The effect of cold shock on the free amino acid pool of rotan pondfish <Emphasis Type="Italic">Perccottus glehni</Emphasis> (<Emphasis Type="Italic">Eleotridae,Perciformes</Emphasis>)

The effect of cold shock on the free amino acid pool of the pondfish P. glehni not adapted to hibernation has been studied. In the beginning of September, after four days of cold shock, a decrease in the blood level of essential amino acids and an increase in the level of alanine and three-fold increase in the level of γ-aminobutyric acid (GABA) were observed. Changes in the free amino acid pool in the mussels occur in two stages. Namely, in a considerable decrease in their concentration after two days of cold shock followed by an even more significant increase in their amount after four days of shock. In the brain changes in the free amino acid pools are associated with their total decrease followed by continuous significant decrease in the level of taurine during the first four days of cold shocking. The obtained results are compared with previously obtained data on the free amino acid content in muscles and blood of P. glehni during preparation for winter sleep and the beginning of hibernation.     

Free amino acid composition in blood and muscle of the gobi <Emphasis Type="Italic">Precottus glehni</Emphasis> at the period of preparation and completion of hibernation

The freshwater fish gobi Preccottus glehni survives after wintering in ponds frozen in winter till the very bottom. In adaptation of poikilothermal animals to wintering at near-zero temperatures, an essential role is played by free amino acids; accumulation of a large amount of some particular acid at the period of preparation to the state of hibernation can indicate the protective role of this acid in the low-temperature adaptation. In the present work it has been shown that as soon as by the end of August, in the gobi muscle, the taurine concentration increases three times as compared with that in July, the taurine pool after this reaching 50% of the total pool of free amino acids in the muscle tissue. During December and after the 3-month hibernation in ice, it exceeds the April and July levels 8 and 4 times, respectively, and amounts to 50% of the total free amino acid pool for muscle and to 40% for blood. Several days prior to the beginning of winter hibernation under natural conditions, both in blood and in muscle, there is revealed disappearance or a sharp fall of concentration of essential amino acids. An essential peculiarity of the change in the free amino acid composition after hibernation was a significant rise of alanine concentration in muscle—3.5 times as compared with July and 1.4–1.8 times as compared with changes in December. The total free amino acid pool in muscle in December as compared with that in July increased almost 1.5 times (34.76 ± 1.12 μmol/g wet weight), while after hibernation—2 times. Peculiarities of taurine accumulation long before the beginning of winter cold allow suggesting that role of taurine consists in providing a possibility of existence of eurythermal fish at near-zero temperature.     

Pool of phosphoethanolamine and phosphoserine in the brain of the snail <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> L. in summer and before winter dormancy

Small amounts of phospholipid metabolites, phosphoethanolamine and phosphoserine, were discovered at a ratio of 1:9 in the brain of a freshwater mollusk, the pond snail Lymnaea stagnalis L., collected both in summer and autumn. The phosphoethanolamine pool increased by 15% in autumn relative to the summer level (up to 625 ± 44 nmol per g of wet tissue), although this value still constituted 6% of the total pool of free amino acids and ninhydrin-positive substances. These findings are in striking contrast with our previous results that showed adaptive modifications of the amino acid and phosphoethanolamine pools in the brain of eurythermal freshwater fish at low temperatures. At the same time, these data demonstrate the presence of phosphoethanolamine and phosphoserine in the central nervous system at comparatively early stages of its evolution.     

Seasonal temperature compensation in the horse mussel, Modiolus modiolus: metabolic enzymes, oxidative stress and heat shock proteins

Seasonal collections of the subtidal horse mussel, Modiolus modiolus, from a depth of 10 m were made at the Isles of Shoals, New Hampshire to assess changes in overall energetic demand, measured as respiration, the maximal activities of rate-limiting enzymes of intermediate metabolism, level of oxidative stress, and the expression of heat shock proteins (HSP). Weighted respiration rates of mussels from winter collections were significantly lower than summer rates but decreased by less than 20%. Specific activities of several rate-limiting enzymes were measured in mussels from the summer and winter collections at the temperature of collection and the reciprocal seasonal temperature (15 and 5 degrees C). Comparisons of these enzyme activities and the protein concentrations of hexokinase and citrate synthase show that a quantitative strategy is used to acclimatize to winter temperatures by these rate-limiting enzymes of intermediate metabolism. The activities and protein concentrations of the antioxidant enzyme, Cu/Zn superoxide dismutase (SOD) is seasonally indistinguishable while the concentration of HSP 70 was greater in winter than in summer samples. These results show that mussels seasonally compensate for decreases in temperature by increasing the concentration of rate-limiting metabolic enzymes while maintaining the same level of antioxidant protection in summer and winter consistent with high aerobic metabolism in both winter and summer. Lastly, the significantly greater concentrations of HSP70 in winter samples suggests that protein chaperone functions must be maintained while other seasonal adjustments to cold temperatures are occurring.     

Seasonal variation in metabolic rate of a medium-sized frugivore, the Knysna Turaco (Tauraco corythaix)

Many seasonal thermoregulation studies have been conducted on Holarctic birds that live in predictable, highly seasonal climates with severe winters. However, relatively few studies have been conducted on their southern hemisphere Afrotropical counterparts that encounter less predictable climates with milder winters. These latter birds are expected to conserve energy in winter by downregulating their metabolic rates. Therefore in this study, metabolic rate was measured during summer and winter in Knysna Turaco, Tauraco corythaix (Musophagiformes, Musophagidae) (c. 310 g), a non-passerine, in order to test whether there is energy conservation in winter. No overall significant differences in resting metabolic rates over a range of ambient temperatures were observed between winter and summer. However, whole-organism basal metabolic rates were 18.5% higher (p=0.005) in winter than in summer (210.83±15.97 vs. 186.70±10.52 O₂ h⁻¹). Knysna Turacos had broad thermoneutral zones ranging from 20 to 28 °C in winter and 10 to 30 °C in summer. These results suggest that Knysna Turacos show seasonal thermoregulatory responses that represent cold defense rather than energy conservation, which is contrary to what was expected.     

Physical and chemical changes associated with seasonal alterations in freezing tolerance in the adult northern tenebrionid, Upis ceramboides

The adult tenebrionid beetle Upis ceramboides overwinters in the northern taiga forests of North America in a hibernaculum typically just beneath loose tree bark above the snowline. The beetles may be exposed to temperatures as low as ?55°C, which is approximately the lower limit of cold tolerance found in specimens collected in mid-winter. Supercooling points average ?6.3°C throughout the year and, contrary to expectation, show no seasonal variation in spite of major alterations in haemolymph composition and freezing tolerance. Summer beetles are incapable of withstanding temperatures below the supercooling point but freezing tolerance increases during the fall (September–November) and the lower lethal temperature (LLT) is maintained at ca. ?55°C until March, after which it gradually rises to the summer level of ?6°C. Changes in freezing tolerance are closely associated with seasonal alterations in the polyhydric alcohols sorbitol and threitol. Neither polyol is present in measureable amount during summer; sorbitol accumulates to an average haemolymph concentration of 0.44 M/l in winter and threitol reaches 0.25 M/l. Summer beetles contain about 14% more water than beetles collected during the other seasons. Upis ceramboides thus undergoes unique seasonal changes in physical and chemical characteristics that enable it to tolerate severe, prolonged subfreezing temperatures.     

Seasonal variation in the metabolism-temperature relation of House Sparrows (Passer domesticus) in KwaZulu-Natal,South Africa

Many birds exhibit considerable phenotypic flexibility in metabolism to maintain thermoregulation or to conserve energy. This flexibility usually includes seasonal variation in metabolic rate. Seasonal changes in physiology and behavior of birds are considered to be a part of their adaptive strategy for survival and reproductive success. House Sparrows (Passer domesticus) are small passerines from Europe that have been successfully introduced to many parts of the world, and thus may be expected to exhibit high phenotypic flexibility in metabolic rate. Mass specific Resting Metabolic Rate (RMR) and Basal Metabolic Rate (BMR) were significantly higher in winter compared with summer, although there was no significant difference between body mass in summer and winter. A similar, narrow thermal neutral zone (25–28 °C) was observed in both seasons. Winter elevation of metabolic rate in House Sparrows was presumably related to metabolic or morphological adjustments to meet the extra energy demands of cold winters. Overall, House Sparrows showed seasonal metabolic acclimatization similar to other temperate wintering passerines. The improved cold tolerance was associated with a significant increase in VO₂ in winter relative to summer. In addition, some summer birds died at 5 °C, whereas winter birds did not, further showing seasonal variation in cold tolerance. The increase in BMR of 120% in winter, compared to summer, is by far the highest recorded seasonal change so far in birds.     

Seasonal thermoregulation in the burrowing parrot (Cyanoliseus patagonus)

Birds exposed to seasonal environments are faced with the problem of maintaining thermogenic homoeostasis. Previous studies have established that birds native to the Holarctic increase their Resting Metabolic Rate at different ambient temperatures (RMR_Ta) and Basal Metabolic Rate (BMR) in winter as an adaptation to cold temperature since winters are more severe, while their non-Holarctic counterparts generally decrease their winter BMR as an energy saving mechanism during unproductive and dry winter months. In this study, we examined seasonal thermoregulation in the burrowing parrot (Cyanoliseus patagonus), a colonial psittacine native to the Patagonian region of Argentina, a region with an unpredictable environment. We found significantly higher mass specific RMR_Ta and BMR in summer than in winter. Both summer and winter BMR of the species fell within the predicted 95% confident interval for a parrot of its size. Body mass was significantly higher in winter than in summer. The burrowing parrot had broad thermo-neutral zones in winter and summer. The circadian rhythm of core body temperature (T_b) of burrowing parrots was not affected by season, showing that this species regulated its T_b irrespective of season. These results suggest that the burrowing parrots' seasonal thermoregulatory responses represent that of energy conservation which is important in an unpredictable environment.     

Anthelmintic efficacy of genistein, the active principle of Flemingia vestita (Fabaceae): alterations in the free amino acid pool and ammonia levels in the fluke, Fasciolopsis buski

The crude root-peel extract of Flemingia vestita, its active principle genistein and the reference flukicide oxyclozanide were tested against Fasciolopsis buski, the giant intestinal trematode. The amino acid composition of F. buski was demonstrated using HPLC and it was observed that the free amino acid (FAA) pool of the control worm consisted of aspartate, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, lysine, histidine, arginine, phosphoserine, taurine, citrulline, ornithine, β-alanine, and γ-amino butyric acid (GABA). Of the amino acids detected valine was found to be the maximum in quantitative analysis. In qualitative analysis the FAA pool of the parasites under various treatments remained same as that of the control; however, quantitatively the level of various FAAs in the parasite was significantly affected. The treated parasites showed a marked decrease in the levels of arginine, ornithine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, serine, threonine, and taurine following treatment with 20 mg/ml of crude peel extract, 0.5 mg/ml of genistein and 20 mg/ml of the reference drug, though an increase in the levels of glutamic acid, glutamine, phosphoserine, citrulline and GABA was noticeable. Enhanced levels of GABA and citrulline under the influence of genistein may be implicated in alterations of nitric oxide release and consequent neurological change (e.g. paralysis) in the parasite. Ammonia in the tissue homogenate as well as in the incubation medium showed a quantitative increase compared to the controls after treatment with the various test materials. The ammonia level increased by 40.7%, 66.4% and 18.16% in treatments with F. vestita, genistein and oxyclozanide, respectively, at the mentioned dosages. The changes in the levels of the amino acids and nitrogen components post treatment suggest that the amino acid metabolism in the parasite may have been altered under the influence of the test materials.     

Cryoprotection in dampwood termites (Termopsidae, Isoptera)

In contrast to the majority of the Order, the dampwood termites of the family Termopsidae found in colder regions can experience frost and snow, either in cool temperate areas at high latitudes (45°), or alpine areas at high elevations (>1000 m). This suggests that dampwood termites are adapted to cold climates. We investigated this hypothesis in two dampwood termites, Porotermes adamsoni Froggatt and Stolotermes victoriensis Hill. We measured nest temperatures and atmospheric temperatures of their alpine habitat during winter, and measured survival and recovery at subzero temperatures. We also determined the minimum temperature at which these species remain active and the LT50 values. We used a novel gas chromatographic strategy to examine eight metabolites from individuals of both species collected in winter and summer to identify possible cryoprotectants. Both P. adamsoni and S. victoriensis had significantly higher levels of trehalose, a known cryoprotectant, in winter than in summer; in addition S. victoriensis also had higher levels of unsaturated fatty acid ligands in winter than in summer, consistent with patterns observed for cold adaptation in other organisms. These results are the first to reveal that dampwood termites are adapted to cold climates and use trehalose and unsaturated lipids as cryoprotectants.     

Seasonal changes in thermal responses of urban residents to cold exposure

To determine whether urban circumpolar residents show seasonal acclimatisation to cold, thermoregulatory responses and thermal perception during cold exposure were examined in young men during January-March (n=7) and August-September (n=8). Subjects were exposed for 24 h to 22 and to 10 degrees C. Rectal (T(rect)) and skin temperatures were measured throughout the exposure. Oxygen consumption (VO(2)), finger skin blood flow (Q(f)), shivering and cold (CDT) and warm detection thresholds (WDT) were assessed four times during the exposure. Ratings of thermal sensations, comfort and tolerance were recorded using subjective judgement scales at 1-h intervals. During winter, subjects had a significantly higher mean skin temperature at both 22 and 10 degrees C compared with summer. However, skin temperatures decreased more at 10 degrees C in winter and remained higher only in the trunk. Finger skin temperature was higher at 22 degrees C, but lower at 10 degrees C in the winter suggesting an enhanced cold-induced vasoconstriction. Similarly, Q(f) decreased more in winter. The cold detection threshold of the hand was shifted to a lower level in the cold, and more substantially in the winter, which was related to lower skin temperatures in winter. Thermal sensations showed only slight seasonal variation. The observed seasonal differences in thermal responses suggest increased preservation of heat especially in the peripheral areas in winter. Blunted vasomotor and skin temperature responses, which are typical for habituation to cold, were not observed in winter. Instead, the responses in winter resemble aggravated reactions of non-cold acclimatised subjects.     

Influence d'une microsporidiose sur les acides aminés libres de Carcinus mediterraneus Czerniavsky, 1884 soumis à diverses salinités et à des valeurs extrêmes de température

The influence of a microsporidian, Thelohania maenadis Pérez, 1904 (Protozoa: Microspora), on the free amino-acid content of the haemolymph and muscle tissue of Carcinus mediterraneus Czerniavsky, 1884 (Crustacea: Decapoda: Brachyura) is studied.Analysis of free amino acids reveals the presence in affected crabs, whose muscle tissue is 70–80% destroyed, of four additional, non-identified compounds in the haemolymp and eleven in the muscle. Parasitization does not provoke a substantial variation in the total free amino acids of the haemolymph and the muscle. In the latter, methionine sulphoxide, methionine and serine levels increase considerably; glycine, proline and arginine levels fall while alanine and taurine levels rise.The desalination at moderate temperatures of healthy and parasitized crabs alike results in a reduction of the total free amino acids of the haemolymph, mainly due to the lowering of proline, glycine and alanine concentrations. Supersalination leads to only a slight reduction of the total free amino acids in healthy specimens, and has no effect on parasitized crabs. The sharp increase in proline and alanine is offset by a reduction in the levels of other amino acids - arginine, glutamic acid, tryptophan and taurine in healthy crabs; and arginine, tryptophan, taurine and serine in parasitized specimes.The influence of the combined factors of salinity and temperature was studied in conditions comparable to those pertaining in winter and summer in the lagoon which is the crab's natural habitat. At low and high extremes of temperature, the effect of salinity on healthy crabs is reversed: desalination results in increased free amino-acid levels. Parasitization reduces this effect of temperature extremes on the influence exercised by salinity, and may annul it completely at high temperatures.     

Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis

Two populations of the gall fly Eurosta solidaginsis utilize different strategies to endure seasonal exposure to temperatures below freezing. Both populations are freezing tolerant. In north temperate populations, supercooling points rise from ?10.2°C to ?6.2°C following exposures to temperatures below freezing. This level is maintained throughout winter and ensures frequent and prolonged periods of tissue freezing. South temperate populations depress the supercooling point to ?14.2°C during autumn and early winter, and this depression precludes extracellular ice formation during periods of supra-optimal temperature fluctuations. During mid-winter, supercooling points rise to the same level as in northern groups.Both populations accumulate three principal cryoprotective agents following first frost exposures (glycerol, sorbitol and trehalose). Cryoprotectants levels do not peak in northern populations until 4–6 weeks after first frost. In southern populations the accumulation profile is characterized by a high initial rate of synthesis, a protective overshoot and pronounced seasonal fluctuations. The relative survival advantages of each strategy are discussed.     

Photoprotective role of rhodoxanthin during cold acclimation in Cryptomeria japonica

To examine the role of rhodoxanthin in long‐term acclimation to low temperatures, we monitored seasonal changes in pigment composition, photosynthesis, chlorophyll fluorescence and the level of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in needles of wild‐type and mutant forms of Cryptomeria japonica. In winter, rhodoxanthin accumulated in sun‐exposed needles of wild‐type plants, but not in those of the mutant. The level of chlorophyll decreased in both types of plant in winter. In contrast, the level of the xanthophyll cycle pool increased in both cases. The level of the pool in the mutant was twice that in the wild type in winter, on a Chl basis, even though the levels in both were similar in summer. The synthesis of rhodoxanthin might be triggered by photo‐inhibitory conditions, as suggested by the sustained elevated levels of zeaxanthin (Z) and antheraxanthin (A). In the wild type and the mutant, the quantum yield of CO₂ fixation (φ), the photosynthetic capacity, the photochemical efficiency of photosystem II (PSII), the photochemical quenching and the level of Rubisco in summer were similar. However, all these values for the wild type were higher than those for the mutant in winter. The non‐photochemical quenching (NPQ) in the mutant in winter increased rapidly even under low light conditions due to the high sustained levels of Z and A. In contrast, in the wild type, the conversion of Z via A to rhodoxanthin prevented the rapid increase in NPQ to maintain the relatively high level of φ. These findings suggest that rhodoxanthin might play an important photoprotective role in long‐term acclimation to cold. The dynamic regulation of the amount of rhodoxanthin relative to the level of the xanthophyll cycle pool might act to maintain an appropriate balance between light absorption, photosynthesis and the thermal dissipation of energy due to excess absorbed light in winter.     

Seasonal variability of free amino acids in two marine bivalves, Macoma balthica and Mytilus spp., in relation to environmental and physiological factors

The seasonal variability of the intracellular free amino acid (FAA) concentration was studied in 5 Macoma balthica populations and 7 Mytilus spp. populations along their European distribution. Because of the well known physiological role of FAA as organic osmolytes for salinity induced cell volume regulation in marine osmoconformers, FAA variations were compared in bivalve populations that were exposed to high vs. low intraannual salinity fluctuations. In general, seasonal FAA variations were more pronounced in M. balthica than in Mytilus spp. In both bivalve taxa from different locations in the Baltic Sea, highest FAA concentrations were found in autumn and winter and low FAA concentrations were measured in summer. Seasonal patterns were less pronounced in both taxa at locations with constant salinity conditions. In contrast to Baltic Sea populations, Atlantic and Mediterranean bivalves showed high FAA concentrations in summer and low values in winter, regardless of seasonal salinity fluctuations. Significant seasonal FAA variations at locations with constant salinity conditions showed that salinity appeared not to be the main factor in determining FAA concentrations. The seasonal patterns of the main FAA pool components, i.e. alanine, glycine and taurine, are discussed in the context of seasonal variations in environmental factors (salinity, temperature) and physiological state (glycogen content, reproductive stage).     

Seasonal composition of lipids, fatty acids, and sterols in the edible red alga Grateloupia turuturu

Composition of lipids, sterols, fatty acids (FA), and phospholipids in the edible Rhodophyta Grateloupia turuturu from Britanny, France, was investigated over four seasons in order to identify compounds with potential benefits in health and nutrition. The lipid content was found to vary from 3.3 to 4.1 % dry weight. No marked variations were observed for glycolipids accounting for 42.3–46.8 %, whereas neutral lipids and phospholipids fluctuated from 20.1 % (summer) to 41.8 % (winter), and 11.2 % (winter) to 33.4 % (summer), respectively. Polyunsaturated FA of the total lipids were found from 20.4 % (winter) to 31.1 % (summer), including 20:5 ω3 acid as the major one (up to 16.3 % in summer). Phosphatidylcholine (20.0–43.7 %) and phosphatidylserine (24.6–37.5 %) were the dominant phospholipids in all seasons. Compounds of interest were identified in minor amounts such as squalene, α-tocopherol, phytonadione (vitamin K₁), cholesteryl formate, cholest-4-en-3-one, and cholesta-4,6-dien-3-one. Cholesterol was the major sterol with a lower content in spring and summer.     

Changes in free amino acid content and frost resistance in Nothofagus dombeyi leaves

The annual course of frost resistance and free proline content was studied in leaves at different stages of development of a woody species (Nothofagus dombeyi) from Southern Chile. The freezing resistance reached a minimum in late spring or summer and a maximum in the autumn-winter period. Adult and juvenile trees showed a similar degree of resistance; meanwhile, cold resistance was maximum at the seedling stage. Free proline levels and frost resistance in leaves changed throughout the seasonal cycle, increasing in winter and decreasing in summer. Artificial hardening caused changes in amino acid content of leaves; while valine, proline, lysine, histidine, serine and alanine increased upon hardening, aspartic acid, glutamic acid and arginine decreased. The nature of cold-induced metabolic adjustments is discussed as well as its ecological significance.     

Seasonal variation of behavior and brain size in a freshwater fish

1. Teleost fishes occupy a range of ecosystem, and habitat types subject to large seasonal fluctuations. Temperate fishes, in particular, survive large seasonal shifts in temperature, light availability, and access to certain habitats. Mobile species such as lake trout (Salvelinus namaycush) can behaviorally respond to seasonal variation by shifting their habitat deeper and further offshore in response to warmer surface water temperatures during the summer. During cooler seasons, the use of more structurally complex nearshore zones by lake trout could increase cognitive demands and potentially result in a larger relative brain size during those periods. Yet, there is limited understanding of how such behavioral responses to a seasonally shifting environment might shape, or be shaped by, the nervous system.
2. Here, we quantified variation in relative brain size and the size of five externally visible brain regions in lake trout, across six consecutive seasons in two different lakes. Acoustic telemetry data from one of our study lakes were collected during the study period from a different subset of individuals and used to infer relationships between brain size and seasonal behaviors (habitat use and movement rate).
3. Our results indicated that lake trout relative brain size was larger in the fall and winter compared with the spring and summer in both lakes. Larger brains coincided with increased use of nearshore habitats and increased horizontal movement rates in the fall and winter based on acoustic telemetry. The telencephalon followed the same pattern as whole brain size, while the other brain regions (cerebellum, optic tectum, olfactory bulbs, and hypothalamus) were only smaller in the spring.
4. These findings provide evidence that flexibility in brain size could underpin shifts in behavior, which could potentially subserve functions associated with differential habitat use during cold and warm seasons and allow fish to succeed in seasonally variable environments.

     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.