Data from PhD thesis

The ocean quahog, Arctica islandica is the longest-lived non-colonial animal known to science. A maximum individual age of this bivalve of 405 years has been found in a population off the north western coast of Iceland. Conspicuously shorter maximum lifespan potentials (MLSPs) were recorded from other populations of A. islandica in European waters (e.g. Kiel Bay: 30 years, German Bight: 150 years) which experience wider temperature and salinity fluctuations than the clams from Iceland. The aim of my thesis was to identify possible life-prolonging physiological strategies in A. islandica and to examine the modulating effects of extrinsic factors (e.g. seawater temperature, food availability) and intrinsic factors (e.g. species-specific behavior) on these strategies. Burrowing behavior and metabolic rate depression (MRD), tissue-specific antioxidant and anaerobic capacities as well as cell-turnover (= apoptosis and proliferation) rates were investigated in A. islandica from Iceland and the German Bight. An inter-species comparison of the quahog with the epibenthic scallop Aequipecten opercularis (MLSP = 8-10 years) was carried out in order to determine whether bivalves with short lifespans and different lifestyles also feature a different pattern in cellular maintenance and repair. The combined effects of a low-metabolic lifestyle, low oxidative damage accumulation, and constant investment into cellular protection and tissue maintenance, appear to slow-down the process of physiological aging in A. islandica and to afford the extraordinarily long MLSP in this species. Standard metabolic rates were lower in A. islandica when compared to the shorter-lived A. opercularis. Furthermore, A. islandica regulate mantle cavity water PO2 to mean values < 5 kPa, a PO2 at which the formation of reactive oxygen species (ROS) in isolated gill tissues of the clams was found to be 10 times lower than at normoxic conditions (21 kPa). Burrowing and metabolic rate depression (MRD) in Icelandic specimens were more pronounced in winter, possibly supported by low seawater temperature and food availability, and seem to be key energy-saving and life-prolonging parameters in A. islandica. The signaling molecule nitric oxide (NO) may play an important role during the onset of MRD in the ocean quahog by directly inhibiting cytochome-c-oxidase at low internal oxygenation upon shell closure. In laboratory experiments, respiration of isolated A. islandica gills was completely inhibited by chemically produced NO at low experimental PO2 <= 10 kPa. During shell closure, mantle cavity water PO2 decreased to 0 kPa for longer than 24 h, a state in which ROS production is supposed to subside. Compared to other mollusk species, onset of anaerobic metabolism is late in A. islandica in the metabolically reduced state. Increased accumulation of the anaerobic metabolite succinate was initially detected in the adductor muscle of the clams after 3.5 days under anoxic incubation or in burrowed specimens. A ROS-burst was absent in isolated gill tissue of the clams following hypoxia (5 kPa)-reoxygenation (21 kPa). Accordingly, neither the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), nor the specific content of the ROS-scavenger glutathione (GSH) was enhanced in different tissues of the ocean quahog after 3.5 days of self-induced or forced hypoxia/anoxia to prepare for an oxidative burst. While reduced ROS formation compared to routine levels lowers oxidative stress during MRD and also during surfacing, the general preservation of high cellular defense and the efficient removal and replacement of damaged cells over lifetime seem to be of crucial importance in decelerating the senescent decline in tissues of A. islandica. Along with stable antioxidant protection over 200 years of age, proliferation rates and apoptosis intensities in most investigated tissues of the ocean quahog were low, but constant over 140 years of age. Accordingly, age-dependent accumulations of protein and lipid oxidation products are lower in A. islandica tissues when compared to the shorter-lived bivalve A. opercularis. The short-lived swimming scallop is a model bivalve species representing the opposite life and aging strategy to A. islandica. In this species permanently high energy throughput, reduced investment into antioxidant defense with age, and higher accumulation of oxidation products are met by higher cell turnover rates than in the ocean quahog. The only symptoms of physiological change over age ever found in A. islandica were decreasing cell turnover rates in the heart muscle over a lifetime of 140 years. This may either indicate higher damage levels and possibly ongoing loss of functioning in the heart of aging clams, or, the opposite, lower rates of cell damage and a reduced need for cell renewal in the heart tissue of A. islandica over lifetime. Basic physiological capacities of different A. islandica populations, measured at controlled laboratory conditions, could not explain considerable discrepancies in population specific MLSPs. For example, levels of tissue-specific antioxidant capacities and cell turnover rates were similarly high in individuals from the German Bight and from Iceland. Rather than genetic differences, the local impacts of environmental conditions on behavioral and physiological traits in the ocean quahog seem to be responsible for differences in population-specific MLSPs.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Gene expression and physiological changes of the Antarctic krill Euphausia superba under different hypoxia intensities

Antarctic krill (Euphausia superba) from South Georgia comprise one of the most northern and abundant krill stocks. South Georgia waters are undergoing rapid warming, as a result of climate change, which in turn could alter the oxygen concentration of the water. We investigated gene expression in Antarctic krill related to aerobic metabolism, antioxidant defence, and heat-shock response under severe (2.5% O2 saturation or 0.6 kPa) and threshold (20% O2 saturation or 4 kPa) hypoxia exposure compared to in situ levels (normoxic; 100% O2 saturation or 21 kPa). Biochemical metabolic and oxidative stress indicators complemented the genic expression analysis to detect in vivo signs of stress during the hypoxia treatments. Expression levels of the genes citrate synthase (CS), mitochondrial manganese superoxide dismutase (SODMn-m) and one heat-shock protein isoform (E) were higher in euphausiids incubated 6 h at 20% O2 saturation than in animals exposed to control (normoxic) conditions. All biochemical antioxidant defence parameters remained unchanged among treatments. Levels of lipid peroxidation were raised after 6 h of severe hypoxia. Overall, short-term exposure to hypoxia altered mitochondrial metabolic and antioxidant capacity, but did not induce anaerobic metabolism. Antarctic krill are swarming organisms and may experience short periods of hypoxia when present in dense swarms. A future, warmer Southern ocean, where oxygen saturation levels are decreased, may result in smaller, less dense swarms as they act to avoid greater levels of hypoxia.

(Table 1) Antioxidant concentrations in liver of wild sea bird chicks from the Norwegian arctic region

The efficiency of antioxidant defenses and relationship with body burden of metal and organic contaminants has not been previously investigated in arctic seabirds, neither in chicks nor in adults. The objective of this study was to compare such defenses in chicks from three species, Black-legged kittiwake (Rissa tridactyla), Northern fulmar (Fulmarus glacialis), and Herring gull (Larus argentatus), and the relationship with tissue concentrations of essential metals such as selenium and iron and halogenated organic compounds, represented by polychlorinated biphenyl (PCB). The results showed significant species-specific differences in the antioxidant responses which also corresponded with metal and PCB levels in different ways. The capability to neutralize hydroxyl radicals (TOSC-HO°) and the activities of catalase and Se-dependent glutathione peroxidases (GPX) clearly increased in species with the higher levels of metals and PCBs, while the opposite trend was observed for Se-independent GPX, TOSC against peroxyl radicals (ROO°) and peroxynitrite (ONOOH). Less clear relationships were obtained for glutathione levels, GSH/GSSG ratio, glutathione reductase and superoxide dismutase. The results showed differences in antioxidant efficiency between the species, and some of these defenses exhibited dose-response-like relationships with measured levels of selenium, iron and XPCBs. PCBs, selenium and iron levels were positively related to the responses of antioxidants with potential to reduce HO°/H2O2 (Se-dependent GPX, CAT and TOSC against HO°). However, direct causal relationships between antioxidant responses and contaminant concentrations could not be shown on individual level. Varying levels of metals and contaminants due to different diet and age were probably the main explanations for the species differences in antioxidant defense.

Response of three krill species to hypoxia and warming: An experimental approach to oxygen minimum zones expansion in coastal ecosystems

To understand the adaptation of euphausiid (krill) species to oxygen minimum zones (OMZ), respiratory response and stress experiments combining hypoxia/reoxygenation exposure with warming were conducted. Experimental krill species were obtained from the Antarctic (South Georgia area), the Humboldt Current system (HCS, Chilean coast), and the Northern California Current system (NCCS, Oregon). Euphausia mucronata from the HCS shows oxyconforming or oxygen partial pressure (pO2)-dependent respiration below 80% air saturation (18 kPa). Normoxic subsurface oxygenation in winter posed a "high oxygen stress" for this species. The NCCS krill, Euphausia pacifica, and the Antarctic krill, Euphausia superba maintain respiration rates constant down to low critical pO2 values of 6 kPa (30% air saturation) and 11 kPa (55% air saturation), respectively. Antarctic krill had the lowest antioxidant enzyme activities, but the highest concentrations of the molecular antioxidant glutathione (GSH) and was not affected by 6 h exposure to moderate hypoxia. Temperate krill species had higher SOD (superoxide dismutase) values in winter than in summer, which relate to higher winter metabolic rate (E. pacifica). In all species, antioxidant enzyme activities remained constant during hypoxic exposure at habitat temperature. Warming by 7°C above habitat temperature in summer increased SOD activities and GSH levels in E. mucronata (HCS), but no oxidative damage occurred. In winter, when the NCCS is well mixed and the OMZ is deeper, +4°C of warming combined with hypoxia represents a lethal condition for E. pacifica. In summer, when the OMZ expands upwards (100 m subsurface), antioxidant defences counteracted hypoxia and reoxygenation effects in E. pacifica, but only at mildly elevated temperature (+2°C). In this season, experimental warming by +4°C reduced antioxidant activities and the hypoxia combination again caused mortality of exposed specimens. We conclude that a climate change scenario combining warming and hypoxia represents a serious threat to E. pacifica and, as a consequence, NCCS food webs.

Physiological and biochemical characteristics of polar cod (Boreogadus saida) from Kongsfjorden

Seasonality of biomarker baseline levels were studied in polar cod (Boreogadus saida), caught in Kongsfjorden, Svalbard, in April, July, September and December, 2006-2007. Physiological parameters (condition factor, gonado- and hepato-somatic indexes, energy reserves, potential metabolic activity and antifreeze activity) in polar cod were used to interpret the seasonality of potential biomarkers. The highest levels of ethoxyresorufin-O-deethylase (EROD) activity occurred concomitantly with the highest potential metabolic activity in July due to e.g. intense feeding. During pre-spawning, EROD showed significant inhibition and gender differences. Hence, its potential use in environmental monitoring should imply gender differentiation at least during this period. Glutathione S-transferase and catalase activities were stable from April to September, but changed in December suggesting a link to low biological activity. Knowledge of the biomarker baseline levels and their seasonal trends in polar cod is essential for a trustworthy interpretation of forthcoming toxicity data and environmental monitoring in the Arctic.

Nitrate limitation and ocean acidification interact with UV-B to reduce photosynthetic performance in the diatom

It has been proposed that ocean acidification (OA) will interact with other environmental factors to influence the overall impact of global change on biological systems. Accordingly we investigated the influence of nitrogen limitation and OA on the physiology of diatoms by growing the diatom Phaeodactylum tricornutum Bohlin under elevated (1000 µatm; high CO2- HC) or ambient (390 µatm; low CO2-LC) levels of CO2 with replete (110 µmol/L; high nitrate-HN) or reduced (10 ?mol/L; low nitrate-LN) levels of NO3- and subjecting the cells to solar radiation with or without UV irradiance to determine their susceptibility to UV radiation (UVR, 280-400 nm). Our results indicate that OA and UVB induced significantly higher inhibition of both the photosynthetic rate and quantum yield under LN than under HN conditions. UVA or/and UVB increased the cells' non-photochemical quenching (NPQ) regardless of the CO2 levels. Under LN and OA conditions, activity of superoxide dismutase and catalase activities were enhanced, along with the highest sensitivity to UVB and the lowest ratio of repair to damage of PSII. HC-grown cells showed a faster recovery rate of yield under HN but not under LN conditions. We conclude therefore that nutrient limitation makes cells more prone to the deleterious effects of UV radiation and that HC conditions (ocean acidification) exacerbate this effect. The finding that nitrate limitation and ocean acidification interact with UV-B to reduce photosynthetic performance of the diatom P. tricornutum implies that ocean primary production and the marine biological C pump will be affected by OA under multiple stressors.

Effects of ocean acidification on the swimming ability, development and biochemical responses of sand smelt larvae

Ocean acidification, recognized as a major threat to marine ecosystems, has developed into one of the fastest growing fields of research in marine sciences. Several studies on fish larval stages point to abnormal behaviours, malformations and increased mortality rates as a result of exposure to increased levels of CO2. However, other studies fail to recognize any consequence, suggesting species-specific sensitivity to increased levels of CO2, highlighting the need of further research. In this study we investigated the effects of exposure to elevated pCO2 on behaviour, development, oxidative stress and energy metabolism of sand smelt larvae, Atherina presbyter. Larvae were caught at Arrábida Marine Park (Portugal) and exposed to different pCO2 levels (control: 600 µatm, pH = 8.03; medium: 1000 µatm, pH = 7.85; high: 1800 µatm, pH = 7.64) up to 15 days, after which critical swimming speed (Ucrit), morphometric traits and biochemical biomarkers were determined. Measured biomarkers were related with: 1) oxidative stress-superoxide dismutase and catalase enzyme activities, levels of lipid peroxidation and DNA damage, and levels of superoxide anion production; 2) energy metabolism - total carbohydrate levels, electron transport system activity, lactate dehydrogenase and isocitrate dehydrogenase enzyme activities. Swimming speed was not affected by treatment, but exposure to increasing levels of pCO2 leads to higher energetic costs and morphometric changes, with larger larvae in high pCO2 treatment and smaller larvae in medium pCO2 treatment. The efficient antioxidant response capacity and increase in energetic metabolism only registered at the medium pCO2 treatment may indicate that at higher pCO2 levels the capacity of larvae to restore their internal balance can be impaired. Our findings illustrate the need of using multiple approaches to explore the consequences of future pCO2 levels on organisms.

Size, blood organochlorine pesticide concentrations and biochemical indicators measured in hawksbill sea turtles (Eretmochelys imbricata) nesting at Punta Xen (Campeche, Mexico) in August 2010

The impact of environmental pollution on the homeostasis of sea turtles remains scarce, particularly in the southern Gulf of Mexico. As many municipalities do not rely on a waste treatment plant along the coastline of the Yucatan Peninsula, the vulnerability of these specimens could results enhanced. We searched for relationships between presence of organochlorine pesticides (OCP) and the level of several oxidative and pollutant stress indicators of the hawksbill sea turtle (Eretmochelys imbricata) during the egg-laying period 2010 at Punta Xen (Campeche, Mexico). Endosulfans, aldrin related (aldrin, endrin, dieldrin, endrin ketone, endrin aldehyde) and dichlorodiphenyldichloroethylene (DDT) families were detected in 17, 21 and 26 of the 30 sampled sea turtles, respectively. Significant correlation existed between the size of sea turtles with the concentration of methoxychlor, cholinesterase activity in plasma and heptachlors family, and catalase activity and hexachlorohexane family. Cholinesterase activity in washed erythrocytes and lipid peroxidation were positively correlated with glutathione reductase activity. Antioxidant enzyme actions seem adequate as no lipids damages were correlated with any OCPs. Future studies are necessary to evaluate the effect of OCPs on males of the area because of the significant detection of methoxychlor that target endocrine functioning and increase its concentration with size of the sea turtles.