Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.     

Freeze fitness in alpine Tiger moth caterpillars and their parasitoids

The adaptive fitness of a freeze-tolerant insect may be mediated by both endogenous and exogenous interactions. The aim of the study presented here was to characterize the freeze tolerance of alpine Tiger moth caterpillars (Metacrias huttoni) and highlight two poorly explored indices of the potential attrition of fitness: (1) downstream development and reproduction; (2) parasitism. Caterpillars survived temperatures as low as −16°C and demonstrated >90% 72-h survival after exposures to −10°C. Two-week acclimations at 5, 10, and 20°C had no effect on body water content, haemolymph osmolality or survival of equilibrium freezing, but there was a significant elevation of the temperature of crystallization (T _c) in those caterpillars acclimated to 5°C. Cell viability of fat body tissue was resilient to freezing (−10 to −16°C), but midgut and tracheal cells showed significant degradation. Pupation and eclosion were unaffected by freezing at −5 or −10°C. Likewise, there were no significant differences in egg production or the proportion of eggs that hatched between control and frozen insects. By contrast, the ability of tachinid larvae to survive freezing within their hosts means that parasitism plays an important role in regulating population size. Mean parasitism of caterpillars by tachinids was 33.3 ± 7.2%. Pupation and imago emergence of tachinids after host ‘endo-nucleation’ was >75%. Eclosed adult tachinids showed a non-significant increase in the incidence of wing abnormalities in relation to low temperature exposure.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

The oatmeal nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> survives moderately low temperatures by freezing tolerance and cryoprotective dehydration

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S ₅₀) of −2.5°C after cooling at 0.5°C min⁻¹ and freezing for 1 h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7 days at 5°C), with the S ₅₀ being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S ₅₀ is lowered by 0.55°C, compared to the control, after 4 h freezing at −1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.     

A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L⁻¹ Kinetin (Kn), 0.5 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.5 mg L⁻¹ 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl₂ solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS₂ solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m⁻² s⁻¹ provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Thermal physiology and energetics in male desert hamsters (<Emphasis Type="Italic">Phodopus roborovskii</Emphasis>) during cold acclimation

The adjustments in thermal physiology and energetics were investigated in male desert hamsters (Phodopus roborovskii) which were acclimated to 5°C for 4 weeks. Mean core body temperature in cold acclimated animals decreased by 0.21°C compared with controls. Further analysis revealed that the decrease mainly occurred in the scotophase, while in the photophase core body temperature remained constant during the whole cold acclimation. Thermogenic capacity, represented by resting metabolic rate and nonshivering thermogenesis increased in cold acclimated hamsters from initial values of 1.38 ± 0.05 and 5.32 ± 0.30 to 1.77 ± 0.08 and 8.79 ± 0.31 mlO₂ g⁻¹ h⁻¹, respectively. After cold acclimation, desert hamsters maintained a relative stable body mass of 21.7 ± 0.1 g very similar to the controls kept at 23°C (21.8 ± 0.1 g). The mean values of food intake and digestible energy (metabolisable energy) in cold acclimated hamsters were 5.3 ± 0.1 g day⁻¹ and 76.3 ± 0.9 kJ day⁻¹ (74.8 ± 0.9), respectively, which were significantly elevated by 76.7 and 80.4% compared to that in control group. The apparent digestibility was 81.0 ± 0.3% in cold acclimated animals which was also higher than the 79.7 ± 0.2% observed in controls. This increase corresponded with adaptive adjustments in morphology of digestive tracts with 20.2 and 36.8% increases in total length and wet mass, respectively. Body fat mass and serum leptin levels in cold acclimated hamsters decreased by 40.7 and 67.1%, respectively. The wheel running turns and the onset of wheel running remained unchanged. Our study indicated that desert hamsters remained very active during cold acclimation and displayed adaptive changes in thermal physiology and energy metabolism, such as enhanced thermogenic and energy processing capacities.     

Equilibrium,quasi-equilibrium,and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.     

Freezing tolerance of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F _v/F _m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F _v/F _m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F _v/F _m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F _v/F _m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F _v/F _m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Effect of seawater temperature on the productivity of <Emphasis Type="Italic">Laminaria japonica</Emphasis> in the Uwa Sea,southern Japan

Recent studies on global climate change report that increase in seawater temperature leads to coastal ecosystem change, including coral bleaching in the tropic. In order to assess the effect of increased seawater temperature on a temperate coastal ecosystem, we studied the inter-annual variation in productivity of Laminaria japonica using long-term oceanographic observations for the Uwa Sea, southern Japan. The annual productivity estimates for L. japonica were 2.7 ± 2.5 (mean ± SD) kg wet wt. m⁻¹ (length of rope) (2003/2004), 1.0 ± 0.6 kg wet wt. m⁻¹ (2004/2005) and 12.1 ± 12.5 kg wet wt. m⁻¹ (2005/2006). Our previous study using the same methodology at the same locality reported that the productivity was estimated for the 2001/2002 (33.3 ± 15.2 kg wet wt. m⁻¹) and 2002/2003 (34.0 ± 8.7 kg wet wt. m⁻¹) seasons. Productivity in 2003/2004 and 2004/2005 was significantly lower than in years 2001/2002, 2002/2003 and 2005/2006. A comparison of oceanographic conditions among the 5 years revealed the presence of threshold seawater temperature effects. When the average seawater temperature during the first 45 days of each experiment exceeded 15.5°C, productivity was reduced to about 10 % of that in cooler years. Moreover the analysis of growth and erosion rates indicates that when the seawater temperature was over 17.5°C, erosion rate exceeded growth rate. Thus, an increase of seawater temperature of just 1°C during winter drastically reduces the productivity of L. japonica in the Uwa Sea.     

Differences in cold tolerance,desiccation resistance,and cryoprotectant production between three populations of <Emphasis Type="Italic">Eurosta solidaginis</Emphasis> collected from different latitudes

Possible links between cold-tolerance and desiccation resistance were examined between larvae of the goldenrod gall fly collected from Michigan, southern Ohio, and Alabama locations as their host plant senesced. After acclimation to 5°C, Michigan-collected larvae were more cold-tolerant (25% survival after a 96 h exposure to −40°C) than larvae from Ohio (10% survival) and Alabama (0% survival). Increased cold-tolerance was partially linked to higher concentrations of the cryoprotectant glycerol (Michigan: 500 ± 30 mmol; Ohio: 270 ± 20; Alabama: 220 ± 20). Moreover, cryoprotectants may have functioned to reduce rates of overall and cuticular water loss for Michigan larvae, 0.10 ± 0.01 and 0.037 ± 0.003 μg mm⁻² h⁻¹, respectively, values that were 40-44% lower than those for Ohio and Alabama larvae and may represent a link between desiccation resistance and cold-tolerance. After acclimation to 20°C, Alabama-collected larvae had metabolic rates that were 40% lower than those from Ohio and Michigan that averaged 0.100 ± 0.006 μl of CO₂ produced g⁻¹ h⁻¹. The lower metabolic rate of Alabama-collected larvae at 20°C likely resulted in reduced respiratory transpiration that may represent a mechanism to maintain water balance at the higher overwintering temperatures they typically experience.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Delayed onset of adult antifreeze activity in juveniles of the Antarctic icefish <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis>

Many Antarctic notothenioid species endemic to the Seasonal Pack-ice Zone have converged on adult blood serum freezing points that are several tenths of a degree above the freezing point of seawater. While these fishes share high adult serum freezing points, the development of their freeze avoidance during ontogeny has not been studied. We investigated this in wild caught juveniles of one such species, Chaenocephalus aceratus (family Channichthyidae), using blood serum antifreeze activity as a proxy for their freeze avoidance. Juvenile serum antifreeze activity was significantly below that of adults through the oldest year 2+ specimens collected. This increased at an estimated rate of 0.368 × 10⁻³ ± 0.405 × 10⁻⁴°C day⁻¹ which, if sustained, would leave C. aceratus below their adult serum antifreeze activity levels of 0.57 ± 0.08°C until 4.2 years after hatching. Underlying the 2.7-fold increase in their serum antifreeze activity from late year 0+ juveniles to adults was an even greater 10.4-fold increase in the concentration of their serum antifreeze glycopeptides, which increased proportionally across all of their serum AFGP size isoforms. With insufficient antifreeze activity to avoid freezing in the ice-laden surface waters, both adult and juvenile C. aceratus are most likely restricted to the year round ice-free waters where a metastable supercooled state can be maintained.     

Dehydration and cold hardiness in the Arctic Collembolan Onychiurus arcticus Tullberg 1876

Specimens of the Arctic Collembolon Onychiurus arcticus were exposed to desiccation at several subzero temperatures over ice and at 0.5 °C over NaCl solutions. The effects of desiccation on water content (WC), body fluid melting point (MP), supercooling point (SCP) and survival were studied at several acclimation temperatures and relative humidities. Exposure to temperatures down to −19.5 °C caused a substantial and increasing dehydration. At the lowest exposure temperature unfrozen individuals lost 91.6% of the WC at full hydration but more than 80% of the individuals survived when rehydrated. Exposure at 0.5 °C to decreasing relative humidities (RH) from 100% to 91.3% caused increasing dehydration and increasing mortality. Survival of equally dehydrated individuals was higher at subzero temperatures than at 0.5 °C. Concurrent with the decline in WC a lowering of the MP was observed. Animals exposed to −3 °C and −6 °C over ice for 31 days had a MP of −3.8 and < −7.5 °C, respectively. Specimens from a laboratory culture had a mean SCP of −6.1 °C, and acclimation at 0 or −3 °C had little effect on SCPs. Exposure at −8.2 °C over ice for 8 days, however, caused the mean SCP to decline to −21.8 °C due to the severe dehydration of these individuals. Dehydration at 0.5 °C in 95.1 and 93.3% RH also caused a decline in SCPs to about −18 °C. Individuals that had been acclimated over ice at −12.4 °C or at lower temperatures apparently did not freeze at all when cooled to −30 °C, probably because all freezeable water had been lost. These results show that O. arcticus will inevitably undergo dehydration when exposed to subzero temperatures in its natural frozen habitat. Consequently, the MP and SCP of the Collembola are substantially lowered and in this way freezing is avoided. The increased cold hardiness by dehydration is similar to the protective dehydration mechanism described in earthworm cocoons and Arctic enchytraeids. Accepted: 5 January 1998     

Impact of In-Situ CO2 Nano-Bubbles Generation on Freezing Parameters of Selected Liquid Foods

The impact of in-situ CO₂ nano-bubbles generation on the freezing properties of soft serve, milk, and apple juice was investigated. Carbonated (0, 1000, and 2000 ppm) liquid foods contained in a tube were submerged and cooled for 90 min in a pre-set ethylene glycol bath (−15 °C). Before the enclosed liquid reached 0 °C, the vibration was discharged through ultrasound in the bath to create nano-bubbles within the carbonated food samples, and the changes in temperature for 90 min of each food were recorded as a freezing curve. The time for onset of nucleation of control soft serve mix was halved in samples with 2000-ppm CO₂ due to the presence of nano-bubbles. Likewise, the nucleation time for milk with and without nano-bubbles at the same CO₂ concentration of 2000 ppm was 7.9 ± 0.1 and 2.8 ± 0.8 min_, respectively. The generation of CO₂ nano-bubbles from 2000-ppm CO₂ level in 10 ^oBx apple juice displayed −9.3 ± 0.3 °C nucleation temperature while the control one had −11.7 ± 0.9 °C.

     

Comparative physiology of Australian quolls (Dasyurus; Marsupialia)

Quolls (Dasyurus) are medium-sized carnivorous dasyurid marsupials. Tiger (3,840 g) and eastern quolls (780 g) are mesic zone species, northern quolls (516 g) are tropical zone, and chuditch (1,385 g) were once widespread through the Australian arid zone. We found that standard physiological variables of these quolls are consistent with allometric expectations for marsupials. Nevertheless, inter-specific patterns amongst the quolls are consistent with their different environments. The lower T _b of northern quolls (34°C) may provide scope for adaptive hyperthermia in the tropics, and they use torpor for energy/water conservation, whereas the larger mesic species (eastern and tiger quolls) do not appear to. Thermolability varied from little in eastern (0.035°C °C⁻¹) and tiger quolls (0.051°C oC⁻¹) to substantial in northern quolls (0.100°C oC⁻¹) and chuditch (0.146°C oC⁻¹), reflecting body mass and environment. Basal metabolic rate was higher for eastern quolls (0.662 ± 0.033 ml O₂ g⁻¹ h⁻¹), presumably reflecting their naturally cool environment. Respiratory ventilation closely matched metabolic demand, except at high ambient temperatures where quolls hyperventilated to facilitate evaporative heat loss; tiger and eastern quolls also salivated. A higher evaporative water loss for eastern quolls (1.43 ± 0.212 mg H₂O g⁻¹ h⁻¹) presumably reflects their more mesic distribution. The point of relative water economy was low for tiger (−1.3°C), eastern (−12.5°C) and northern (+3.3) quolls, and highest for the chuditch (+22.6°C). We suggest that these differences in water economy reflect lower expired air temperatures and hence lower respiratory evaporative water loss for the arid-zone chuditch relative to tropical and mesic quolls.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Physiological constraints on the life cycle and distribution of the Antarctic fairy shrimp <Emphasis Type="Italic">Branchinecta gaini</Emphasis>

Maturation to adulthood and successful reproduction in the Antarctic fairy shrimp, Branchinecta gaini, must be completed within a physiologically challenging temporal window of ca. 2.5 months in the southern Antarctic Peninsula. Although adults show considerable metabolic opportunism at positive temperatures, little is known of their tolerance of two physiological insults potentially typical to pool life in the maritime Antarctic: sub-zero temperatures and salinity. B. gaini are freeze-avoiding crustaceans with temperatures of crystallisation (T _cs) of −5°C. No antifreeze proteins were detected in the haemolymph. Adults osmoregulate in relation to temperature, but rapid mortality in saline solutions of even low concentration, indicate they cannot osmoregulate in relation to salinity. Survival of ice encasement at temperatures above their T _c was found to be pressure but not time dependent: at severe inoculative ice pressures, there was little immediate survival and none survived after 48 h below −2°C; at mild inoculative ice pressures, immediate survival was ca. 100% at −3°C, but <20% after 48 h. There was no significant difference in survival after 1 and 6 h encasement at −3°C. Observations of ventilation suggest that it is not low temperature per se, but ice that represents the primary cryo-stress, with ventilatory appendages physically handcuffed below the freezing point of pool water. Both sub-zero temperatures and salinity represent real physiological constraints on adult fairy shrimp.     

Geographic range and cold hardiness of the earthworm <Emphasis Type="Italic">Drawida ghilarovi</Emphasis> (Oligochaeta,Moniligastridae)

The earthworm Drawida ghilarovi Gates 1969 is a typical representative of the Amur fauna and the only species of the tropic family Moniligastridae on the territory of Russia. The northern boundary of its range passes from the Khingan (Hinggan) River on the west, along the mountain framing of the Amur plains, to Evoron Lake (or, probably, to the Amgun River valley) on the east. Drawida ghilarovi is widespread in the Sikhote Alin, but the northern boundary of its distribution in the northern part of this mountain range has not been delimited; the same applies to the left bank of the Amur downstream of Komsomolsk-on-Amur and its lower reaches. These earthworms lay cocoons in summer in the surface soil horizon (0–20 cm) and overwinter also at a depth of only 15–20 cm, although D. ghilarovi is classified as an anecic (deep burrower) species (Vsevolodova-Perel, 1997). The median lethal temperature (LT_50%) is about −15°C for cocoons and −12°C for worms; the minimum tolerable temperature, about −20 and −16°C, respectively. The mechanism of protection against freezing in cocoons involves a decrease in water content from an average of 71.1 ± 0.8% to a minimum of 39.8%; this decrease in worms is less significant: from 85.5 ± 0.8% (feeding worms) to 75.3 ± 0.7% (wintering worms). Since the development of juveniles in cocoons is completed by autumn, the critical factor is the minimum temperature to which the worms are exposed. In woodless areas near Khabarovsk, the average soil temperature at a depth of 40 cm during the coldest month is only 2–3°C higher than LT_50% (the difference is greater in forest habitats), and the minimal temperature should be still lower. Under current climatic conditions, D. ghilarovi could have inhabited the area extending over the Zeya River basin and, in the north, to the sources of left-bank tributaries of the middle and lower reaches of the Amur (not everywhere, but in the warmest habitats). The present-day boundaries of the D. ghilarovi appear to reflect past changes in climatic and soil conditions.