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.     

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.     

Dual 13C, 15N labelling of terrestrial slugs (Deroceras reticulatum)

A simple, rapid and cost-effective laboratory method is described for labelling terrestrial slugs simultaneously with 13C and 15N. Slugs (Deroceras reticulatum) were provided with a mixture of [U-13C6]glucose, 15N-enriched lettuce powder, and wheat bran. Assimilation efficiencies for 13C (24.2%) and 15N (27.4%) were not affected by feeding regimes offering ad libitum or restricted access to unlabelled food during the labelling period. Body tissue was significantly more highly enriched in 13C but significantly less in 15N than cutaneous mucus after 15 days.     

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.     

Relationships between cold hardiness, and ice nucleating activity, glycerol and protein contents in the hemolymph of caterpillars, Aporia crataegi L

Insects in Siberia must tolerate some of the coldest conditions on earth. The relationship between hemolymph ice nucleating activity, glycerol and total protein concentrations, and cold hardiness was explored in Aporia crataegi L. (Lepidoptera: Pieridae). Cold-hardened overwintering caterpillars were collected at a time of year when temperatures are regularly below -50 degree C, and warm-acclimated at +22 degree C, to see how changes in the physical and chemical properties of the hemolymph influence their cold hardiness potential. Warm acclimation led to a decrease in glycerol and proteins content in the hemolymph, which was associated with the decrease in ice nucleating activity and dramatic loss of cold hardiness potential of the caterpillars. It is suggested that one of the effects of cryoprotection in the freeze tolerant insects, caused by glycerol, might be associated with its ability to form larger aggregates of ice nucleating polypeptides that initiate the ice nucleation at high subzero temperatures. Such ice nucleating structures seem to ensure a high probability of ice nucleation at relatively high temperatures, which may contribute to the extraordinary cold hardiness of A. crataegi caterpillars, which may tolerate temperatures below -85 degree С.     

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.     

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.     

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 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.     

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     

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.     

Cryopreservation of winter-dormant apple buds: I -Variation in recovery with cultivar and winter conditions

The widely-adopted protocol for the cryopreservation of winter buds of fruit trees, such as Malus and Pyrus, was developed in a region with a continental climate, that provides relatively hard winters with a consequent effect on adaptive plant hardiness. In this study the protocol was evaluated in a typical maritime climate (eastern Denmark) where milder winters can be expected. The survival over two winters was evaluated, looking at variation between seasons and cultivars together with the progressive reduction in survival due to individual steps in the protocol. The study confirms that under such conditions significant variation in survival can be expected and that an extended period of imposed dehydration at -4 degree C is critical for bud survival. The occurrence of freezing events during this treatment suggests that cryodehydration may be involved, as well as evaporative water loss. To optimize the protocol for maritime environments, further investigation into the water status of the explants during cryopreservation is proposed.     

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.     

Rapid cold-hardening in Zaprionus vittiger (Coquillett) (Diptera: Drosophilidae)

Low temperature injury can be significantly reduced by pre-treatment at sub-lethal low temperatures of only a few hours--a phenomenon termed 'rapid cold-hardening' (RCH). However, most studies to date have focused extensively on only a few key insect taxa, e.g. Drosophila melanogaster within the Drosophilidae family. Further studies on other closely-related species are required to better understand evolutionary and ecological variation in the magnitude of the RCH response in terrestrial arthropods. Here, we investigated RCH in a previously unstudied fruit fly, Zaprionus vittiger, following a range of high and low pretreatment temperatures. There was a significant improvement in Z. vittiger survival of lethal temperatures (2 h at -3 degrees C) following a 2 h pretreatment at 4, 7 and 10 degrees C as well as 30 degrees C for 2 h. Maximum survival (60-70%) during RCH was achieved following hardening at 7 degrees C and 10 degrees C but is lower than some Drosophila species under similar treatment conditions. Therefore, since RCH was detected in a confamilial species, we propose that RCH might be a widely conserved response to temperature variation in the family Drosophilidae, although some variation in the magnitude of the response can be detected.     

The effects of desiccation and exposure to cryogenic temperatures on embryonic axes of Landolphia kirkii

The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates on survival, assessed by the ability for both root and shoot development, of embryonic axes excised with a small portion of each cotyledon, from mature, recalcitrant seeds of Landolphia kirkii. All axes withstood rapid (flash) drying to a water content of c. 0.28 g water per g dry mass; however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g water per g dry mass, reducing survival to 10% and 0% after exposure to -196 degree C and -210 degree C, respectively. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent rewarming and rehydration. Cooling at 1 degree C per min facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g water per g dry mass after exposure to -70 degree C, with 45% viability retention when further cooled at 15 degree C per min to -180 degree C. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.     

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.     

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.     

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.     

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.     

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.