Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999     

Latitudinal differences in somatic energy storage: adaptive responses to seasonality in an estuarine fish (Atherinidae: Menidia menidia)

This study focuses on the seasonal accumulation and depletion of somatic energy in the Atlantic silverside (Menidia menidia), an annual estuarine fish. Previous research revealed that northern silversides are subject to strong size-dependent winter mortality, while southern fish suffer no appreciable winter mortality. To examine whether there was geographic differentiation in allocation strategies, we compared temporal patterns of energy storage and utilization among three populations along this gradient in seasonality. The comparative design used monthly or biweekly samples of fish collected in the wild, as well as samples of fish from each population reared in a common environment, where genetic differences can be clarified. Somatic energy stores were quantified via gravimetric analysis of neutral storage lipids and lean tissue. Analysis revealed that small individuals maintained relatively low levels of lipid reserves, which may account for their lower survival in winter. Wild fish in the north rapidly accumulated large somatic reserves, which were depleted over the winter and then increased again during the subsequent spring breeding season. In wild southern fish, relatively small reserves accumulated slowly until breeding commenced in the spring. The common-environment comparison of somatic storage patterns revealed a genetic basis for among-population differences in reserve accumulation rates, but no differences in the amount of reserves stored. We conclude that the overwinter depletion of somatic reserves has a significant selective impact on energy accumulation and allocation strategies in seasonal environments. Received: 1 November 1995 / Accepted: 13 September 1996     

Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata

Incubation temperature and the amount of water taken up by eggs from the substrate during incubation affects hatchling size and morphology in many oviparous reptiles. The Brisbane river turtle Emydura signata lays hard-shelled eggs and hatchling mass was unaffected by the amount of water gained or lost during incubation. Constant temperature incubation of eggs at 24 °C, 26 °C, 28 °C and 31 °C had no effect on hatchling mass, yolk-free hatchling mass, residual yolk mass, carapace length, carapace width, plastron length or plastron width. However, hatchlings incubated at 26 °C and 28 °C had wider heads than hatchlings incubated at 24 °C and 31 °C. Incubation period varied inversely with incubation temperature, while the rate of increase in oxygen consumption during the first part of incubation and the peak rate of oxygen consumption varied directly with incubation temperature. The total amount of oxygen consumed during development and hatchling production cost was significantly greater at 24 °C than at 26 °C, 28 °C and 31 °C. Hatchling mass and dimensions and total embryonic energy expenditure was directly proportional to initial egg mass. Accepted: 18 March 1998     

Thermal acclimation of locomotor performance in tadpoles of the frog Limnodynastes peronii

Previous analyses of thermal acclimation of locomotor performance in amphibians have only examined the adult life history stage and indicate that the locomotor system is unable to undergo acclimatory changes to temperature. In this study, we examined the ability of tadpoles of the striped marsh frog (Limnodynastes peronii) to acclimate their locomotor system by exposing them to either 10 °C or 24 °C for 6 weeks and testing their burst swimming performance at 10, 24, and 34 °C. At the test temperature of 10 °C, maximum velocity (U_max) of the 10 °C-acclimated tadpoles was 47% greater and maximum acceleration (A_max) 53% greater than the 24 °C-acclimated animals. At 24 °C, U_max was 16% greater in the 10 °C-acclimation group, while there was no significant difference in A_max or the time taken to reach U_max (T-U_max). At 34 °C, there was no difference between the acclimation groups in either U_max or A_max, however T-U_max was 36% faster in the 24 °C-acclimation group. This is the first study to report an amphibian (larva or adult) possessing the capacity to compensate for cool temperatures by thermal acclimation of locomotor performance. To determine whether acclimation period affected the magnitude of the acclimatory response, we also acclimated tadpoles of L. peronii to 10 °C for 8 months and compared their swimming performance with tadpoles acclimated to 10 °C for 6 weeks. At the test temperatures of 24 °C and 34 °C, U_max and A_max were significantly slower in the tadpoles acclimated to 10 °C for 8 months. At 10 °C, T-U_max was 40% faster in the 8-month group, while there were no differences in either U_max or A_max. Although locomotor performance was enhanced at 10 °C by a longer acclimation period, this was at the expense of performance at higher temperatures. Accepted: 25 June 1999     

Direct and size-mediated effects of temperature and ration-dependent growth rates on energy reserves in juvenile anadromous alewives (Alosa pseudoharengus)

Growth rate and energy reserves are important determinants of fitness and are governed by endogenous and exogenous factors. Thus, examining the influence of individual and multiple stressors on growth and energy reserves can help estimate population health under current and future conditions. In young anadromous fishes, freshwater habitat quality determines physiological state and fitness of juveniles emigrating to marine habitats. In this study, the authors tested how temperature and food availability affect survival, growth and energy reserves in juvenile anadromous alewives (Alosa pseudoharengus), a forage fish distributed along the eastern North American continent. Field-collected juvenile anadromous A. pseudoharengus were exposed for 21 days to one of two temperatures (21°C and 25°C) and one of two levels of food rations (1% or 2% tank biomass daily) and compared for differences in final size, fat mass-at-length, lean mass-at-length and energy density. Increased temperature and reduced ration both led to lower growth rates, and the effect of reduced ration was greater at higher temperature. Fat mass-at-length decreased with dry mass, and energy density increased with total length, suggesting size-based endogenous influences on energy reserves. Lower ration also directly decreased fat mass-at-length, lean mass-at-length and energy density. Given the fitness implications of size and energy reserves, temperature and food availability should be considered important indicators of nursery habitat quality and incorporated in A. pseudoharengus life-history models to improve forecasting of population health under climate change.     

Effects of hypothermic hypoxia on anaerobic energy metabolism in isolated anuran livers

Many lower vertebrates (reptilian and amphibian species) are capable of surviving natural episodes of hypoxia and hypothermia. It is by specific metabolic adaptations that anurans are able to tolerate prolonged exposure to harsh environmental stresses. In this study, it was hypothesized that livers from an aquatic frog would possess an inherent metabolic ability to sustain high levels of ATP in an isolated organ system, providing insight into a metabolic system that is well-adapted for low temperature in vitro organ storage. Frogs of the species, R. pipiens were acclimated at 20 °C and at 5 °C. Livers were preserved using a clinical preservation solution after flushing. Livers from 20 °C-acclimated frogs were stored at 20 °C and 5 °C and livers from 5 °C-acclimated frogs were stored at 5 °C. The results indicated that hepatic adenylate status was maintained for 96 h during 5 °C storage, but not longer than 4–10 h during 20 °C storage. In livers from 5 °C-acclimated animals subjected to 5 °C storage, ATP was maintained at 100% throughout the 96-h period. Warm acclimation (20 °C) and 20 °C storage resulted in poorer maintenance of ATP; energy charge values dropped to 0.50 within 2 h and by 24 h, only 24% of control ATP remained. Lactate levels remained less than 25 μ mol/g dry weight in all 5 °C-stored livers; 20 °C-stored livers exhibited greater accumulation of this anaerobic end-product (lactate reached 45–50 μ mol/g by 10 h). The data imply that hepatic adenylate status is largely dependent on exposure to hypothermic hypoxia and although small amounts of ATP were accounted for by anaerobic glycolysis, there must have been either a substantial reduction in cellular energy-utilization or an efficient use of low oxygen tensions. Accepted: 24 August 1998     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

The relationship between environmental temperature and clothing insulation across a year

People adapt to thermal environments, such as the changing seasons, predominantly by controlling the amount of clothing insulation, usually in the form of the clothing that they wear. The aim of this study was to determine the actual daily clothing insulation on sedentary human subjects across the seasons. Thirteen females and seven males participated in experiments from January to December in a thermal chamber. Adjacent months were grouped in pairs to give six environmental conditions: (1) January/February = 5°C; (2) March/April = 14°C; (3) May/June = 25°C; (4) July/August = 29°C; (5) September/October = 23°C; (6) November/December = 8°C. Humidity(45 ± 5%) and air velocity(0.14 ± 0.01 m/s) were constant across all six experimental conditions. Participants put on their own clothing that allowed them to achieve thermal comfort for each air temperature, and sat for 60 min (1Met). The clothing insulation (clo) required by these participants had a significant relationship with air temperature: insulation was reduced as air temperature increased. The range of clothing insulation for each condition was 1.87–3.14 clo at 5°C(Jan/Feb), 1.62–2.63 clo at 14°C(Mar/Apr), 0.87–1.59 clo at 25°C(May/Jun), 0.4–1.01 clo at 29°C(Jul/Aug), 0.92–1.81 clo at 23°C (Sept/Oct), and 2.12–3.09 clo at 8°C(Nov/Dec) for females, and 1.84–2.90 clo at 5°C, 1.52–1.98 clo at 14°C, 1.04–1.23 clo at 25°C, 0.51–1.30 clo at 29°C, 0.82–1.45 clo at 23°C and 1.96–3.53 clo at 8°C for males. The hypothesis was that thermal insulation of free living clothing worn by sedentary Korean people would vary across seasons. For Korean people, a comfortable air temperature with clothing insulation of 1 clo was approximately 27°C. This is greater than the typical comfort temperature for 1 clo. It was also found that women clearly increased their clothing insulation level of their clothing as winter approached but did not decrease it by the same amount when spring came.     

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     

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C

A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C₁₄ to C₁₇) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry. Received: 5 October 1998 / Received revision: 4 December 1998 / Accepted: 5 December 1998     

Thermal acclimation of locomotor performance in tadpoles and adults of the aquatic frog Xenopus laevis

Among amphibians, the ability to compensate for the effects of temperature on the locomotor system by thermal acclimation has only been reported in larvae of a single species of anuran. All other analyses have examined predominantly terrestrial adult life stages of amphibians and found no evidence of thermal acclimatory capacity. We examined the ability of both tadpoles and adults of the fully aquatic amphibian Xenopus laevis to acclimate their locomotor system to different temperatures. Tadpoles were acclimated to either 12 °C or 30 °C for 4 weeks and their burst swimming performance was assessed at four temperatures between 5 °C and 30 °C. Adult X. laevis were acclimated to either 10 °C or 25 °C for 6 weeks and their burst swimming performance and isolated muscle performance was determined at six temperatures between 5 °C and 30 °C. Maximum swimming performance of cold-acclimated X. laevis tadpoles was greater at cool temperatures and lower at the highest temperature in comparison with the warm-acclimated animals. At the test temperature of 12 °C, maximum swimming velocity of tadpoles acclimated to 12 °C was 38% higher than the 30 °C-acclimation group, while at 30 °C, maximum swimming velocity of the 30 °C-acclimation group was 41% faster than the 12 °C-acclimation group. Maximum swimming performance of adult X. laevis acclimated to 10 °C was also higher at the lower temperatures than the 25 °C acclimated animals, but there was no difference between the treatment groups at higher temperatures. When tested at 10 °C, maximum swimming velocity of the 10 °C-acclimation group was 67% faster than the 25 °C group. Isolated gastrocnemius muscle fibres from adult X. laevis acclimated to 10 °C produced higher relative tetanic tensions and decreased relaxation times at 10 °C in comparison with animals acclimated to 25 °C. This is only the second species of amphibian, and the first adult life stage, reported to have the capacity to thermally acclimate locomotor performance. Accepted: 28 October 1999     

Temperature-dependent conduction properties in Arctic fish peripheral nerves

The conduction properties of peripheral nerves from the Arctic fish species Arctic eelpouts (Lycodes sp.), snake blenny (Lumpenus lampretaeformis) and polar cod (Boreogadus saida), permanently adapted to low temperatures, were studied. Nerves of these fishes have two types of fibres, characterised by extracellular compound action potentials with fast (7 m/s) and slow (4 m/s) conduction velocities, as measured at 12 °C. The temperature dependence of the conduction velocity was bimodal, changing its slope at about 16 °C. The Q ₁₀ above 16 °C was 1.12–1.49, while below 16 °C it was 1.82–2.16. Irreversible deterioration of the nerve was observed at temperatures around 19–27 °C. A comparison with data previously obtained from Mediterranean fishes indicates that Arctic fishes have similar temperature sensitivity of nerve conduction and a slight vertical displacement on the conduction velocity-temperature curves, which is insufficient to compensate the decrease of the conduction velocity at their physiological temperature, the conduction velocity of Arctic fishes being about one-half of that of temperate fishes. This suggests that this neurophysiological function is not fully cold-adapted in these Arctic fish species. Accepted: 3 June 2000     

How incubation temperature influences the physiology and growth of embryonic lizards

Eggs of two small Australian lizards, Lampropholis guichenoti and Bassiana duperreyi, were incubated to hatching at 25 °C and 30 °C. Incubation periods were significantly longer at 25 °C in both species, and temperature had a greater effect on the incubation period of B. duperreyi (41.0 days at 25 °C; 23.1 days at 30 °C) than L. guichenoti (40.1 days at 25 °C; 27.7 days at 30 °C). Patterns of oxygen consumption were similar in both species at both temperatures, being sigmoidal in shape with a fall in the rate of oxygen consumption just prior to hatching. The higher incubation temperature resulted in higher peak and higher pre-hatch rates of oxygen consumption in both species. Total amount of oxygen consumed during incubation was independent of temperature in B. duperreyi, in which approximately 50 ml oxygen was consumed at both temperatures, but eggs of L. guichenoti incubated at 30 °C consumed significantly more (32.6 ml) than eggs incubated at 25 °C (28.5 ml). Hatchling mass was unaffected by either incubation temperature or the amount of water absorbed by eggs during incubation in both species. The energetic production cost of hatchling B. duperreyi (3.52 kJ · g⁻¹) was independent of incubation temperature, whereas in L. guichenoti the production cost was greater at 30 °C (4.00 kJ · g⁻¹) than at 25 °C (3.47 kJ · g⁻¹). Snout-vent lengths and mass of hatchlings were unaffected by incubation temperature in both species, but hatchling B. duperreyi incubated at 30 °C had longer tails (29.3 mm) than those from eggs incubated at 25 °C (26.2 mm). These results indicate that incubation temperature can affect the quality of hatchling lizards in terms of embryonic energy consumption and hatchling morphology. Accepted: 27 January 2000     

Thermal tolerance and acclimation response of larvae of the sub-Antarctic beetle Hydromedion sparsutum (Coleoptera: Perimylopidae)

Hydromedion sparsutum is a locally abundant herbivorous beetle on the sub-Antarctic island of South Georgia, often living in close association with the tussock grass Parodiochloa flabellata. Over a 4-day period in mid-summer when the air temperature varied from 0 to 20°C, the temperature in the leaf litter 5–10 cm deep at the base of tussock plants (the microhabitat of H. sparsutum) was consistently within the range of 5–7.5°C. Experiments were carried out to assess the ability of H. sparsutum larvae collected from this thermally stable environment to acclimate when maintained at lower (0°C) and higher (15°C) temperatures. The mean supercooling points (freezing temperature) of larvae collected in January and acclimated at 0°C for 3 and 6 weeks and 15°C for 3 weeks were all within the range of −2.6 to −4.6°C. Larvae in all treatment groups were freeze tolerant. Acclimation at 0°C significantly increased survival in a 15-min exposure at −8°C (from 27 to 96%) and −10°C (from 0 to 63%) compared with the field-fresh and 15°C-treated larvae. Similarly, survival of 0°C-acclimated larvae in a 72-h exposure at −6°C increased from 20 to 83%. Extending the acclimation period at 0°C to 6 weeks did not produce any further increase in cold tolerance. The concentrations of glucose and trehalose in larval body fluids increased significantly with low temperature acclimation. Larvae maintained at 15°C for 3 weeks (none survived for 6 weeks) were less able to survive 1-h exposures between 30 and 35°C than the 0°C-treated samples. Whilst vegetation and snow cover are an effective buffer against low winter temperatures in many polar insects, the inability of H. sparsutum larvae to acclimate or survive at 15°C suggests that protection against high summer temperatures is equally important for this species. Accepted: 2 August 1999     

Effect of developmental temperature on swimming performance of zebrafish (<Emphasis Type="BoldItalic">Danio rerio</Emphasis>) juveniles

It is widely known that water temperature affects the swimming capacity of fish. But the effect of the rearing temperature on the swimming ability of the fish at later stages, has not had similar attention. In this study, four populations of zebrafish, were reared in different water temperatures (22, 25, 28 and 31°C) and after being acclimatized in a common temperature (26.5°C) for over a month, they were subjected to swimming trials in order to evaluate the maximum relative critical velocity (RU _crit ) in each case. Fish that were reared in 22°C showed statistically significant lower performance than the ones reared in 31°C (7.72 ± 0.17 vs. 8.79 ± 0.28, means ± S.E.). Possible explanations for the observed differentiation could be the effect of early life temperature on fish muscle ontogeny or on body shape.     

Food requirements of breeding king penguins at Heard Island and potential overlap with commercial fisheries

The diet composition of king penguins (Aptenodytes patagonicus) at Heard Island (53°05′S; 73°30′E) was determined from stomach contents of 98 adults captured as they returned to the island throughout 1992. During the two growth seasons, the diet was dominated by the myctophid fish Krefftichthys anderssoni (94% by number, 48% by mass). The paralepidid fish Magnisudis prionosa contributed <1% by numbers but 17% by mass. Mackerel icefish (Champsocephalus gunnari) accounted for 17% by mass of chick diet in late winter, when chicks were malnourished and prone to starvation, although its annual contribution to the penguins' diet was only 3%. Squid was consumed only between April and August; Martialia hyadesi was the commonest squid taken, comprising 40–48% of the winter diet. The remainder of the diet consisted of the squid Moroteuthis ingens and fish other than K. anderssoni. The energy content of the diet mix fed to the chicks varied seasonally being highest during the growth seasons (7.83 ± 0.25 kJ g⁻¹) and lowest in winter (6.58 ± 0.19 kJ g⁻¹). From energetic experiments we estimated that an adult penguin consumed 300 kg of food each, of which its chick received 55 kg during the 1992 season. The chicks received large meals at the beginning of winter (1.2 ± 0.3 kg) and during the middle of the second growth season (1.2 ± 0.3 kg), and their smallest meals in late winter (0.4 ± 0.1 kg). The gross energy required to rear a king penguin chick was estimated to be 724 MJ. The potential impact of commercial fisheries on the breeding activities of king penguins is discussed. Received: 20 October 1997 / Accepted: 27 April 1998     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-adrenergic signals, leading to a rise in intracellular 3′,5′-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 ± 0.08. A change in assay temperature from a euthermic value (37 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) K_m values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH₄Cl was reduced. However, temperature change had little or no effect on K_m values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 °C and activation energies that were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the rabbit enzyme showed a breakpoint at 17 °C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 ± 0.18 at 37 °C to 2.19 ± 0.07 at 5 °C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with ³²P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 °C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. Accepted: 10 July 1998     

The influence of physiological age of Argas reflexus larvae (Acari: Argasidae) and of temperature and photoperiod on induction and duration of diapause

The occurrence of diapause and quiescence was investigated in Argas reflexus engorged larvae, nymphs I and nymphs II. For diapause experiments, larvae were maintained at five different locations: at constant 20°C long day (LD; 17 h light:7 h dark) or short day (SD; 10 h light:14 h dark), at two locations with natural photoperiod and temperature and at one location with natural photoperiod but constant 15°C. At 20°C, diapause incidence was low in physiologically young larvae, increased with larval age, and then decreased to zero in specimens of increased physiological age. This pattern, observed both at constant LD and SD, suggests that the propensity to diapause changes with the physiological age of the unfed larva. The duration of diapause decreased with increasing larval physiological age at all locations, resulting in a seasonally synchronized moulting pattern. The results suggest that A. reflexus larvae are photoperiodically sensitive both before and after feeding and that decreasing daylengths may be particularly strong inductive stimuli. The developmental zero and thermal constant of the larvae were determined as 13.24°C and 220 degree-days, respectively. Degree-day measurements revealed that larval A. reflexus may enter a diapause of different length when fed between August and December and kept at natural daylength. Development of engorged nymphs I and nymphs II, but not of larvae, was ultimatively restricted at a temperature of 37.5°C, but immediately resumed at 25°C, demonstrating the occurrence of quiescence at high temperatures. Similarly, at a low temperature of 15°C, many nymphs I and II did not develop within 58 months, but did so successfully after transfer to 25°C, without additional food intake. Received: 20 May 1997 / Accepted: 4 August 1997     

Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass

A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 °C, and even a small increase in temperature can have a deleterious effect. This points to a need for a more thermotolerant yeast. To this end, S. cerevisiae D₅A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 °C, 40 °C, and 42 °C using dilute-acid-pretreated poplar as substrate. At 40 °C, C. acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S.cerevisiae D₅A at either 37 °C or 40 °C. At 42 °C, C. acidothermophilum showed a slight drop in performance. On the basis of preliminary estimates, SSF with C. acidothermophilum at 40 °C can reduce cellulase costs by about 16%. Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible. This demonstrates the advantage of using thermophilic or thermotolerant yeasts. Received: 20 February 1997 / Received revision: 24 June 1997 / Accepted: 4 July 1997     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997