Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.     

Sublethal effects of subzero temperatures on the light brown apple moth,Epiphyas postvittana: fitness costs in response to partial freezing

Population responses to envir on mental extremes often dictate the bounds to species' distributions. However, population dynamics at, or near, those range limits may also be affected by sublethal effects. We exposed late instars and pupae of an invasive leafroller, Epiphyaspostvittana (Walker)(Lepidoptera: Tortricidae), to cold temperatures and measured the effects of exposure on subsequent survivorship, development, and reproduction. Cold temperature was applied as acute exposure to -10 °C (a low, but not immediately lethal temperature for this species) or the onset of freezing (the peak of the supercooling point exotherm). Survival was defined as the ability to successfully eclose as an adult. We measured immature development times, pupal mass, and adult longevity as proxies of fitness in survivors. Additionally, surviving insects were mated with individuals that had not been exposed to cold to measure fertility. There was no difference between the proportion of larvae or pupae that survived acute exposure to -10 °C and those exposed to the control temperature. Approximately 17% of larvae and 8% of pupae survived brief periods with internal ice formation and continued development to become reproductively viable adults. Importantly, surviving the onset of freezing came with significant fitness costs but not to exposure to -10 °C;most insects that survived partial freezing had lower fertility and shorter adult lifespans than either the -10 °C or control group. These results are discussed within the context of forecasting invasive in sect distributions.     

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.     

An inexpensive replacement for dry ice in the laboratory

A reusable inexpensive replacement for dry ice in laboratory use is presented. Commercially available small pellets of stone or metal can be stored in a −80 °C freezer and used for quickly freezing small samples with a freezing rate that is actually somewhat faster than with dry ice itself. Following use, the material is returned to the freezer to re-chill until the next usage.     

A molecular marker to select for freezing tolerance in Gramineae

Summary We isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat. The gene product was purified and used to produce antibodies. Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance. This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5. The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance. Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one. In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals. The antibody did not cross-react with any of the non-cereal species examined. The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.     

Climatic variability and the evolution of insect freeze tolerance

Insects may survive subzero temperatures by two general strategies: Freeze-tolerant insects withstand the formation of internal ice, while freeze-avoiding insects die upon freezing. While it is widely recognized that these represent alternative strategies to survive low temperatures, and mechanistic understanding of the physical and molecular process of cold tolerance are becoming well elucidated, the reasons why one strategy or the other is adopted remain unclear. Freeze avoidance is clearly basal within the arthropod lineages, and it seems that freeze tolerance has evolved convergently at least six times among the insects (in the Blattaria, Orthoptera, Coleoptera, Hymenoptera, Diptera and Lepidoptera). Of the pterygote insect species whose cold-tolerance strategy has been reported in the literature, 29% (69 of 241 species studied) of those in the Northern Hemisphere, whereas 85 % (11 of 13 species) in the Southern Hemisphere exhibit freeze tolerance. A randomization test indicates that this predominance of freeze tolerance in the Southern Hemisphere is too great to be due to chance, and there is no evidence of a recent publication bias in favour of new reports of freeze-tolerant species. We conclude from this that the specific nature of cold insect habitats in the Southern Hemisphere, which are characterized by oceanic influence and climate variability must lead to strong selection in favour of freeze tolerance in this hemisphere. We envisage two main scenarios where it would prove advantageous for insects to be freeze tolerant. In the first, characteristic of cold continental habitats of the Northern Hemisphere, freeze tolerance allows insects to survive very low temperatures for long periods of time, and to avoid desiccation. These responses tend to be strongly seasonal, and insects in these habitats are only freeze tolerant for the overwintering period. By contrast, in mild and unpredictable environments, characteristic of habitats influenced by the Southern Ocean, freeze tolerance allows insects which habitually have ice nucleators in their guts to survive summer cold snaps, and to take advantage of mild winter periods without the need for extensive seasonal cold hardening. Thus, we conclude that the climates of the two hemispheres have led to the parallel evolution of freeze tolerance for very different reasons, and that this hemispheric difference is symptomatic of many wide-scale disparities in Northern and Southern ecological processes.     

异色瓢虫成虫耐寒能力的季节性变化

为研究异色瓢虫Harmonia axyridis自然种群耐寒能力的季节性变化,测定了其过冷却点、体内含水量及总脂肪含量和低温存活力。结果表明：异色瓢虫成虫低温存活力呈现出明显的季节性变化,越冬前成虫的耐寒性显著强于夏季成虫和越冬后成虫。冷驯化（5℃, 5 d）可以显著提高夏季成虫的低温存活力。雌雄成虫过冷却点和体内含水量随气温的降低而降低,升高而升高。过冷却点7月份最高,分别为−7.6℃和−8.0℃;越冬中期（2008-01-15）最低,分别为−18.1℃ 和−16.9℃。雌雄成虫体内含水量9月份最高,分别为66.87%和68.01%,10月份显著降低,越冬后期（2008-02-19）最低,分别为52.94%和51.53%。越冬期间过冷却点和体内含水量显著低于其他时期。而雌雄成虫体内总脂肪含量在越冬开始就达到最高,分别为50.07%和47.93%,随后又逐渐降低,越冬期间显著高于其他时期。由此可知异色瓢虫自然种群的耐寒性呈现出明显的季节性变化,文中还就异色瓢虫自然种群耐寒性影响因素及其越冬策略进行了讨论。     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.     

Survival at low temperature of larvae of the pine processionary moth Thaumetopoea pityocampa from an area of range expansion

1 Larvae of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) develop throughout the winter, although their feeding activity and survival can be impaired by adverse climatic factors. The present study investigated the survival at low temperature of larvae originating from a population with range expansion in an alpine valley in Northern Italy.
2 The supercooling point of individually analysed larvae averaged at −7 °C. This value insufficiently described the cold hardiness of the larvae; 39% of the tested larvae were alive when returned to room temperature immediately after freezing. When larval colonies inside their nest were exposed to −17 °C for 1 h after gradual temperature decrease, survival was 70.4%.
3 Rearing of larvae in the laboratory at different day/night temperatures indicated an effect of cumulative chill injury on larvae. A logistic regression explained the relationship between negative thermal sum (h°C below 0 °C) received in the laboratory experiment and larval survival. A similar relationship was demonstrated between negative thermal sum and survival of larval colonies in the field.
4 In the laboratory experiment, some tested larvae were able to survive for up to 8 weeks without feeding depending on rearing temperature. As expected, feeding occurred only when larvae were reared at temperatures of 9 °C day/0 °C night.
5 We classify the larvae of T. pityocampa as being moderate freezing tolerant. The winter behaviour allows this species to track climate warming by a rapid expansion into those areas that become compatible with the insect's development.     

Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode Globodera rostochiensis

Perry R. N. and Wharton D. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Globodera rostochiensis. International Journal for Parasitology15: 441–445. Hatched second-stage juveniles of Globodera rostochiensis can survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling.     

Intracellular freezing, viability, and composition of fat body cells from freeze-intolerant larvae of Sarcophaga crassipalpis.

Although it is often assumed that survival of freezing requires that ice formation must be restricted to extracellular compartments, fat body cells from freeze-tolerant larvae of the gall fly, Eurosta solidaginis (Diptera, Tephritidae) survive intracellular freezing. Furthermore, these cells are highly susceptible to inoculative freezing by external ice, undergo extensive lipid coalescence upon thawing, and survive freezing better when glycerol is added to the suspension medium. To determine whether these traits are required for intracellular freeze tolerance or whether they are incidental and possessed by fat body cells in general, we investigated the capacity of fat body cells from nondiapause-destined and diapause-destined (i.e., cold-hardy) larvae of the freeze-intolerant flesh fly Sarcophaga crassipalpis (Diptera, Sarcophagidae) to survive intracellular freezing. Fat body cells from both types of larvae were highly susceptible to inoculative freezing; all cells froze between -3.7 to -6.2 degrees C. The highest rates for survival of intracellular freezing occurred at -5 degrees C. The addition of glycerol to the media markedly increased survival rates. Upon thawing, the fat body cells showed little or no lipid coalescence. Fat body cells from E. solidaginis had a water content of only 35% compared to cells from S. crassipalpis larvae that had 52-55%; cells with less water may be less likely to be damaged by mechanical forces during intracellular freezing.     

Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at -10 degrees C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at -10 degrees C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at -20 degrees C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.     

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.     

Effect of antibiotics on thermodynamic properties of freezing media in rabbit species: A first calorimetric approach

Semen freezing is an effective and safe solution for the cryopreservation of animal genetic resources and for the diffusion of the genetic progress. Actually, these techniques are not yet under control for the rabbit species partly because methods are not clearly defined. Thus, the aim of this work is to study the effect of antibiotics (Penicillin G, Streptomycin) routinely used in freezing semen on the thermodynamic properties of freezing media mainly used in rabbit species. Measurements realized by differential scanning calorimetry show that these antibiotics may change the temperature of crystallization and the quantity of ice formed in the freezing media considered. Our calorimetric approach underlined that the composition and the properties of the cryoprotective solutions should be studied more precisely.     

Drivers of apoplastic freezing in gymnosperm and angiosperm branches

It is not well understood what determines the degree of supercooling of apoplastic sap in trees, although it determines the number and duration of annual freeze–thaw cycles in a given environment. We studied the linkage between apoplastic ice nucleation temperature, tree water status, and conduit size. We used branches of 10 gymnosperms and 16 angiosperms collected from an arboretum in Helsinki (Finland) in winter and spring. Branches with lower relative water content froze at lower temperatures, and branch water content was lower in winter than in spring. A bench drying experiment with Picea abies confirmed that decreasing branch water potential decreases apoplastic ice nucleation temperature. The studied angiosperms froze on average 2.0 and 1.8°C closer to zero Celsius than the studied gymnosperms during winter and spring, respectively. This was caused by higher relative water content in angiosperms; when branches were saturated with water, apoplastic ice nucleation temperature of gymnosperms increased to slightly higher temperature than that of angiosperms. Apoplastic ice nucleation temperature in sampled branches was positively correlated with xylem conduit diameter as shown before, but saturating the branches removed the correlation. Decrease in ice nucleation temperature decreased the duration of freezing, which could have an effect on winter embolism formation via the time available for gas escape during ice propagation. The apoplastic ice nucleation temperature varied not only between branches but also within a branch between consecutive freeze–thaw cycles demonstrating the stochastic nature of ice nucleation.     

Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria

An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.     

Experimental studies on the cold tolerance of Alaskozetes antarcticus

The cold tolerance mechanism of the Antarctic terrestrial mite Alaskozetes antarcticus (Michael) was investigated in cultured animals. Freezing is fatal in this species and winter survival occurs by means of supercooling, which is enhanced by the presence of glycerol in the body. There is an inverse, linear relationship between the concentration of glycerol and the supercooling point, which may be as low as ?30°C. Feeding detracts from supercooling ability by providing ice nucleators in the gut which initiate freezing at relatively high sub-zero temperatures. Experiments on the effects of various environmental factors showed that low temperature acclimation gave rise to increased glycerol concentrations and suppressed feeding, while desiccation also stimulated glycerol production. Photoperiod had no effect on cold tolerance in this species. The juvenile instars of A. antarcticus were found to possess a greater degree of low temperature tolerance than adults.     

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.