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The species delimitation of the marine bivalve species complex Aequiyoldia eightsii in South America and Antarctica is complicated by mitochondrial heteroplasmy and amplification bias in molecular barcoding. In this study, we compare different data sources (mitochondrial cytochrome c oxidase subunit I (COI) sequences; nuclear and mitochondrial SNPs). Whilst all the data suggest that populations on either side of the Drake Passage belong to different species, the picture is less clear within Antarctic populations, which harbor three distinct mitochondrial lineages (p-dist ≈ 6%) that coexist in populations and in a subset of individuals with heteroplasmy. Standard barcoding procedures lead to amplification bias favoring either haplotype unpredictably and thus overestimate the species richness with high confidence. However, nuclear SNPs show no differentiation akin to the trans-Drake comparison, suggesting that the Antarctic populations represent a single species. Their distinct haplotypes likely evolved during periods of temporary allopatry, whereas recombination eroded similar differentiation patterns in the nuclear genome after secondary contact. Our study highlights the importance of using multiple data sources and careful quality control measures to avoid bias and increase the accuracy of molecular species delimitation. We recommend an active search for mitochondrial heteroplasmy and haplotype-specific primers for amplification in DNA-barcoding studies.

The limpet Nacella concinna is a dominant macroinvertebrate along the coastal Antarctic Peninsula with two ecotypes inhabiting intertidal and subtidal areas, respectively. The ecological aim of the study was to understand whether higher stress competence and migratory energy expenses in intertidal Antarctic limpets shorten their lifetime and limit the shell growth rate compared to their sublittoral conspecific. We evaluated shell morphometry, age and internal shell growth bands in a large number of intertidal and subtidal N. concinna shells in Potter Cove, South Shetland Islands. Comparisons of their morphometrics showed that intertidal limpets are relatively shorter and less wide, and have higher shell mass, i.e. at common shell height, intertidal shells are relatively thicker and heavier than those of subtidal specimens. Internal shell growth bands showed alternating wide opaque (faster growth in summer) and thin translucent bands (slow growth in winter). The maximum age read was close to 20 years for both groups. Comparisons of von Bertalanffy growth curves showed for shell length and shell width lower growth rate k in intertidal animals than in subtidal ones associated to a great variability, with no differences in other growth parameters. However, when shell height vs. age is considered, no differences were observed for any growth parameter. Curtailed variability of growth rates in the intertidal population reflects either a limitation of the food reserves or feeding time, or an energy gap for shell growth due to the costs for migratory movements and stress defense.

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 free radical nitric oxide (NO) is a powerful metabolic regulator in vertebrates and invertebrates. At cellular concentrations in the nanomolar range, and simultaneously reduced internal oxygen partial pressures (pO2), NO completely inhibits cytochrome-c-oxidase (CytOx) activity and hence mitochondrial- and whole-tissue respiration. The infaunal clam Arctica islandica regulates pO2 of hemolymph and mantle cavity water to mean values of

The west Antarctic Peninsula (WAP) region has undergone significant changes in temperature and seasonal ice dynamics since the mid-twentieth century, with strong impacts on the regional ecosystem, ocean chemistry and hydrographic properties. Changes to these long-term trends of warming and sea ice decline have been observed in the 21st century, but their consequences for ocean physics, chemistry and the ecology of the high-productivity shelf ecosystem are yet to be fully established. The WAP shelf is important for regional krill stocks and higher trophic levels, whilst the degree of variability and change in the physical environment and documented biological and biogeochemical responses make this a model system for how climate and sea ice changes might restructure high-latitude ecosystems. Although this region is arguably the best-measured and best-understood shelf region around Antarctica, significant gaps remain in spatial and temporal data capable of resolving the atmosphere-ice-ocean-ecosystem feedbacks that control the dynamics and evolution of this complex polar system. Here we summarise the current state of knowledge regarding the key mechanisms and interactions regulating the physical, biogeochemical and biological processes at work, the ways in which the shelf environment is changing, and the ecosystem response to the changes underway. We outline the overarching cross-disciplinary priorities for future research, as well as the most important discipline-specific objectives. Underpinning these priorities and objectives is the need to better define the causes, magnitude and timescales of variability and change at all levels of the system. A combination of traditional and innovative approaches will be critical to addressing these priorities and developing a co-ordinated observing system for the WAP shelf, which is required to detect and elucidate change into the future. Fil: Henley, Sian F.. University of Edinburgh; Reino Unido Fil: Schofield, Oscar M.. State University of New Jersey; Estados Unidos Fil: Hendry, Katharine R.. University Of Bristol; Reino Unido Fil: Schloss, Irene Ruth. Universidad Nacional de Tierra del Fuego; Argentina. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Steinberg, Deborah K.. Virginia Institute of Marine Science. College of William and Mary; Estados Unidos Fil: Moffat, Carlos. University of Delaware; Estados Unidos Fil: Peck, Lloyd S.. British Antarctic Survey; Reino Unido Fil: Costa, Daniel P.. University of California Santa Cruz; Estados Unidos Fil: Bakker, Dorothee C.E.. University of East Anglia; Reino Unido Fil: Hughes, Claire. University Of California, Santa Cruz; Fil: Rozema, Patrick D.. University of East Anglia; Reino Unido Fil: Ducklow, Hugh W.. Columbia University; Estados Unidos Fil: Abele, Doris. Alfred Wegener Institute; Alemania Fil: Stefels, Jacqueline. University of Groningen; Países Bajos Fil: Van Leeuwe, Maria A.. University of Groningen; Países Bajos Fil: Brussaard, Corina P.D.. Royal Netherlands Institute for Sea Research; Países Bajos Fil: Buma, Anita G.J.. University of Groningen; Países Bajos Fil: Kohut, Josh. Royal Netherlands Institute For Sea Research - Nioz; Fil: Sahade, Ricardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. University Of Groningen; Fil: Friedlaender, Ari S.. State University of New Jersey; Estados Unidos Fil: Stammerjohn, Sharon E.. State University of Colorado at Boulder; Estados Unidos Fil: Venables, Hugh J.. British Antarctic Survey; Reino Unido Fil: Meredith, Michael. British Antarctic Survey; Reino Unido

Glacier melting sediment inputs affect coastal ecosystems on the Antarctic Peninsula. In Potter Cove (South Shetland Islands, Antarctica), the shift from an “ascidian dominated” to a “mixed” assemblage has been linked to sedimentation. However, in recently described newly ice-free areas ascidians became dominant in spite of total suspended particulate matter (TSPM) concentrations, which are the highest measured in Potter Cove. Here, we compared the gut content and energy reserve of three ascidian species at three stations under different TSPM regimes. All analysed species had a higher gut content with lower %OM at these newly areas. A theoretical relationship between the scope for growth for the targeted ascidians and TSPM explained assemblages’ recorded change but failed to explain current ascidians distribution. The results may indicate the existence of a TSPM threshold that allows the spatial coexistence of alternative stable states at benthic Potter Cove system. Fil: Torre, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Alurralde, Roque Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Lagger, Cristian Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Abele, D.. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Schloss, Irene Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina Fil: Sahade, Ricardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina

Multiple toxic and bioactive compounds produced by Alexandrium spp. cause adverse effects on bivalves, but these effects are frequently difficult to attribute to a single compound class. To disentangle the effect of neurotoxic vs lytic secondary metabolites, we exposed blue mussels to either a paralytic shellfish toxin (PST) producing Alexandrium spp. strain, or to an exclusively lytic compound (LC) producing strain, or a strain containing both compound classes, to evaluate the time dependent effects after 3 and 7 days of feeding. Tested parameters comprised signs of paralysis, feeding activity, and immune cell integrity (hemocyte numbers and viability; lysosomal membrane destabilization) and function (ROS production). Both compound classes caused paralysis and immune impairment. The only effect attributable exclusively to PST was increased phagocytic activity after 3 days and impaired feeding activity after 7 days, which curtailed toxin accumulation in digestive glands. Lysosomal membrane destabilization were more closely, but not exclusively, matched with LC exposure. Effects on circulating hemocyte integrity and immune related functions were mostly transient or remained stable within 7 days; except for increased lysosomal labialization and decreased extracellular ROS production when mussels were exposed to the toxin combination. M. edulis displays adaptive fitness traits to survive and maintain immune capacity upon prolonged exposure to environmentally relevant concentrations of PST and/or LC producing Alexandrium strains. Fil: Bianchi, Virginia Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; Argentina Fil: Langeloh, Hendrick. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania Fil: Tillmann, Urban. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania Fil: Krock, Bernd. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania Fil: Müller, Annegret. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania Fil: Bickmeyer, Ulf. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania. Alfred-Wegener-Institut. Helmholtz-Zentrum für Polar und Meeresforschung; Alemania Fil: Abele, Doris. Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung; Alemania

The West Antarctic Peninsula (WAP) is a climatically sensitive region where periods of strong warming have caused significant changes in the marine ecosystem and food-web processes. Tight coupling between phytoplankton and higher trophic levels implies that the coastal WAP is a bottom-up controlled system, where changes in phytoplankton dynamics may largely impact other food-web components. Here, we analysed the inter-decadal time series of year-round chlorophyll-a (Chl) collected from three stations along the coastal WAP: Carlini Station at Potter Cove (PC) on King George Island, Palmer Station on Anvers Island and Rothera Station on Adelaide Island. There were trends towards increased phytoplankton biomass at Carlini Station (PC) and Palmer Station, while phytoplankton biomass declined significantly at Rothera Station over the studied period. The impacts of two relevant climate modes to the WAP, the El Niño-Southern Oscillation and the Southern Annular Mode, on winter and spring phytoplankton biomass appear to be different among the three sampling stations, suggesting an important role of local-scale forcing than large-scale forcing on phytoplankton dynamics at each station. The inter-annual variability of seasonal bloom progression derived from considering all three stations together captured ecologically meaningful, seasonally co-occurring bloom patterns which were primarily constrained by water-column stability strength. Our findings highlight a coupled link between phytoplankton and physical and climate dynamics along the coastal WAP, which may improve our understanding of overall WAP food-web responses to climate change and variability. This article is part of the theme issue 'The marine system of the West Antarctic Peninsula: status and strategy for progress in a region of rapid change'. Fil: Kim, Hyewon. Columbia University; Estados Unidos Fil: Ducklow, Hugh W.. Columbia University; Estados Unidos Fil: Abele, Doris. Alfred Wegener Institute for Polar and Marine Research; Alemania Fil: Ruiz Barlett, Eduardo M.. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Buma, Anita G. J.. University of Groningen; Países Bajos Fil: Meredith, Michael. British Antarctic Survey; Reino Unido Fil: Rozema, Patrick D.. University of Groningen; Países Bajos Fil: Schofield, Oscar M.. Rutgers University. School of Environmental and Biological Sciences. Department of Marine and Coastal Sciences. Center for Ocean Observing Leadership; Estados Unidos Fil: Venables, Hugh J.. British Antarctic Survey; Reino Unido Fil: Schloss, Irene Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina. Universidad Nacional de Tierra del Fuego; Argentina

The coasts of the West Antarctic Peninsula are strongly influenced by glacier meltwater discharge. The spatial structure and biogeochemical composition of inshore habitats are shaped by large quantities of terrigenous particulate material deposited in the vicinity of the coast, which impacts the pelagic and benthic ecosystems. We used a multitude of geochemical and environmental variables to identify the radius extension of the meltwater impact from the Fourcade Glacier into the fjord system of Potter Cove, King George Island. The k -means cluster algorithm, canonical correspondence analysis, variance analysis and Tukey's post hoc multiple comparison tests were applied to define and cluster coastal meltwater habitats. A minimum of 10 clusters were needed to classify the 8 km 2 study area into meltwater fjord habitats (MFHs), fjord habitats and marine habitats. Strontium content in surface sediments is the main geochemical indicator for lithogenic creek discharge in Potter Cove. Furthermore, bathymetry, glacier distance and geomorphic positioning are the essential habitats explaining variables. The mean and maximum MFH extent amounted to 1 km and 2 km, respectively. Extrapolation of the identified meltwater impact ranges to King George Island coastlines, which are presently ice-covered bays and fjord areas, indicated an overall coverage of 200–400 km 2 MFH, underpinning the importance of better understanding the biology and biogeochemistry in terrestrial marine transition zones. This article is part of the theme issue ‘The marine system of the West Antarctic Peninsula: status and strategy for progress in a region of rapid change’.

Glacial meltwater discharge from Antarctica is a key influence on the marine environment, impacting ocean circulation, sea level and productivity of the pelagic and benthic ecosystems. The responses elicited depend strongly on the characteristics of the meltwater releases, including timing, spatial structure and geochemical composition. Here we use isotopic tracers to reveal the time-varying pattern of meltwater during a discharge event from the Fourcade Glacier into Potter Cove, northern Antarctic Peninsula. The discharge is strongly dependent on local air temperature, and accumulates into an extremely thin, buoyant layer at the surface. This layer showed evidence of elevated turbidity, and responded rapidly to changes in atmospherically driven circulation to generate a strongly pulsed outflow from the cove to the broader ocean. These characteristics contrast with those further south along the Peninsula, where strong glacial frontal ablation is driven oceanographically by intrusions of warm deep waters from offshore. The Fourcade Glacier switched very recently to being land-terminating; if retreat rates elsewhere along the Peninsula remain high and glacier termini progress strongly landward, the structure and impact of the freshwater discharges are likely to increasingly resemble the patterns elucidated here. This article is part of the theme issue 'The marine system of the West Antarctic Peninsula: status and strategy for progress in a region of rapid change'. Fil: Meredith, Michael. British Antarctic Survey; Reino Unido Fil: Ulrike Falk. Universitat Bremen; Alemania. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Bers, Anna Valeria. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Mackensen, Andreas. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Schloss, Irene Ruth. Universidad Nacional de Tierra del Fuego; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Austral de Investigaciones Científicas; Argentina. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Ruiz Barlett, Eduardo Mariano. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Jerosch, Kerstin. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Silva Busso, Adrián. Universidad de Buenos Aires; Argentina Fil: Abele, Doris. Helmholtz Centre for Polar and Marine Research; Alemania

Background: Scavenger guilds are composed of a variety of species, co-existing in the same habitat and sharing the same niche in the food web. Niche partitioning among them can manifest in different feeding strategies, e.g. during carcass feeding. In the bentho-pelagic realm of the Southern Ocean, scavenging amphipods (Lysianassoidea) are ubiquitous and occupy a central role in decomposition processes. Here we address the question whether scavenging lysianassoid amphipods employ different feeding strategies during carcass feeding, and whether synergistic feeding activities may influence carcass decomposition. To this end, we compared the relatively large species Waldeckia obesa with the small species Cheirimedon femoratus, Hippomedon kergueleni, and Orchomenella rotundifrons during fish carcass feeding (Notothenia spp.). The experimental approach combined ex situ feeding experiments, behavioural observations, and scanning electron microscopic analyses of mandibles. Furthermore, we aimed to detect ecological drivers for distribution patterns of scavenging amphipods in the Antarctic coastal ecosystems of Potter Cove. In Potter Cove, the climate-driven rapid retreat of the Fourcade Glacier is causing various environmental changes including the provision of new marine habitats to colonise. While in the newly ice-free areas fish are rare, macroalgae have already colonised hard substrates. Assuming that a temporal dietary switch may increase the colonisation success of the most abundant lysianassoids C. femoratus and H. kergueleni, we aimed to determine their consumption rates (g food x g amphipods−1 x day−1) and preferences of macroalgae and fish.Results: We detected two functional groups with different feeding strategies among scavenging amphipods during carcass feeding: carcass 'opener' and 'squeezer'. Synergistic effects between these groups were not statistically verified under the conditions tested. C. femoratus switched its diet when fish was not available by consuming macroalgae (about 0.2 day−1) but preferred fish by feeding up to 80% of its own mass daily. Contrary, H. kergueleni rejected macroalgae entirely and consumed fish with a maximal rate of 0.8 day−1.Conclusion: This study reveals functional groups in scavenging shallow-water amphipods and provides new information on coastal intraguild niche partitioning. We conclude that the dietary flexibility of C. femoratus is a potential ecological driver and central to its success in the colonisation of newly available ice-free Antarctic coastal habitats.

A diesel spill occurring at Carlini Station (King George Island (Isla 25 de Mayo), South Shetland Islands) in 2009 started the study of the fate of the hydrocarbons and their effect on the bacterial communities of the Potter Cove ecosystem. Soils and sediments were sampled across the 200-meter long diesel plume towards Potter Cove four and 15 months after the spill. The sampling revealed a second fuel leakage from an underground pipeline at the spill site. The hydrocarbon fraction spilt over frozen and snow-covered ground reached the sea and dispersed with the currents. Contrary, diesel that infiltrated unfrozen soil emained detectable for years, and was seeping with ground water towards coastal marine sediments. Structural changes of the bacterial communities as well as hydrocarbon, carbon and nitrogen contents were investigated in sediments in front of the station, two affected terrestrial sites, and a terrestrial non-contaminated reference site. Bacterial communities (16S rRNA gene clone libraries) changed over time in contaminated soils and sediments. At the underground seepage site of highest contamination (5812 to 366 μgg−1dw hydrocarbons fromsurface to 90-cm depth), communities were dominated by Actinobacteria (18%) and a betaproteobacteriumclosely related to Polaromonas naphthalenivorans (40%). At one of the spill sites, affected exclusively at the surface, contamination disappeared within one year. The same bacterial groups were enriched at both contaminated sites. This response at community level suggests that the cold-adapted indigenous microbiota in soils of the West Antarctic Peninsula have a high potential for bioremediation and can support soil cleaning actions in the ecosystem. Intensive monitoring of pollution and site assessment after episodic fuel spills is required for decision-making towards remediation strategies. Fil: Vázquez, Susana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Monien, Patrick. Institute for Chemistry and Biology of the Marine Environment; Alemania. Universitat Bremen; Alemania Fil: Pepino Minetti, Roberto. Universidad Tecnológica Nacional. Facultad Regional Córdoba; Argentina Fil: Jürgens, Jutta. Alfred-Wegener-Institut Helmholtzzentrum für Polar und Meeresforschung; Alemania Fil: Curtosi, Antonio. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Villalba Primitz, Julia Elena Rosina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Frickenhaus, Stephan. Alfred-Wegener-Institut Helmholtzzentrum für Polar und Meeresforschung; Alemania Fil: Abele, Doris. Alfred-Wegener-Institut Helmholtzzentrum für Polar und Meeresforschung; Alemania Fil: Mac Cormack, Walter Patricio. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Helmke, Elisabeth. Alfred-Wegener-Institut Helmholtzzentrum für Polar und Meeresforschung; Alemania

Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (< 0.2 mg O2/L), hypoxia (2 mg O2/L), and normoxia (9 mg O2/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks. Fil: Yusseppone, Maria Soledad. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Enzimología, Estrés Oxidativo y Metabolismo; Argentina Fil: Rocchetta, Iara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; Argentina Fil: Sabatini, Sebastian Eduardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Enzimología, Estrés Oxidativo y Metabolismo; Argentina Fil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Ecotoxicología Acuática; Argentina Fil: Rios, Maria del Carmen. 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. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Enzimología, Estrés Oxidativo y Metabolismo; Argentina Fil: Held, Christoph. Helmholtz Centre for Polar and Marine Research; Alemania Fil: Abele, Doris. Helmholtz Centre for Polar and Marine Research; Alemania

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.

Marine habitats worldwide are increasingly pressurized by climate change, especially along the Antarctic Peninsula. Well-studied areas in front of rapidly retreating tidewater glaciers like Potter Cove are representative for similar coastal environments and, therefore, shed light on habitat formation and development on not only a local but also regional scale. The objective of this study was to provide insights into habitat distribution in Potter Cove, King George Island, Antarctica, and to evaluate the associated environmental processes. Furthermore, an assessment concerning the future development of the habitats is provided. To describe the seafloor habitats in Potter Cove, an acoustic seabed discrimination system (RoxAnn) was used in combination with underwater video images and sediment samples. Due to the absence of wave and current measurements in the study area, bed shear stress estimates served to delineate zones prone to sediment erosion. On the basis of the investigations, two habitat classes were identified in Potter Cove, namely soft-sediment and stone habitats that, besides influences from sediment supply and coastal morphology, are controlled by sediment erosion. A future expansion of the stone habitat is predicted if recent environmental change trends continue. Possible implications for the Potter Cove environment, and other coastal ecosystems under similar pressure, include changes in biomass and species composition.

Owing to its extremely long life span and occurrence in the entire North Atlantic, the Arctic boreal Arctica islandica has become of particular significance for monitoring the environment, because information on past environmental conditions is archived in morphological and biogeochemical properties of the calcareous shell. To evaluate whether such properties are comparable between different localities, shell and soft body morphometry of six A. islandica populations, Norwegian Coast, Kattegat, Kiel Bay (Baltic), White Sea, German Bight (North Sea), and off NE Iceland, were compared. Discriminant analysis indicated distinct differences between populations, albeit not related to geographical distance, but more likely to local hydrography, bottom morphology, and food regime.

Ensemble habitat modeling is a tool in the multivariate analysis of arbitrary species or community distribution which combines models of best fit to an optimized model (ensemble model, EM). To simulate spatial variation of communities and predict the impact of climate change, it is essential to identify the distribution-controlling factors. Macroalgae biomass production in polar regions is determined by environmental factors such as irradiance, which are modified under climate change impact. In coastal fjords of King George Island/Isla 25 de Mayo, Antarctica, suspended particulate matter (SPM) from glacial melting causes shading of algal communities during summer. Ten different species distribution models (SDMs) were applied to predict macroalgae distribution based on their statistical relationships with environmental variables. The suitability of the SDMs was assessed by two different evaluation methods. Those SDMs based on a multitude of decision trees such as Random Forest and Classification Tree Analysis reached the highest predictive ability followed by generalized boosted models and maximum-entropy approaches. We achieved excellent results for the current status EM (true scale statistics 0.833 and relative operating characteristics 0.975). The environmental variables hard substrate and SPM were identified as the best predictors explaining more than 60 % of the modelled distribution. Additional variables distance to glacier, total organic carbon, bathymetry and slope increased the explanatory power proved by cross-validation. Presumably, the SPM load of the meltwater streams on the Potter Peninsula will continue to increase at least linearly. We therefore coupled the EM with changing SPM conditions representing enhanced or reduced melt water input. Increasing SPM by 25% decreased predicted macroalgal coverage by approximately 38%. The ensemble species distribution modelling helps to identify the important factors controlling spatial distribution and can be used to link causes to effects in (Antarctic) coastal community change.

Molecular technologies are more frequently applied in Antarctic ecosystem research and the growing amount of sequence-based information available in databases adds a new dimension to understanding the response of Ant- arctic organisms and communities to environmental change. We apply molecular techniques, including finger- printing, and amplicon and metagenome sequencing, to understand biodiversity and phylogeography to resolve adaptive processes in an Antarctic coastal ecosystem from microbial to macrobenthic organisms and communities. Interpretation of the molecular data is not only achieved by their combination with classical methods (pigment analyses or microscopy), but furthermore by combining molecular with environmental data (e.g., sediment characteristics, biogeochemistry or oceanography) in space and over time. The studies form part of a long-term ecosystem investigation in Potter Cove on King-George Island, Antarctica, in which we follow the effects of rapid retreat of the local glacier on the cove ecosystem. We formulate and encourage new approaches to integrate molecular tools into Antarctic ecosystem research, environmental conservation ac- tions, and polar ocean observatories. Fil: Abele, Doris. Helmholtz Centre For Polar And Marine Research; Fil: Vázquez, Susana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Buma, A. G. J.. University of Groningen; Países Bajos Fil: Hernández, Edgardo. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Quiroga, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Held, Christoph. Helmholtz Centre For Polar And Marine Research; Fil: Frickenhaus, Stephan. Helmholtz Centre For Polar And Marine Research; Fil: Harms, L.. Helmholtz Centre For Polar And Marine Research; Fil: Lopez, Jose Luis. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina Fil: Helmke, E.. Helmholtz Centre For Polar And Marine Research; Fil: Mac Cormack, Walter Patricio. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina

Longevity and ageing are two sides of a coin, leaving the question open as to which one is the cause and which one the effect. At the individual level, the physiological rate of ageing determines the length of life (= individual longevity, as long as death results from old age and not from disease or other impacts). Individual longevity depends on the direct influence of environmental conditions with respect to nutrition, and the possibility for and timing of reproduction, as well as on the energetic costs animals invest in behavioural and physiological stress defence. All these environmental effectors influence hormonal and cellular signalling pathways that modify the individual physiological condition, the reproductive strategy, and the rate of ageing. At the species level, longevity (= maximum lifespan) is the result of an evolutionary process and, thus, largely determined by the species' behavioural and physiological adaptations to its ecological niche. Specifically, reproductive and breeding strategies have to be optimized in relation to local environmental conditions in different habitats. As a result of adaptive and evolutionary processes, species longevity is genetically underpinned, not necessarily by a few ageing genes, but by an evolutionary process that has hierarchically shaped and optimized species genomes to function in a specific niche or environmental system. Importantly, investigations and reviews attempting to unravel the mechanistic basis of the ageing process need to differentiate clearly between the evolutionary process shaping longevity at the species level and the regulatory mechanisms that alter the individual rate of ageing.

Recent rapid changes of air temperature on the western side of the Antarctic Peninsula results in increased sediment discharge and ice scouring frequencies in coastal regions. These changes are bound to especially affect slow growing, sessile filter feeders such as the Antarctic bivalve, Laternula elliptica, a long-lived and abundant key species with circumpolar distribution. We investigated the effect of sedimentation and ice scouring on small/young and large/old individuals at two closely located stations, distinctly influenced by both types of disturbance. Small individuals dealt better with disturbance in terms of their respiratory response to sediment exposure, reburrowing ability, and survival after injury, compared to larger animals. At the more disturbed station L. elliptica population density was lower, but larger animals reburrowed faster after iceberg disturbance and reduced their metabolic rate under strong sediment coverage, compared to larger animals of the less disturbed station, indicating that an adaptation or learning response to both types of disturbance may be possible. Smaller individuals were not influenced. Laternula elliptica seems capable of coping with the rapidly changing environmental conditions. Due to a decrease in population density and mean population lifespan, L. elliptica could however lose its key role in the bentho-pelagic carbon flux in areas of high sediment deposition.

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.

Owing to its extraordinary lifespan and wide geographical distribution along the continental margins of the North Atlantic Ocean, the ocean quahog Arctica islandica may become an important indicator species in environmental change research. To test for applicability and ''calibrate'' the Arctica-indicator, metabolic properties of A. islandica specimens were compared across different climatic and oceanographic regions. Fully saline populations from Iceland to the North Sea as well as animals from polyhaline and low salinity, environments, the White Sea and the Baltic were included in the study. This calibration centrally includes recordings of growth-age relationships in different populations. Shells were used as age recorders by counting annual growth bands. As a result of this study, we propose a general respiration model that links individual metabolic rates of A. islandica from five populations: Norwegian coast, Kattegat, Kiel Bay (Baltic Sea), White Sea and German Bight (North Sea), to body mass, water temperature and site. Temperature exerts distinct site specific effects on respiration rate, which is indicated by Q10 values ranging from 4.48 for German Bight to 1.15 for Kiel Bay animals. Individual age, occurrence of apneal respiratory gaps, parasite infestation and salinity do not affect respiration rate. Respiration of Arctica islandica is significantly below the average of 59 bivalve species when compared at the same temperature and animal mass. This respiration model principally enables the coupling of A. islandica life history and population dynamics to regional oceanographic temperature models.

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.

Antarctic intertidal zones are extremely harsh environments, and the Antarctic limpet Nacella concinna is one of the most conspicuous components of the macrofauna, colonizing such areas at King George Island, South Shetlands. The limpet subpopulation encompasses an intertidal and a subtidal ecomorph; these differ with respect to shell height and inner volume. We studied mor- phological, behavioural and physiological traits that distinguish both N. concinna subpopulations. We found a higher volume to circumference ratio of intertidal versus subtidal limpets to go along with a drastically reduced loss of body water during air exposure in intertidal specimens. Smaller inter- tidal limpets were less susceptible to desiccation than larger individuals. Further, the intertidal snails avoided accumulation of anaerobic metabolites (succinate, lactate and propionate), whereas subtidal limpets switched to anaerobic fermentation when exposed to air for >12 h. Only acetate accumulated in both types of snails after 12 h. Mean pO2 in shell water was higher in intertidal (2.6 kPa) than sub- tidal individuals (1.3 kPa) when submersed under water, and only the intertidal snails appeared able to respire air on emersion. These differences document strong physiological diversification between shore levels and shell morphs in the Antarctic limpet subpopulation at King George Island.

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.

Background: Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. Most analytical work uses tissue homogenates, and lacks spatial information on the tissue specific sites of actual ROS formation. Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes at cellular level. Results: Together with oxidative stress measurements, here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of ROS and RNS formation in the living tissue. Our results indicate that a) mitochondria located in the basal parts of the epithelial cells close to the blood vessels are hyperpolarized with high Delta Psi m, whereas b) the peripheral mitochondria close to the cilia have low (depolarized)Delta Psi m. These mitochondria are densely packed (mitotracker Deep Red 633 staining), have acidic pH (Ageladine-A) and collocate with high formation of nitric oxide (DAF-2DA staining). NO formation is also observed in the endothelial cells surrounding the filament blood sinus. ROS (namely H2O2, HOO center dot and ONOO- radicals, assessed through C-H(2)DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O-2 is approximately equal to than the rest of the tissue. Whether this O-2 is approximately equal to production is instrumental to mucus formation or serves antimicrobial protection of the gill surface is unknown. Cells of the ventral bends contain the superoxide forming mucocytes and show significantly higher protein carbonyl formation than the rest of the gill tissue. Conclusions: In summary, ROS and RNS formation is highly compartmentalized in bivalve gills under unstressed conditions. The main mechanisms are the differentiation of mitochondria membrane potential and basal ROS formation in inner and outer filament layers, as well as potentially antimicrobial ROS formation in the central blood vessel. Our results provide new insight into this subject and highlight the fact that studying ROS formation in tissue homogenates may not be adequate to understand the underlying mechanism in complex tissues.