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: 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: Arctica islandica is known as the longest-lived non-colonial metazoan species on earth and is therefore increasingly being investigated as a new model in aging research. As the mitochondrial genome is associated with the process of aging in many species and bivalves are known to possess a peculiar mechanism of mitochondrial genome inheritance including doubly uniparental inheritance (DUI), we aimed to assess the genomic variability of the A. islandica mitochondrial DNA (mtDNA). We sequenced the complete mitochondrial genomes of A. islandica specimens from three different sites in the Western Palaearctic (Iceland, North Sea, Baltic Sea). We found the A. islandica mtDNA to fall within the normal size range (18 kb) and exhibit similar coding capacity as other animal mtDNAs. The concatenated protein sequences of all currently known Veneroidea mtDNAs were used to robustly place A. islandica in a phylogenetic framework. Analysis of the observed single nucleotide polymorphism (SNP) patterns on further specimen revealed two prevailing haplotypes. Populations in the Baltic and the North Sea are very homogenous, whereas the Icelandic population, from which exceptionally old individuals have been collected, is the most diverse one. Homogeneity in Baltic and North Sea populations point to either stronger environmental constraints or more recent colonization of the habitat. We also detected potential signs of recombination between these two haplotypes. Our analysis lays the foundation for further studies on A. islandica population structures, age research with this organism, and for phylogenetic studies. Accessions for the mitochondrial genome sequences: KC197241Iceland KF363951Baltic Sea KF363952North Sea

Description: 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 〈5 kPa, even in a completely oxygen-saturated environment of 21 kPa. These low internal pO2 values support a longer NO lifespan and NO accumulation in the body fluids and can thus trigger a depression of metabolic rate in the clams. Measurable amounts of NO formation were detected in hemocyte cells (~110 pmol NO 100−1 hemocytes h-1 at 6 kPa), which was not prevented in the presence of the NO synthase inhibitor L-NAME, and in the gill filaments of A. islandica. Adding a NO donor to intact gills and tissue homogenate significantly inhibited gill respiration and CytOx activity below 10 kPa. Meanwhile, the addition of the NO-oxidation product nitrite did not affect metabolic rates. The high nitrite levels found in the hemolymph of experimental mussels under anoxia do not indicate cellular NO production, but could be an indication of nitrate reduction by facultative anaerobic bacteria associated with tissue and/or hemolymph biofilms. Our results suggest that NO plays an important role in the initiation of metabolic depression during self-induced burrowing and shell closure of A. islandica. Furthermore, NO appears to reduce mitochondrial oxygen radical formation during surfacing and cellular reoxygenation after prolonged periods of hypoxia and anoxia.