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.     

Bioenergetics and survival of the marine snail Thais lima during long-term oil exposure

The carnivorous snail Thais lima was fed Mytilus edulis during a 28-d exposure to the water soluble fraction (WSF) of Cook Inlet crude oil. The LC-50 of T. lima declined from >3000 ppb aromatic hydrocarbons on Day 7 to 818±118 ppb on Day 28. The LC-50 of M. edulis declined from >3 000 ppb aromatic hydrocarbons on Day 7 to 1 686±42 ppb on Day 28. Predation rate declined linearly with increasing aromatic hydrocarbon concentration up to 302 ppb; little predation occurred at 538 ppb and none at 1 160 or 1 761 ppb. Snail absorption efficiency averaged 93.5% and did not vary as a function of WSF dose. Total energy expenditure (R+U) increased at 44 ppb aromatics and declined at lethal WSF exposures. At sublethal WSF exposures, percentages of total energy expenditure were: respiration (87%), ammonia excretion (9%) and primary amine loss (4%). These percentages did not vary as a function of WSF dose or time. Oxygen:nitrogen ratios were not affected by WSF concentration or time and indicated that T. lima derived most of its energy from protein catabolism. The uptake of aromatic hydrocarbons into the soft tissues of snails and mussels was directly related to the WSF concentration. Naphthalenes accounted for 67 to 78% of the aromatic hydrocarbons in T. lima and 56 to 71% in M. edulis. The scope for growth was negative above 150 ppb WSF aromatic hydrocarbons and above 1 204 ppb soft-body aromatic hydrocarbons. These snails were physiologically stressed at an aromatic hydrocarbon concentration which was 19% of the 28-d WSF LC-50 (818±118 ppb) and/or 48% of the 28-d LC-50 of soft tissue aromatics (2 502 ppb).     

Apoptosis in marine sponges: a biomarker for environmental stress (cadmium and bacteria)

The marine demosponge Suberites domuncula is abundantly present on muddy sand bottoms, both in the open sea and in harbors. In the present study it is shown that exposure of S. domuncula to cadmium (CdCl₂) in concentrations ranging from 0.01 to 5.0 g ml⁻¹ for up to 5 d results in apoptotic fragmentation of DNA. Kinetics experiments revealed that after 24 h a significant increase of DNA fragmentation already occurred. Besides cadmium a second stimulus was identified to also cause apoptosis in this species, namely exposure to heat-treated Escherichia coli. In order to support the finding that both cadmium and E. coli induce apoptosis in the sponge, expression of the apoptotic gene MA-3 was studied. The cDNA, SDMA3, was isolated and found to be 2247 nucleotides long. The deduced amino acid sequence (M_r 50 765) shares high similarity with the corresponding mouse molecule. Like the mouse gene, the sponge MA-3 gene undergoes increased expression in response to apoptotic stimuli. While the specimens remained alive after treatment with cadmium, the sponges treated with E. coli died after approximately 12 d. The E. coli-treated animals started to form gemmules 10 to 12 d after addition of the bacteria. Hence, the process of apoptosis in sponges is triggered by two different pathways, one which is initiated by exogenous factors, e.g. heavy metals, and a second one, caused by endogenous factors, which leads to gemmule formation and a shift of the presumably immortal cells to mortal cells. The latter assumption is supported by the finding that during the process of bacteria-induced apoptosis, which results in the death of the specimens, the activity of the telomerase drops. It is concluded that the cells which appear to be immortal and telomerase-positive undergo apoptosis during the process of gemmule formation. In consequence cells not involved in the production of gemmules become mortal. Based on these data, it is proposed that apoptosis is a suitable biomarker in the bioindicator organism S. domuncula to monitor unfavorable environmental conditions, at least in this animal phylum. Received: 10 November 1997 / Accepted: 6 March 1998     

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.     

Chitinolytic enzymes in the digestive system of marine fishes

Chitinase, exo-N-acetyl--D-glycosaminidase (NAGase) and lysozyme activities were assayed in the digestive tract of 6 species of marine fishes: Myxine glutinosa (cyclostome), Chimaera monstrosa (holocephalan), Squalus acanthias, Etmopterus spinax, Raja radiata (elasmobranchs) and Coryphaenoides rupestris (teleost). Strong chitinase activity was found in the gastric mucosa of the elasmobranchs (S. acanthias, E. spinax and R. radiata) and the teleost (Coryphaenoides rupestris). A remarkably high chitinase activity occurred in the pancreas of the stomachless holocephalan fish Chimaera monstrosa. NAGase activity was strong in the digestive tract of all species. It could be concluded that marine fishes with diets consisting largely of chitinous invertebrates may display high chitinase and NAGase activities in their digestive system; however, only low chitinase activity was found in the intestine of the cyclostome Myxine glutinosa. Coryphaenoides rupestris gastric mucosa chitinase had one optimum activity at pH 1.25, whereas S. acanthias chitinase had two optima, at pH 1.6 and 3.6. The NAGase pH-activity curves from S. acanthias and R. radiata gastric mucosa displayed similar optima, at pH 4.5 and 4.25 respectively. Chimaera monstrosa pancreatic chitinase had a very strong optimum around pH 8 to 10, and one less strong at pH 3. These enzyme activities could not be separated by gel filtration or isoelectric focusing. The pI (isoelectric point) was approximately 4.9 for both enzymes. The molecular weight of the C. monstrosa pancreatic chitinase was estimated to be approximately 43 000. Lysozyme activity was absent or extremely weak in the material studied.     

Studies on the resistance of marine bottom invertebrates to oxygen-deficiency and hydrogen sulphide

Oxygen-deficient and H₂S-containing marine areas are characterized by a decline in the number of species. In laboratory experiments with bottom invertebrates from various biotopes of the North Sea and the Baltic, comparative measurements of the resistance to oxygen-deficiency alone, and to the simultaneous presence of H₂S, were carried out. The resistance values obtained show relations to the substratum on which the species naturally occur. The resistance to H₂S is greater in those macrofauna species which show higher survival rates under oxygen-deficiency. Further experiments with isolated tissues demonstrate that the species specific differences in resistance occurring in whole animals are already based on the cell metabolism. In general, oxygen-deficiency and simultaneous presence of H₂S were endured better in cold than in warmth, and at somewhat reduced pH-values (around 7). The dependence of this resistance on the salinity was only minimal in euryhaline species.     

Effect of hydrogen sulfide in comparison to atorvastatin on endothelial damage in cholesterol-fed rabbits

Hydrogen sulfide (H₂S) is an endogenous gaseous molecule, functioning as a physiological regulator. This study aimed to investigate the role of H₂S in atherosclerosis. Rabbits receiving high cholesterol were given atorvastatin (10 mg/kg/day, orally) or sodium sulfide (1.1 mg/kg/day, intramuscular) 2 weeks after commencement of high-cholesterol diet and continued for 4 weeks. Serum lipid profiles, lipid peroxidation indexed as malondialdehyde, glutathione, superoxide dismutase, the vascular adhesion molecule-1, and aortic vascular endothelial growth factor were determined. Rabbits receiving a high-cholesterol diet showed elevated levels of total serum cholesterol, triglycerides, low-density lipoprotein cholesterol, malondialdehyde and vascular adhesion molecule-1, and aortic vascular endothelial growth factor expression and lower levels of high-density lipoprotein cholesterol, glutathione, and superoxide dismutase. Treatment with atorvastatin and sodium sulfide has beneficial effects on serum lipid profile, oxidative damage, vascular adhesion molecule-1, and aortic expression of vascular endothelial growth factor.     

Metabolic adaptation of Zostera marina (eelgrass) to diurnal periods of root anoxia

The temperate seagrass Zostera marina L. is common in coastal marine habitats characterized by the presence of reducing sediments. The roots of this seagrass grow in these anoxic sediments, yet eelgrass is highly productive. Through photosynthesis-dependent oxygen transport from leaves to roots, aerobic respiration is supported in eelgrass roots only during daylight; consequently, roots are subjected to diurnal periods of anoxia. Under anoxic root conditions, the amino acids alanine and -amino butyric acid accumulate within a few hours to account for 70% of the total amino acid pool, while glutamate and glutamine decline. Little ethanol is produced, and the pool size of the organic acid malate changes little or declines slowly. Upon the resumption of shoot photosynthesis and oxygen transport to the roots, the accumulated -amino butyric acid declines rapidly, glutamate and glutamine pools increase, and alanine declines over a 16-h period. These adaptive metabolic responses by eelgrass to diurnal root anoxia must contribute to the successful exploitation of shallow-water marine sediments that have excluded nearly all vascular plant groups. A metabolic scheme is presented that accounts for the observed changes in organic and amino acid pool sizes in response to anoxia.     

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     

Simultaneous removal of hydrogen sulfide and ammonia in the gas phase: a review

Environmental Chemistry Letters - Hydrogen sulfide (H2S) and ammonia (NH3) are malodorous and toxic gases occurring in gas emissions from industrial processes such as fuel production and anaerobic...     

A meiofaunal “thiobios” limited to the anaerobic sulfide system of marine sand does not exist

The spatial relationship of interstitial metazoans to sources of oxygen has been studied on a tidal flat in the Wadden Sea near Sylt (eastern part of the North Sea). Consistently, meiofauna remains in close proximity to oxygenated layers or pocket areas within the sediment. This is exemplified by the pattern of meiofaunal abundance and species composition alongside oxic burrows of the lugworm Arenicola marina L. A specific meiofauna confined to oxygen-deficient horizons of the sediment does not exist.     

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.     

Tolerance to low oxygen and sulfide in Amphiura filiformis and Ophiura albida (Echinodermata: Ophiuroidea)

The brittle stars Amphiura filiformis (Müller) and Ophiura albida (Forbes) were exposed to different oxygen saturations (100, 10, 5, 3, and <1% oxygen saturation) and to physiological anoxia (<1% oxygen saturation) at different total sulfide concentrations (0, 2, 20, 200 μM). The mortality was followed during experiments and the median survival time (LT₅₀) was determined. The infaunal A. filiformis had a significantly higher tolerance to both hypoxia and sulfide than did the epibenthic O. albida. After exposure to 10% oxygen saturation for a month, only 2.0% A. filiformis and 0% O. albida were dead. In oxygen saturations <1% A. filiformis and O. albida had a LT₅₀ of 7.5 and 2.5 d, respectively. The presence of even very small concentrations of sulfide decreased the survival significantly. Sulfide is shown to be the key factor for the survival of the two species. Received: 11 October 1996 / Accepted: 12 November 1996     

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     

Regulation of respiration during juvenile development of Sepia officinalis (Mollusca: Cephalopoda)

A culture of juvenile Sepia officinalis L. was kept during summer 1985 in the aquaria of the Station Marine, Wimereux, France. During the first four months of juvenile development, oxygen consumption under increasing hypoxia was measured with a closed respirometer. The experiments revealed a high regulatory capacity of juvenile S. officinalis. The critical oxygen concentrations were calculated and their ontogenetical evolution was studied. The critical oxygen concentration increased with increasing development. A linear relationship emerged between the critical oxygen concentration and the logarithm of the wet weight [CO_c (mg O₂ l^-1)=-0.393+0.893×log₁₀(W _w )]. The decreasing regulatory capacity of growing S. officinalis is most probably related to adaptations to a changing ecological environment during development. Another possibility is a physiological change, most probably related to the shift from embryonic to adult hemocyanin.     

Effects of exposure to petroleum hydrocarbons on the gill functions and ciliary activities of a marine bivalve

The effects of long-term exposure to low levels of water-accommodated fractions of Kuwait Crude oil, or to shortterm exposure to chemically dispersed oil, on the gill performance of the clam Venus verrucosa were investigated. Reduced pumping activities of the lateral cilia as well as interference with the normal beating activities of the eulaterofrontal cirri led to reduced clearance rates and retention efficiencies of food particles less than 6 m in diameter. On the other hand, frontal ciliary activities were significantly accelerated, while any retained oil droplets were conducted to the mouth region as food particles. The activities of terminal and sensory cilia were also enhanced and mucus production increased. The significance of these responses to the clam's energy budget is discussed.     

Growth of juvenile Macrocystis pyrifera (Laminariales) in relation to environmental factors

Instantaneous relative growth rates, (d^-1), were measured for juveniles of the giant kelp Macrocystis pyrifera transplanted to study sites in Southern California kelp forests between 1978 and 1982. Growth rates ranged from negative values (indicating loss of tissue) to 0.03 (doubling of total frond length every 19 d). Multiple regression analysis of growth versus irradiation, temperature, nitrogen concentration and amount of fouling revealed that all these factors had significant effects, together accounting for about 50% of the total variance. Elevated irradiation and nitrogen levels had strongly stimulatory effects (tissue nitrogen may have been more critical than ambient nitrogen for growth), while high temperature and fouling had strongly inhibitory effects. Irradiation was the most important factor influencing growth in 6 of the 8 transplant experiments. During these 6 experiments, the compensating irradiation level (below which there was no growth) was between 0.4 and 0.7 E m^-2 d^-1, and saturating irradiation was between 2 and 3 E m^-2 d^-1. During two of the experiments, growth was apparently limited by extremely high temperatures or low nitrogen levels. Quantum irradiation levels in the kelp forest were generally between the compensation and saturation levels. However, irradiation levels occasionally dropped below the compensation point for several months. Irradiation was occasionally low enough to limit the distribution of juvenile kelp by inhibiting growth, especially in the deeper portions of the kelp forest and under dense canopies formed by adult plants.     

Human exposure to methylmercury from crayfish (Procambarus clarkii) in China

Methylmercury (MeHg) accumulation in aquatic food raises global concerns about human exposure to MeHg. Crayfish is the world’s third largest farmed crustacean species and a favorite aquatic food in many countries. However, human health hazard due to MeHg exposure via crayfish consumption is unclear, partly because appropriate survey data are lacking. We report on mercury concentrations and speciation in edible tail muscle of crayfish collected from restaurants in 23 Chinese cities. On average, MeHg constituted 99.1 % of mercury in tail muscle, and MeHg concentrations were comparable with those reported for fish in China. Variation in MeHg concentrations was not attributable to broad geographic region (i.e., provinces) or tail length. For different populations, potential health risk (characterized by hazard quotient or HQ) of MeHg exposure through crayfish consumption depended largely on crayfish consumption rates. In particular, a health hazard (HQ > 1) was found for high-rate consumers (i.e., 95 %ile or higher) in some cities in the middle and lower reaches of the Yangtze River (MLYR), during the peak consumption season. Our results suggest that more attention should be paid to dietary MeHg intake via crayfish consumption in China, particularly for communities with high consumption in MLYR.