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.     

Ammonia production and kinetic properties of glutamate dehydrogenase in the sipinculid Phascolosoma arcuatum exposed to anoxia

The amounts of total NH ₄ ⁺ detected in the external media in which Phascolosoma arcuatum had been exposed to various periods of anoxia were significantly greater than those in which the worms were exposed to normoxia for a similar period. The increased NH ₄ ⁺ production by P. arcuatum during anoxic exposure was unlikely to be due to an increased catabolism of adenine nucleotides or urea. In contrast, there were significant decreases in the concentrations of several free amino acids in the coelomic plasma and body tissues of individuals during the 48 h of anoxic exposure. The amount of NH ₄ ⁺ produced by the anoxic P. arcuatum could be accounted for by the decreases in the concentrations of aspartate or glycine. Increases in the catabolism of free amino acids (FAA), leading to the increased production of NH ₄ ⁺ , in P. arcuatum during anoxia were supported by the detection of significant changes in the kinetic properties of glutamate dehydrogenase (GDH), in the deaminating direction, from worms exposed to anoxia for 48 h. The apparent increase in the affinity of GDH from the anoxic worm to glutamate would bring about a greater deaminating activity at physiological concentrations of ths substrate. P. arcuatum used in these experiments were collected from the mangrove swamp at Mandai, Singapore between 1990 and 1993.     

Biochemical adaptations of the mudskipper Boleophthalmus boddaerti to a lack of oxygen

Specimens of the mudskipper Boleophthalmus boddaerti, collected along the estuarine canal at Pasir Ris, Singapore between April 1987 and December 1989, were examined in the laboratory. After being exposed to environmental hypoxia for 6 h, no accumulation of lactate, alanine or succinate was observed in the muscle tissue of B. boddaerti. In addition, the blood lactate content and muscle creatine phosphate (CrP), adenosine triphosphate (ATP) and glycogen contents were not significantly different from those of the control. During normoxic recovery, oxygen debt repayment was only a small fraction of the oxygen deficit incurred during the 6 h of hypoxic exposure. Therefore, it would appear that B. boddaerti coped with environmental hypoxia by undergoing metabolic rate reduction. After anoxic exposure, the CrP, ATP and adenosine diphosphate (ADP) contents in the muscle tissue of B. boddaerti were significantly lower than the corresponding control values. Lactate accumulated in the blood and muscle tissue of the anoxic fish though the muscle energy charge and glycogen content remained constant. Hence, B. boddaerti was able to cope with such a period of anoxia without increasing its glycolytic rate. Succinate, which was not detected in the muscle tissue of the control and hypoxia-exposed fish, accumulated in significant quantities in the muscle tissue of B. boddaerti exposed to environmental anoxia and functional hypoxia. In the case of functional hypoxia, there was a significant decrease in the muscle glycogen content, and the muscle lactate content increased 17.3-fold, indicating that glycolysis was activated to provide the source of energy during muscular exercise.     

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.     

An investigation on the anaerobic metabolism of Nephtys hombergii (Annelida: Polychaeta)

In Nephtys hombergii Savigny (Annelida: Polychaeta: Errantia), succinate and less pronounced L-alanine are accumulated as endproducts during an initial phase of anaerobiosis. In this phase aspartate is utilized as substrate for anaerobic energy production in addition to glycogen. Prolonged anaerobiosis results in the formation of propionate and acetate which, to a large extent, are excreted into the water. The concentrations of aspartate and succinate were found to remain unchanged down to a Pw_{O 2}(oxygen partial pressure in incubation water) of approximately 20 torr, indicating a fully aerobic metabolism. At a Pw_{O 2}of 15 torr characteristic changes can be observed: energy production becomes partially anaerobic. At a Pw_{O 2}of 7 torr the level of aspartate is largely reduced, but the accumulation of succinate is still significantly less than at anoxia. [In the habitat of N. hombergii, the intertidal mud flats, a Pw_{O 2}in the range from 0 torr (Pamatmat, 1968) to 12 torr (Wells and Warren, 1975) was measured.] The activities of some important enzymes involved in anaerobic energy production (among them pyruvate kinase, phosphoenolpyruvate carboxykinase and lactate dehydrogenase) were measured. Although N. hombergii has a high glycolytic capacity, no lactate dehydrogenase was detected. Instead strombine and alanopine dehydrogenases were present in comparatively high activities.     

Anaerobic metabolism in the shrimp Crangon crangon exposed to hypoxia,anoxia and hydrogen sulfide

The brown shrimp Crangon crangon was collected in the Penzé estuary, Bretagne, in April 1994 and exposed to hypoxia, anoxia and combinations of hypoxia and sulfide. Exposure to sulfide induced total anaerobic metabolism even at an oxygen saturation which would otherwise permit totally aerobic metabolism. In addition to preventing aerobic metabolism there was a direct toxic effect of sulfide. Haemocyanin oxygen affinity (p₅₀) values from non-stressed C. crangon were relatively low. The p₅₀ values all exceed those where environmentally induced lactate accumulation occurs. It seems unlikely that lactate is an affinity modulating factor under environmental hypoxia.     

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.     

Functional and environmental anaerobiosis in the razor-clam Ensis directus (Mollusca: Bivalvia)

In Ensis directus (Conrad) metabolic changes accompanying muscular activity and experimental anoxia were investigated. Octopine dehydrogenase, strombine dehydrogenase, alanopine dehydrogenase and lactic dehydrogenase were shown to be present in different parts of the body, but the activity of ODH always exceeded that of the other enzymes by far. Muscular activity connected with escape digging or evoked by electrical stimulation resulted in a decrease of stored phosphagen and in an accumulation of octopine. During recovery the normal concentrations of energy-rich phosphates were restored quickly, whereas octopine was removed more slowly. In experimental anoxia only minor quantities of L-and D-alanine, succinate, propionate, and acetate were formed. Quantitatively the most important endproduct was strombine, which rose linearly with the duration of anoxic incubation.     

Lactate formation in Callianassa californiensis and Upogebia pugettensis (Crustacea: Thalassinidea)

The ability to accumulate lactate as a result of laboratory exposure to anoxia was examined in 2 species of mud-dwelling shrimp, the ghost shrimp Callianassa californiensis and the mud shrimp Upogebia pugettensis. Hemolymph lactate accumulated to a much greater degree in the mud shrimp, even though the overall levels of lactate dehydrogenase activity were similar in tissues of the two species. Carbohydrate reserves, estimated as glycogen, were not significantly depleted in muscle or midgut gland of either species as a result of anoxic stress. The results are discussed in relation to possible metabolic strategies employed to cope with the hypoxic environment of these crustaceans.     

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.     

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.     

Free amino acids and osmoregulation in the intertidal pulmonate Onchidium tumidium

The aim of this study is to elucidate the osmoregulatory capabilities of the intertidal pulmonate Onchidium tumidium. Our results indicate that O. tumidium could tolerate hyperosmotic stress more effectively than hypoosmotic stress. In 10% seawater (SW), it was capable of maintaining its plasma hyperosmotic and hyperionic to the external medium. However, it rapidly gained weight in 10% SW, and no restoration of body weight was observed. In contrast, in 90% SW it was able to recover its body weight partially. In a hyperosmotic medium, accumulation of intracellular free amino acids (FAA) was responsible for cell volume regulation. The accumulated FAA might originate from protein catabolism, which occurred in the internal organs (IO). In contrast, the relatively smaller amination:deamination ratio of glutamate dehydrogenase in the body wall (BW) and IO of specimens exposed to hypoosmotic stress might lead to a higher rate of amino acid catabolism. This was supported by the greater ammonia contents in the BW and IO of, and higher rate of ammonia excretion by, specimens exposed to 10% SW. Received: 11 March 1998 / Accepted: 2 May 1999     

Anaerobic respiration in the polychaete Euzonus (Thoracophelia) mucronata

The intertidal polychaete Euzonus mucronata (Treadwell) is found in great numbers within beach sands that undergo periodic anoxia. Short-term exposures (2 to 4 h) to anaerobicity are withstood through the use of a supply of oxyhemoglobin to support continued aerobic metabolism. Longer periods of environmental anoxia activate an anaerobic metabolism capable of sustaining life for up to 20 days, and characterized by the excretion of succinate and propionate. These two mechanisms appear to be adaptations to the variations in occurrence and duration of anoxia within the intertidal sands inhabited by E. mucronata.Supported by an NSF Graduate Fellowship, 1973–1974.     

Cyclic AMP-dependent protein kinase: role in anoxia and freezing tolerance of the marine periwinkle Littorina littorea

A key regulatory mechanism underlying the switch between aerobic and anaerobic metabolism amongst anoxia-tolerant marine molluscs is reversible protein phosphorylation. To assess the role of cAMP-dependent protein kinase (PKA) in aerobic–anaerobic transitions, the effects of anoxia on the activity and subcellular distribution of PKA were assessed in foot and hepatopancreas of the marine periwinkle, Littorina littorea. Exposure to N₂ gas at 5 °C caused a rapid decline in the percentage of total enzyme present as the free catalytic subunit (PKAc) in both tissues; the percentage of PKAc fell from ∼30% in controls to 3% after 1 h anoxia and remained low over 72 h. Total PKA also fell by 30% after 72 h anoxia in hepatopancreas but rebounded during aerobic recovery. Freezing at −8 °C elicited parallel results for both percentage of PKAc and total PKA, suggesting that PKA responses to freezing were stimulated by the ischemia that develops when hemolymph freezes. Anoxia also led to a shift in PKA subcellular distribution in hepatopancreas (but not in foot), the percentage of total PKA activity associated with the nuclear fraction dropping from 25% in controls to 8% in 12 h anoxic snails with opposite changes in the cytosolic fraction. The catalytic subunit (PKAc) of foot PKA was purified to a final specific activity of 63.5 nmol phosphate transferred per minute per milligram protein. Enzyme properties included a molecular weight of 33 to 35 kDa, an activation energy from Arrhenius plots of 65.1 ± 4.8 kJ mol⁻¹, and substrate affinity constants of 151 ± 6 μM for the phosphate acceptor, Kemptide, and 72 ± 9 μM for Mg.ATP. Activity was strongly reduced by mammalian PKA inhibitors (H-89, PKA-I), by neutral chloride salts (I₅₀ values 165 to 210 mM) and by NaF (I₅₀ 62 mM). Reduced PKA activity under anoxic or freezing conditions would facilitate the observed suppression of the activities of numerous enzymes that are typically PKA-activated and thereby contribute to the overall anoxia-induced metabolic rate depression. Received: 19 November 1997 / Accepted: 30 September 1998     

The fluffy sea anemone Metridium senile in periodically oxygen depleted surroundings

In SCUBA-diving monitored field experiments (Flensburg Fjord, 15 m, 1981) and parallel aquarium tests,the behavior of Metridium senile under anoxic conditions and its oxygen-deficiency resistance were studied. When oxygen is lacking the fluffy sea anemone diminishes body surface, and then, successively, shuts off most energy consuming activities. By means of this strategy, 50% of the tested individuals survived 3 weeks of total anoxia.     

Metabolic responses of the prawns Palaemon elegans and P. serratus (Crustacea: Decapoda) to acute hypoxia and anoxia

Using prawns, Palaemon elegans (Rathke) from intertidal pools on the Isle of Cumbrae, Scotland, and P. serratus (Pennant) from the subtidal at Plymouth, England, some metabolic responses to hypoxia and anoxia have been studied. P. elegans was found to have a greater tolerance of severe hypoxia than P. serratus. Tolerance of totally anoxic conditions, however, was limited to only 4 h in P. elegans and to approximately 1 h in P. serratus. exposure to moderate hypoxia (30 torr) resulted in little change in the concentration of L-lactate in the blood or in the tissues of either P. elegans or P. serratus. When exposed to extreme hypoxia (10 or 5 torr for P. elegans), however, there was a progressive increase in the concentration of L-lactate in the blood and in the tissues of both species. After normoxic conditions had been restored, the concentration of L-lactate in the blood and in the tissues returned to normal resting levels more rapidly in P. elegans than in P. serratus. Under hypoxic conditions, both P. elegans and P. serratus showed an increase in the concentration of blood glucose and a slight reduction in the glycogen content of the tissues. The concentrations of blood glucose and of tissue glycogen returned to normal levels within 6 h of the prawns being returned to normoxic conditions. The results of an in situ study in April and August 1986 to examine the metabolic responses of P. elegans to the hypoxic conditions normally experienced in high-shore rock pools are also presented. The ecological significance of the differing abilities of these species to survive hypoxic exposure is discussed.     

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     

Sea anemones and brittle stars: unexpected predatory interactions during induced in situ oxygen crises

During oxygen crises, benthic faunas exhibit a series of behavioural patterns that reflect the duration and severity of the event. During artificially induced oxygen deficiencies at 24 m depth in the Northern Adriatic Sea, we photographically documented predation by the sea anemones Cereus pedunculatus (Pennant, 1777) and Calliactis parasitica (Couch, 1842) on the brittle star Ophiothrix quinquemaculata (DelleChiaje, 1828). Five predatory events were recorded with four anemones during nine deployments totalling 817 h of observation. Under near-anoxic conditions, individuals of both actinians made contact with, pulled in and consumed the brittle stars. The duration of each predatory event was 1.5–7.5 h. In three of the five events, brittle star remains were regurgitated after an additional 2.0–12.5 h of digestion by the anemones. Our time-lapse sequences demonstrate that oxygen deficiency, beyond eliciting a series of specific behaviours in members of each species, also promotes previously unobserved interspecific interactions. Our results show that sea anemones are not only highly resistant to anoxia, but may also benefit by taking advantage of prey that are more vulnerable to anoxic conditions.     

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     

Effects of anoxia on the viability of subitaneous eggs of planktonic copepods

The effect of anoxia on the viability (as evidenced by hatching) of newly spawned and fully developed subitaneous eggs of three species of copepods, Acartia tonsa (Dana), Centropages hamatus (Lilljeborg), and Labidocera aestiva (Wheeler) was determined for eggs produced in the laboratory by adults collected from inshore waters of the northeastern Gulf of Mexico between January and August 1992. Hatching success decreased to 50% or less when eggs were exposed to oxygen concentrations <0.06 mll^-1 for more than 12 d, except for newly spawned eggs of L. aestiva, which still showed 50% survival after 32 d of exposure to anoxia. For all three species, newly spawned eggs survived exposure to anoxia longer than fully developed eggs. The results indicate that the increasing occurrence of anoxia in estuarine and coastal waters could have a major impact on the population growth of these important food web organisms.