Hypoxically Induced Nitric Oxide: Potential Role as a Vasodilator in Mytilus edulis Gills

Intertidal Mytilus edulis experience rapid transgression to hypoxia when they close theirvalves during low tide. This induces a physiological stress response aiming to stabilizetissue perfusion against declining oxygen partial pressure in shell water.We hypothesizedthat nitric oxide (NO) accumulation supports blood vessel opening in hypoxia andused live imaging techniques to measure NO and superoxide anion (O?−2 ) formationin hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO2) and severehypoxia (1 kPa pO2) caused 1.6- and 2.4-fold increase, respectively, of NO accumulationin the endothelial muscle cells of the hemolymphatic vessels of the gill filaments. This ledto a dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which facilitatesblood flow. Experiments in which we applied the chemical NO-donor Spermine NONOate(concentrations ranging from 1 to 6mM) under normoxic conditions corroborate thedilatational effect of NO on the blood vessel. The formation of O?−2 within the filamentepithelial cells increased 1.5 (7 kPa) and 2-fold (1 kPa) upon treatment. Biochemicalanalysis of mitochondrial electron transport complexes in hypoxia-exposed gill tissueindicates decreased activity of complexes I and III in both hypoxic conditions; whereascomplex IV (cytochrome-c oxidase) activity increased at 7 kPa and decreased at 1kPa compared to normoxic exposure conditions. This corresponds to the pattern ofpO2-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, sinceless O2 is available for NO oxidation to nitrite/nitrate. Hypoxia thus supports theNO dependent inhibition of complex IV activity, a mechanism that could fine tunemitochondrial respiration to the local O2 availability in a tissue. Our study highlights abasal function of NO in improving perfusion of hypoxic invertebrate tissues, which couldbe a key mechanism of tolerance toward environmental O2 variations.Fil: González, Paula Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: 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; ArgentinaFil: Abele, Doris. Alfred Wegener Institute For Polar And Marine Research; AlemaniaFil: Rivera Ingraham, Georgina A.. Alfred Wegener Institute For Polar And Marine Research; Alemani

Paleoenvironmental reconstruction using stable isotopes and trace elements from archaeological freshwater bivalve shell fragments in Northwest Patagonia, Argentina

Stable oxygen and carbon isotopes (18O and 13C) and the trace elements, manganese (Mn) and strontium (Sr) were analyzed in archaeological bivalve (Diplodon chilensis) shell fragments recovered from Parque Diana Cave, Patagonia, Argentina, to analyze the temporal trends of human occupation of the North Patagonia forest with climatic conditions recorded at regional scale but poorly known at local scale for this site. We established a stratigraphic sequence of thirteen 10 cm thick layers spanning the period c. 2370 BP to c. 580 BP and grouped these layers into three cultural components.According to this region´s climate, with rain and snow concentrated in winter and dry summers, our data suggest that these components are contemporary with three climatic periods. A dry period indicated by high values of δ18Oshell, δ13Cshell and Sr/Ca ratio (Mn/Ca ratio does not show any significant trend), in the Lower component is associated with brief incursions of hunter-gatherers from the eastern steppe. In the Middle component, low δ18Oshell, δ13Cshell and Sr/Ca ratios, suggest a humid climate with increased supply of meteoric and/or melt-water along with terrestrial plants organic matter and soil carbonates to the river. According to the archaeological evidence, in this period, hunter gatherers from the Pacific coast and forest started to colonize the area across the Andes cordillera. At the beginning of the Upper component, which is mostly contemporary with the expression of the Medieval Climatic Anomaly (MCA), the proxies are not totally coincident but a tendency to progressively drier conditions could be inferred. Except for layer 4 for δ18Oshell and layer 5 for Sr/Ca ratio, δ18Oshell, δ13Cshell and Sr/Ca ratio show consistently high levels in the Upper than in the Middle component. In layer 5, δ18Oshell and δ13Cshell are elevated with respect to the Middle component while Sr/Ca ratio remains low. In layer 4, δ13Cshell and Sr/Ca ratio are high while δ18Oshell shows a negative peak. In layers 3 to 1, δ18Oshell, δ13Cshell and Sr/Ca ratio remain at high levels, indicating an arid-warm climate with high aquatic plant productivity, in coincidence with the expression of the MCA reported for this region in a partially contemporaneous period (1080?1250 AD). At this time, the occupation of the site by groups of the same origin as those present in the Middle component became permanent. Extensive and intensive use of local resources in this period can be inferred from the quality and quantity of artifacts recovered.Fil: Perez, Alberto Enrique. Universidad Católica de Temuco; ChileFil: Batres, Daniel Alejandro. Universidad Católica de Temuco; ChileFil: 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; ArgentinaFil: Eppis, María. Comisión Nacional de Energía Atómica; ArgentinaFil: Bianchini, María Laura. Comisión Nacional de Energía Atómica; ArgentinaFil: 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; Argentin

Does shell shape variation play a role in conservation of the long‐lived freshwater bivalve Diplodon chilensis (Bivalvia, Hyriidae)?

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.

Hypoxically Induced Nitric Oxide: Potential Role as a Vasodilator in Mytilus edulis Gills

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 hypothesized 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. This led to a dilatation of blood vessel diameter by 43% (7 kPa) and 56% (1 kPa), which 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 toward environmental O2 variations

Biomarker responses to sewage pollution in freshwater mussels (Diplodon chilensis) transplanted to a Patagonian river

Field and laboratory experiments were combined to evaluate biomarker responses of Diplodon chilensis to sewage pollution. Mussels from an unpolluted area in Lacar lake (S0) were caged at a reference site (S1) and at two sites with increasing sewage pollution (S2, S3) in Pocahullo river (all in Argentina). After 1 month, gill (g) glutathione S-transferase (GST) and catalase (CAT) activities, and lipid peroxidation (TBARS) were found to be significantly elevated in S3, gGST being positively correlated with fecal bacteria (FC) concentration. Digestive gland (dg) enzyme activities were depressed and dgTBARS were increased in all transplanted mussels. After 3 mo, most variables returned to control levels in S1 mussels except for dgCAT and dgTBARS. After seven months, GST and CAT activities of S0 and S3 mussels were evaluated in the laboratory, before and after acute exposure (8 h) to high fecal bacteria concentration ([FC] in S3x 2). gGST increased in both groups, while dgGST responded only in S3 mussels. gCAT and dgCAT activities were similarly increased by acute exposure in both groups. Our results suggest that gGST and gCAT are suitable biomarkers for high FC pollution regardless of previous exposure history. In addition, we show that dgCAT is sensitive to the acute increase in FC load, both in naive and long-term exposed individuals, while dgGST becomes responsive after long-term acclimatization.Fil: Bianchi, Virginia Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; ArgentinaFil: Rocchetta, Iara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Luquet, Carlos Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación En Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; Argentin

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

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

INSIGHTS ON LOCALIZED ROS FORMATION AND PH IN ISOLATED BIVALVE GILLS: A LIVE-IMAGING APPROACH

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

Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: Foe or phantom? Experiments with a hypoxia tolerant bivalve

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 imagin