Description: 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 Δψm, whereas b) the peripheral mitochondria close to the cilia have low (depolarized) Δψm. 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• 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. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O2 •- than the rest of the tissue. Whether this O2 •- 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.

Description: Background: Scavenger guilds, composed of a variety of species, co-existing in the same habitat, are responsible for biomass transformation throughout 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 of the speciose superfamily Lysianassoidea are amongst the ubiquitous taxa and occupy an essential role in decomposition processes. First, we addressed the question whether scavenging lysianassoid amphipods have different feeding strategies during carcass feeding, and if their potential synergistic feeding activities influence carcass decomposition. To this end, we compared the relatively large-sized species Waldeckia obesa with the small-sized species Cheirimedon femoratus, Hippomedon kergueleni, and Orchomenella rotundifrons during carcass feeding (Notothenia spp.). Our approach combines ex situ feeding experiments, behavioural observations, and scanning electron microscopic analyses of mandibles. Secondly, we aimed to detect ecological drivers for succession patterns of scavenging amphipods in Antarctic coastal ecosystems affected by environmental disturbances. 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 records are rare, macroalgae have already colonised hard substrates. Therefore, we carried out feeding assays of the most abundant lysianassoids in Potter Cove C. femoratus and H. kergueleni, to determine their consumption rates (mg food x mg amphipods-1 x day-1) and preferences of macroalgae and fish. Results We detected two functional groups with different feeding strategies among the investigated scavenging amphipods: the 'outside-insider' (openers) and 'inside-outsider' (squeezers). Synergistic effects during carcass feeding was not statistical evident. C. femoratus showed a flexible diet when fish was not available by consuming macroalgae with a consumption about 0.2 day-1 but preferred fish with feedings rates up to 0.8 day-1. Contrary, H. kergueleni rejected macroalgae entirely and consumed fish with consumption rates up to 0.8 day-1. Conclusion This study reveals functional groups in scavenging shallow water amphipods and provides new information on coastal intraguild niche partitioning. Moreover, we conclude that dietary flexibility of scavenging amphipods is a potential ecological driver for succession and colonisation of newly available ice-free Antarctic coastal habitats.

Description: Melting of coastal glaciers at the West Antarctic Peninsula (WAP) causes shorter winter sea ice duration, intensified ice scouring, sediment erosion and surface freshening in summer, which alters coastal productivity and feeding conditions for the benthos. The soft shell clam Laternula elliptica is a fast growing and abundant filter feeder in coastal Antarctica and a key element for bentho-pelagic carbon recycling. Our aim was to assess the cellular growth and maintenance capacity of small and large clams during natural winter food shortage (seasonal sampling) and in response to experimental starvation exposure. We measured tissue specific proliferation rates and the expression of cell cycling genes and the iron binding protein Le-ferritin in freshly collected specimens in spring (Nov 2008) and at the end of summer (March 2009). For the experimental approach, we focused on 14 cell cycling and metabolic genes using the same animal size groups. Mantle tissue of young bivalves was the only tissue showing accelerated proliferation in summer (1.7 % of cells dividing per day in March) compared to 0.4% dividing cells in animals collected in November. In mantel, siphon and adductor muscle proliferation rates were higher in younger compared to older indiciduals. At transcript level, Le-cyclin D was upregulated in digestive gland of older animals collected in spring (Nov) compared to March indicating enhanced cell proliferation. Likewise, during experimental starvation Le-cyclin D expression increased in large clam digestive gland, whereas Le-cyclin D and the autophagic factor beclin1 decreased in digestive gland of smaller starved clams. The paper corroborates earlier findings of size and age dependent differences in the metabolic response and gene expression patterns in L. elliptica under energetic deprivation. Age structure of shallow water populations can potentially change due to differences in cellular response between young and old animals as environmental stress levels increase.

Description: Rising temperatures and other environmental factors influenced by global climate change can cause increased physiological stress for many species and lead to range shifts or regional population extinctions. To advance the understanding of species’ response to change and establish links between individual and ecosystem adaptations, physiological reactions have to be compared between populations living in different environments. Although changes in expression of stress genes are relatively easy to quantify, methods for reliable comparison of the data remain a contentious issue. Using normalization algorithms and further methodological considerations, we compare cellular stress response gene expression levels measured by RT-qPCR after air exposure experiments among different subpopulations of three species of the intertidal limpet Nacella. Results: Reference gene assessment algorithms reveal that stable reference genes can differ among investigated populations and / or treatment groups. Normalized expression values point to differential defence strategies to air exposure in the investigated populations, which either employ a pronounced cellular stress response in the inducible Hsp70 forms, or exhibit a comparatively high constitutive expression of Hsps (heat shock proteins) while showing only little response in terms of Hsp induction. Conclusions: This study serves as a case study to explore the methodological prerequisites of physiological stress response comparisons among ecologically and phylogenetically different organisms. To improve the reliability of gene expression data and compare the stress responses of subpopulations under potential genetic divergence, reference gene stability algorithms are valuable and necessary tools. As the Hsp70 isoforms have been shown to play different roles in the acute stress responses and increased constitutive defences of populations in their different habitats, these comparative studies can yield insight into physiological strategies of adaptation to environmental stress and pr 47 ovide hints for the prudent use of the cellular stress response as a biomarker to study environmental stress and stress adaptation of populations under changing environmental conditions.

Description: Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O2•- and hydrogen peroxide, H2O2), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this “natural stress” would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO2 in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO2 range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical pc values (pc1: onset of oxyregulation in gills, pc2: switch from oxyregulation to oxyconformity) to higher pO2. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions. Keywords: blue mussel, oxidative stress, reoxygenation, live imaging