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Metazoans normally possess a single lineage of mitochondria inherited from the mother (♀-type mitochondria) while paternal mitochondria are absent or eliminated in fertilized eggs. In doubly uniparental inheritance (DUI), which is specific to the bivalve clade including the ocean quahog, Arctica islandica, ♂-type mitochondria are retained in male gonads and, in a few species, small proportions of ♂-type mitochondria co-exist with ♀-type in somatic tissues. To the best of our knowledge, we report, for the first time in metazoan, the natural occurrence of male and female individuals with exclusively ♂-type mitochondria in somatic tissues of the bivalve A. islandica. Mitochondrial genomes differ by ~5.5% at DNA sequence level. Exclusive presence of ♂-type mitochondria affects mitochondrial complexes partially encoded by mitochondrial genes and leads to a sharp drop in respiratory capacity. Through a combination of whole mitochondrial genome sequencing and molecular assays (gene presence and expression), we demonstrate that 1) 11% of individuals of an Icelandic population appear homoplasmic for ♂-type mitochondria in somatic tissues, 2) ♂-type mitochondrial genes are transcribed and 3) individuals with ♂-type mitochondria in somatic cells lose 30% of their wild-type respiratory capacity. This mitochondrial pattern in A. islandica is a special case of DUI, highlighted in individuals from both sexes with functional consequences at cellular and conceivably whole animal level., Dégletagne et al. report on an unusual case of doubly uniparental inheritance in the ocean quahog, Arctica isladica. Some individuals in an Icelandic population present homoplasmic presence of male-type mitochondrial genes in somatic tissues, which appears to confer some changes in respiratory capacity in some environmental conditions.

Bivalve sclerochronological records with annually resolved growth bands are applicable proxies in reconstructing features of the hydro-climate system. Here we evaluate the relationship between growth indices of A. islandica, previously collected at approximately 82 m depth in the North Atlantic, and seasonal subsurface temperature at various depths for the 1900–2005 period. Correlations with sea surface temperature at the collection site are not significant during winter and weak for the remaining seasons. The strongest in-phase correlations persist for summer and autumn below 56 m water depth, whereas weaker correlations are lagged by one or two years. We also observe similarities with distant water bodies in the North Atlantic sector, and a corresponding large-scale oceanographic pattern that increases significantly with water depth along the trajectory of the North Atlantic Current. We suggest that by investigating the relationship with the temperature signal at various depths locally and at large-scale increases the reliability and application of bivalve shells as marine archives.

Benthic organisms of the Southern Ocean are particularly vulnerable to ocean acidification (OA), as they inhabit cold waters where calcite-aragonite saturation states are naturally low. OA most strongly affects animals with calcium carbonate skeletons or shells, such as corals and mollusks. We exposed the abundant cold-water coral Malacobelemnon daytoni from an Antarctic fjord to low pH seawater (LpH) (7.68 ± 0.17) to test its physiological responses to OA, at the level of gene expression (RT-PCR) and enzyme activity. Corals were exposed in short- (3 days) and long-term (54 days) experiments to two pCO2 conditions (ambient and elevated pCO2 equaling RCP 8.5, IPCC 2019, approximately 372.53 and 956.78 μatm, respectively). Of the eleven genes studied through RT-PCR, six were significantly upregulated compared with control in the short-term in the LpH condition, including the antioxidant enzyme superoxide dismutase (SOD), Heat Shock Protein 70 (HSP70), Toll-like receptor (TLR), galaxin and ferritin. After long-term exposure to low pH conditions, RT-PCR analysis showed seven genes were upregulated. These include the mannose-binding C-Lectin and HSP90. Also, the expression of TLR and galaxin, among others, continued to be upregulated after long-term exposure to LpH. Expression of carbonic anhydrase (CA), a key enzyme involved in calcification, was also significantly upregulated after long-term exposure. Our results indicated that, after two months, M. daytoni is not acclimatized to this experimental LpH condition. Gene expression profiles revealed molecular impacts that were not evident at the enzyme activity level. Consequently, understanding the molecular mechanisms behind the physiological processes in the response of a coral to LpH is critical to understanding the ability of polar species to cope with future environmental changes. Approaches integrating molecular tools into Antarctic ecological and/or conservation research make an essential contribution given the current ongoing OA processes.

The gills and mantle are the first tissues exposed to altered oxidative conditions during dissolved Fe uptake. The aim of this work was to characterize Fe affected oxidative metabolism on the respiratory tissues of Mya arenaria. After 9 days of exposure to elevated Fe levels (500 μM of Fe as Fe-EDTA), a significant increase in the total Fe and labile Fe pool content was found in the gills. Dichlorofluorescein diacetate oxidation rate was higher after 17 days of exposure as compared to controls. Thiobarbituric acid reactive substances content increased 1.9- and 3.7-fold over controls on day 9 and 17, respectively. Both, catalase activity and nitrate and nitrite content decreased significantly on days 9 and 17 compared with controls. Similar effects were observed in mantle, but catalase activity was not affected. The results showed that in respiratory tissues the labile Fe pool is critically controlled to avoid radical-dependent cellular deterioration, but when the endogenous protection mechanisms are overw...

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Increase in oxidative damage and decrease in cellular maintenance is often associated with aging, but, in marine ectotherms, both processes are also strongly influenced by somatic growth, maturation and reproduction. In this study, we used a single cohort of the short-lived catarina scallop Argopecten ventricosus, to investigate the effects of somatic growth, reproduction and aging on oxidative damage parameters (protein carbonyls, TBARS and lipofuscin) and cellular maintenance mechanisms (antioxidant activity and apoptosis) in scallops, caged in their natural environment. The concentrations of protein carbonyls and TBARS increased steeply during the early period of fast growth and during reproduction in one-year-old scallops. However, oxidative damage was transient, and apoptotic cell death played a pivotal role in eliminating damage in gill, mantle and muscle tissues of young scallops. Animals were able to reproduce again in the second year, but the reduced intensity of apoptosis impaired subsequent removal of damaged cells. In late survivors low antioxidant capacity and apoptotic activity together with a fast accumulation of the age pigment lipofuscin was observed. Rates of oxygen consumption and oxidative stress markers were strongly dependent on somatic growth and reproductive state but not on temperature. Compared to longer-lived bivalves, A. ventricosus seems more susceptible to oxidative stress with higher tissue-specific protein carbonyl levels and fast accumulation of lipofuscin in animals surviving the second spawning. Superoxide dismutase activity and apoptotic cell death intensity were however higher in this short-lived scallop than in longer-lived bivalves. The life strategy of this short-lived and intensely predated scallop supports rapid somatic growth and fitness as well as early maturation at young age at the cost of fast cellular degradation in second year scallops.

A quatic ecosystems house a large biosphere of marine and freshwater organisms, highly diverse in their tolerance of fluctuations in PO2 and temperature, two major modulators of metabolism. Often, both factors act in concert, and some of the most hypoxia-tolerant fish and molluskan species are indeed from cold-water environments. Other marine invertebrate and fish specialists thrive in the mixed waters at hydrothermal vent sites, underwater volcanic outflows where warm and hydrogen-sulfide-enriched, deoxygenated vent waters mix with colder and oxygenated oceanic waters, and temperatures and oxygen concentrations are extremely variable. Many vent species can even deal with toxic hydrogen sulfide that threatens to inhibit their mitochondrial electron transporters. More than 700 Myr of aquatic evolution have fostered a huge variety of ectothermic life-forms that can deal with the most extreme and fluctuating environmental conditions. The discovery of many fascinating underwater biota has raised an interest in the respiratory capacities of aquatic organisms and in how they deal with, from our air breathing perspective

In Arctica islandica, a long lifespan is associated with low metabolic activity, and with a pronounced tolerance to low environmental oxygen. In order to study metabolic and physiological responses to low oxygen conditions vs. no oxygen in mantle, gill, adductor muscle and hemocytes of the ocean quahog, specimens from the German Bight were maintained for 3.5 days under normoxia (21 kPa = controls), hypoxia (2 kPa) or anoxia (0 kPa). Tissue levels of anaerobic metabolites octopine, lactate and succinate as well as specific activities of octopine dehydrogenase (ODH) and lactate dehydrogenase (LDH) were unaffected by hypoxic incubation, suggesting that the metabolism of A. islandica remains fully aerobic down to environmental oxygen levels of 2 kPa. PO2-dependent respiration rates of isolated gills indicated the onset of metabolic rate depression (MRD) below 5 kPa in A. islandica, while anaerobiosis was switched on in bivalve tissues only at anoxia. Tissue-specific levels of glutathione (GSH), a scavenger of reactive oxygen species (ROS), indicate no anticipatory antioxidant response takes place under experimental hypoxia and anoxia exposure. Highest specific ODH activity and a mean ODH/LDH ratio of 95 in the adductor muscle contrasted with maximal specific LDH activity and a mean ODH/LDH ratio of 0.3 in hemocytes. These differences in anaerobic enzyme activity patterns indicate that LDH and ODH play specific roles in different tissues of A. islandica which are likely to economize metabolism during anoxia and reoxygenation.

Marine bivalves, such as the Antarctic soft shell clam Laternula elliptica (King and Broderip 1831), experience a wide range of environmental influences including the permanent contact to the surrounding microbial community due to filter feeding or strong physiochemical disturbances in a changing environment. Such impacts are anticipated to influence L elliptica physiology, including the immune system, especially under the current climate change conditions at the Western Antarctic Peninsula (WAP). To understand basal immune defence reactions in L elliptica, we investigated in vitro hemocyte responses upon bacterial challenge or after stimulation with different substances of microbial origin (pathogen associated molecular patterns, PAMPS). To study the age-dependent in vivo response of hemocyte abundance, starvation and injury experiments were undertaken with young and old individuals. Hemocytes of L. elliptica feature common immune functions such as phagocytosis and ROS generation, as well as agglutination of bacterial cells. ROS generation response to different stimuli was generally low and lower in larger/older animals compared to smaller/younger individuals. Physiological conditions such as size/age, starvation and injury modulated the abundance of hemocytes in L. elliptica. Implications are that current and future climate change conditions in West Antarctic coastal ecosystems scenarios may lead to changes in species survival and population composition. (C) 2010 Elsevier B.V. All rights reserved.

The effect of in vivo Fe exposure on the oxidative metabolism of the bivalve Myaarenaria was studied. Fe was supplemented in natural seawater and resulted in a significant increase in the total Fe content in the bivalve digestive gland (DG) between 9 to 17days of exposure. Mortality of treated animals increased drastically after day 18. Oxidative stress conditions were characterized in DG through assessment of the generation of reactive oxygen species (ROS) and ascorbyl radical (A) content. Both parameters were affected following a biphasic profile showing significant increases by days 2 and 9 of Fe exposure. The content of 2-thiobarbituric acid reactive substances (TBARS) was significantly increased over control values by days 2, 9 and 17 of treatment. The labile Fe pool (LIP) in isolated DG was elevated over control values by day 7, and maintained this increase until day 17 of Fe exposure. The content of NO, assessed by EPR spin trapping, was 60% lower in DG of animals exposed for 2days to Fe than in control values, with no further changes. The biphasic profile of oxidative stress response to Fe exposure in DG suggests that at early stages of Fe supplementation the cellular control mechanisms, such as CAT activity, were operative to limit oxidative damage, but further Fe exposure overwhelmed these abilities. Moreover, the second phase could be understood as the consequence of the exhaustion of cellular protective systems that could also involve NO.

Summary 1. The oxidative stress theory of ageing predicts that animals living longer will have less cumulative oxidative damage together with structural characteristics that make them more resistant to oxidative damage itself. 2. Although a general relationship between body size, metabolism and longevity does not exist in marine invertebrates, they are generally characterized by low rates of metabolism and reactive oxygen species (ROS) formation associated with lower antioxidant enzyme activities compared to vertebrates. 3. Birds and mammals have very similar size-affected metabolic rates and their metabolic intensity explains only some of the variation in maximum lifespan potential (MLSP). Within each class, smaller animals have higher rates of metabolism and ROS production and membranes that are more susceptible to oxidative damage and autocatalytic propagation of free radicals than larger ones. 4. Although the high variation in life-history strategies is accompanied by substantial variation in MLSP, there is a consistent positive correlation between rates of ROS formation and antioxidant levels among most animals examined so far for these traits. The consensus of these studies is that ROS and antioxidant levels are inversely related to MLSP. 5. The lack of a clear stoichiometric relation between variables contributing to oxidative stress limits our capacity to infer longevity consequences from measures of pro-oxidant or antioxidant status among or within species.

Marine invertebrates with open circulatory system establish low and constant oxygen partial pressure (Po2) around their tissues. We hypothesized that as a first step towards maintenance of low haemolymph and tissue oxygenation, the Po2in molluscan mantle cavity water should be lowered against normoxic (21 kPa) seawater Po2, but balanced high enough to meet the energetic requirements in a given species. We recorded Po2in mantle cavity water of five molluscan species with different lifestyles, two pectinids ( Aequipecten opercularis , Pecten maximus ), two mud clams ( Arctica islandica , Mya arenaria ), and a limpet ( Patella vulgata ). All species maintain mantle cavity water oxygenation below normoxic Po2. Average mantle cavity water Po2correlates positively with standard metabolic rate (SMR): highest in scallops and lowest in mud clams. Scallops show typical Po2frequency distribution, with peaks between 3 and 10 kPa, whereas mud clams and limpets maintain mantle water Po2mostly 2time series. Adjustment of mantle cavity Po2to lower than ambient levels through controlled pumping prevents high oxygen gradients between bivalve tissues and surrounding fluid, limiting oxygen flux across the body surface. The patterns of Po2in mantle cavity water correspond to molluscan ecotypes.

During the austral spring and summer months, the Antarctic limpet Nacella concinna colonizes intertidal environments in the Western Antarctica Peninsula region. The species is divided into a permanently sub-littoral and a seasonally intertidal, migratory subpopulation. We investigate the physiological differentiation between the two limpet groups to identify cellular and molecular changes that accompany adaptation of stenothermal Antarctic invertebrates to life under more stressful intertidal habitat conditions. A major difference between the two groups is the significantly higher concentrations of heavy metals (Fe, Al, Zn) from ingested sediments in sub-littoral limpet digestive glands (DG), associated with higher rates of reactive oxygen species (ROS) formation in this organ. ROS formation is accompanied by significantly higher SOD activity in sub-littoral limpet DG. These high SOD activities are, however, not conserved during either air exposure or hypoxic stress exposure of the sub-littoral limpets, when ROS production is slowed due to the absence of oxygen. The intertidal animals maintain higher levels of SOD and also conserve catalase activity at higher levels during hypoxia or air exposure compared to sub-littoral individuals under the same exposure conditions. More oxidized redox potential in gills and foot muscle and higher antioxidant enzyme activities in gills indicate that intertidal limpets maintain more oxygenated tissues during air exposure, in keeping with shell-lifting for oxygen up-take by the gills of intertidal limpets which migrate up the shore in the spring and down in the autumn. An increase of the redox ratio (GSSG/GSH) and accumulation of the lipid oxidation derived malonedialdehyde in intertidal limpet foot muscle during 12 h of exposure to air shows that indeed this tissue becomes more oxidized before the limpets eventually contract their shells tightly to minimize water loss and eventually become anaerobic. Intertidal limpets obviously avoid early onset of anaerobic energy production seen in their sub-littoral congeners when exposed to air and are still able to maintain tissue redox ratio balance when exposed to air.

SUMMARY The decline of cellular and especially mitochondrial functions with age is,among other causes, held responsible for a decrease in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10 years)from the Isle of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the change in physiology with age and size might result in a changed escape response behaviour towards predators.

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella ( P. ) magellanica . Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O 2 h − 1 g − 1 ; summer: 0.31 ± 0.06 μmol O 2 h − 1 g − 1 ). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH − ). The A / AH − ratio showed minimum values in winter (3.7 ± 0.2 10 − 5 AU) and increased in summer (18 ± 5 10 − 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg − 1 fresh mass [FW] in winter and 314 ± 45 pmol mg − 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg − 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg − 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. ( P. ) magellanica are strongly dependent on season.

Effects of acute cold exposure (at 1 degrees C and 5 degrees C) on tissue redox state and oxidative stress parameters, as well as the onset of hypoxic signaling were investigated in the North Sea eelpout, Zoarces viviparus. Activation of the transcription factor HIF-1 (hypoxia inducible factor) was detected in liver samples after acute cold exposure. At this temperature the cellular redox milieu was significantly reduced (below -270 mV) as compared to controls (-250 to -267 mV). Increased levels of oxidative stress parameters (TBARS and protein carbonyls) were observed mainly during recovery at control temperature (12 degrees C). This increase in oxidative stress parameters, in spite of maintained antioxidant capacity, indicates that acute cold stress and recovery mimic ischemia/reperfusion events as found in mammals. Notably the non-enzymatic antioxidant defense (e.g. glutathione) may play an important role for eelpout ROS scavenging capacity under cold stress.

We compared physiological ageing parameters in 2 scallops, the temperate Aequipecten opercularis and the Antarctic Adamussium colbecki. These 2 species are phylogeneti- cally closely related and display a similar lifestyle but have distinctly different maximum lifespans (MLSP). A. opercularis does not live longer than 8 to 10 yr, whereas A. colbecki lives over 18 yr. The development of several physiological ageing parameters over time, chosen according to the 'free rad- ical theory of ageing', was compared in the 2 species to identify differences in the ageing process. In the shorter-lived A. opercularis, activities of the mitochondrial enzymes citrate synthase and cytochrome c oxidase and of the antioxidant enzyme catalase showed a more pronounced decrease with increasing age than in the longer-lived A. colbecki. In line with this finding, lipofuscin accumu- lation increased more distinctly in A. opercularis than in A. colbecki, while tissue protein content decreased in A. opercularis but increased in A. colbecki. Its better preservation of mitochondrial and antioxidant enzyme activities and the avoidance of waste accumulation may enable A. colbecki to live longer than A. opercularis. Mitochondrial function investigated in A. opercularis showed only minor changes with age, and mitochondrial H2O2 generation rates were low at all ages. We relate our findings to the 'free radical-rate of living' theory, to the 'uncoupling to survive' hypothesis, and to the particular lifestyle of these scallops.

SUMMARYThe interplay between antioxidants, heat shock proteins and hypoxic signaling is supposed to be important for passive survival of critical temperature stress, e.g. during unfavorable conditions in hot summers. We investigated the effect of mild (18°C), critical (22°C) and severe(26°C) experimental heat stress, assumed to induce different degrees of functional hypoxia, as well as the effect of recovery following heat stress on these parameters in liver samples of the common eelpout Zoarces viviparus.Upon heat exposure to critical and higher temperatures we found an increase in oxidative damage markers such as TBARS (thiobarbituric reactive substances)and a more oxidized cellular redox potential, combined with reduced activities of the antioxidant enzyme superoxide dismutase at 26°C. Together, these point to higher oxidative stress levels during hyperthermia. In a recovery-time series, heat-induced hypoxia and subsequent reoxygenation upon return of the fishes to 12°C led to increased protein oxidation and chemiluminescence rates within the first 12 h of recovery, therein resembling ischemia/reperfusion injury in mammals.HSP70 levels were found to be only slightly elevated after recovery from sub-lethal heat stress, indicating minor importance of the heat shock response in this species. The DNA binding activity of the hypoxia-inducible transcription factor (HIF-1) was elevated only during mild heat exposure(18°C), but appeared impaired at more severe heat stress. We suppose that the more oxidized redox state during extreme heat may interfere with the hypoxic signaling response.

High oxygen solubility at cold-water temperature is frequently considered to be responsible for an apparently elevated level of antioxidant protection in marine ectotherms from polar environments. However, tissue oxidative stress is in most cases a function of elevated or variable pO2, rather than of an elevated tissue oxygen concentration. This review summarizes current knowledge on pro- and antioxidant processes in marine invertebrates and fish, and relates reactive oxygen species (ROS) formation in polar ectotherms to homeoviscous adaptations of membrane and storage lipids, as well as to tissue hypoxia and re-oxygenation during physiological stress.

Lipid peroxidation in Laternula elliptica was assessed by detecting lipid radicals by electronic paramagnetic resonance. The values were compared with data from the temperate mud clam Mya arenaria. Lipid radical content was higher in the Antarctic bivalve than in the temperate mud clam, even within the range of its habitat temperature. The rate of generation of lipid radicals was affected by the iron content in the samples. The iron content in individual samples of digestive glands in L. elliptica ranged from 3 to 6 nmol g(-1) fresh weight (fwt) and in M. arenaria from 0.6 to 2.7 nmol g(-1) fwt. Arrhenius plots, developed from the rates obtained in the presence of 25 microM iron, showed no significant differences between the activation energy calculated for digestive glands of L. elliptica and M. arenaria. The Fe3+ reduction rate in L. elliptica was higher than in M. arenaria (4.7 +/- 0.9 vs. 1.8 +/- 0.4 nmol mg(-1) protein min(-1), respectively). L. elliptica had a higher content of alpha-tocopherol and beta-carotene than M. arenaria. Our data suggest that increased lipid radical content in the membranes of cold-adapted organisms could be related to iron content.

SUMMARY Mitochondrial respiration, energetic coupling to phosphorylation and the production of reactive oxygen species (ROS) were studied in mitochondria isolated from the eurythermal bivalve Mya arenaria (Myoidea) from a low-shore intertidal population of the German Wadden Sea. Measurements were conducted both within the range of the habitat temperatures (5-15 °C) and when subjected to heat exposure at 20 °C and 25 °C. Experimental warming resulted in an increase in the rate of state 3 and state 4 respiration in isolated mitochondria. The highest respiratory coupling ratios (RCR) were found at 15 °C; at higher temperatures mitochondrial coupling decreased,and release of ROS doubled between 15 and 25 °C. ROS production was 2-3%of total oxygen consumption in state 3 (0.3-0.5 nmol ROS mg-1protein min-1) at the habitat temperature, reaching a maximum of 4.3 % of state 3 respiration and 7 % of oligomycin-induced state 4+respiration under heat stress. Thus, state 4 respiration, previously interpreted exclusively as a measure of proton leakage, included a significant contribution from ROS formation in this animal, especially under conditions of heat stress. Oxygen radical formation was directly dependent on temperature-controlled respiration rates in states 3 and 4 and inversely related to mitochondrial coupling (RCR+) in state 4. Mitochondrial ROS formation is therefore involved in cellular heat stress in this eurythermal marine ectotherm.

In this research, three lines of investigation were pursued. (1) In vivo effects of temperature and H2O2 were evaluated in the limpet Nacella concinna. Production of reactive oxygen species was assessed with the dihydrorhodamine 123 fluorescent probe, lysosome alterations with histochemical techniques, and antioxidant responses through Superoxide dismutase and catalase assays. Data suggest that Nacella spring migrations to intertidal levels, where rises in temperature occur, induce oxidative stress and antioxidant response. (2) In the scallop Adamussium colbecki, the sarcoplasmic reticulum Ca2+ ATPase was characterized and heavy metal effects were assessed. An IC50 of 0.9 μ? was found for Hg2+ and of 3 μ? for Cd2+. (3) Effects of Cu2+, Cd2+, and Hg2+ (2.5 μ?), and of temperature on Ca2+ homeostasis and cell viability were assessed in the ciliate Euplotes focardii. Ca2+ homeostasis and cell viability were altered by Hg2+ at a temperature of 0° C and above, and by Cu2+ and Cd2+ at 10° C and above. ...

This study deals with the occurrence of oxidative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna, as an effect of temperature increments and H2O2 exposure under controlled laboratory conditions. Experiments were designed to simulate transient conditions of increased T and:or H2O2 accumulation met by the limpets in intertidal rockpool habitats [5]. Specimens were collected at Jubany Station, South Shetland Islands, transferred to the Alfred-Wegener Institute, Bremerhaven and maintained in seawater aquaria at 0°C. Different groups of animals were acclimated at 4 and 9°C for 24‐48 h (controls at 0°C). The effect of starvation was studied at 0°C and of H2O2 exposure at 4°C. Temperature acclimation above 0°C resulted in a progressive alteration of the lysosomal compartment in digestive gland cells, as shown by cytochemical analyses (lipofuscin and neutral lipid accumulation and lysosomal membrane destabilization). Concurrently, real activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (i.e. measured at the respective experimental temperature or calculated by means of previously determined Q10 values) increased in gills and digestive gland tissues. Measurements of intracellular pH at the different temperatures showed a rise from pH 7.21 at 0°C to 7.36 at 9°C. These changes in pH are indicated to increase SOD activity by approximately 10% in both kinds of tissue at 9° as compared to 0°C. H2O2 exposure at 4°C produced physiological alterations at the systemic (lowered O2 consumption) and at the cellular levels (enhanced lysosome damage). Starvation induced lysosomal alterations in animals kept at 0°C and inhibited CAT activation under H2O2 exposure at 4°C. The complex of data suggests that when Nacella migrates to intertidal levels during the Austral Spring it experiences oxidative stress which induces an antioxidant response, which is facilitated by higher temperatures and increasing intracellular pH and the exploitation of intertidal food resources. Yet, the occurrence of cellular damage and systemic alterations shows that the limpets approach their boundaries of physiological tolerance during prolonged exposure to higher temperatures and H2O2 in intertidal habitats. © 1998 Elsevier Science Inc. All rights reserved.

UV-dependent hydrogen peroxide formation can lead to high levels of hydrogen peroxide in tidepool waters. The effect of ambient hydrogen peroxide on the rate of oxygen consumption and on the levels of intracellular pH, lactate and high energy phosphates was studied in the shrimp, Crangon crangon. Exposure to elevated hydrogen peroxide levels caused a decrease in metabolic rate by an average value of 26% in intact animals and by up to 60% in isolated preparations of the tail muscle. Muscle intracellular pH decreased by 0.1–0.2 pH units in both isolated tissue and intact animal. The maintenance of lactate and ATP at control levels strongly suggests that the acidosis was not associated with anaerobic proton production and, as a corollary, must be explained through an effect of hydrogen peroxide on proton equivalent ion exchange mechanisms. Future studies will attempt to elucidate the nature of the effect and whether the associated acidosis leads to the observed decrease in aerobic metabolic rate.

Arctica islandica is known as the longest-lived non-colonial metazoan species on earth and is therefore increasingly being investigated as a new model in aging research. As the mitochondrial genome is associated with the process of aging in many species and bivalves are known to possess a peculiar mechanism of mitochondrial genome inheritance including doubly uniparental inheritance (DUI), we aimed to assess the genomic variability of the A. islandica mitochondrial DNA (mtDNA). We sequenced the complete mitochondrial genomes of A. islandica specimens from three different sites in the Western Palaearctic (Iceland, North Sea, Baltic Sea). We found the A. islandica mtDNA to fall within the normal size range (18 kb) and exhibit similar coding capacity as other animal mtDNAs. The concatenated protein sequences of all currently known Veneroidea mtDNAs were used to robustly place A. islandica in a phylogenetic framework. Analysis of the observed single nucleotide polymorphism (SNP) patterns on further specimen revealed two prevailing haplotypes. Populations in the Baltic and the North Sea are very homogenous, whereas the Icelandic population, from which exceptionally old individuals have been collected, is the most diverse one. Homogeneity in Baltic and North Sea populations point to either stronger environmental constraints or more recent colonization of the habitat. Our analysis lays the foundation for further studies on A. islandica population structures, age research with this organism, and for phylogenetic studies. Accessions for the mitochondrial genome sequences: KC197241 Iceland; KF363951 Baltic Sea; KF363952 North Sea; KF465708 to KF465758 individual amplified regions from different speciemen.

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O2·− and hydrogen peroxide, H2O2), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this “natural stress” would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO2 in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO2 range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical pc values (pc1: onset of oxyregulation in gills, pc2: switch from oxyregulation to oxyconformity) to higher pO2. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions. Fil: Rivera Ingraham, Georgina A.. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Rocchetta, Iara. Helmholtz Centre for Polar and Marine Research; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Meyer, Stefanie. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Abele, Doris. Helmholtz Centre for Polar and Marine Research; Alemania

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.

Fil: Joyner Matos, Joanna. Eastern Washington University; Estados Unidos Fil: Puntarulo, Susana Angela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Vásquez Medina, José Pablo. University of California Merced; Estados Unidos Fil: Zenteno Zabin, Tania. Instituto Politécnico Nacional; México

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.

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 bivalve Arctica islandica is extremely long lived (>400 years) and can tolerate long periods of hypoxia and anoxia. European populations differ in maximum life spans (MLSP) from 40 years in the Baltic to >400 years around Iceland. Characteristic behavior of A. islandica involves phases of metabolic rate depression (MRD) during which the animals burry into the sediment for several days. During these phases the shell water oxygen concentrations reaches hypoxic to anoxic levels, which possibly support the long life span of some populations. We investigated gene regulation in A. islandica from a long-lived (MLSP 150 years) German Bight population and the short-lived Baltic Sea population, experimentally exposed to different oxygen levels. A new A. islandica transcriptome enabled the identification of genes important during hypoxia/anoxia events and, more generally, gene mining for putative stress response and (anti-) aging genes. Expression changes of a) antioxidant defense: Catalase, Glutathione peroxidase, manganese and copper-zinc Superoxide dismutase; b) oxygen sensing and general stress response: Hypoxia inducible factor alpha, Prolyl hydroxylase and Heat-shock protein 70; and c) anaerobic capacity: Malate dehydrogenase and Octopine dehydrogenase, related transcripts were investigated. Exposed to low oxygen, German Bight individuals suppressed transcription of all investigated genes, whereas Baltic Sea bivalves enhanced gene transcription under anoxic incubation (0 kPa) and, further, decreased these transcription levels again during 6 h of re-oxygenation. Hypoxic and anoxic exposure and subsequent re-oxygenation in Baltic Sea animals did not lead to increased protein oxidation or induction of apoptosis, emphasizing considerable hypoxia/re-oxygenation tolerance in this species. The data suggest that the energy saving effect of MRD may not be an attribute of Baltic Sea A. islandica chronically exposed to high environmental variability (oxygenation, temperature, salinity). Contrary, higher physiological flexibility and stress hardening may predispose these animals to perform a pronounced stress response at the expense of life span.

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.

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.

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28 degrees C to 18 degrees C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%-90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%-100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18 degrees C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.

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.

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.

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.

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.