Adaptation of the polychaete worm Scoloplos armiger to hypoxic conditions

The anaerobic metabolism of the intertidal polychaete Scoloplos armiger, its recovery from anaerobiosis and the importance of anaerobic energy production during low tide in the field were investigated. Under anaerobic conditions S. armiger produces energy in the same manner as Arenicola marina, a prototype of an euryoxic invertebrate from the intertidal. Energy is produced from the phosphagen stores and from the breakdown of glycogen to volatile fatty acids, mainly propionate and to a lesser extend acetate. However, S. armiger cannot reduce its energy demand to the same degree as A. marina. This and the relatively small pool of glycogen may be the reason for its only moderate resistance to anoxia. The recovery from anaerobiosis proceeds in S. armiger significantly slower than in A. marina. S. armiger is able to maintain a fully aerobic metabolism down to a Pw_{O 2}of ca. 20 torr and even at a Pw_{O 2}of 10 torr a partly aerobic metabolism was retained. In the field during low tide S. armiger ascends into the oxidative layer, where it is able to maintain an aerobic metabolism even at parts without remaining puddels on the surface.     

Nephtys hombergii, a free-living predator in marine sediments: energy production under environmental stress

Nephtys hombergii is a free-living, burrowing predator in marine sediments. The worm is, therefore, exposed to various environmental conditions which tube-dwelling polychaetes of the same habitat most likely do not encounter. The worms have to survive periods of severe hypoxia and sulphide exposure, while at the same time, they have to maintain agility in order to feed on other invertebrates. N. hombergii is adapted to these conditions by utilising several strategies. The species has a remarkably high content of phosphagen (phosphoglycocyamine), which is the primary energy source during periods of environmental stress. With increasing hypoxia, energy is also provided via anaerobic glycolysis (pO₂<7 kPa), with strombine as the main end-product. Energy production via the succinate pathway becomes important only under severe hypoxia (<2 kPa), suggesting a biphasic response to low oxygen conditions which probably is related to the worm's mode of life. The presence of sulphide resulted in a higher anaerobic energy flux and a more pronounced energy production via glycolysis than in anoxia alone. Nevertheless, after sulphide exposure under anaerobic conditions of <24 h, N. hombergii is able to recover completely. Although N. hombergii appears to be well adapted to a habitat with short-term fluctuations in oxygen and appearance of hydrogen sulphide, its high energy demand as a predator renders it likely to limit its survival in an environment with longer lasting anoxia and concomitant sulphide exposure. Received: 28 May 1997 / Accepted: 21 June 1997     

Anaerobic and aerobic energy metabolism in ovaries of the sea urchin Strongylocentrotus droebachiensis

The gonads of sea urchins undergo large changes in mass during their gametogenic cycle. In addition, they have relatively low aerobic capacities and are poorly perfused by the circulatory system and thus are continually hypoxic or anoxic. The present study of Strongylocentrotus droebachiensis investigates seasonal changes in the relationships among mass of the ovaries, pH and P_O2 of the perivisceral coelomic fluid which bathes the ovaries, and partitioning of ovary energy metabolism into its anaerobic and aerobic components. S. droebachiensis were collected at Blue Hill Falls, Maine, USA, from August 1982 to March 1984. We found that from 76 to 92% of the heat dissipated by isolated ovaries of the sea urchin S. droebachiensis derives from anaerobic energy metabolism at partial pressures of oxygen prevailing in vivo. Ovaries from S. droebachiensis have the capacity to produce large amounts of lactate under imposed anoxia, but lactate accounts for only 37% of the total anoxic heat dissipation, which suggests that other end products of anaerobiosis are present. Seasonal changes in pH and P_O2 of the perivisceral coelomic fluid can be explained by a complex relationship among changes in temperature, reproductive condition, and anaerobic and aerobic metabolism in the ovaries, gut and body wall. Seasonal changes in the buffering capacity of the perivisceral coelomic fluid must be determined before the effects of respiratory and metabolic acid production on the acid-base status of the coelomic fluid can be fully understood.     

Lactate and succinate oxidoreductases in marine invertebrates

Nineteen species of littoral marine invertebrates, representing Porifera, Cnidaria, Ctenophora, Brachiopoda, Mollusca, and Arthropoda, were studied with respect to the ability of tissue extracts to catalyze the lactate and succinate dehydrogenase reactions in both directions. Pyruvate reductase (PR) activity varied tremendously with species, from 0.014 mole/min/g of tissue in the etenophore Mnemiopsis leidyi to 145 -moles/min in leg muscle of the horseshoe crab Limulus polyphemus, a 10,000-fold range. Lactate dehydrogenase (LD) activity varied from 0.010 in the ctenophore to 2.91 in the squid Loligo pealei, a 300-fold range. The ratio PR/LD, indicating the probability of lactic acid production, was 1,968 in muscle of the flounder, the only vertebrate studied. It was very much lower in all invertebrates; in a brachiopod, Terebratulina septentrionalis, the ratio was only 0.68. Fumarate reductase (FR) and succinate dehydrogenase (SD) activities varied less widely. The extremes of the ratio FR/SD, indicating the probability of succinic acid production, were 0.24 in the clam Mercenaria mercenaria, and 7.6 in the oyster Crassostrea virginica. PR/LD appears related to the capacity for vigorous muscular activity, sustaining rapid movement of larger animals, and FR/SD appears related to tolerance of temporary anaerobiosis, such as found in sessile animals that close their valves tightly on exposure to air during low tide.     

Metabolic adaptation of the intertidal worm Sipunculus nudus to functional and environmental hypoxia

The scope of anaerobic metabolism of Sipunculus nudus L. was assessed from the maximal activities of some enzymes of the intermediary metabolism and from the concentration of some metabolites accumulated during enhanced muscular activity and during prolonged experimental hypoxia.

1. Maximal enzyme activities demonstrate that the scope of anaerobic glycolysis, as indicated by maximal activities of glycogen phosphorylase (0.84 U g^-1 fresh wt), far exceeds the aerobic capacity, which is assumed not to surpass the activity of succinate dehydrogenase (0.09 U g^-1 fresh wt). Three pyruvate reductase activities (alanopine-, strombine- and octopine dehydrogenase) can possibly terminate anaerobic glycolysis.
2. During muscular activity, energy is provided by the degradation of phospho-L-arginine and by anaerobic glycolysis. Octopine is the major endproduct during functional anaerobiosis while the formation of strombine is less pronounced.
3. During exposure to a nitrogen atmosphere, several anaerobic endproducts are found to accumulate. Anaerobic glycolysis is terminated by strombine synthesis. This opine accumulates in concentrations much higher than octopine. In addition the concentrations of succinate, propionate and acetate are found to increase in tissues, and/or in the coelomic fluid and the incubation water.
4. The relative contribution of energy by the different anaerobic metabolic pathways are estimated during functional and environmental hypoxia.

     

Energy metabolism in the foot of the marine gastropod Nassa mutabilis during environmental and functional anaerobiosis

This study concerns the effects of oxygen deprivation due to incubation in oxygen free sea water (environmental anoxia) or exercise (functional anoxia) and of exposure to air on the mode of energy production in the foot of the whelk Nassa mutabilis. Additionally, energy metabolism of the foot muscle was investigated during exercise after different anoxia periods and during the subsequent recovery period. During environmental anoxia, phosphoarginine, glycogen and aspartate are broken down as substrates and alanine and succinate are formed as products. There was no production of D-lactate or octopine. The energy charge value fell after 24-h anoxia. Exposure to air resulted in only small changes in phosphoarginine and alanine levels, suggesting that oxygen uptake was impaired in the first phase of air exposure but that, later, aerial respiration kept pace with the energy demand. Exercise caused a dramatic decrease of phosphoarginine concentration, coupled with glycolytic ATP production via octopine formation. In the recovery period (after exercise), the level of phosphoarginine was rapidly restored. An anaerobic component was evident during recovery as shown by the accumulation of D-lactate. Thus, both terminal dehydrogenases, octopine- and lactate dehydrogenase, are active in the muscle, but under different physiological conditions. Octopine formation also took place when the whelks were subjected to exercise after 4 or 24 h of anoxia. In this case, glycolysis provided between 70 and 90% of the energy required since the phosphagen store had already been depleted during the anoxic period. When the work load was increased (greater number of leaps), it became evident that the action of arginine kinase and octopine dehydrogenase are not closely linked. First there was an increase of arginine and then later a condensation of arginine with pyruvate to form octopine.     

Anaerobic metabolism in Arenicola marina and Nereis diversicolor during low tide

In order to observe the metabolic response of two marine polychaetes, Arenicola marina L. and Nereis diversicolor Müller, at various times during low tide, the concentrations of adenine nucleotides, phosphagens, D- and L-alanine, succinate, acetate, propionate and lactate were measured. During 8h of exposure ATP does not change significantly in the body-wall musculature of the lug-worm A. marina, whereas ADP and AMP concentrations double the first hour of exposure. Correspondingly, the energy charge falls from 0.91 to about 0.84. The concentration of the phosphagen phosphotaurocyamine decreases by about 20% during the first 2 h and after 6 h only about 50% of the initial concentration was measured. During exposure, aspartate decreases by approximately 6 mol g^-1 fresh weight within 6 h (from 23 to 17 mol g^-1 fresh weight). During the first 4 h of exposure succinate concentration doubles and within the next 4 h it reaches values about 10-fold the initial value. The concentrations of acetate and propionate only increase during the last 2 h (6–8) of exposure. After several hours of exposure typical changes in the concentrations of all these metabolites could also be demonstrated in N. diversicolor. The concentrations of the two phosphagens phosphoglycocyamine and phosphocreatine and of aspartate decrease by about 25% during 9 h of exposure, on the other hand succinate and especially D-lactate accumulate. In both species, the partial anaerobic energy production during exposure does not have a lasting detrimental effect. After one high tide the worms have fully recovered.     

Effects of hypoxia on the respiratory and circulatory regulation of Nephrops norvegicus

The burrowing decapod Nephrops norvegicus (L.) was kept under various degrees of hypoxia in order to measure respiration, heart rate, scaphognathite rate, haemolymph oxygen content and pH. An emergence reaction to hypoxia occurred only in dim light (<10^-2 m-c) or darkness, but after 10 d of moderate hypoxia the decapods showed no emergence response at all. The weight specific respiration of quiescent individuals was relatively low and increased only slightly in hypoxia (P_wO₂=40 torr). Heart rate, about 50 beats min^-1, changed little during hypoxia, down to P_wO₂=40 torr, whereas scaphognathite rates rose from about 60 beats min^-1 at normoxia to peak at 120 beats min^-1 at P_wO₂=40 torr. The oxygen extraction efficiency (E) remained at 20 to 30% during the first hour of hypoxia then rose gradually to maximum values of 30 to 40%. A small respiratory alkalosis of the blood became evident only after 4h of hypoxia (P_wO₂=50 torr). Normoxic postbranchial O₂ tensions (P_aO₂) were low (25–30 torr) and showed only a small decline during hypoxia. Over 10 to 13 d in moderate hypoxia an effective biosynthesis of 0.024 mM haemocyanin individual^-1 d^-1 occurred in fed decapods, whereas controls (normoxic) showed no significant change in pigment levels. A linear relationship between oxygen carrying capacity and haemocyanin concentration was found. It is contended that N. norvegicus is better able to cope with periodic exposure to hypoxia when food of sufficient quantity and quality is available.     

Population and gender-based differences in tissue-specific enzyme activities in brackish water and freshwater threespine sticklebacks, Gasterosteus aculeatus

Sticklebacks (Gasterosteus aculeatus) from a brackish water and a freshwater population were compared immediately after removal from their habitat and after being maintained in the laboratory. Glutamate dehydrogenase activities in the liver and both lactate dehydrogenase and aspartate aminotransferase activities in the axial muscle were higher in the brackish water sticklebacks than in the freshwater specimens. Liver glutamate dehydrogenase activities were higher in males than in females, whereas lactate dehydrogenase activities were distinctly higher in females. A significant positive correlation was found between size and both aspartate aminotransferase and lactate dehydrogenase. The population-specific activities of muscle enzymes are interpreted as an example of evolutionary adaptation of enzyme regulation. Received: 28 February 1997 / Accepted: 5 August 1997     

Ultrastructure and anaerobic metabolism of mitochondria in the marine oligochaete Tubificoides benedii : effects of hypoxia and sulfide

It has often been suggested that ultrastructural properties of mitochondria are correlated with oxygen and sulfide levels from the environment, although careful analyses of this question are rare. In this study the ultrastructure and distribution of mitochondria in Tubificoides benedii, a marine oligochaete from sulfide-rich sediments, were investigated after a series of oxic, hypoxic and hypoxic–sulfidic (200 μM H₂S) incubations up to 24 h. Succinate, one of the key endproducts of an anaerobic metabolism, was used as an indicator of mitochondrial anaerobiosis. Consistent differences in mitochondrial ultrastructure were not observed in any of the incubations, even after 24 h. Stereological parameters of mitochondria (volume density, surface density of the outer mitochondrial membrane, and specific surface) in epidermal and intestinal tissues of T. benedii were not affected by hypoxia or sulfide either. On the other hand, succinate concentrations increased significantly within 24 h under hypoxic and hypoxic–sulfidic conditions. Thus, experimental hypoxia and sulfide clearly caused mitochondrial anaerobiosis without affecting ultrastructure or distribution of mitochondria in T. benedii. Distinct differences in ultrastructural and stereological parameters were common between different tissues and between individuals, showing that different forms of mitochondria can occur within one species. Our results imply that a mitochondrial ultrastructure specific to thiobiotic animals does not appear to exist. Received: 4 August 1996 / Accepted: 20 September 1996     

Effects of anaerobiosis on root metabolism of Zostera marina (eelgrass): implications for survival in reducing sediments

The temperate seagrass Zostera marina L. typically grows in highly reducing sediments. Photosynthesis-mediated O₂ supplied to below-ground tissues sustains aerobic respiration during photosynthetic periods. Roots, however, experience daily periods of anoxia and/or hypoxia at night and under conditions that reduce photosynthesis. Rhizosphere cores of Z. marina were collected in August 1984 from Great Harbor, Massachusetts, USA. We examined short-term anaerobic metabolism of [U-¹⁴C]sucrose in excised roots and roots of intact plants. Under anaerobic conditions roots showed appreciable labeling of CO₂, ethanol and lactate, and slight labeling of alanine and other metabolites. Over 95% of the ¹⁴C-ethanol was recovered in the root exudate. Release of other metabolites from the roots was minimal. Ethanol was also released from hypoxic/anoxic roots of intact plants and none of this ethanol was transported to the shoot under any experimental conditions. Loss of ethanol from roots prevented tissue levels of this phytotoxin from increasing during anaerobiosis despite increased synthesis of ethanol. Anaerobic metabolism of [U-¹⁴C]glutamate in excised roots led to appreciable labelling of -aminobutyrate, which was known to accumulate in eelgrass roots. Roots recovered to fully aerobic metabolism within 4 h after re-establishment of aerobic conditions. The contributions of these root metabolic responses to the ability of Z. marina to grow in reducing marine sediments are related to light-regulated interactions of shoots and roots that likely dictate depth penetration, distribution and ecological success of eelgrass.     

Sodium cyanide-induced modulations in the activities of some oxidative enzymes and metabolites in the fingerlings of Cyprinus carpio (Linnaeus)

Carp fingerlings exposed to a sublethal concentration (0.5?mg?L^?1) of sodium cyanide showed a steady decrement over a 7-day period in respiratory rate, rise in lactate dehydrogenase (LDH), and fall in succinate dehydrogenase (SDH) activities followed by variations in lactic and pyruvate levels. Changes in these enzyme activities might be due to impaired oxidative metabolism and severe cellular damage leading to the release of these enzymes. Decline in the activities of SDH and LDH clearly represents a shift from aerobic to anaerobic metabolism as evidenced by elevated lactate and decline in pyruvate levels. The shift to anaerobic metabolism is also reflected by severe drop in the respiratory rate of the fish. This may be a consequence of the blockage of electron transfer from cytochrome c oxidase to molecular oxygen, thus ceasing cellular respiration and it can lead to cellular hypoxia even in the presence of normal hemoglobin oxygenation. Hence, we indirectly reconfirm the inhibition of oxidative metabolism by sodium cyanide. Alterations in behavioral pattern induced by sublethal sodium cyanide exposure may be due to the combination of cytotoxic hypoxia with lactate acidosis, which depresses the central nervous system (CNS); as the brain is the most sensitive site to anoxia, it results in impaired CNS function.     

Effects of anoxia on the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase,and the production of lactate and succinate in the intertidal pulmonate Onchidium tumidium

Onchidium tumidium, an intertidal pulmonate, has evolved to depend mainly on the formation of succinate, rather than lactate and opines, to survive in anoxia. For our study O. tumidum were collected from the mud flats of the mangrove swamp at Mandai, Singapore between 1988 and 1991. After 24 h of anoxic exposure, the lactate and succinate contents of the anoxic individuals were approximately 10 and 150 times, respectively, the corresponding values of the normoxic individuals. Alanine and acetate accumulations also occurred during anoxia, though to a much lesser extent. No propionate or octopine was detected. The depletion in aspartate content in O. tumidium could not account for the amount of succinate accumulated during anoxia. The succinate formed might have originated from glycogen involving the flow of carbon through the phosphoenolpyruvate (PEP) branch point of glycolysis. In support of such a hypothesis, results indicate that there was a decrease in the affinity of pyruvate kinase from O. tumidium exposed to 24h of anoxia to PEP to facilitate succinate formation through phosphoenolpyruvate carboxykinase (PEPCK). In comparison, the affinity of PEPCK from O. tumidium exposed to anoxia to PEP was apparently unaltered.     

Alterations in Carbohydrate Metabolism in Selected Tissues of Crab,Oziotelphusa Senex Senex (Fabricius) Under Fenvalerate-Induced Stress

Changes in carbohydrate metabolism in hepatopancreas and muscle of the crab, Oziotelphusa senex senex exposed to a sublethal concentration (0.2 ppm) of fenvalerate, a pyrethroid insecticide, were studied. the glycogen and total carbohydrate levels decreased significantly in the tissues of crab exposed to fenvalerate. an increase in phosphorylase ‘a’ and decrease in aldolase activity levels suggested increased glycogenolysis, and decreased glycolysis during fenvalerate toxicity. Krebs cycle enzymes such as NAD-isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were decreased, suggesting reduced mitochondrial oxidative metabolism. Glucose-6-phosphate dehydrogenase activity was increased significantly, indicating enhanced oxidation of glucose through the hexose monophosphate shunt pathway. Lactate dehydrogenase activity was elevated indicating the development of anaerobic conditions at tissue level in the stressed crab. Cytochrome C oxidase and Mg2+ ATPase activity levels were also decreased, indicating the impaired energy synthesis and prevalence of energy crisis. These results suggest that fenvalerate has a profound effect on the glucose metabolism of crab.     

Some effects of hypoxia and medium ammonia enrichment on efflux rates and circulating levels of ammonia inNephrops norvegicus

Eutrophication has been reported for autumn months in regions of the Kattegat/Skagerrak, causing stress to bottom-living organisms. The present studies, undertaken in April (1989), investigated the effects of hypoxia and high ammonia levels in the burrowing decapodNephrops norvegicus (L.). The net ammonia efflux rates and circulating ammonia levels at 6 and 12°C, at normoxia [partial pressure of O₂ in the water (torr),P _wO₂ = 155 torr)] and hypoxiaP _wO₂ = 24 torr) in normal seawater and ammonia-enriched (300µmol ammonia l^–1) seawater were examined. The hourly weight-specific efflux rates were very variable and in all groups included some individuals which showed periods of no net efflux, or even a net uptake of ammonia. At each temperature, net efflux-rate differences due to treatments were not significant (P>0.05; ANOVA, in all cases) and only the differences between the net efflux rates of the normoxic groups were significantly affected by temperature (P<0.05; ANOVA). Circulating ammonia levels were also variable, and at 6°C the ammonia-enriched groups had significantly higher weight-specific blood ammonia content values than the normoxic group (P<0.05 in both cases). A net uptake of ammonia occurred in ammoniaenriched conditions — probably along a reversed NH ₄ ⁺ gradient, as downhill pNH₃ gradients were maintained in all groups — and may represent the only means by which some branchial efflux of ammonia could proceed.     

Effects of long-term acclimation to environmental hypercapnia on extracellular acid–base status and metabolic capacity in Mediterranean fish <Emphasis Type="Italic">Sparus aurata</Emphasis>

In the context of future scenarios of anthropogenic CO₂ accumulation in marine surface waters, the present study addresses the effects of long-term hypercapnia on a Mediterranean fish, Sparus aurata. By equilibration with elevated CO₂ levels seawater pH was lowered to a value of 7.3, close to the maximum pH drop expected in marine surface waters from atmospheric CO₂ accumulation. Intra- and extracellular acid–base parameters as well as changes in enzyme profiles were studied in red and white muscles and the heart under both normocapnia and hypercapnia. The activities of pyruvate kinase (PK), lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase and and 3-hydroxyacyl CoA dehydrogenase (HOAD) reflect the pathways and capacity of oxidative processes in metabolism. Long-term hypercapnia caused a transient reduction in blood plasma pH (pH_e) as well as in intracellular pH (pH_i). Compensation of the acidosis occurred through increased plasma and cellular bicarbonate levels. Changes in enzymatic activities, especially the increase in the activity of L-LDH, paralleled by a drop in CS activity in white and red muscles reflect a shift from aerobic to anaerobic pathways of substrate oxidation during long-term acclimation under hypercapnia. The present results suggest that moderate environmental hypercapnia changes the metabolic profile in tissues of S. aurata. Consequences for slow processes like growth and reproduction potential as well as potential harm at population, species and ecosystem levels require further investigation.     

L-Lactate dehydrogenase in the longitudinal muscle of the sea cucumber Sclerodactyla briareus (Echinodermata: Holothuroidea)

The infaunal holothurian Sclerodactyla briareus (Thyone briareus) is able to tolerate exposure to hypoxic conditions for over 2 days. Since the in vitro anaerobic degradation of glucose-U-C¹⁴ by longitudinal muscle preparations leads to an accumulation of labeled lactic acid, it is apparent that lactate dehydrogenase plays a key role during anoxia. Disc electrophoresis resolved one major band of lactate dehydrogenase (LDH) activity in longitudinal muscle extracts. Muscle extracts of s. briareus oxidized L-lactate only, as is the case with most other echinoderms. The apparent Michaelis constants for lactate oxidation and pyruvate reduction are dissimilar from values reported for other marine invertebrates. The LDH activity in this holothurian is higher than the activities reported for other marine invertebrates with the exception of some active, large arthropods. Substrate inhibition of LDH maximal activity occurs at substrate concentrations (pyruvate) exceeding 10 mM, and then to only a minor extent. The characteristics of lactate dehydrogenase from the longitudinal muscle of S. briareus support the hypothesis that it functions to maintain glycolytic flux during prolonged periods of exposure to anoxic or hypoxic conditions.     

Survival of the intertidal pulmonate Onchidium tumidium during short term and long term anoxic stress

Onchidium tumidium showed a triphasic response to anoxia. Twelve hours of anoxic exposure had no effect on the glycogen content in O. tumidium. However, there were significant increases in the alanine, lactate and succinate contents in the anoxic individuals. These were accompanied by a significant decrease in the ATP content. These results suggest that O. tumidium survived the first 12 h of anoxic exposure without increasing the glycolytic flux to compensate for the lower efficiency of ATP production through anaerobic pathways. Indeed, the fructose-2,6-bisphosphate (F-2,6-P₂) content and the percentage of phosphofruc-tokinase (PFK) associated with the subcellular particles remain unchanged in O. tumidium exposed to 12 h of anoxia. Hence, a reduction in the metabolic rate of these individuals might have occurred during such a period of anoxia. In contrast, in between 12 and 24 h of anoxic exposure, the glycogen content O. tumidium decreased significantly, and levelled off thereafter. A significant increase in the percentage of PFK associated with the subcellular particles was observed in individuals exposed to 24 h of anoxia. In addition, the F-2,6-P₂ content of these anoxic individuals increased significantly. Taken together, these two mechanisms could activate PFK and lead to a greater glycolytic flux. Beyond 24 h of anoxic exposure, survival of O. tumidium must have required considerable suppression of metabolism as accumulation of end products and depletions of glycogen and ATP had reached constant levels.     

Respiratory quotient and ammonia excretion in Tilapia mossambica

Tilapia mossambica (Peters), acclimated to and tested in fresh water at 30°C, maintained a routine respiratory quotient (R Q) of about unity and an ammonia quotient (A Q) (Vol. NH₂/Vol. O₂) of about 0.2 at high ambient oxygen concentrations. At low oxygen concentrations (below 2 ppm) R Q and A Q increased sharply to values of 8 and 1, respectively (at 0.6 ppm), indicating a close relationship of increase in anaerobic energy utilization and increase in protein metabolism at inadequate oxygen concentrations. T. mossambica (8 cm), exercised continuously with intervening sampling and flushing stops for 6 h, at a swimming speed of about 2 body lengths/sec, derived some anaerobic energy throughout the exercise (R Q: 1.2), utilizing more protein the longer the exercise. The coupling of the increased protein metabolism and anaerobic energy utilization may be of advantage in preventing acidosis and also in conserving sodium (Na⁺) in fish.Part of this work was included in a paper presented at the Centennial Meeting of The American Fisheries Society, September 13–16, 1970, New York.     

Molecular mechanisms of adaptation to low temperature in marine poikilotherms. Some regulatory properties of dehydrogenases from two arctic species

The properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from gill tissue of the tanner crab Chionocetes bairdi, and lactate dehydrogenase (LDH) and glyceraldehyde dehydrogenase from skeletal muscle of C. bairdi and the yellowfin sole Limanda aspera were examined over the physiological temperature range of the animals. Both animals were obtained in the Bering Sea in winter, and their enzymes appear to be remarkably cold-adapted. Affinity of sole LDH for substrate appears to increase with decreasing temperature, thus keeping reaction rate essentially independent of temperature at physiological concentrations of the substrate. Calculated values of activation energy are low, in keeping with the argument that organisms from cold environments have enzymes with a reduced energy of activation. In addition, Hill plots of the substrate saturation curves for lactate dehydrogenase from muscle of sole indicate that there is a facilitation of allosteric behaviour at low temperatures. Maximum affinity of sole LDH for substrate in the absence of univalent cations occurs at 3°C, while in the presence of 150 mN K⁺, it occurs between 0° to-2°C. The effects of Mg²⁺ on enzyme activity appear to be determined by concentration of substrate and temperature. Thus, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are stimulated more effectively by Mg²⁺ at low temperature and at low substrate levels whereas, at high concentrations of substrate, they are relatively independent of the bivalent cation. All four dehydrogenases are affected by the univalent cations Na⁺, K⁺ and NH₄ ⁺ in a manner which appears to be determined, in part at least, by concentration of substrate and by temperature. These findings suggest mechanisms for the maintenance and regulation of enzyme activity in poikilothermic tissues at low and changing temperatures.