Doris Abele

Production of reactive oxygen species by isolated mitochondria of the Antarctic bivalve Laternula elliptica (King and Broderip) under heat stress.

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.

Exposure to elevated temperatures and hydrogen peroxide elicits oxidative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna

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.

Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparus L.

SUMMARY The 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.

Bivalve models of aging and the determination of molluscan lifespans

Bivalves are newly discovered models of natural aging. This invertebrate group includes species with the longest metazoan lifespan approaching 400 y, as well as species of swimming and sessile lifestyles that live just for 1 y. Bivalves from natural populations can be aged by shell growth bands formed at regular intervals of time. This enables the study of abiotic and biotic environment factors (temperature, salinity, predator and physical disturbance) on senescence and fitness in natural populations, and distinguishes the impact of extrinsic effectors from intrinsic (genetic) determinants of animal aging. Extreme longevity of some bivalve models may help to analyze general metabolic strategies thought to be life prolonging, like the transient depression of metabolism, which forms part of natural behaviour in these species. Thus, seasonal food shortage experienced by benthic filter feeding bivalves in polar and temperate seas may mimic caloric restriction in vertebrates. Incidence of malignant neoplasms in bivalves needs to be investigated, to determine the implication of late acting mutations for bivalve longevity. Finally, bivalves are applicable models for testing the implication of heterozygosity of multiple genes for physiological tolerance, adaptability (heterozygote superiority), and life expectancy.

Masters of longevity: lessons from long-lived bivalves--a mini-review.

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.

Seasonality of energetic functioning and production of reactive oxygen species by lugworm (Arenicola marina) mitochondria exposed to acute temperature changes

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. marina mitochondria.

Imperceptible senescence: Ageing in the ocean quahog Arctica islandica

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 (∼32 years). Thereafter all three parameters kept rather stable levels for > 150 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.

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

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°C and after long-term warming to 5°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°C for the polar and 6°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.

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

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°C to 18°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°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.

Climate change and glacier retreat drive shifts in an Antarctic benthic ecosystem

Climatic change in the Antarctic Peninsula has driven profound shifts in the seabed. The Antarctic Peninsula (AP) is one of the three places on Earth that registered the most intense warming in the last 50 years, almost five times the global mean. This warming has strongly affected the cryosphere, causing the largest ice-shelf collapses ever observed and the retreat of 87% of glaciers. Ecosystem responses, although increasingly predicted, have been mainly reported for pelagic systems. However, and despite most Antarctic species being benthic, responses in the Antarctic benthos have been detected in only a few species, and major effects at assemblage level are unknown. This is probably due to the scarcity of baselines against which to assess change. We performed repeat surveys of coastal benthos in 1994, 1998, and 2010, analyzing community structure and environmental variables at King George Island, Antarctica. We report a marked shift in an Antarctic benthic community that can be linked to ongoing climate change. However, rather than temperature as the primary factor, we highlight the resulting increased sediment runoff, triggered by glacier retreat, as the potential causal factor. The sudden shift from a “filter feeders–ascidian domination” to a “mixed assemblage” suggests that thresholds (for example, of tolerable sedimentation) and alternative equilibrium states, depending on the reversibility of the changes, could be possible traits of this ecosystem. Sedimentation processes will be increasing under the current scenario of glacier retreat, and attention needs to be paid to its effects along the AP.