response can also be induced by gradual cooling at rates of between 0.05 and 0.1 degreess C min(-1). Rapid cold hardening also elevates the Ltime50 of first instar hoppers at -7 degrees C by approximately 3 fold, and reduces the lethal temperature from -10 degrees C to -12 degrees C. However, the protection from cold shock gained through rapid cold hardening was transient and easily lost within 2 h of hoppers being returned to 30 degrees C. The rapid cold hardening response is possibly advantageous to first instar hoppers that are often exposed to large temperature fluctuations in spring or early summer.     

Chilling sensitivity in zebrafish (Brachydanio rerio) oocytes is related to lipid phase transition

Oocytes of zebrafish were used to study chilling sensitivity and membrane lipid phase transitions in tropical fish. The oocytes were divided into two groups: small (without yolk, <0.1mm) and large (with yolk, >0.1mm). After exposure of the oocytes to different temperatures (25, 22, 19, 16, 12, 8, 0, -8+0.5 degree C) for 15 minutes, the integrity of their membranes was determined by carboxyfluorescein diacetate (CFDA) staining. At 16 and 12 degree C, damage was maximum (membrane integrity decreased by 50%) for small and large sizes, respectively. Lipid phase transition (LPT), which was evaluated using Fourier transform infrared (FTIR) microscopy, indicated phase transitions at the same temperatures at which damage was maximal (between 22 and 12 degree C).In another series of experiments, the chilling sensitivity of oocytes taken from zebrafish which had been held at 16 degree C for different periods of time (0, 15, 30, 60 minutes) was determined as described above. In small oocytes membrane integrity decreased after 15 minutes, and in large oocytes integrity decreased after 30 minutes. Chilling sensitivity was also measured in oocytes from zebrafish that had been held at 16 degree C for 30 minutes and then rewarmed to 28 degree C for 2 hours. Despite this recovery period, the integrity of the oocytes remained low. We suggest that chilling sensitivity in zebrafish oocytes is related to lipid phase transition of their membranes and starts at 10 degree C below the physiological temperature     

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.