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The respiration rate of meiofauna is difficult to measure, and the response to variations in the environmental oxygen concentration has so far been mainly addressed through behavioral investigation. We investigated the effect of different oxygen concentrations on the physiology of the marine platyhelminth Macrostomum lignano. Respiration was measured using batches of 20 animals in a glass microtiter plate equipped with optical oxygen sensor spots. At higher oxygen saturations (>12 kPa), the animals showed a clear oxyconforming behavior. However, below this value, the flatworms kept respiration rates constant at 0.064±0.001 nmol O2 l(-1) h(-1) individual(-1) down to 3 kPa PO2, and this rate was increased by 30% in animals that were reoxygenated after enduring a period of 1.5 h in anoxia. Physiological changes related to tissue oxygenation were assessed using live imaging techniques with different fluorophores in animals maintained in normoxic (21 kPa), hyperoxic (40 kPa) or near-anoxic (~0 kPa) conditions and subjected to anoxia-reoxygenation. The pH-sensitive dyes Ageladine-A and BCECF both indicated that pHi under near-anoxia increases by about 0.07-0.10 units. Mitochondrial membrane potential, Δψm, was higher in anoxic and hyperoxic than in normoxic conditions (JC1 dye data). Staining with ROS-sensitive dyes - DHE for detection of superoxide anion (O2•(-)) formation and C-H DFFDA for other ROS species aside from O2•(-) (H2O2, HOO• and ONOO) - showed increased ROS formation following anoxia-reoxygenation treatment. Animals exposed to hyperoxic, normoxic and anoxic treatments displayed no significant differences in O2•(-) formation, whereas mitochondrial ROS formation as detected by C-H2DFFDA was higher after hyperoxic exposure and lowest under near-anoxia conditions compared with the normoxic control group. Macrostomum lignano seems to be a species that is tolerant of a wide range of oxygen concentrations (being able to maintain aerobic metabolism from extremely low PO2 up to hyperoxic conditions), which is an essential prerequisite for successfully dealing with the drastic environmental oxygen variations that occur within intertidal sediments.

Exposure to environmental sulfide has been related to formation of reactive oxygen species (ROS) in vertebrate and invertebrate tissues. The present paper compares activity levels of the oxygen radical quencher superoxide dismutase (SOD) in benthic polychaetes and bivalves of differing sulphide tolerance. Total SOD activity showed no correlation to biotope related sulphide exposure of different species. Sulfide-insensitive MnSOD is present in tissues and body fluids of some sulphide adapted species. Total SOD activity was generally higher in haemoglobin containing animals, than in species lacking respiratory pigments. The sulphide-resistant MnSOD was the dominating enzymatic form in respiratory fluids of haemoglobin containing species, whereas sulphide-sensitive CuZnSOD dominated in the body tissues. Bivalves without respiratory pigments had mostly SOD deficient haemolymph. The results indicate, that release of reactive oxygen species in tissues and body fluids of invertebrates could be connected to the presence of haemoglobin, rather than to sulphide oxidation.

Antioxidant enzymes were analysed in spawn, larvae and in adult individuals of the polychaete Phyllodoce mucosa from an intertidal sandflat of the German Wadden coast. The analyses were carried out during two successive years (1992 and 1993) which were markedly different with respect to the weather conditions during the spawning period of P. mucosa. Extremely sunny weather conditions in 1993 led to hyperoxia and maximum peroxide concentrations of 4000 nmol l-1 in intertidal pools during daytime. Egg masses deposited into these intertidal pools were directly exposed to solar radiation and to hyperoxia stress. They had higher levels of the oxygen radical scavenger superoxide-dismutase (SOD) and of glutathione-reductase (GR, analysed only in 1993), a constituent of the glutathione antioxidant cycle, than the adults. Moreover, SOD and GR activities in the spawn were higher in 1993 than in 1992. We postulate that high UV irradiation during early spring, which causes elevated H2O2 concentrations in tid...

Haemoglobin autoxidation and the simultaneous liberation of H2O2 was studied in the blood of the lugworm, Arenicola marina, and the haemolymph of the clam, Astarte borealis, under normoxia, hypoxia, and hypoxia with 200 μmol·l−1 sulphide. In both species Hb autoxidation and hydrogen peroxide liberation into the respiratory fluid increased under hypoxia. Even higher hydrogen peroxide levels were measured under sulphidic hypoxia conditions. Under normoxia and hypoxia, autoxidation of the respiratory pigment seems to be a major source of H2O2 in blood and haemolymph. Whereas the body fluids of both species lack the hydrogen peroxide scavenger catalase, they contain considerable amounts of the antioxidant enzymes glutathione reductase (Astarte borealis only) and superoxide-dismutase (SOD). SOD activity in Arenicola marina blood was unaffected by sulphide in vivo and in vitro, while haemolymph SOD activity was significantly reduced during sulphide treatment in Astarte borealis. Catalase activity in the gills of Astarte borealis and the chloragog storage tissue of Arenicola marina were reduced by prolonged hypoxia, while the SOD activities in the tissues were unaffected. A possible involvement of H2O2, partly deriving from autoxidation of respiratory pigments, in H2S oxidation in invertebrate body fluids is discussed.

Summary The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome-activity assays, represented by the trypsin-like, the chymotrypsin-like, or the caspase-like site, lies in the fact that most commonly-used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we could show that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for the chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.

Predation is known to impact growth and reproduction, and the physiological state of the prey, including its susceptibility to oxidative stress. In this study, we investigated how prolonged exposure to predators modulates tissue specific antioxidant defense and oxidative damage in the short-lived epibenthic scallop Argopecten ventricosus (2 years maximum lifespan). Scallops that were experimentally exposed to predators had not only lower antioxidant capacities (superoxide dismutase and catalase), but also lower oxidative damage (protein carbonyls and TBARS = thiobarbituric acid reactive substances including lipid peroxides) in gills and mantle compared to individuals not exposed to predators. In contrast, oxidative damage in the swimming muscle was higher in predator-exposed scallops. When predator-exposed scallops were on the verge of spawning, levels of oxidative damage increased in gills and mantle in spite of a parallel increase in antioxidant defense in both tissues. Levels of oxidative damage increased also in the swimming muscle whereas muscle antioxidant capacities decreased. Interestingly, post-spawned scallops restored antioxidant capacities and oxidative damage to immature levels, suggesting they can recover from spawning-related oxidative stress. Our results show that predator exposure and gametogenesis modulate oxidative damage in a tissue specific manner and that high antioxidant capacities do not necessarily coincide with low oxidative damage.

The Western Antarctic Peninsula is one of the most rapidly warming regions on earth. It is therefore important to analyze long-term trends and inter-annual patterns of change in major environmental parameters to understand the process underlying climate change in Western Antarctica. Since many polar long-term data series are fragmented and cannot be analysed with common time series analysis tools, we present statistical approaches that can deal with missing values. We applied U-statistics after Pettit and Buishand to detect abrupt changes, dynamic factor analysis to detect functional relationships, and additive modelling to detect patterns in time related to climatic cycles such as the Southern Annular Mode and El Nino Southern Oscillation in a long-term environmental data set from King George Island (WAP), covering 20 years. Our results not only reveal sudden changes for sea surface temperature and salinity, but also clear patterns in all investigated variables (sea surface temperature, salinity, suspended particulate matter and Chlorophyll a) that can directly be related to climatic cycles. Our results complement previous findings on climate related changes in the King George Island Region and provide insight into the environmental conditions and climatic drivers of system change in the study area. Hence, our statistical analyses may prove valuable for other polar environmental data sets and contribute to a better understanding of the regional variability of climate change and its impact on coastal systems.

Fish gills are target organs for waterborne metal ions and this work aimed to investigate the effects of waterborne Ni(2+) (10, 25 and 50 mg L(-1)) on goldfish gills. A special focus was on the relationship between Ni uptake and the homeostasis of reactive oxygen species (ROS) in the gills, the tissue, in direct contact with the metal pollutant. Ni-accumulation in the gills occurred as a function of exposure concentration (R(2)=0.98). The main indices of oxidative stress, namely carbonyl proteins (CP) and lipid peroxides (LOOH), decreased by 21-33% and 21-24%, as well as the activities of principal antioxidant enzymes superoxide dismutase and glutathione-dependent peroxidase, by 29-47% and 41-46%, respectively, in gills of Ni-exposed fish. One of the main players in the antioxidant defense of gills seems to be catalase, which increased by 23-53% in Ni-treated fish, and low molecular mass thiol-containing compounds (L-SH), exceeding untreated controls by 73-105% after fish exposure to 10-50 mg L(-1) of Ni(2+). The increased level of L-SH, mainly represented by reduced glutathione, was supported by enhanced activities of glutathione reductase (by 27-38%), glutathione-S-transferase (56-141%) and glucose-6-phosphate dehydrogenase (by 96-117%) and demonstrates the ability of the antioxidant system of gills to resist Ni-induced oxidative stress.

Toxic and carcinogenic effects of nickel compounds are suggested to result from nickel-mediated oxidative damage to macromolecules and/or inhibition of cellular antioxidant defenses. We investigated the effects of waterborne Ni(2+) (10, 25 and 50 mg/L) on the blood and blood-producing tissues (kidney and spleen) of goldfish to identify relationships between Ni accumulation and oxidative stress. Whereas the main hematological parameters (total hemoglobin and hematocrit) were unaffected, Ni(2+) exposure had substantial influence on goldfish immune system, causing lymphopenia. Ni accumulation increased renal iron content (by 49-78%) and resulted in elevated lipid peroxide (by 29%) and protein carbonyl content (by 274-278%), accompanied by suppression of the activities of superoxide dismutase (by 50-53%), glutathione peroxidase (15-45%), glutathione reductase (31-37%) and glucose-6-phosphate dehydrogenase (20-44%), indicating development of oxidative stress in kidney. In contrast to kidney, in spleen the activation of glutathione peroxidase (by 34-118%), glutathione-S-transferase (by 41-216%) and glutathione reductase (by 47%), as well as constant levels of low molecular mass thiols and metals together with enhanced activity of glucose-6-phosphate dehydrogenase (by 41-94%) speaks for a powerful antioxidant potential that counteracts Ni-induced ROS production. Further, as Ni accumulation in this organ was negligible, Ni-toxicity in spleen may be minimized by efficient exclusion of this otherwise toxic metal.

This chapter focuses on the adaptive physiological and ecological responses to warming in cold-temperate marine ectotherms. To understand centrally important traits of thermal tolerance, adaptive traits that restrict the capacities of cold stenotherms from Antarctic environments to acclimatize to the present warming trend in Western Antarctica are studied. The present knowledge of regulatory pathways that support metabolic adaptations to warmer temperatures is also discussed.

Environmental factors such as temperature and salinity regimes shape lifespan in marine ectotherms. We investigated whether the effect occurs through modification of metabolic reactive oxygen species (ROS)-producing processes and is thus in line with the rate of living-free radical theory of aging. We compared 6 biogeographically and climatically distinct populations of the extremely long-lived ocean quahog Arctica islandica for age-dependent differences in meta- bolic rates and antioxidant capacities (superoxide dismutase, catalase activity and total glu- tathione concentration). The temperature and salinity ranges covered by the sampling locations (Norwegian coast, White Sea, Iceland, Kattegat, Kiel Bay and German Bight) were 3.7 to 9.3°C and 20 to 35 ppt. Bivalve shells were used as age recorders by counting annual growth bands. Maximum determined age in different populations varied between 29 and 192 yr. Extreme longevity observed in some North Atlantic A. islandica populations seems to be based on their very low lifetime mass-specific respiration, in combination with stable maintenance of antioxidant protection throughout life in mature specimens. While the antioxidant capacity was similar among all populations, the shorter-lived populations exhibited the highest metabolic rates and showed no metabolic response (Q10) when warmed to higher temperature. Low and fluctuating salinity in the Baltic may further exert stress, which enhances respiration rates and reduces longevity in the Baltic Sea population. The exceptionally long lifespan of A. islandica cannot be exclusively explained by a well-established antioxidant defense system, and the long lifespan of some popu- lations may rather be a function of low ROS formation (low metabolic rate) and high damage repair/removal capacities.

[1] We analyzed shell cuts of five individuals of the Antarctic bivalve Laternula elliptica from three locations along the Antarctic Peninsula by means of Confocal Raman Microscopy (CRM) as well as Electron Microprobe (EMP). The shell of L. elliptica has been previously described as being composed of aragonite exclusively. Now, CRM mapping reveals that three polymorphs of calcium carbonate – aragonite, calcite, and vaterite – are present in the chondrophore region of the examined individuals. Annual shell growth layers continue through aragonite and vaterite, suggesting simultaneous mineralization of both polymorphs. Spatially congruent EMP scans showed that the calcium carbonate polymorph affects the distribution of magnesium and strontium within the chondrophore. This is, to our knowledge, the first report of the coexistence of these three calcium carbonate polymorphs within the mineralized structures of a marine calcifying organism. Particularly the presence of vaterite is unexpected, but shows striking similarities to some fish otoliths. The strong effect of the calcium carbonate polymorph on trace element incorporation restrict the suitability of magnesium and strontium based proxies for the chondrophore area of L. elliptica.

Climate warming involves not only a rise of air temperature means, but also more frequent heat waves in many regions on earth, and is predicted to intensify physiological stress especially in extremely changeable habitats like the intertidal. We investigated the heat-shock response (HSR) and enzymatic antioxidant defense levels of Patagonian shallow-water limpets, adapted to distinct tidal exposure conditions in the sub- and intertidal. Limpets were sampled in the temperate Northern Patagonia and the subpolar Magellan region. Expression levels of two Hsp70 genes and activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in submerged and 2- and 12-h air-exposed specimens. Air-exposed Patagonian limpets showed a tiered HSR increasing from South to North on the latitudinal gradient and from high to low shore levels on a tidal gradient. SOD activities in the Magellan region correlated with the tidal rhythm and were higher after 2 and 12 h when the tide was low at the experimental site compared to the 6 h value taken at high tide. This pattern was observed in intertidal and subtidal specimens, although subtidal individuals are little affected by tides. Our study shows that long-term thermal adaptation shapes the HSR in limpets, while the oxidative stress response is linked to the tidal rhythm. Close to the warm border of their distribution range, energy expenses to cope with stress might become overwhelming and represent one cause why the limpets are unable to colonize the shallow intertidal zone.

SUMMARYArctica islandica is the longest-lived non-colonial animal found so far, and reaches individual ages of 150 years in the German Bight (GB) and more than 350 years around Iceland (IC). Frequent burrowing and physiological adjustments to low tissue oxygenation in the burrowed state are proposed to lower mitochondrial reactive oxygen species (ROS) formation. We investigated burrowing patterns and shell water partial pressure of oxygen (PO2) in experiments with live A. islandica. Furthermore, succinate accumulation and antioxidant defences were recorded in tissues of bivalves in the normoxic or metabolically downregulated state, as well as ROS formation in isolated gills exposed to normoxia, hypoxia and hypoxia/reoxygenation. IC bivalves burrowed more frequently and deeper in winter than in summer under in situ conditions, and both IC and GB bivalves remained burrowed for between 1 and 6 days in laboratory experiments. Shell water PO2 was

Female and male individuals of the same species often differ with respect to their susceptibility to toxicant stress. In the present study, sea urchins (Psammechinus miliaris) of both sexes were exposed to high (150 μg L⁻¹) and environmentally relevant (5 μg L⁻¹) concentrations of phenanthrene over 10 days. While food intake was significantly decreased following exposure to 150 μg L⁻¹ phenanthrene, histological indices (lipofuscin accumulation, fibrosis, oocyte atresia), energetic status (energy charge, sum adenylates, AMP/ATP ratio) as well as ascorbate levels in the gonads showed either little or no effect upon phenanthrene exposure. However, most parameters (vitamin C, energy charge, sum adenylates, AMP/ATP ratio, ATP and ADP concentrations, lipofuscin content, fibrosis) significantly differed between male and female animals. This study illustrates the difficulties to identify toxic injury in reproductive tissue as it may be superimposed by gametogenesis and spawning of gametes.

We compared lifetime and population energy budgets of the extraordinary long-lived ocean quahog Arctica islandica from 6 different sites—the Norwegian coast, Kattegat, Kiel Bay, White Sea, German Bight, and off northeast Iceland—covering a temperature and salinity gradient of 4-10C (annual mean) and 25-34, respectively. Based on von Bertalanffy growth models and size-mass relationships, we computed organic matter production of body (PSB) and of shell (PSS), whereas gonad production (PG) was estimated from the seasonal cycle in mass. Respiration (R) was computed by a model driven by body mass, temperature, and site. A. islandica populations differed distinctly in maximum life span (40 y in Kiel Bay to 197 y in Iceland), but less in growth performance (f# ranged from 2.41 in the White Sea to 2.65 in Kattegat). Individual lifetime energy throughput, as approximated by assimilation, was highest in Iceland (43,730 kJ) and lowest in the White Sea (313 kJ). Net growth efficiency ranged between 0.251 and 0.348, whereas lifetime energy investment distinctly shifted from somatic to gonad production with increasing life span; PS/PG decreased from 0.362 (Kiel Bay, 40 y) to 0.031 (Iceland, 197 y). Population annual energy budgets were derived from individual budgets and estimates of population mortality rate (0.035/y in Iceland to 0.173/y in Kiel Bay). Relationships between budget ratios were similar on the population level, albeit with more emphasis on somatic production; PS/ PG ranged from 0.196 (Iceland) to 2.728 (White Sea), and P/B ranged from 0.203-0.285/y. Life span is the principal determinant of the relationship between budget parameters, whereas temperature affects net growth efficiency only. In the White Sea population, both growth performance and net growth efficiency of A. islandica were lowest. We presume that low temperature combined with low salinity represent a particularly stressful environment for this species.

Digestion of the phaeophyte carotenoids fucoxanthin, violaxanthin and neoxanthin and the chlorophylls a and c by the phytophagous snail Littorina littorea was studied in feeding experiments. Gut-passage results in alterations of pigment composition: (i) Chlorophylls are rapidly turned into pheophytin and, to a lesser extent, into pheophorbide in the snails' foregut and digestive gland. (ii) The mono-epoxide pigments fucoxanthin and neoxanthin were not (or only negligibly) reduced in the digestive system. (iii) The di-epoxide violaxanthin was completely de-epoxidized and converted into zeaxanthin. This means that the cyanophyte marker zeaxanthin (3,3′-dihydroxy-β-β-carotene), analysed in sediment samples with possible phaephyte input, may derive from this source material as a r eduction product of violaxanthin. (iv) Absorption of algal pigments in the digestive system seems to be a function of the nutritional state of the animals. Prolonged periods of starvation result in a more efficient digestion of later ingested food material and a practically complete destruction of the algal pigments.

The individual ages of bivalve molluscs can be inferred from the age rings laid down every year in the shell, especially in species inhabiting areas with seasonal variability in environmental factors such as food supply and temperature. Animals obtained from different environmental settings can therefore be used to investigate how specific environmental factors shape the process of ageing in this animal class. Some bivalves have extraordinary long life spans. Species like the ocean quahog Arctica islandica and the freshwater pearl mussel Margaritifera margaritifera live for over hundreds of years. Few studies so far have attempted to study the process of ageing, either specifically in long-lived bivalves or generally in very long-lived species. This review summarizes the current knowledge of cellular ageing in bivalves with a focus on the antioxidant system, as well as tissue repair and metabolic capacities of extremely long-lived species. We discuss the applicability of these animals as models for different ageing theories. We recommend a focus of future research on the molecular mechanisms potentially involved in supporting longevity in these species, including evolutionary old cellular mechanisms such as autophagy and apoptosis, as well as diverse cellular repair pathways.

The ocean quahog Arctica islandica is the longest-lived of all bivalve and molluscan species on earth. Animals close to 400 years are common and reported maximum live span around Iceland is close to 400 years. High and stable antioxidant capacities are a possible strategy to slow senescence and extend lifespan and this study has investigated several antioxidant parameters and a mitochondrial marker enzyme in a lifetime range spanning from 4-200 years in the Iceland quahog. In gill and mantle tissues of 4-192 year old A. islandica, catalase, citrate synthase activity and glutathione concentration declined rapidly within the first 25 years, covering the transitional phase of rapid somatic growth and sexual maturation to the outgrown mature stages (approximately 32 years). Thereafter all three parameters kept rather stable levels for150 years. In contrast, superoxide dismutase activities maintained high levels throughout life time. These findings support the 'Free Radical-Rate of Living theory', antioxidant capacities of A. islandica are extraordinarily high and thus may explain the species long life span.

Physiological fitness and exercise capacity decrease with age in humans and rodents, a finding for which, among other causes, the decline of cellular and especially mitochondrial functions with age is held responsible. We investigated changes of escape response to predator attacks in swimming scallops (Aequipecten opercularis) with size using the natural predator Asterias rubens. Aequipecten opercularis swim mainly to avoid predators and a decrease in swimming abilities would increase risk of capture and lower survival. The queen scallop A. opercularis is a very active, epibenthic species with a short lifespan of 8–10 years and a shell height up to 90 mm in the investigated fishing ground around the Isle of Man. No difference in clap rate was found between smaller and bigger A. opercularis individuals, but differences were found in swimming mode (jumping and swimming) and valve opening and closure behaviour between the two size groups in response to predator attack. Smaller individuals showed les...

The objective of this work was to analyze oxidative metabolism in Mya arenaria. Total Fe content in M. arenaria collected in the German Wadden Sea was 1.9+/-0.7, 0.7+/-0.1 and 0.17+/-0.01 nmol/mg fresh weight (FW), in digestive glands (DG), mantle and gills, respectively. Labile Fe pool, assessed by electronic paramagnetic resonance (EPR), was 146+/-10 pmol/mg FW, and by the fluorescence method employing calcein it was 118+/-9 pmol/mg FW. The lipid radical content in the DG, assessed by EPR, was 27+/-7 pmol/mg FW, and the thiobarbituric reactive substances content amounted to 57+/-8 pmol/mg FW. Ascorbyl radical (A) content, assessed by quantification of EPR signals, was 0.04+/-0.01 pmol/mg FW, and the ascorbate content (AH(-)) was 478+/-12 pmol/mg FW. The ratio A/AH(-) was (8+/-1)x10(-5)AU, suggesting a minimum oxidative stress even under physiological conditions, presumably depending on basal metabolic functions. The content of nitric oxide (NO), assessed by EPR, was 99+/-3 pmol/mg FW. The generation rate of NO by nitric oxide synthase-like activity (NOS-like) was assayed as NO production detected by EPR in the presence of l-arginine and NADPH, and was 3.16+/-0.06 pmol/(mg FW min). The data presented here document the detectable presence of highly reactive species in M. arenaria.

Calorie restriction (CR) without undernutrition has been found to enhance stress resistance and life span in endotherms and ectotherms. We investigated the eiect of 30% reduction in food oiering on growth, aerobic capacities and oxidative stress parameters in young turbot (Scophthalmus maximus ,L .). No diierences in body weight, length and hepatosomatic index between the ad libitum (AL)-fed and the CR group occurred after 55 days of diet application. Of the mitochondrial marker enzymes, only citrate synthase (CS) activity in the liver was reduced under CR, whereas muscle CS activity and cytochrome oxidase (CCO) activity in both tissues remained the same in both feeding groups. The concentration of reduced glutathione increased signi¢cantly during feeding in muscle of CR ¢sh, resulting in a more reduced glutathione redox ratio (GSH/GSSG) compared with AL ¢sh muscle. Thiobarbituric acidreactive lipid hydroperoxides (lipid peroxidation) but not protein carbonyl content (protein oxidation) was signi¢cantly reduced in CR ¢sh muscle. Liver oxidative stress parameters did not vary signi¢cantly between experimental feeding groups. We conclude that 30% CR over 8 weeks has no adverse eiect on young turbot. On the contrary, CR supports a reduced tissue oxidation state and reduces accumulation of lipid peroxidation products in muscle at sustained muscular aerobic capacity.

Heavy metal accumulation into bivalve soft tissues has received increasing interest in recent years with respect to biomonitoring of environmental change including pollution. To a lesser extent, accretion of elements from the environment into bivalve hard structures (shells) has been investigated, although the importance of the shells as environmental archives has been acknowledged. Here we report element distribution within consecutive growth bands in the shells of the Antarctic soft shell clam Laternula elliptica, which is currently exposed to vast environmental change in Antarctic Peninsula coastal environments that undergo rapid climate warming. We performed a high spatial resolution analysis for Al, Fe, Mn, Cu, Pb and U in the shell umbo, by means of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Element ratios within the umbo did not resemble either the ratios in the surrounding seawater, the sedimenting material in Potter Cove, or even the Earth's crust basal composition. Mn and Cu were preferentially incorporated into the umbo. A strong decrease of element accretion with time could be related to lifetime respiration mass (R) of the animals. This indicates element accretion into the umbo and shell matrix to be largely a function of animal ecophysiology and life history, and these effects need to be considered in the context of potential usefulness of L. elliptica shells as environmental archives.

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0 degrees C and after long-term warming to 5 degrees C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0 degrees C for the polar and 6 degrees C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.

We investigated mitochondrial ageing in a temperate (Mya arenaria) and an Antarctic (Laternula elliptica) mud clam, with similar lifestyle (benthic filter feeders) but different maximum life spans (MLSP), 13 and 36 years, respectively. The short-lived temperate M. arenaria showed a more pronounced decrease in mitochondrial function (respiration, respiratory control ratio, proton leak, membrane potential) with age than the long-lived Antarctic L. elliptica. H2O2 generation rates at habitat temperature were far higher in the short-lived M. arenaria compared to L. elliptica. Reactive oxygen species (ROS) production as proportion of the mitochondrial oxygen consumption rate (%H2O2/O2) increased significantly with age in M. arenaria, whereas in L. elliptica the proportion remained unchanged. Lower rates of mitochondrial H2O2 generation were presumably due to mild uncoupling as L. elliptica mitochondria showed higher proton leak compared to M. arenaria mitochondria. The results are discussed in to the light of the “Free Radical-Rate of Living theory”, (Pearl, R., 1928. The Rate of Living. Alfred Knopf, New York; Harman, D., 1956. Aging: a theory based on free radical and radiation biology. J. Gerontol. 11, 298–300) and the “Uncoupling to Survive” hypothesis (Brand, M.D., 2000. Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp. Gerontol. 35, 811–820).

The paper investigates tolerance to UV radiation (UVR) in 3 amphipod species from the Arctic Kongsfjord, Spitsbergen: the herbivore Gammarellus homari (0- to 5-m water depth), the strictly carnivore scavenger Anonyx nugax (2- to 5-m water depth) and the detritivore/carnivore Onisimus edwardsi (2- to 5-m water depth). In previous radiation exposure experiments, both carnivore species displayed elevated mortality rates already at moderate UVR levels. Therefore, the concentrations of sunscreening compounds (mycosporine-like amino acids, MAAs, and carotenoids) and two antioxidant enzymes (superoxide dismutase, catalase) were studied in the animals under control conditions and following moderate as well as high UVR exposure. In both carnivore amphipods elevated sensitivity to experimental UVR exposure went along with a degradation of the tissue carotenoid and MAAs and a decrease of the enzymatic antioxidant defence, which resulted in increased lipid peroxidation in exposed animals. In contrast, the herbivore G. homari seems well protected by high concentrations of MAAs absorbed from its algal diet, and no oxidative stress occurred under experimental UVR. The species-specific degree of UV tolerance correlates well with the animals' typical vertical distribution in the water column.

We investigated chronological and physiological ageing of two mud clams with regard to the "rate of living theory" (Pearl, 1928) and the "free radical theory of ageing" (Harman, 1956). The Antarctic Laternula elliptica (Pholadomyoida) and the temperate Mya arenaria (Myoida) represent the same ecotype (benthic infaunal filter feeders), but differ in maximum life span, 36 and 13 years, respectively. L. elliptica has a two-fold lower standard metabolic rate than M. arenaria, but its life long energy turnover at maximal age is three times higher. When comparing the two species within the lifetime window of M. arenaria, antioxidant capacities (glutathione, catalase) are higher and tissue oxidation (ratio of oxidised to reduced glutathione, lipofuscin accumulation) is lower in the polar L. elliptica than in the temperate mud clam. Tissue redox state in L. elliptica remained stable throughout all ages, whereas it increased dramatically in aged M. arenaria. Our results indicate that metabolic rates and maintenance of tissue redox state are major factors determining maximum lifespan in the investigated mud clams.

SUMMARY The influence of seasonal and acute temperature changes on mitochondrial functions were studied in isolated mitochondria of the eurythermal lugworm Arenicola marina (Polychaeta), with special emphasis on the interdependence of membrane potential and radical production. Acclimatisation of lugworms to pre-spawning/summer conditions is associated with rising mitochondrial substrate oxidation rates, higher proton leakage rates, elevated membrane potentials, and increased production of reactive oxygen species (ROS)in isolated mitochondria, compared with mitochondria from winter animals. However, a high ROS production was compensated for by higher activities of the antioxidant enzymes catalase and superoxide dismutase, as well as lower mitochondrial densities in summer compared with winter animals. In summer animals, a higher sensitivity of the proton leakage rate to changes of membrane potential will confer better flexibility for metabolic regulation(mild uncoupling) in response to temperature change. These seasonal alterations in mitochondrial functions suggest modifications of energy metabolism in eurythermal and euryoxic organisms on intertidal mudflats during summer. In winter, low and less changeable temperatures in intertidal sedimentary environments permit higher respiratory efficiency at low aerobic metabolic rates and lower membrane potentials in A. marinamitochondria.

Formation of reactive oxygen species (ROS) in mitochondrial isolates from gill tissues of the Antarctic polar bivalve Laternula elliptica was measured fluorimetrically under in vitro conditions. When compared to the rates measured at habitat temperature (1 degrees C), significantly elevated ROS formation was found under temperature stress of 7 degrees C and higher. ROS formation correlated significantly with oxygen consumption in individual mitochondrial preparations over the entire range of experimental temperatures (1-12 degrees C). ROS generation per mg of mitochondrial protein was significantly higher in state 3 at maximal respiration and coupling to energy conservation, than in state 4+, where ATPase-activity is inhibited by oligomycin and only proton leakage is driving the residual oxygen consumption. The percent conversion of oxygen to the membrane permeant hydrogen peroxide amounted to 3.7% (state 3) and 6.5% (state 4+) at habitat temperature (1 degrees C), and to 7% (state 3) and 7.6% (state 4+) under experimental warming to 7 degrees C. This is high compared to 1-3% oxygen to ROS conversion in mammalian mitochondrial isolates and speaks for a comparatively low control of toxic oxygen formation in mitochondria of the polar bivalve. However, low metabolic rates at cold Antarctic temperatures keep absolute rates of mitochondrial ROS production low and control oxidative stress at habitat temperatures. Mitochondrial coupling started to fall beyond 3 degrees C, closely to pejus temperature (4 degrees C) of the bivalve. Accordingly, the proportion of state 4 respiration increased from below 30% at 1 degrees C to over 50% of total oxygen consumption at 7 degrees C, entailing reduced ADP/O ratios under experimental warming. Progressive mitochondrial uncoupling and formation of hazardous ROS contribute to bias mitochondrial functioning under temperature stress in vitro. Deduced from a pejus temperature, heat stress commences already at 5 degrees C, and is linked to progressive loss of phosphorylation efficiency, increased mitochondrial oxygen demand and elevated oxidative stress above pejus temperatures.

A primary function of mitochondria may have been to compartmentalize respiration, thus protecting cells from the damaging side-effects of oxygen metabolism.

The effect of hydrogen peroxide on the rate of tissue oxygen consumption, on intracellular pH (pH i ) and on malondialdehyde (MDA) accumulation was studied in isolated body wall tissue of the lugworm Arenicola marina (L.). H 2 O 2 effects were investigated at various levels of pH i by changing medium pH (pH e ). The largest decrease of tissue oxygen consumption (by 17% below controls), as well as the highest degree of MDA accumulation (four-fold compared to control values) after H 2 O 2 exposure were found at acidic pH e of 6.4. This was attributed to the higher redox potential of H 2 O 2 in acidic solutions. Oxygen consumption at alkaline pH e (8.5) was not affected by H 2 O 2 . MDA accumulation in the tissue was considerably lower than at pH e 7.4 or 6.4. Despite pH dependent alterations of H 2 O 2 redox potential, we observed more or less constant pH e independent acidification of the tissue upon exposure to H 2 O 2 . We attributed the acidification to an inhibition of ATP consuming proton equivalent ion transport across the cellular membrane. Inactivation of carrier proteins is discussed to be responsible for the decrease in tissue oxygen consumption. However, with a larger effect on oxygen consumption at acidic pH e values, the latter may not be the only explanation, but additional impairment of other energy demanding processes may be involved.

Short and long-term exposure to elevated temperatures were studied in the Antarctic stenothermal protobranch bivalve Yoldia eightsi (Courthouy) from Potter Cove, King George Island (South Shetland Islands, Antarctica). Above a breakpoint temperature of 2°C – the upper habitat temperature for the Potter Cove Y. eightsi stock – both routine (RMR) and standard metabolic rate (SMR) increased steeply. The fraction of metabolism allocated to SMR, as well as the number of intervals of elevated activity per hour increased significantly with temperature. During acute exposure, ATP concentrations in the foot muscle peaked at 2°C and fell at 5°C, whereas superoxide dismutase (SOD) activity decreased upon warming. Slow stepwise warming to a final temperature of 11°C resulted in a significant decrease of SOD activity. Malondialdehyde concentration increased compared with controls at 0°C. In contrast to the effect of short-term exposure, tissue adenylate concentrations displayed a mild increase at higher temperatures during slow warming, indicating an acclimation response. A switch to anaerobic energy production could not be observed up to 11°C, demonstrating a higher level of thermal tolerance than in other Antarctic ectotherms, or a failure of the relevant pathways in Y. eightsi. The imbalance between pro- and antioxidant processes upon warming indicate oxidative stress to be one feature accompanying early heat stress in Y. eightsi.

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.

The rates of oxygen uptake of the marine polychaete Nereis pelagica and the bivalve Arctica islandica depend on the availability of ambient oxygen. This is manifest both at the tissue level and in isolated mitochondria studied between oxygen tensions of 6.3 and 47.6 kPa (47–357 mmHg). Oxyconformity was found in both Baltic Sea (Kiel Bight) and cold-adapted White Sea populations of the two species. However, mitochondria isolated from White Sea specimens of N. pelagica and A. islandica showed a two-to threefold higher aerobic capacity than mitochondria prepared from Baltic Sea specimens. We tested whether mitochondrial oxyconformity can be explained by an additional electron pathway that is directly controlled by . Mitochondrial respiration of both invertebrate species was inhibited by cyanide (KCN) and by salicylhydroxamic acid (SHAM). The overall rate of mitochondrial oxygen consumption increased at high . Phosphorylation efficiency (ADP/O ratio) decreased at elevated (27.5–47.6 kPa, 206–357 mmHg), regardless of whether malate or succinate was used as a substrate. In contrast to the invertebrate mitochondria studied, mitochondria isolated from bovine heart, as an oxyregulating control species, did not show an elevated rate of oxygen uptake at high in any respiratory state, with the exception of state 2 malate respiration. In addition, rates of ATP formation, respiratory control ratios (RCR) and ADP/O ratios remained virtually unchanged or even tended to decreased. In conclusion, the comparison between mitochondria from oxyregulating and oxyconforming organisms supports the existence of an alternative oxidase in addition to the classical cytochrome c oxidase. In accordance with models discussed previously, oxidative phosphorylation does not explain the rate of mitochondrial oxygen consumption during progressive activation of the alternative electron transport system. We discuss the alternative system, thought to be adaptive in confined, usually hypoxic environments, where excess oxygen can be eliminated and oxygen levels can be kept low by an increase in the rate of oxygen consumption, thereby minimizing the risk of oxidative stress.

Background Rising temperatures and other environmental factors influenced by global climate change can cause increased physiological stress for many species and lead to range shifts or regional population extinctions. To advance the understanding of species’ response to change and establish links between individual and ecosystem adaptations, physiological reactions have to be compared between populations living in different environments. Although changes in expression of stress genes are relatively easy to quantify, methods for reliable comparison of the data remain a contentious issue. Using normalization algorithms and further methodological considerations, we compare cellular stress response gene expression levels measured by RT-qPCR after air exposure experiments among different subpopulations of three species of the intertidal limpet Nacella. Results Reference gene assessment algorithms reveal that stable reference genes can differ among investigated populations and / or treatment groups. Normalized expression values point to differential defense strategies to air exposure in the investigated populations, which either employ a pronounced cellular stress response in the inducible Hsp70 forms, or exhibit a comparatively high constitutive expression of Hsps (heat shock proteins) while showing only little response in terms of Hsp induction. Conclusions This study serves as a case study to explore the methodological prerequisites of physiological stress response comparisons among ecologically and phylogenetically different organisms. To improve the reliability of gene expression data and compare the stress responses of subpopulations under potential genetic divergence, reference gene stability algorithms are valuable and necessary tools. As the Hsp70 isoforms have been shown to play different roles in the acute stress responses and increased constitutive defenses of populations in their different habitats, these comparative studies can yield insight into physiological strategies of adaptation to environmental stress and provide hints for the prudent use of the cellular stress response as a biomarker to study environmental stress and stress adaptation of populations under changing environmental conditions. Keywords: Ecological physiology; Cellular stress response; Hsp70; RT-qPCR; Gene expression; Normalization; Intertidal; Nacella

The aim of this work was to study the oxidative profile of gills of two limpet species (Nacella (Patinigera) magellanica and Nacella (Patinigera) deaurata) (Gmelin, 1971) exposed to different environmental conditions. Due to the tidal characteristics of the Beagle Channel, N. magellanica are exposed to air twice daily for 3 to 5 hours each time, whereas N. deaurata are exposed to air for 3 hours only during spring tides. The different regime of exposure includes extreme temperatures under 0ºC during winter and more than 20°C in summer for N. magellanica, whereas N. deaurata are usually covered by more than 0.3 m of water at 4°C in winter and 11°C in summer. No significant differences were found between the two molluscs regarding the oxygen uptake, the content of ?-tocopherol and ?-carotene and the activities of the antioxidant enzymes catalase and superoxide dismutase. Lipid peroxidation in gills was estimated as the content of lipid radicals, assessed by electron paramagnetic resonance (EPR). Lipid radical content and total iron content were respectively 80.6 and 62% lower in N. magellanica than in N. deaurata. A typical EPR spectrum of ascorbyl radical in gills from both limpets was observed. Both the ascorbyl radical content and the ascorbyl radical content/ascorbate content ratio were significantly lower in N. magellanica than in N. deaurata. In the Antarctic Nacella concinna inhabits all levels of the littoral zone. Limpets at the highest level in the intertidal showed significantly increased activities of both catalase and superoxide dismutase as compared to their intertidal and subtidal relatives. Thus, it seems that Antarctic high intertidal conditions, involving regular exposure to air and presumably also thermal stress on sunny days during the Antarctic summer, cause a necessity for N. concinna to ward off higher oxygen radical species production by increasing its antioxidant defence. Taken as a whole, the data presented here indicate that coping with environmentally demanding conditions requires a complex adjustment of the physiological metabolic pathways to ensure survival by minimising intracellular damage. El objetivo del presente trabajo fue estudiar el perfil oxidativo en branquias de dos especies de lapas (Nacella (Patinigera) magellanica and Nacella (Patinigera) deaurata) (Gmelin, 1971) expuestas a diferentes condiciones ambientales. Debido a las características de las mareas en el Canal del Beagle, las lapas N. magellanica están expuestas diariamente al aire dos veces durante 3 a 5 h cada vez, pero las lapas N. deaurata están expuestas al aire durante 3 h, solamente en las mareas de primavera. El diferente régimen de exposición incluye temperaturas extremas debajo de 0°C durante el invierno y más de 20°C en verano para N. magellanica, mientras que las lapas N. deaurata están habitualmente cubiertas por más de 0.3 m de agua que alcanza una temperatura de 4°C en invierno y 11°C en verano. No se observaron cambios significativos en ambos moluscos con respecto al consumo de oxígeno, el contenido de ?-tocoferol, ?-caroteno y la actividad de las enzimas antioxidantes catalasa y superóxido dismutasa. La peroxidación lipídica fue estimada como el contenido de radicales lipídicos, determinados por resonancia paramagnética electrónica (EPR). El contenido de radicales lipídicos y de hierro total fue de 80,6 y 62% menor en N. magellanica en comparación con N. deaurata. Se observó un típico espectro de EPR del radical ascorbilo en branquias de ambas lapas. Tanto el contenido de radical ascorbilo como el cociente contenido de radical ascorbilo/contenido de ascorbato fue significativamente menor en N. magellanica en comparación con N. deaurata. Estudios realizados en Nacella concinna (Antártida) indican que esta especie cuenta con mayor actividad de catalasa y superóxido dismutasa que sus congéneres de la región subantártica. Por lo tanto, condiciones de baja marea en la región antártica, con una exposición continua al aire y probablemente estrés térmico en días soleados durante el verano, podrían ser responsables de la necesidad de N. concinna de contar con una mayor protección antioxidante. Tomados en su conjunto, los datos presentados aquí indican que soportar condiciones ambientales demandantes requiere un complejo ajuste de las vías metabólicas fisiológicas para asegurar la supervivencia minimizando el daño intracelular.

The aim of this study is to elucidate the impact of age and temperature on metabolic key parameters (respiration rate and mitochondrial marker enzymes) controlling maximal size and longevity in the blue mussel Mytilus edulis L. from geographically separated populations of Northern European seas. Mussels from the Barents Sea attain similar maximal sizes as animals from the warmer North Sea. Young animals (

The age dependence of growth, metabolic rate, the degree of lipid peroxidation and antioxidative defence was studied in 3 different size groups of White Sea (Russia) blue mussels Mytilus edulis L.: small (S, wet tissue weight = 0.23 g, length 20-25 mm), medium (M, wet tissue weight = 0.52 g, length 30-35 mm) and large (L, wet tissue weight = 1.05 g, length 40-50 mm). Sg roup mussels were 2-8 yr old, M group mussels 2-9 yr old, and L group mussels 3-10 yr old. Absolute and weight-specific growth rates were determined from the beginning of the growth sea- son (May) to the time of sampling (August). Respiration rates were measured in individuals from all size groups. Net growth efficiency coefficients were calculated. Malondialdehyde (MDA) and fluo- rescent age pigment (FAP) concentrations were determined as a measure of lipid peroxidation. The level of antioxidant defence was estimated from superoxide dismutase (SOD) and catalase (CAT) activities in whole body homogenates. Metabolic performance was found to be a function of both age and body size. Weight-specific growth rates and net growth efficiencies decreased with age until mussels reached a 'critical' age, beyond which growth virtually stopped. Respiration rates were size dependent, but did not show a clear correlation with age. MDA levels remained unrelated to both age and body size, whereas FAP accumulation increased exponentially with age. However, FAP levels correlated negatively with size (growth rate). At constant body size, SOD and CAT activities did not display clear age-related changes; however, CAT activity decreased significantly with increasing body size. As a corollary, size effects potentially mimic age effects on key physiological functions of continuously growing species, and this needs to be considered in physiological studies of the ageing process.