Georgina A.
Rivera-Ingraham
Latest Publications
27-Nov-2016
New contribution: Metabolic depression and consequent reoxygenation: conciling energy-saving processes and recovery anticipation
Aude Nommick, Eva Blondeau-Bidet, Anne-Laure Charles, Bernard Geny, Jehan-hervé Lignot, Georgina A. Rivera-Ingraham
Abstract:
Intertidal organisms must face daily cycles of environmental variations but also unpredictable events, which occurrence should increase due to global changes. As an interstitial organism dealing with such challenges, Macrostomum lignano is a suitable model species for studies focusing on physiological adaptations to environmental constraints. In this context, we can identify two physiological responses to stress: those entailing an increased metabolic demand whilst others induce metabolic depression. The latter is a common strategy that allows survival until the arrival of better environmental conditions. Our work concentrates on this last physiological response and our recent results demonstrate that metabolic depression is induced in M.lignano by hypo-salinity and hypoxia. However, the challenge comes with recovery and the reoxygenation at the mitochondrial level, which results in a burst in free radical production. If uncontrolled, this redox unbalance can lead to cell damage and, eventually, death.
We demonstrate that M. lignano is probably capable of anticipating this reoxygenation by inducing antioxidant defenses under conditions of restricted metabolism. Among the different antioxidants tested, these worms responded by significantly increasing the gene expression of glutathione S transferase-Pi (GST-Pi), a powerful antioxidant that providing protection against oxidative damage. It is especially interesting to note that this was observed for both hyposaline and hypoxic conditions. However, this anticipation is completely abolished when worms are exposed to an antioxidant (ascorbic acid) during a hypoxic event. This suggests that free radicals are mediating in this process, which is not the case for animals exposed to hyposaline conditions.
These results lead to interesting bioenergetic questions. How this anticipation process is achieved under conditions of restricted energy availability? What is the benefit of increasing GST-Pi compared to other antioxidants? Given that this preferential use has also been described in other invertebrates, what is the driving force for such an evolutionary convergence?
In 10th International Macrostomum Meeting, November 2016, Basel (Switzerland). You may view the TALK by clicking HERE.
27-Nov-2016
New contribution: Salinity changes, free radical production and physiological acclimation: the coping strategy of Macrostomum lignano.
Georgina A. Rivera-Ingraham; Aude Nommick, Eva Blondeau-Bidet, Peter Ladurner, Jehan-hervé Lignot
Abstract:
In this era of increasing environmental changes, studying how species cope with this stress arises as a key research issue. Macrostomum lignano, as other intertidal organisms, are adapted to face variations in their environmental conditions, and are thus interesting models for these studies. We here analyse their physiological strategy to face salinity variations. Acclimation processes necessarily come with a bioenergetic cost and thus, a mitochondrial/oxidative stress approach emerges as the most relevant way of analysing whole-animal responses. We conducted this study by using microrespirometric measurements, in-vivo confocal microscopy and gene expression quantification through RT-qPCR. The changes in body volume at different salinities suggest that M. lignano is a hyper-/iso-regulator. Hypersalinity seems to be the most energetically expensive conditions leading to higher respiration rates (RR), a strong production of superoxide anion, increased mitochondrial density, and higher expression of antioxidant enzymes (such as superoxide dismutase) to deal with increased cellular damage. On the other hand, platyhelminths under hyposanility showed metabolic depression, characterized by low swimming activity and decreased RR. This was accompanied by a strong nitric oxide (NO) formation, likely responsible for the metabolic arrest since NO competes with O2 at the cytochrome c (responsible for most of the O2 consumption). Such conditions of reduced ATP production also explain the increased mitochondrial membrane potential observed in these animals. Surprisingly, worms under hyposaline conditions showed an upregulation of gluthathione-S-transferase pi 1 (GSTP1) expression. If animals at low salinity are indeed facing metabolic depression, the return to seawater may result in an oxidative burst. We hypothesize that this increase in GSTP1 could be a “preparation for oxidative stress”, i.e. a mechanism to counteract the production of free radicals upon returning to seawater. Our results open new questions regarding the bioenergetics of stress responses under conditions of depressed metabolism.
In 10th International Macrostomum Meeting, November 2016, Basel (Switzerland). You may view the TALK by clicking HERE.
22-Sept-2016
New article: Salinity stress from the perspective of the energy-redox axis: lessons from a marine intertidal flatworm
Georgina A. Rivera-Ingraham; Aude Nommick, Eva Blondeau-Bidet, Peter Ladurner, Jehan-hervé Lignot
Abstract:
In the context of global change, there is an urgent need for researchers in conservation physiology to understand the physiological mechanisms leading to the acquisition of stress acclimation phenotypes. Intertidal organisms continuously cope with drastic changes in their environmental conditions, making themoutstanding models for the study of physiological acclimation. As the implementation of such processes usually comes at a high bioenergetic cost, a mitochondrial/oxidative stress approach emerges as the most relevant approach when seeking to analyze whole-animal responses. Here we use the intertidal flatworm Macrostomum lignano to analyze the bioenergetics of salinity acclimation and its consequences in terms of reactive oxygen/nitrogen species formation and physiological response to counteract redox imbalance. Measures of water fluxes and body volume suggest that M. lignano is a hyper-/iso-regulator. Higher salinities were revealed to be the most energetically expensive conditions, with an increase in mitochondrial density accompanied by increased respiration rates. Such modifications came at the price of enhanced superoxide anion production, likely associated with a high caspase 3 upregulation. These animals nevertheless managed to live at high levels of environmental salinity through the upregulation of several mitochondrial antioxidant enzymes such as superoxide dismutase. Contrarily, animals at low salinities decreased their respiration rates, reduced their activity and increased nitric oxide formation, suggesting a certain degree of metabolic arrest. A contradictory increase in dichlorofluorescein fluorescence and an upregulation of pi-gluthathione-S-transferase (GST-pi) expression were observed in these individuals. If animals at low salinity are indeed facing metabolic depression, the return to seawater may result in an oxidative burst. We hypothesize that this increase in GST-pi could be a “preparation for oxidative stress”, i.e. a mechanism to counteract the production of free radicals upon returning to seawater. The results of the present study shed new light on how tolerant organisms carry out subcellular adaptations to withstand environmental change.
Redox Biology 10, 53-64.
21-Jul-2016
New contribution: Trace metals and oxidative damage in the endangered mollusc Patella ferruginea (Gastropoda: Patellidae)
Free Espinosa; Georgina A. Rivera-Ingraham.
Abstract:
Intertidal species, living in the realm between terrestrial and marine environments, are more vulnerable to anthropogenic disturbances than others inhabiting subtidal and offshore habitats. Coastal development frequently results in trace-metal pollution, a type of impact considered as one of the main threats to Mediterranean marine environments.
The high sensitivity of certain endangered species to pollution events can lead local populations to extinction. Such is the case of Patella ferruginea, considered as the most endangered marine invertebrate in the Mediterranean and which is in clear regression since the early 1900s. Surprisingly, the knowledge about its vulnerability to deteriorated environmental quality from an eco-physiological perspective is non-existent. The fact that the species frequently settles on artificial substrata, thus being potentially affected by diverse pollution sources, makes it urgent to obtain data on the physiological status of such populations. Specimens of P. ferruginea were collected from three different localities in Ceuta: 1) a natural rocky shore from a pristine area; 2) an artificial breakwater outside of the harbor; 3) an artificial breakwater within the harbor. Within each locality three sites were established and six limpets of similar size were collected per site to perform metal content analyses and assessments on the oxidative balance (antioxidant defenses and cell damage on gills).
The most abundant heavy metals found in Patella ferruginea soft tissues were Fe, Zn and As, but only Hg and As showed significant differences among localities. In fact, the specimens collected inside the harbor reached 3-fold higher Hg content than those incoming from the natural area. Additionally, ANCOVA test showed that females had highest values for Zn, Cd and As, while PERMANOVA test indicated that metal composition of the specimens from inside the harbor was different from the rest. On the other hand, antioxidant defenses differed between natural and harbor areas, but no differences were recorded between sexes, whereas cell damage was lower in the natural locality compared to the harbor area.
Our results support those from several other authors that have reported enhanced contents of several heavy metals such as Cu, Pb and Zn for other limpets in areas under pollution. However, to our knowledge this is the first report on differential trace-metal bioaccumulation between males and females in limpets. Taking into account that females of Patella ferruginea are intensively harvested by humans, which is dramatically affecting the reproductive output of several populations, the fact that they accumulate higher contents of some heavy metals could be problematic from a conservation perspective. In addition, results from the present study indicate that pollutant exposure is inducing significant cellular damage and perturbed redox status, a matter which could potentially threaten the future viability of the population settled in Ceuta’s harbor, one of Patella ferruginea most important remaining populations. This highlights the urgency of undertaking a physiological evaluation of some of the most vulnerable populations, and most importantly, of establishing eco-physiological protocols for monitoring and managing populations settled on artificial substrata in the context of the implementation of the recently proposed AMMRs (Artificial Marine Micro Reserves).
In 9th XIX SIEBM, September 2016, Porto (Portugal). You may view the POSTER by clicking HERE.
1-Jul-2016
New contribution: Facing salinity changes in the intertidal environment: a platyhelminth's strategy for preparing for oxidative stress
Georgina A. Rivera-Ingraham, Eva Blondeau-Bidet, Aude Nommick, Jehan-Hervé Lignot
Abstract:
Intertidal organisms must daily cope with drastic changes in their environmental conditions (temperature, oxygenation, salinity, radiation…). This is usually accompanied by increased formation in reactive oxygen (ROS) and nitrogen (RNS) species, which, if not controlled, leads to oxidative stress. Intertidal organisms are usually capable to counteract these deleterious effects of ROS and RNS formation, through behavioral and/or physiological mechanisms (e.g. enhanced production of antioxidants). Macrostomum lignano is used to test this hypothesis when exposed to environmental salinity changes. Animals demonstrated to be confortable in a wide range of salinities, ranging from freshwater to hypersaline seawater (>60 ppt). Energetically speaking, higher salinities were the most expensive conditions, since we detected an increase in mitochondrial density accompanied by increased respiration rates. However, such modifications come at the price of an enhanced superoxide anion production (DHE staining), which is likely associated with a high caspase 3 upregulation (detected by RTqPCR). However, animals are still able to live at high environmental salinity, likely through the upregulation of several mitochondrial antioxidants. However, animals at low salinities decrease their respiration rates, have reduced activity and enter metabolic depression, but, show an upregulation of their GST-pi. If animals at low salinity are indeed facing metabolic depression (and, thus, functional hypoxia), the return to seawater may result in an oxidative burst, as it happens in fasting/re-feeding, hibernation/arousal or ischemia/re-perperfusion situations. This increase in GST-pi could be interpreted as a “preparation for oxidative stress”, a mechanism to fight the free radical production that occurs upon returning to seawater.
In SEB16, July 2016, Brighton (UK). You may view the TALK by clicking HERE
1-Jul-2016
New publication: Subcellular evidences of redox imbalance in well-established populations of an endangered limpet. Reasons for alarm?
Free Espinosa & Georgina A. Rivera-Ingraham
Abstract:
Intertidal species aremore vulnerable to anthropogenic disturbances than others inhabiting subtidal and offshore habitats. Coastal development frequently results in trace-metal pollution. For endangered species such as Patella ferruginea it can be a high risk that leads local populations to extinction. Three localitieswere surveyed, onewithin a natural and unpolluted area and the other twowithin the harbor of Ceuta (Strait of Gibraltar), on breakwaters
outside and inside. he specimens collected inside the harbor reached 3-fold higher Hg content than for those incoming from the natural area. PERMANOVA test indicated thatmetal composition of the specimens frominside the harborwas different from the rest. In addition, evidence of cell damage was detected in the specimens from the harbor area. This highlights the urgency of undertaking a physiological evaluation of some of themost vulnerable populations, establishing eco-physiological protocols formonitoring and managing populations settled on artificial substrata.
Marine Pollution Bulleting in press. Click here to download.
18-Nov-2015
New contribution: Disentangling the physiological response of Macrostomum lignano exposed to environmental salinity change
Georgina A. Rivera-Ingraham, Jehan-Hervé Lignot.
Abstract:
Macrostomum lignano, as other intertidal organisms, must daily cope with drastic changes in their environmental conditions, such as temperature, oxygenation, salinity or radiation. This type of changes are usually accompanied by increased formation in reactive oxygen (ROS) and nitrogen (RNS) species, which, if not controlled, leads to the so-called oxidative stress: protein carbonylation, lipid peroxidation, aging, mutagenesis or even cell death. Intertidal organisms are usually capable to put in place the necessary mechanisms to counteract these deleterious effects of ROS and RNS formation, normally consisting in behavioral and physiological mechanisms (e.g. enhanced production of antioxidants). We use M. lignano to test this hypothesis when exposed to environmental salinity changes. Animals demonstrated to be confortable in a wide range of salinities, ranging from sweet water to hypersaline seewater (>60 ppt). Microrespirometric measurements on animals acclimated to seawater (SW, 35 ppt) indicate that 6h exposure to diluted SW and concentrated SW involve an increase in respiration rates, but it is the higher salinities that pose the higher degrees of stress. This is supported by the in-vivo measurement of ROS (C-H2DFFDA and DHE stainings) and animals show higher fluorescence intensities in hypersaline SW. A time analysis upon salinity change evidences that ROS formation (DCF fluorescence) significantly increases until 1h after the environmental change, and progressively decreases after this time, suggesting that this is the time needed to put in place oxidative stress regulation mechanisms. However, the mechanisms signaling and triggering these pathways remain mostly unknown, and a growing body of evidence indicates that small amounts of free radicals may be involved in triggering this process. Further research will be oriented to study the effects of the inhibition of ROS/RNS formation on the antioxidant response of M. lignano upon environmental change to further disentangle the mechanisms that trigger the antioxidant response.
In 9th International Macrostomum Meeting, Novemeber 2015, Innsbruck (Austria). You may view the TALK by clicking HERE.
10-Nov-2015
New contribution: Free radical metabolism in the Mediterranean green crab: consequences for respiratory and osmoregulatory gill tissues
Georgina A. Rivera-Ingraham, Kiam Barry, Mélanie Boël, Emilie Farcy, Anne-Laure Charles, Bernard Geny, Jehan-Hervé Lignot.
Abstract:
Osmoregulating brachyura possess two groups of spatially segregated gills: anterior gills (AGs) serve purely respiratory purposes, while posterior gills (PGs) contain osmoregulatory structures. The presence of similar tissues serving partially different purposes provides a unique opportunity to study differential adaptation, in terms of free radical metabolism, upon salinity change, a subject which has only started to receive more attention in the later years. Here, we use Carcinus aestuarii, which osmoconforms down to 25ppm and hyper-regulates below this point. Crabs were immersed for two weeks in sea water (SW) (37ppm), diluted SW (10ppm) and concentrated SW (45ppm) and their AGs and PGs were analyzed for respiration rates, antioxidant activities (SOD, CAT), ROS production (measured through electron paramagnetic resonance (EPR) and fluorometrically using C-H2DFFDA on fresh hemolymph) and oxidative damage (measurement of lipofuscin-like granule accumulation and caspase 3/7 activities). Exposure to diluted-SW is the most stressful condition, elevating respiration rates of whole animals and ROS formation in hemolymph. PGs are the main tissues fueling osmoregulatory-related processes since their respiration rates in diluted-SW are 3.2-fold higher than in AGs, likely also determining their increased ROS-formation (1.4-fold greater) (EPR). Paradoxically, it is the AGs which suffer the consequences of diluted-SW exposure, as shown by the lipofuscin-like accumulation and caspase 3/7 activities, enhanced in AGs by 2–fold and 2.2–fold, respectively. This may only be possible due to the fact that PGs benefit from a high antioxidant protection (SOD in PGs is over 6 times higher than in AGs). In conclusion, we evidence that only PGs are clearly adapted to diluted-SW exposure and are capable of controlling the deleterious effects of the ROS production resulting from this insult. Furthermore, we speculate that as previously suggested in other models, ROS are involved in triggering this upregulation in antioxidant defense, a hypothesis that will be tested in further studies.
In 2nd International Conference on oxidative stress in aquatic ecosystems, Novemeber 2015, La Paz (Mexico). You may view the TALK by clicking HERE.
30-Aug-2015
New contribution: How do osmoregulating tissues cope with oxidative stress? A Mediterranean crab as a study model
Georgina A. Rivera-Ingraham, Kiam Barry, Mélanie Boël, Emilie Farcy, Anne-Laure Charles, Bernard Geny, Jehan-Hervé Lignot.
Abstract:
Green crabs are one of the most osmotically tolerant species in estuaries, and thus represent an interesting physiological model in osmoregulation studies. The Mediterranean crab Carcinus aestuarii, possess two groups of spatially segregated gills: anterior gills (pairs 1-6) serve purely respiratory purposes, while posterior gills (pairs 7-9) contain osmoregulatory structures. The presence of similar tissues serving partially different purposes provides a unique opportunity to study differential adaptation, in terms of free radical metabolism, upon salinity change. C. aestuarii were immersed for two weeks in sea water (SW) (37ppm), diluted SW (dSW, 10ppm) and concentrated SW (cSW, 45ppm). Measurements of hemolymph osmotic pressure indicate that the species is an osmoconformer down to 25ppm and hyper-regulates below this point. Animals acclimated at cSW show the lowest respiration rates (RR) and reactive oxygen species (ROS) formation (measured fluorometrically though C-H2DFFDA on hemolymph and through electron spin resonance, EPR, on tissues). Exposure to decreased environmental salinity leads to increased RR, reduced tolerance to hypoxia and increased circulating ROS levels. Further analyses on excised tissues show how posterior gills are responsible for the high RR and also ROS production (EPR). However, these posterior gills are able to avoid cellular damage by enhancing antioxidant activities (catalase and superoxide dismutase). Anterior gills, on the other hand, show no differences in antioxidant capacity among treatments which translates into a dramatic increase in caspase 3/7 activity (which for dSW-acclimated animals is 2.4-fold and 5-fold higher than those placed in SW and cSW, respectively). Our results support the hypothesis that a “preparation for oxidative stress” mechanism is put in place in osmoregulating gill tissues to avoid the negative consequences of FR formation derived from the increased metabolic rates fueling active ion pumping. Further research will be directed towards identifying which molecules are involved in signaling and triggering such an adaptation.
In International Conference on Comparative Physiology and Biochemistry, August 2015, Krakow (Poland). You may view the poster by clicking HERE.
30-Aug-2015
New contribution: Determination of free radical formation under environmental stress in aquatic invertebrates
Georgina A. Rivera-Ingraham, Iara Rocchetta, Emilie Farcy, Ulf Bickmeyer, Doris Abele.
Abstract:
In oxidative and nitrosative stress studies, the quantification of antioxidant defenses and oxidative damage are often not easily interpretable without a proper determination of reactive oxygen (ROS) and nitrogen (RNS) species production. The only quantitative way to measure ROS production is electron paramagnetic resonance (EPR). As EPR is often not available, live-imaging (LIT) techniques using ROS sensitive fluorescent dyes have become popular in free radical research. Using life imaging the cellular source structures of ROS formation can be identified and the underlying physiological processes can be experimentally determined. We studied the oxidative metabolism related to environmental change (notably salinity variations and hypoxia-reoxygenation) in invertebrate models. In our talk we provide an overview of results ranging from fluorometry of crab hemocytes, to confocal analysis of complete platyhelminths and ex-vivo bivalve gills. Based on our experience, we will also make an overview of the pros and cons of using such techniques, a matter which is strongly debated since some years.
In International Conference on Comparative Physiology and Biochemistry, August 2015, Krakow (Poland). You may view the presentation by clicking HERE.
3-Jul-2015
New contribution: Oxidative adaptation in osmoregulating tissue: how crab gills prepare for salinity-related stress?
Georgina A. Rivera-Ingraham, Kiam Barri, Mélanie Boël, Emilie Farcy, Jehan-Hervé Lignot.
Abstract:
Brachyuran green crabs are one of the most tolerant species in estuaries regarding salinity changes. The Mediterranean crab Carcinus aestuarii possess anterior gills (pairs 1-6) serving purely respiratory purposes and posterior gills (pairs 7-9) with well-developed osmoregulatory structures. Here, we describe how these gills respond to salinity changes. Crabs were left for two weeks in seawater (SW, 37ppm), diluted SW (dSW, 10ppm) and concentrated SW (cSW, 45ppm). Measurements of hemolymph osmotic pressure indicate that the species is osmoconforming down to 25ppm and hyper-regulates below this point. Animals in cSW show the lowest respiration rates (RR). Lower salinity leads to increased RR and reduced tolerance to hypoxia. Free radical (FR) formation (C-H2DFFDA staining of hemocytes) also increases with reduced salinity. There is no difference in antioxidant capacity (catalase and superoxide dismutase activities) between treatments in the anterior gills. However, posterior gills show a significant induction of these two enzymes with decreasing salinity, allowing them to avoid cellular damage. Therefore, anterior gills suffer from the osmoregulation activity of the posterior gills. It induces a dramatic increase in caspase 3/7 activity in the anterior gills of dSW animals with a 2.4-fold and 5-fold increase compared to SW and cSW animals, respectively.
We hypothesize that a “preparatory mechanism for oxidative stress” is put in place in osmoregulating gill tissue to avoid the negative consequence of FR formation derived from ion concentration changes in the hemolymph. Further research will be directed towards identifying which molecules are involved in signaling and triggering this adaptive mechanism.
In Society of Experimental Biology Meeting, June 2015, Prague (Chech). You may view the presentation by clicking HERE.
28-Nov-2014
New contribution: Exploring the role of free radicals in triggering physiological adaption to environmental changes
Georgina A. Rivera-Ingraham, Jehan-Hervé Lignot, Ulf Bickmeyer, Doris Abele.
Abstract:
A common consequence of exposure to abiotic changes is that they result, at some stage of exposure, in an increased production of free radicals (FR), reactive-oxygen species (ROS) and reactive-nitrogen species (RNS) that occur in aerobically functioning cells as by-products of cellular respiration and of several cellular defense and detoxification mechanisms. Accumulation of FR derives in “oxidative stress” (OS), which has been widely demonstrated to be directly linked with organismal fitness and cellular/organismal senescence. But since the beginning of the 21st century, many works have demonstrated that FRs are more than harmful molecules and that can also be involved in important physiological functions, such as muscle relaxation, immune reactions, signal transduction, apoptotic cell death, homeostasis and induction of adaptive mechanisms.
In this presentation I will introduce our new ongoing project that aims to explain the mechanisms underlying the magnificent capabilities of Macrostomum lignano to withstand changes in environmental salinity, but also explore if ROS or RNS may be involved in signaling such changes. M. lignano being an intertidal species is highly tolerant to physical environmental changes such as oxygen, temperature and salinity variations. Furthermore, making use of this small and transparent organism allows us to avoid the traditional studies using isolated cells and tissues and instead conduct in-vivo live-imaging. Using these techniques we expect to provide a unique and organismic perspective in the subject of FR signaling. We will describe the preliminary results, using not only salinity but also hypoxia as environmental stressors, which have set the basis and scope of the project, and we will make an overview of our methodological approach to the subject and our expected results.
In 8th International Macrostomum Meeting, November 2014, Basel (Switzerland). You may view the presentation by clicking HERE.
05-Nov-2014
New contribution: Live-imaging of oxygen radical formation in ex-vivo gills after exposure to anoxia and anoxia-reoxygenation: physiological consequences for a hypoxia tolerant bivalve.
Georgina A. Rivera-Ingraham, Iara Rocchetta, Stephanie Meyer, Doris Abele.
Abstract:
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. In a first approach, we applied live-imaging techniques 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 under normoxic conditions. Our results indicate that in the ciliated epithelial cells composing the gill filaments, two mitochondrial clusters with distinct membrane potential (Δψm) (JC-1 staining) coexist, where cilia-associated mitochondria show the weakest polarization. Along average gill filaments, superoxide (O2•-: DHE staining, 2-OH-E+ fluorescence) concentrates in the outermost areas of the ciliated epithelial cells of the filaments, which also have the highest mitochondrial densities (MTK Deep Red 633 staining). Contrary, peroxide (H2O2), hydroxyl radical (HOO•) and peroxinitrite (ONOO-) (assessed through C-H2DFFDA staining, DCF fluorescence) mainly formed within the blood sinus of the gill filaments and are likely to be produced by hemocytes as defense against invading pathogens.
In a second approach, we exposed mussels to experimental short (48h) and prolonged (72h) anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on ROS and RNS formation. 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 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. Both 2-OH-E+ and DCF fluorescence decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS/RNS burst induced lipid peroxidation in the mantle (assessed by TBARS measurement), 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 work that directly measures ROS/RNS formation ex-vivo during anoxia reoxygenation in isolated mussel gills.
Our results open new perspectives for unraveling the different ecophysiological roles of ROS in gills and we conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under experimental hypoxia reoxygenation exposure.
In Physiomar 14, November 2014, La Serena (Chile). You may download the poster by clicking HERE.
07-Nov-2014
New contribution: Adaptations to low tissue pO2 and the molecular stress response in bivalves.
Doris Abele, Georgina A. Rivera-Ingraham, Cyril Dégletagne, Iara Rocchetta, Julia Strahl, Silvia Hardenberg, Eva Philipp.
Abstract: Maximum performance, such as highest respiration and heart rates, or pumping activities are often interpreted as marking the range of “optimal physiological performance” in marine species. However, many bivalves are highly tolerant of hypoxic conditions, and often self-induce low oxygen partial pressure in shell water and hemolymph compared to the outside medium. Maximum heart beat and pumping rates are seldom observed and either mark stress conditions or episodic high scope for activity. We have recently studied patterns of shell water oxygenation in different mobile and sessile bivalve ecotypes and found all species to reduce internal shell water pO2 against normoxic levels in the surrounding seawater, often with a species specific oscillation pattern between high and low pO2. Alternations of tissue pO2 between hypoxia and reoxygenation produce oxidative stress in mammals. Contrary, hypoxia tolerant species are hypothesized to upregulate antioxidant systems during hypoxia in “preparation for reoxygenation” to avoid oxidative stress and damage.
We have investigated mitochondrial functioning, reactive oxygen species production, and stress gene expression in response to hypoxia and reoxygenation in bivalve gills and mantle tissues. Mitochondria of ciliated cells in the gill periphery of blue mussels appear loosely coupled contrary to mitochondria in the central gills. [G1] Somewhat counterintuitively we observed that ADP addition reduces oxygen uptake of mitochondria in permeabilized gills, whereas trials with permeabilized mantle tissue show the expected increase of respiration after ADP addition. ROS formation in gills decreased in hypoxia and increased upon reoxygenation, but we found no evidence for the upregulation of antioxidants during reoxygenation. We further tested the response in stress and antioxidant gene expression to hypoxia and anoxia in two populations of the ocean quahog Arctica islandica from environments with differing natural stress levels and found that only animals from the high stress environment (Baltic Sea) induce stress genes under severe anoxic stress, whereas no gene induction occurred in North Sea Arctica.
In my talk I will summarize knowledge of the stress response to hypoxia/anoxia and reoxygenation, and discuss our concept of the functioning of the gill in the adjustment of tissue oxygenation against the fluctuating oxygen content in shell water.
In Physiomar 14, November 2014, La Serena (Chile).
12-Sept-2013
New publication: Oxygen radical formation in anoxic transgression and anoxia-reoxygenation:Foe or phantom? Experiments with a hypoxia tolerant bivalve.
Georgina A. Rivera-Ingraham, Iara Rocchetta, Stefanie Meyer & Doris Abele