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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.     

Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686

The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.     

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.     

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.     

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.     

气体传热对多层绝热性能影响的试验研究

文中通过建立的能进行夹层气体置换的稳态量热器试验系统,试验分析了夹层气体传热对多层绝热材料有效热导率的影响,重点对置换气体种类、气体压强、材料层数及冷热边界温度对多层材料的影响进行试验研究。试验表明在10—60层/cm层密度范围,真空度低于100Pa时,Kn数属于自由分子状态区域和中间压强区域,此时材料的有效热导率随残留气体热适应系数的增大而减小,并随着真空度的降低而增大,当残留气体为空气时,为保证多层材料的绝热性能,应尽量维持真空度不低于10-2Pa。同时,分析表明为有效降低低真空下稀薄气体传热对多层绝热性能的影响,可以采用综合热适应系数较低的气体置换夹层中的空气,以减少低真空多层绝热材料的有效热导率,改善绝热性能。     

Determination of molecular orientational order in cold-stretched poly(p-phenylene vinylene) thin films by DECODER 13C NMR

The properties of poly(p-phenylene vinylene) (PPV) films depend on the degree of orientational order present in the films. Recently, Dermaut et al. reported a novel cold-stretching technique (Macromolecules 33, 5634-5637 (2000)) in which chain alignment can be introduced into PPV precursor films by uniaxially stretching them prior to the thermal elimination reaction that forms PPV. The two-dimensional direction exchange with correlation for orientation-distribution evaluation and reconstruction (DECODER) 13C NMR technique was applied to both unstretched PPV films and PPV films that were uniaxially cold stretched to a draw ratio lambda = l/l0 = 5. The unstretched films were found to be moderately ordered, comprised of a component present at 80% with a Gaussian distribution of 60 degrees fwhm, while the remaining 20% is isotropically distributed. A distribution of 9 degrees +/- 3 degrees fwhm was measured by NMR in good agreement with IR dichroism measurements for the uniaxially cold-stretched films, establishing that a high degree of orientational order can be introduced by cold stretching PPV films.     

Investigation of Hg resonance 184.9 nm line profile in a low-pressure mercury-argon discharge

The line profiles of 184.9 nm Hg resonance line emitted from a Hg low-pressure high-frequency electrodeless discharge lamp, containing Hg 198 isotope, have been measured by means of a Zeeman scanning spectrometer at the mercury cold spot temperature values in the range of 0-23 °C. Two different methods were used to determine the real spectral line profile and to separate the instrument function: (i) solving the ill-posed inverse problem by means of the Tikhonov's regularization method; and (ii) the mathematical modelling by means of a non-linear multi-parameter chi-square fit. The real Hg 184.9 nm spectral line profiles, determined by both methods, are compared. Influence of instrumental function, degree of the self-absorption and temperature of the radiating atoms are obtained.     

Mammalian hibernation: lessons for organ preparation?

The adaptations to low environmental temperatures exhibited in mammalian hibernation are many and varied, and involve molecular and cellular mechanisms as well as the systematic physiology of the whole organism. Natural torpidity is characterised by a profound reduction in body temperature and other functions lasting from a few hours to several weeks. Controlled reduction of heart rate, respiration and oxygen consumption is followed by the fall in body temperature. However, thermoregulation persists such that a decrease in ambient temperature below dangerous levels typically triggers arousal, and shivering and non-shivering thermogenesis from brown fat provide the heat to restore body temperature to normal levels. Many of the cellular mechanisms for survival are similar to those brought into play during medium-term storage of organs destined for transplantation. For example maintenance of ionic regulation and membrane fluxes is fundamental to cell survival and function at low body temperatures. Differences between hibernating and non-hibernating species are marked by differences in Na+/K+ transport and Ca++pumps. These in turn are probably associated with alterations in the lipoproteins of the plasma membrane and inner mitochondrial membrane. We have accordingly conducted a series of pilot studies in captured Richardson's ground squirrels kept in laboratory conditions as a model for hypothermic organ preservation. Tissue function was compared during the summer (non-hibernating season) with that in the winter when the animals could be: (i) in deep hibernation in a cold chamber at 4 degree C; (ii) maintained in an ambient temperature of 4 degree C but active and awake; or (iii) active at an ambient temperature of 22 degree C. The studies involved: whole animal monitoring of standard physiological parameters; whole organ (kidney) storage and transplantation for viability assessment; storage and functional assessment on an ex vivo test circuit with capacity for perfusion at normothermic and hypothermic temperatures; measurement of thyroid function; measurements of total nucleotides (ATP, ADP and AMP)and ratios by standard techniques after freeze-clamping of organs; similar nucleotide and pH measurements using31P-NMR as a non-invasive whole animal technique; and measurement of O2 uptake and gluconeogenesis using isolated renal tubules and isolated hepatocytes. Marked differences in cold tolerance were demonstrated between organs taken from hibernating versus non-hibernating individuals. In particular kidneys transplanted from animals in deep hibernation were capable of withstanding up to 72 hours of cold storage as compared with up to 24 hours in non-hibernating squirrels or in comparable sized rats. Adaptations which might provide valuable clues in our attempts to better preserve human organs for transplantation are explored in some depth in this report.