Biochemical strategy of survival of the freshwater mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> at near-zero temperatures

Effect of an autumn-winter decrease of the environmental temperature on changes of the content of free amino acids and reducing sugars was studied in tissues fluids of the freshwater mollusc L. stagnalis. At a decrease of the temperature to 4 and 0°C at the autumn period, concentration of free alanine rose several times; an increase of histidine, glutamate, glycine, and serine concentrations was less pronounced. Accumulation of methionine, leucine, isoleucine, tyrosine, and phenylalanine was revealed at 0°C. In the state of long hypobiosis (at 4°C), pools of all amino acids fell 4–8 times; essential amino acids, except for lysine, were not detected at all. Both at the summer and at the autumn periods, high concentrations of free fructose and glucose were revealed. In the process of hypobiosis the fructose pool decreased almost 35, while the glucose pool—12 times. Maltose that was absent in summer was found at 4 and 0°C as well as after 2.5 months of hypobiosis. The changes of the studied parameters in L. stagnalis in response to hypothermia are compared with the corresponding parameters of changes in cryoresistant animals in response to negative temperatures.     

Seasonal variation in the content of free reducing sugars in body fluids of freshwater mollusk Lymnaea stagnalis

Accumulation of certain mono-and disaccharides in body fluids of ectotherms in autumn and winter suggests their protective role in adaptation to near-zero temperatures. In this context, the effect of seasonal temperature variation on the content of free reducing sugars was studied in freshwater snail Lymnaea stagnalis. The summer and autumn carbohydrate spectrum essentially featured high concentrations of fructose and glucose. After 2.5-month hypobiosis (4°C), the concentration of fructose and glucose decreased 35 and 12 times, respectively. Maltose and other disaccharides (presumably cellobiose) undetectable in summer became detectable in autumn before hypobiosis started. The subsequent level of maltose increased to almost reach the sharply decreased glucose level and to exceed the fructose level after 2.5-month hypobiosis. The involvement of maltose in the mechanisms of L. stagnalis adaptation to near-zero temperatures as a membrane protectant is proposed.     

Biochemical strategy of survival of the freshwater mollusc Lymnaea stagnalis at near-zero temperatures

Effect of an autumn-winter decrease of the environmental temperature on changes of the content of free amino acids and reducing sugars was studied in tissue fluids of the freshwater mollusc L. stagnalis. At a decrease of the temperature to 4 and 0 degrees C at the autumn period, concentration of free alanine rose several times; an increase of histidine, glutamate, glycine, and serine concentrations was less pronounced. Accumulation of methionine, leucine, isoleucine, tyrosine, and phenylalanine was revealed at 0 degree C. In the state of long hypobiosis (at 4 degrees C), pools of all amino acids fell 4-8 times; essential amino acids, except for lysine, were not detected at all. Both at the summer and at the autumn periods, high concentrations of free fructose and glucose were revealed. In the process of hypobiosis the fructose pool decreased almost 35, while the glucose pool--12 times. Maltose that was absent in summer was found at 4 and 0 degree C as well as after 2.5 months of hypobiosis. The changes of the studied parameters in L. stagnalis in response to hypothermia are compared with the corresponding parameters of changes in cryoresistant animals in response to negative temperatures.     

Changes in free amino acid composition in haemolymph of larvae of the wax moth,Galleria mellon ella L., during cold acclimation

• 1.1. Free amino acids were analysed in the haemolymph of Galleria mellonella larvae by HPLC chromatography with o-phthaldialdehyde (OPA)-l-thio-β-d-glucose as derivatization agent.
• 2.2. Fourteen primary amino acids were detected among which glutamine, alanine, γ-aminobutyric acid (GABA) and glycine predominated and constituted 67.7% of the amino acids found.
• 3.3. The concentration of GABA increased significantly with the age of larvae entering the wandering phase and reached a maximum during metamorphosis.
• 4.4. Analysis of cold-acclimated larvae revealed a net increase of free primary amino acids from 96 to 151.8 μmol/ml during consecutive acclimation to 0°C within 20 days and to 205.4μmol/ml during cold shock injury at 0°C (3 hr).
• 5.5. The bulk of this increase was accounted for by alanine, glycine, phenylalanine and lysine.

     

Enhanced granulocyte phagocytosis at low winter temperature and high summer temperature in the tench (Tinca tinca L.)

Tench were captured in summer and winter when water temperature was 30 ± 3°C and 12 ± 2°C, respectively. Blood granulocytes were assayed for mobility capacity and phagocytic and microbicide capability to Candida albicans at 22°C and 30°C in summer and at 22°C and 12°C in winter. The results showed that tench granulocytes were more active in winter than in summer, and that phagocytic function was greatest at the seasonal temperature (30°C in summer and 12°C in winter) with respect to 22°C. This study suggests the importance of seasonally in the study of temperature effects on fish phagocyte function.     

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 changes in phagocytic capacity and superoxide anion production of blood phagocytes from tench (Tinca tinca,L.)

Seasonal variations in the ex vivo phagocytic function of blood cells from tench, including ingestion capacity of inert particles and its destruction (microbicide capacity) assessed by measurement of superoxide anion production, were studied. Tench were maintained under natural conditions throughout the year, and the different assays of samples taken during each season were initially performed in vitro at 22°C and the results compared. Subsequently, assays were performed at the same temperature as that of the water ponds in which the fish were kept (“seasonal temperature”: 12°C in winter, 22°C in spring and autumn and 30°C in summer) and the results compared seasonally. The results at 22°C showed that phagocytic capacity was greatest in spring and summer and lowest in winter. However, when phagocytic capacity was measured at seasonal temperature, highest values appeared in winter and lowest in summer and autumn. Nitroblue tetrazolium reduction by tench phagocytes after phagocytosing latex beads demonstrated a similar seasonal behaviour at both 22°C in each season and at seasonal temperature. The highest values appeared in summer, which suggests a better microbicide capacity in this season. The results obtained in this study suggest that for a correct interpretation of ex vivo phagocytic capacity of fish through the year it is necessary to use the same assay temperature as that of the water in which the fish is kept.     

Phosphoethanolamine in brain of the euritherm lake fish Perccottus glehni (Eleotridae, Perciformes, Dyb. 1877) as a phenomenon depending on temperature factor

The present work studied effect of a seasonal decrease of environmental temperature and cold shock on pools of free amino acids (FA) in brain of the euritherm lake fish P. glehni. For the first time, non-protein amino acid, phosphoethanolamine (PEA), revealed in the great amounts under conditions of near-zero temperatures was found in brain of ectothermic animals. An intensive rise of the PEA pool from 0.3% during the summer period to 33.6% at winter was shown to occur in the brain of P. glehni as a result of a seasonal temperature decrease. In contrast, the level of taurine showing the greatest pool in comparison with other FA (29.0% of the total pool) in summer decreases to 8.9% in the beginning of winter period. Similar negative correlation between the taurine and PEA content was found under effect of acute cold shock (1°C): the taurine level decreased for 4 days from 32.2 to 14.5% of the total pool, whereas the PEA level sharply increased (from 2.1 to 15.3%). Both types of the low-temperature actions have revealed, besides PEA, an intensive rise in the pool of serine and accumulation of phosphoserine. The role of PEA in biochemical evolution and adaptation of the brain to low temperatures is discussed. It is supposed that accumulation of PEA, phosphoserine, and serine is associated with changes in the status of membrane phospholipids at low temperatures.     

Effect of temperature on the termination of diapause in the univoltine almond seed wasp Eurytoma amygdali

Diapausing larvae of Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae) were collected in early August and late September. They were subjected to various photoperiod and temperature regimens for up to 20 weeks, then kept at L16:D8 and 19 °C for another 14 to 26 weeks for diapause to be terminated and pupation to take place. Photoperiod did not affect diapause completion. It was confirmed that the two morphologically distinct diapause stages have different temperature requirements for their completion. The first diapause stage was completed synchronously at temperatures between 16 and 19 °C. A higher temperature of 26 °C delayed diapause development. The second stage required lower temperatures between 4 and 10 °C. Spontaneous termination of diapause was observed at constant 19 °C. When applied to the first diapause stage for 20 weeks, low temperatures made the larvae refractory to subsequent intermediate temperatures. The first stage was thus maintained until a higher temperature of 26 °C made the larvae regain their ability to respond to the intermediate temperatures and complete this stage. Larvae grown in Retsou almonds had a higher diapause intensity than larvae grown in Truoito almonds. The results suggest that, in nature, the high temperatures of late summer and early autumn are likely to maintain the first diapause stage. Subsequently, the less warm temperatures of autumn allow the completion of the first stage by late autumn, and the low temperatures of late autumn and of winter allow the completion of the second diapause stage by mid winter.     

Effects of aging on thermoregulatory responses and hormonal changes in humans during the four seasons in Japan

Physiological functions are impaired in various organs in aged people, as manifest by, e.g., renal and cardiac dysfunction and muscle atrophy. The elderly are also at increased risk of both hypothermia and hyperthermia in extreme temperatures. The majority of those over 65 years old have elevated serum osmolality. Our hypothesis is that the elderly have suppressed osmolality control in different seasons compared to the young. Eight healthy young men and six healthy older men participated in this study. The experiments were performed during spring, summer, autumn and winter in Japan, with average atmospheric temperatures of 15–20°C in spring, 25–30°C in summer, 15–23°C in autumn and 5–10°C in winter. Each subject immersed his lower legs in warm water at 40°C for 30 min. Core (tympanic) temperature and sweat rate at chest were recorded continuously. Blood was taken pre-immersion to measure the concentrations of antidiuretic hormone, serum osmolality, plasma renin activity, angiotensin II, aldosterone, leptin, thyroid stimulating hormone, fT₃ and fT₄. The results suggested that the elderly have suppressed osmolality control compared to the young; osmolality was especially elevated in winter compared to the summer in elderly subjects. Therefore, particularly in the elderly, balancing fluid by drinking water should be encouraged to maintain euhydration status in winter.     

Thermal factors affecting egg development in the wandering glider dragonfly, <Emphasis Type="Italic">Pantala flavescens</Emphasis> (Odonata: Libellulidae)

The wandering glider dragonfly, Pantala flavescens (Fabricius), arrives in Japan from tropical regions every spring. The offspring colonize areas throughout Japan, with rapid increases in populations in the autumn, but all individuals die in the winter, suggesting low tolerance to low temperatures. However, few quantitative data on egg development and water temperature have been reported for this species. Females at the reproductive stage were collected from fields throughout the flying season and their eggs released using an artificial oviposition technique. Almost all of the eggs were fertilized. Egg size was stable throughout the seasons. Most eggs hatched within a period of 5 days at high water temperatures (35 and 30 °C), which were recorded in the shallow ponds and rice paddy fields from summer to early autumn. However, the egg-stage duration increased with declining water temperature. All eggs in water at 15 °C had failed to hatch by 90 days. The calculated critical temperature of water was determined to be approximately 14.3 °C; the total effective temperature for the egg stage was about 80 degree-days. Thus, low water temperatures in winter may prevent P. flavescens overwintering in Japan.     

Studies on Amino Acid Contents of Processed Soybean

The influence of heat treatment on defatted soybean flour was studied. The eighteen kinds of amino acids were determined by microbiological assay method.

The heat destruction of the amino acids was found to occur when defatted soybean flour was autoclaved. At 0 Kg/cm² (100°C) for one, two, and four hours, no remarkable destruction was observed on the amino acids. On the other hand, cystine, lysine and arginine were destroyed under following heat conditions at 0.35 Kg/cm² (108°C), 0.7 Kg/cm² (115°C)and 1.4 Kg/cm² (126°C). Especially, heating at 1.4Kg/cm² (126°C) for four hours, a large amount of cystine, lysine and arginine and a small amount of tryptophan and serine were destroyed, but all other amino acids were not destroyed by any heat treatment.

The different types of heat destruction of cystine, lysine and arginine were observed when defatted soybean flour was autoclaved under the systematically specified heating conditions.

The influences of the added water, the period of heating and the temperature on destruction of amino acids of defatted soybean flour were studied. The eighteen amino acids available to the microbiological assay using lactic acid bacteria were assayed with respect to those products treated under different heating conditions in the presence of water 3 and 6 times the weight of the soybean flour, and in the absence of water under the pressure of 0.7 Kg/cm² (115°C) and 1.4 Kg/cm² (126°C) for various heating periods.

Amino acids except for lysine, arginine, cystine, tryptophan and serine were not destroyed in any heat treatment examined. The destruction of lysine and arginine was mainly due to the conditions of the amount of the added water, and those of cystine, tryptophan and serine were chiefly due to the period and the temperature of heat treatment.     

Seasonal regulation of dormancy in Chrysopa carnea (Neuroptera)

In Ithaca, New York, diapause incidence in Chrysopa carnea exceeds 50 per cent among adults emerging between 31 August and 3 September. Apparently, decreasing late summer day lengths, which exceed the stationary critical photoperiod in the laboratory, act on the sensitive stages and induce diapause. Full diapause intensity occurs about 2 weeks after adult emergence; complete winter coloration appears approximately 2 weeks later. Photoperiodic stimili in combination with the geographic strain largely determine diapause depth.Decreasing autumn day lengths decelerate diapause development and maintain diapause until the winter solstice. Subsequently, short day lengths and cold temperatures slow diapause development. In nature, neither long days, increasing day lengths, nor chilling hasten diapause termination. Diapause duration is largely determined by the interaction between inducing photoperiod, maintaining photoperiodic and thermal stimuli, and geographic strain. In Ithaca, diapause ends (all response to photoperiod ceases) between 22 January and 12 March.After diapause ends, the animals retain their diapause characteristics until temperatures exceed 4°C. Post-diapause reproductive development in females requires approximately 100 heat units above 4°C; however, below 8°C mating and oviposition are absent. Although the winter coloration remains, mating occurs within 1 day after transfer from 4 to 24°C. Temperature determines the rate at which the green summer colour returns.     

SEASONAL CHANGE IN THE DISTRIBUTION OF CELL SIZE OF COCCONEIS SCUTELLUM VAR. ORNATA (BACILLARIOPHYCEAE) IN RELATION TO GROWTH AND SEXUAL REPRODUCTION1

Growth and sexual reproduction of the marine littoral diatom Cocconeis scutellum Ehrenb. var. ornata Grun. were investigated at 30 different combinations of temperature (5, 10, 14, 18, 22° C), irradiance (20, 60, 100 μE·m^?2·s^?1) and daylength (14:10 and 10:14 h LD cycle). Growth occurred at all combinations. The optimal growth was observed at 14–18° C, long daylength and highest-to-moderate irradiance, and at 18° C, short daylength and highest irradiance. Sexual reproduction on the other hand occurred between 5 and 18° C, and the optimal condition was 10–14° C and short daylength. Annual cyclic, and sesonal changes in the distribution of cell size (valve length) were observed in a field population. These changes were characterized by an annual minimum in mean cell size in autumn, an annual maximum in winter, a slight decrease from the mean in spring–middle summer, a rapid decrease from the mean in late summer–early autumn, and appearance of bimodal distribution of cell size in winter. These changes were caused by sexual reproduction in autumn, rapid growth in late summer–early autumn and slow growth in other seasons, and poor viability of small cells near the lower end of the size range.     

Free amino acids in the late embryogenesis and pre-hatching stage of two coregonid fishes

Developing eggs of vendace (Coregonus albula L.) and whitefish (C. lavaretus L.) were experimentally delayed in hatching by incubation at low water temperature (1–2°). Some eggs were taken during this period to a water temperature which was gradually raised up to 8° to provoke mass hatching of embryos. The pattern of free amino acids was followed in eggs incubated at both temperatures. During a 56 days period, the content of several essential amino acids significantly decreased in eggs of both species. For instance, the lysine content dropped from 703 to 270 mg/g dry matter and the arginine content from 257 to 13.3 mg/100 g dry matter in whitefish eggs. A similar pattern of decreasing level of free amino acids in embryonated ova up to hatching was characteristic for essential amino acids and serine. Methionine was exceptional; its level remained approximately the same. On the other hand, non-essential amino acids showed a significant increase in concentration during the experimental period. For instance, the glycine level increased 4.9 and 2.1 times in whitefish and vendace eggs, respectively. Transfer of eggs to 8° accelerated the decrease of nearly all free amino acids before hatching. The consequence of such amino acid metabolism for newly hatched larvae is discussed.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae)

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn. Accepted: 22 February 1999     

Population dynamics, growth and reproduction of Corophium insidiosum (Crustacea: Amphipoda) at low salinities in Monolimni lagoon (Evros Delta, North Aegean Sea)

Distribution, population dynamics, growth and aspects of reproductive biology of Corophium insidiosum were investigated in Monolimni lagoon. Samples were collected in July 1997 (at 30 psu S) and during February 1998–May 1999 (at 0.1–5.7 psu S). Corophium insidiosumwas almost exclusively found in the outer part of the lagoon, which showed a higher water renewal rate. Population density gradually decreased during winter and spring, when salinity was lower than 1 psu and the amphipod finally vanished from the lagoon. Salinity increase during summer (1.2–5.7 psu) was followed by the re-occurrence of C. insidiosum with a time lag of 2–3 months. Population density increased in autumn and peaked in early winter at salinities 1.6–4.2 psu. Three cohorts appeared in the population during September 1998–March 1999. Breeding activity peaked in early autumn (14–21?°C, 4 psu S) and ceased after December (2–6.5?°C, ¡1.5 psu S). The preponderance of females in the large size classes resulted in a female- biased sex ratio in the whole population. The population showed a growth rate of 7.5–11.2 μm d^?1 being faster in autumn (9–21?°C, 3–4 psu S) than in winter (2–12?°C, 0.2–3 psu). An exponential relation existed between body length and cephalic length or dry body weight, while brood size was directly related to body length. Mean brood size was small (4.96 early embryos) and egg loss during development high (53%), possibly as a consequence of low salinities.     

The influence of temperature,sexual condition,and season on the metabolic rate of the lizardPsammodromus hispanicus

Summary Male and femalePsammodromus hispanicus from southern Europe were acclimated to four seasonal conditions of photoperiod and night time temperature. During the dark period, the lizards' body temperatures fell to ambient air temperature but during the light period the lizards were allowed to thermoregulate behaviourally and at such times the lizards' mean body temperature varied from 29.0°C to 32.6°C. The resting metabolic rate of these lizards was measured in 5°C steps from 5°C to 30°C or 35°C. Sexual condition had little effect on resting metabolic rate, but at low temperatures lizards acclimated to winter or spring seasonal conditions had lower resting metabolic rates than those acclimated to summer or autumn conditions. At temperatures above 20°C seasonal acclimation had no effect on resting metabolic rate. It is considered that the reduction in low temperature metabolic rate in spring and winter is induced by low night time temperatures and serves to conserve energy during those seasons when lizards must spend long periods at low temperature without being able to feed.