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

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: 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 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 and succinate dehydrogenase 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 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 exposition to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

Description: Freshwater bivalves of the order Unionoida display an uncommon phenotypic plasticity with high interpopulation and intrapopulation morphological variability, which could be advantageous for coping with habitat modifications. However, unionoids have suffered a marked population decline in different parts of the world in the last decades. A decline in some populations of the South American long‐lived freshwater mussel Diplodon chilensis as a consequence of habitat deterioration has recently been recorded. Ontogenetic allometry and shape variation in shells of D. chilensis from 2 different sites, Paimun lake and Chimehuin river, North Patagonia, Argentina, have been studied. For these purposes, geometric morphometric methods were used. Shell shape shows differences between sites, which the shells from Chimehuin river show less intrapopulation variability; are more elongated, with the anterior part extended upwards and the posterior part downwards; and show a steeper anterior curvature at the umbo compared to those from Paimún lake. These characteristics make shell shape more streamlined to withstand river current. Furthermore, the extended posterior‐ventral part in river shells coincides with higher foot weight that would improve anchoring to the river rocky–sandy substrate. River shells present a bounded eco‐morphotype whereas the higher variability of lake shells includes the “river eco‐morphotype.” Growth is allometric throughout life in both sites and is not sex‐dependent. The success of river repopulation programmes using mussels from lake populations may be increased by transplanting selected individuals that show “river eco‐morphotype.”

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

Description: Live-imaging techniques (LIT), using specific dyes and quantifying the resulting fluorescence signals under a confocal microscope, have traditionally been used with isolated cells and tissues, to study various physiological parameters in-vitro. In the present study we report the successful application of LIT to complete gill filaments for the analysis and characterization of reactive oxygen species (ROS) and nitric oxide (NO) formation. Our results indicate that H2O2 (hydrogen peroxide), HOO• (peroxyl radical) and ONOO- (peroxynitrate anion) radicals (assessed through C-H2DFFDA staining) are mainly formed in the blood sinus of the filaments. Contrary, in the periphery of the filaments, with the lateral cilia and highest mitochondrial densities (MTK Deep Red 633 staining), staining with O2•- (DHE) and NO (DAF-2) sensitive dyes was highest. These peripheral areas also show the most acidic pH values (as observed with the dye ageladine-a) and mitochondria with low membrane potentials (JC-1), suggesting that the high amounts of ATP required for ciliary beating may be produced via a non-mitochondrial pathway such as the enzymatic activity of an ATP-regenerating kinase. On the ventral bend of the gills a high number of subepithelial vesicles were observed, which were especially numerous in the areas close to the mouth. These vesicles were brightly stained with NO and O2•- reactive dyes, while other ROS were absent. We hypothesize that these vesicles may be involved in mucus production in the gills and, given the antimicrobial properties of O2•-, release of this radical together with the mucus may be playing an important role in avoiding the development of microbial biofilms on the gill surface.

Description: Intertidal Mytilus edulis experience rapid transgression to hypoxia when they close their valves during low tide. This induces a physiological stress response aiming to stabilize tissue perfusion against declining oxygen partial pressure in shell water. We hypothesised that nitric oxide (NO) accumulation supports blood vessel opening in hypoxia and used live imaging techniques to measure NO and superoxide anion (O2●−) formation in hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO2) and severe hypoxia (1 kPa pO2) caused 1.6 and 2.4-fold increase, respectively, of NO accumulation in the endothelial muscle cells of the hemolymphatic vessels of the gill filaments. Dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which significantly facilitates blood flow. Experiments in which we applied the chemical NO-donor Spermine NONOate (concentrations ranging from 1 to 6 mM) under normoxic conditions corroborate the dilatational effect of NO on the blood vessel. The formation of O2●− within the filament epithelial cells increased 1.5 (7 kPa) and 2-fold (1 kPa) upon treatment. Biochemical analysis of mitochondrial electron transport complexes in hypoxia-exposed gill tissue indicates decreased activity of complexes I and III in both hypoxic conditions; whereas complex IV (cytochrome-c oxidase) activity increased at 7 kPa and decreased at 1 kPa compared to normoxic exposure conditions. This corresponds to the pattern of pO2-dependent gill respiration rates recorded in ex-vivo experiments. Severe hypoxia (1 kPa) appears to have a stabilizing effect on NO accumulation in gill cells, since less O2 is available for NO oxidation to nitrite/nitrate. Hypoxia thus supports the NO dependent inhibition of complex IV activity, a mechanism that could fine tune mitochondrial respiration to the local O2 availability in a tissue. Our study highlights a basal function of NO in improving perfusion of hypoxic invertebrate tissues, which could be a key mechanism of tolerance towards environmental O2 variations.