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Exposure to environmental changes often results in the production of reactive oxygen species (ROS), which, if uncontrolled, leads to loss of cellular homeostasis and oxidative distress. However, at physiological levels these same ROS are known to be key players in cellular signaling and the regulation of key biological activities (oxidative eustress). While ROS are known to mediate salinity tolerance in plants, little is known for the animal kingdom. In this study, we use the Mediterranean crab Carcinus aestuarii, highly tolerant to salinity changes in its environment, as a model to test the healthy or pathological role of ROS due to exposure to diluted seawater (dSW). Crabs were injected either with an antioxidant [N-acetylcysteine (NAC), 150 mg·kg -1 ] or phosphate buffered saline (PBS). One hour after the first injection, animals were either maintained in seawater (SW) or transferred to dSW and injections were carried out at 12-h intervals. After ≈48 h of salinity change, all animals were sacrificed and gills dissected for analysis. NAC injections successfully inhibited ROS formation occurring due to dSW transfer. However, this induced 55% crab mortality, as well as an inhibition of the enhanced catalase defenses and mitochondrial biogenesis that occur with decreased salinity. Crab osmoregulatory capacity under dSW condition was not affected by NAC, although it induced in anterior (non-osmoregulatory) gills a 146-fold increase in Na + /K + /2Cl - expression levels, reaching values typically observed in osmoregulatory tissues. We discuss how ROS influences the physiology of anterior and posterior gills, which have two different physiological functions and strategies during hyper-osmoregulation in dSW., Competing Interests: Declaration of competing interest No competing interests declared., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypometabolism is a common strategy employed by resilient species to withstand environmental stressors that would be life-threatening for other organisms. Under conditions such as hypoxia/anoxia, temperature and salinity stress, or seasonal changes (e.g. hibernation, estivation), stress-tolerant species down-regulate pathways to decrease energy expenditures until the return of less challenging conditions. However, it is with the return of these more favorable conditions and the reactivation of basal metabolic rates that a strong increase of reactive oxygen and nitrogen species (RONS) occurs, leading to oxidative stress. Over the last few decades, cases of species capable of enhancing antioxidant defenses during hypometabolic states have been reported across taxa and in response to a variety of stressors. Interpreted as an adaptive mechanism to counteract RONS formation during tissue hypometabolism and reactivation, this strategy was coined "Preparation for Oxidative Stress" (POS). Laboratory experiments have confirmed that over 100 species, spanning 9 animal phyla, apply this strategy to endure harsh environments. However, the challenge remains to confirm its occurrence in the natural environment and its wide applicability as a key survival element, through controlled experimentation in field and in natural conditions. Under such conditions, numerous confounding factors may complicate data interpretation, but this remains the only approach to provide an integrative look at the evolutionary aspects of ecophysiological adaptations. In this review, we provide an overview of representative cases where the POS strategy has been demonstrated among diverse species in natural environmental conditions, discussing the strengths and weaknesses of these results and conclusions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Physiological and morphological acclimation capacities of black-chinned tilapia, Sarotherodon melanotheron were studied from fish to gill cell level when fish are maintained in freshwater, seawater, and hypersaline conditions. Fish osmoregulatory capacity, gill ionocyte morphology, osmo-respiratory compromise, O 2 consumption rate, branchial antioxidative defense, and cell apoptosis were considered. Captive juvenile tilapias were maintained in controlled freshwater conditions (FW: 0.4 ppt; 12 mOsm kg -1 ) or gradually transferred to seawater (SW: 32 ppt; 958 mOsm kg -1 ) and concentrated SW (cSW: 65 ppt; 1920 mOsm kg -1 ). After 15 days in these conditions, blood osmolality and chloride ion concentration were determined. Gill ionocyte density and morphology were measured using immunolabelled histological sections to specifically detect the sodium pump (NKA). Gill osmo-respiratory compromise was also calculated along with oxygen consumption rates from normoxic to hypoxic conditions from excised gills (indirect respirometry). Finally, catalase and caspase 3/7activities were recorded from gill extracts. Results indicate that elevated salinity induces an osmotic imbalance and a profound morphological change with proliferating and hypertrophied ionocytes. This thickening of the gill interlamellar cell mass and the shortening of the lamellae induce a reduced osmo-respiratory ratio and reduced respiratory capacity under both normoxic and hypoxic conditions. Although salinity changes do not affect one of the major antioxidative defense mechanism, it strongly affects apoptosis that appears the most elevated in SW. However, in freshwater condition, fish can maintain their osmotic balance with a low ionocyte density, a low apoptotic level and a drastically reduced O 2 consumption in normoxic condition that is maintained in hypoxia. Therefore, S. melanotheron presents the typical functional remodeling due to environmental salinity changes ranging from FW to SW. However, elevated seawater induces major cellular stress inducing a profound gill morphofunctional dysfunctioning. While cell apoptosis is reduced, ionocyte proliferation is massively increased with impaired osmotic regulation and reduced O 2 consumption both in normoxic and hypoxic conditions., (© 2024 The Authors. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

Mangrove crabs are ecosystem engineers through their bioturbation activity. On Mayotte Island, the abundance of Neosarmatium africanum decreased in wastewater-impacted areas. Previous analyses showed that global crab metabolism is impacted by wastewater, with a burst in O 2 consumption that may be caused by osmo-respiratory trade-offs since gill functioning was impacted. As the hepatopancreas is a key metabolic organ, the purpose of this study was to investigate the physiological effects of wastewater and ammonia-N 5-h exposure on crabs to better understand the potential trade-offs underlying the global metabolic state. Catalase, superoxide dismutase, glutathione S-transferase, total digestive protease, and serine protease (trypsin and chymotrypsin) activities were assessed. Histological analyses were performed to determine structural modifications. No effect of short-term wastewater and ammonia-N exposure was found in antioxidant defenses or digestive enzyme activity. However, histological changes of B-cells indicate an increase in intracellular digestive activity through higher vacuolization processes and tubule dilation in wastewater-exposed crabs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The scallop Argopecten purpuratus is an important resource for Chilean and Peruvian aquaculture. Seed availability from commercial hatcheries is critical due to recurrent massive mortalities associated with bacterial infections, especially during the veliger larval stage. The immune response plays a crucial role in counteracting the effects of such infections, but being energetically costly, it potentially competes with the physiological and morphological changes that occur during early development, which are equally expensive. Consequently, in this study, energy metabolism parameters at the individual and cellular levels, under routine-basal status and after the exposure to the pathogenic strain bacteria ( Vibrio splendidus VPAP18), were evaluated during early ontogeny (trochophore, D-veliger, veliger, pediveliger, and early juveniles) of A. purpuratus . The parameters measured were as follows: (1) metabolic demand, determined as oxygen consumption rate and (2) ATP supplying capacity measured by key mitochondrial enzymes activities [citrate synthase (CS), electron transport system (ETS), and ETS/CS ratio, indicative of ATP supplying efficiency], mitochondrial membrane potential (ΔΨm), and mitochondrial density ( ρ m ) using an in vivo image analysis. Data revealed that metabolic demand/capacity varies significantly throughout early development, with trochophores being the most efficient in terms of energy supplying capacity under basal conditions. ATP supplying efficiency decreased linearly with larval development, attaining its lowest level at the pediveliger stage, and increasing markedly in early juveniles. Veliger larvae at basal conditions were inefficient in terms of energy production vs. energy demand (with low ρ m , ΔΨm, enzyme activities, and ETS:CS). Post-challenged results suggest that both trochophore and D-veliger would have the necessary energy to support the immune response. However, due to an immature immune system, the immunity of these stages would rely mainly on molecules of parental origin, as suggested by previous studies. On the other hand, post-challenged veliger maintained their metabolic demand but decreased their ATP supplying capacity, whereas pediveliger increased CS activity. Overall, results suggest that veliger larvae exhibit the lowest metabolic capacity to overcome a bacterial challenge, coinciding with previous works, showing a reduced capacity to express immune-related genes. This would result in a higher susceptibility to pathogen infection, potentially explaining the higher mortality rates occurring during A. purpuratus farming., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rojas, Rivera-Ingraham, Cárcamo, Jeno, de la Fuente-Ortega, Schmitt and Brokordt.)

The role of ecophysiology in mediating marine biological pollution is poorly known. Here we explore how physiological plasticity to environmental stress can explain and predict the context-dependencies of invasive species impacts. We use the case of two sympatric skeleton shrimps, the invader Caprella scaura and its congener C. equilibra, which is currently replaced by the former on the South European coast. We compare their physiological responses to hyposalinity stress under suboptimal low and high temperature, while inferring on hypoxia tolerance. We use an energy-redox approach, analyzing mortality rate, the energetic balance and the consequent effects on the oxidative homeostasis. We found that decreased seawater salinity and/or oxygen levels can weaken biotic resistance, especially in females of C. equilibra, leading to periods of heightened vulnerability to invasion. Our approach provides mechanistic insights towards understanding the factors promoting invader impacts, highlighting the potential of ecophysiology for improving invasive species management., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Space launchers often use aluminized-solid fuel ("propergol") as propellant and its combustion releases tons of Al 2 O 3 and HCl that sink in terrestrial and aquatic environments, polluting and decreasing water pH. We studied the impact of these events on the biochemical/physiological performance of the freshwater shrimp Macrobrachium jelskii, with wild specimens collected from a non-impacted site in French Guiana. In the laboratory, shrimps were exposed for one week to: i) undisturbed conditions; ii) Al 2 O 3 exposure (0.5 mg L -1 ) at normal pH (6.6); iii) decreased pH (4.5) (mimicking HCl release in the environment) with no Al 2 O 3 ; or iv) Al 2 O 3 0.5 mg L -1 and pH 4.5, representing the average conditions found in the water bodies around the Ariane 5 launch pad. Results showed that shrimps bioaccumulated aluminium (Al) regardless of water pH. The combined effect of Al 2 O 3 and low pH caused the most impact: acetylcholinesterase and carboxylesterase activities decreased, indicating neurotoxicity and reduced detoxification capacity, respectively. Animal respiration was enhanced with Al 2 O 3 and pH variations alone, but the synergic interaction of both stressors caused respiration to decrease, suggesting metabolic depression. Oxidative damage followed a similar pattern to respiration rates across conditions, suggesting free radical-mediation in Al toxicity. Antioxidant activities varied among enzymes, with glutathione reductase being the most impacted by Al 2 O 3 exposure. This study shows the importance of addressing space ports' impact on the environment, setting the bases for selecting the most appropriate biomarkers for future monitoring programs using a widespread and sensitive crustacean in the context of an increasing space-oriented activity across the world., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mussels are worldwide bioindicators in pollution monitoring since they fulfil the requirements for being good sentinels. However, some methodological concerns arise in the use of particular biomarkers, particularly those displaying low enzymatic rates and/or limited responsiveness to chemicals and biological-related variability. In the present study, the suitability of oxidative stress and detoxification parameters when using mussels as sentinels of polycyclic aromatic hydrocarbon (PAH) pollution is addressed. Present results show that the S9 subcellular fraction of the digestive gland in mussels is an adequate and convenient matrix where to measure most pollution-related biomarkers. Furthermore, this work constitutes the first evidence of the potential suitability of using particular carboxylesterase (CE) activities in determining PAHs exposure in mussels. This fact could imply the replacement of more controversial cytochrome P450 components (phase I oxidation), which are only measurable in microsomal fractions, by CEs (measured in S9 fractions) as good alternatives for phase I reactions in PAH-exposed mussels. Some methodological considerations, such as the need of including commercial purified proteins in biomarker determinations for quality assurance, are evaluated.

Mangroves are tidal wetlands that are often under strong anthropogenic pressures, despite the numerous ecosystem services they provide. Pollution from urban runoffs is one such threats, yet some mangroves are used as a bioremediation tool for wastewater (WW) treatment. This practice can impact mangrove crabs, which are key engineer species of the ecosystem. Using an experimental area with controlled WW releases, this study aimed to determine from an ecological and ecotoxicological perspective, the effects of WW on the red mangrove crab Neosarmatium africanum. Burrow density and salinity levels (used as a proxy of WW dispersion) were recorded, and a 3-week caging experiment was performed. Hemolymph osmolality, gill Na + /K + -ATPase (NKA) activity and gill redox balance were assessed in anterior and posterior gills of N. africanum. Burrow density decreased according to salinity decreases around the discharged area. Crabs from the impacted area had a lower osmoregulatory capacity despite gill NKA activity remaining undisturbed. The decrease of the superoxide dismutase activity indicates changes in redox metabolism. However, both catalase activity and oxidative damage remained unchanged in both areas but were higher in posterior gills. These results indicate that WW release may induce osmoregulatory and redox imbalances, potentially explaining the decrease in crab density. Based on these results we conclude that WW release should be carefully monitored as crabs are key players involved in the bioremediation process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a "preparation for oxidative stress" (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 ( O 2 ∙ - ) formation in hypoxia-exposed gill filaments. Thirty minutes of moderate (7 kPa pO 2 ) and severe hypoxia (1 kPa pO 2 ) 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 O 2 ∙ - 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 pO 2 -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 O 2 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 O 2 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 O 2 variations.

Immune responses, as well as reproduction, are energy-hungry processes, particularly in broadcast spawners such as scallops. Thus, we aimed to explore the potential reproduction-immunity trade-off in Argopecten purpuratus , a species with great economic importance for Chile and Peru. Hemocytes, key immunological cells in mollusks, were the center of this study, where we addressed for the first time the relation between reproductive stage, hemocyte metabolic energetics and their capacity to support immune responses at cellular and molecular levels. Hemocyte metabolic capacity was assessed by their respiration rates, mitochondrial membrane potential and citrate synthase (CS) activity. Cellular immune parameters such as the number of circulating and tissue-infiltrating hemocytes and their reactive oxygen species (ROS) production capacity were considered. Molecular immune responses were examined through the transcriptional levels of two pattern recognition receptors ( ApCLec and ApTLR ) and two anti-microbial effectors ( ferritin and big defensin ). Their expressions were measured in hemocytes from immature, matured and spawned scallops under basal, and one of the following challenges: (i) in vitro , where hemocytes were challenged with the β glucan zymosan, to determine the immune potentiality under standardized conditions; or (ii) in vivo challenge, using hemocytes from scallops injected with the pathogenic bacteria Vibrio splendidus . Results indicate a post-spawning decrease in the structural components of the immune system (hemocyte number/quality) and their potential capacity of performing immune functions (with reduced ATP-producing machinery and exhaustion of energy reserves). Both in vitro and in vivo challenges demonstrate that hemocytes from immature scallops have, in most cases, the best metabolic potential (increased CS activity) and immune performances, with for example, over threefold higher ROS production and tissue-infiltration capacity than those from mature and spawned scallops after the bacterial challenge. Agreeing with cellular responses, hemocytes from immature individuals induced the highest levels of immune receptors and antimicrobial effectors after the bacterial challenge, while spawned scallops presented the lowest values. Overall, results suggest a trade-off between resource allocation in reproduction and the immune responses in A. purpuratus , with hemocyte energy metabolic capacity potentially underlying cellular and molecular immune responses. Further research would be necessary to explore regulatory mechanisms such as signaling pleiotropy which may potentially be underlying this trade-off.

Dimitri Theuerkauff, Georgina A. Rivera-Ingraham, Jonathan A.C. Roques, Laurence Azzopardi, Marine Bertini, Mathilde Lejeune, Emilie Farcy, Jehan-Hervé Lignot, and Elliott Sucré (2018) Salinity is one of the main environmental factors determining coastal species distribution. However, in the specific case of mangrove crabs, salinity selection cannot be understood through ecological approaches alone. Yet understanding this issue is crucial in the context of mangrove conservation, since this ecosystem is often used as biofilter of (low- salinity) wastewater. Crabs are keystone species in this mangrove ecosystem and are differentially affected by salinity. We hypothesize that crab salinity selection may be partly explained by specific salinity-induced physiological constraints associated with osmoregulation, energy and redox homeostasis. To test this, the response to salinity variation was analysed in two landward mangrove crabs: the fiddler crab Tubuca urvillei , which inhabits low-salinity areas of the mangrove, and the red mangrove crab Neosarmatium meinerti , which lives in areas with higher salinity. Results confirm that both species are strong hypo-/hyper-osmoregulators that deal easily with large salinity variations. Such shifts in salinity do not induce changes in energy expenditure (measured as oxygen consumption) or in the production of reactive oxygen species. However, T. urvillei is physiologically suited to habitats with brackish water, since it presents i) high hemolymph osmolalities over a wider range of salinities and lower osmoregulatory capacity in seawater, ii) high Na+/K+-ATPase (NKA) activity in the posterior osmoregulatory gills and iii) a thicker osmoregulatory epithelium along the posterior gill lamellae. Therefore, while environmental salinity alone cannot directly explain fiddler and red mangrove crab distributions, our data suggest that salinity selection is indeed influenced by specific physiological adjustments.

Mangroves are increasingly used as biofiltering systems of (pre-treated) domestic effluents. However, these wastewater discharges may affect local macrofauna. This laboratory study investigates the effects of wastewater exposure on the mangrove spider crab Neosarmatium meinerti, a key engineering species which is known to be affected by waste waters in effluent-impacted areas. These effects were quantified by monitoring biological markers of physiological state, namely oxygen consumption, the branchial cavity ventilation rate, gill physiology and morphology, and osmoregulatory and redox balance. Adults acclimated to clean seawater (SW, 32 ppt) and freshwater (FW, ∼0 ppt) were compared to crabs exposed to wastewater for 5 h (WW, ∼0 ppt). Spider crabs exposed to WW increased their ventilation and whole-animal respiration rates by 2- and 3-fold respectively, while isolated gill respiration increased in the animals exposed to FW (from 0.5 to 2.3 and 1.1 nmol O 2 min -1  mg DW -1 for anterior and posterior gills, respectively) but was not modified in WW-exposed individuals. WW exposure also impaired crab osmoregulatory capacity; an 80 mOsm kg -1 decrease was observed compared to FW, likely due to decreased branchial NKA activity. ROS production (DCF fluorescence in hemolymph), antioxidant defenses (superoxide dismutase and catalase activities) and oxidative damage (malondialdehyde concentration) responses varied according to animal gender. Overall, this study demonstrates that specific physiological parameters must be considered when focusing on crabs with bimodal breathing capacities. We conclude that spider crabs exposed to WW face osmoregulatory imbalances due to functional and morphological gill remodeling, which must rapidly exhaust energy reserves. These physiological disruptions could explain the ecological changes observed in the field., (Copyright © 2018. Published by Elsevier B.V.)

Osmoregulation is by no means an energetically cheap process, and its costs have been extensively quantified in terms of respiration and aerobic metabolism. Common products of mitochondrial activity are reactive oxygen and nitrogen species, which may cause oxidative stress by degrading key cell components, while playing essential roles in cell homeostasis. Given the delicate equilibrium between pro- and antioxidants in fueling acclimation responses, the need for a thorough understanding of the relationship between salinity-induced oxidative stress and osmoregulation arises as an important issue, especially in the context of global changes and anthropogenic impacts on coastal habitats. This is especially urgent for intertidal/estuarine organisms, which may be subject to drastic salinity and habitat changes, leading to redox imbalance. How do osmoregulation strategies determine energy expenditure, and how do these processes affect organisms in terms of oxidative stress? What mechanisms are used to cope with salinity-induced oxidative stress? This Commentary aims to highlight the main gaps in our knowledge, covering all levels of organization. From an energy-redox perspective, we discuss the link between environmental salinity changes and physiological responses at different levels of biological organization. Future studies should seek to provide a detailed understanding of the relationship between osmoregulatory strategies and redox metabolism, thereby informing conservation physiologists and allowing them to tackle the new challenges imposed by global climate change., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Patellogastropods, also known as true limpets, are distributed throughout the world and constitute key species in coastal ecosystems. Some limpet species achieve remarkable sizes, which in the most extreme cases can surpass 35cm in shell length. In this review, we focus on giant limpets, which are defined as those with a maximum shell size surpassing 10cm. According to the scientific literature, there are a total of 14 species across five genera that reach these larger sizes. Four of these species are threatened or in danger of extinction. Inhabiting the intertidal zones, limpets are frequently affected by anthropogenic impacts, namely collection by humans, pollution and habitat fragmentation. In the case of larger species, their conspicuous size has made them especially prone to human collection since prehistoric times. Size is not phylogeny-dependent among giant limpets, but is instead related to behavioural traits instead. Larger-sized species tend to be nonmigratory and territorial compared to those that are smaller. Collection by humans has been cited as the main cause behind the decline and/or extinction of giant limpet populations. Their conspicuously large size makes them the preferred target of human collection. Because they are protandric species, selectively eliminating larger specimens of a given population seriously compromises their viability and has led to local extinction events in some cases. Additionally, sustained collection over time may lead to microevolutionary responses that result in genetic changes. The growing presence of artificial structures in coastal ecosystems may cause population fragmentation and isolation, limiting the genetic flow and dispersion capacity of many limpet species. However, when they are necessitated, artificial structures could be managed to establish marine artificial microreserves and contribute to the conservation of giant limpet species that naturally settle on them., (© 2017 Elsevier Ltd. All rights reserved.)

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 them outstanding 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 gluthathione-S-transferase pi 1 (GSTP1) 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 GSTP1 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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Intertidal species are more 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 localities were surveyed, one within a natural and unpolluted area and the other two within the harbor of Ceuta (Strait of Gibraltar), on breakwaters outside and inside. The specimens collected inside the harbor reached 3-fold higher Hg content than for those incoming from the natural area. PERMANOVA test indicated that metal composition of the specimens from inside the harbor was 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 the most vulnerable populations, establishing eco-physiological protocols for monitoring and managing populations settled on artificial substrata., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2 weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress., (© 2016. Published by The Company of Biologists Ltd.)

Organisms that tolerate wide variations in oxygen availability, especially to hypoxia, usually face harsh environmental conditions during their lives. Such conditions include, for example, lack of food and/or water, low or high temperatures, and reduced oxygen availability. In contrast to an expected strong suppression of protein synthesis, a great number of these animals present increased levels of antioxidant defenses during oxygen deprivation. These observations have puzzled researchers for more than 20 years. Initially, two predominant ideas seemed to be irreconcilable: on one hand, hypoxia would decrease reactive oxygen species (ROS) production, while on the other the induction of antioxidant enzymes would require the overproduction of ROS. This induction of antioxidant enzymes during hypoxia was viewed as a way to prepare animals for oxidative damage that may happen ultimately during reoxygenation. The term "preparation for oxidative stress" (POS) was coined in 1998 based on such premise. However, there are many cases of increased oxidative damage in several hypoxia-tolerant organisms under hypoxia. In addition, over the years, the idea of an assured decrease in ROS formation under hypoxia was challenged. Instead, several findings indicate that the production of ROS actually increases in response to hypoxia. Recently, it became possible to provide a comprehensive explanation for the induction of antioxidant enzymes under hypoxia. The supporting evidence and the limitations of the POS idea are extensively explored in this review as we discuss results from research on estivation and situations of low oxygen stress, such as hypoxia, freezing exposure, severe dehydration, and air exposure of water-breathing animals. We propose that, under some level of oxygen deprivation, ROS are overproduced and induce changes leading to hypoxic biochemical responses. These responses would occur mainly through the activation of specific transcription factors (FoxO, Nrf2, HIF-1, NF-κB, and p53) and post translational mechanisms, both mechanisms leading to enhanced antioxidant defenses. Moreover, reactive nitrogen species are candidate modulators of ROS generation in this scenario. We conclude by drawing out the future perspectives in this field of research, and how advances in the knowledge of the mechanisms involved in the POS strategy will offer new and innovative study scenarios of biological and physiological cellular responses to environmental stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Patella ferruginea is a giant patellid limpet endemic to the Mediterranean Sea. It presently is in danger of extinction, and many have called for developing conservation measures including the mass production of spats for re-introduction projects. However, so far all attempts have been relatively unsuccessful. Previous work analyzing the effects of gamma-aminobutyric acid (GABA) on the recruitment of patellid limpets has shown that larvae respond to the presence of this signaling molecule. This response could explain the gregarious distribution typical of this species. In the present study, we demonstrated that GABA is naturally secreted by P. ferruginea in the pedal mucus. GABA is preferentially secreted during the summer, coinciding with the reproductive resting period of the species. Further research should aim to analyze the effects of GABA on larval development and metamorphosis in order to assess its potential use to improve conservation efforts.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The respiration rate of meiofauna is difficult to measure, and the response to variations in the environmental oxygen concentration has so far been mainly addressed through behavioral investigation. We investigated the effect of different oxygen concentrations on the physiology of the marine platyhelminth Macrostomum lignano. Respiration was measured using batches of 20 animals in a glass microtiter plate equipped with optical oxygen sensor spots. At higher oxygen saturations (>12 kPa), the animals showed a clear oxyconforming behavior. However, below this value, the flatworms kept respiration rates constant at 0.064±0.001 nmol O2 l(-1) h(-1) individual(-1) down to 3 kPa PO2, and this rate was increased by 30% in animals that were reoxygenated after enduring a period of 1.5 h in anoxia. Physiological changes related to tissue oxygenation were assessed using live imaging techniques with different fluorophores in animals maintained in normoxic (21 kPa), hyperoxic (40 kPa) or near-anoxic (~0 kPa) conditions and subjected to anoxia-reoxygenation. The pH-sensitive dyes Ageladine-A and BCECF both indicated that pHi under near-anoxia increases by about 0.07-0.10 units. Mitochondrial membrane potential, Δψm, was higher in anoxic and hyperoxic than in normoxic conditions (JC1 dye data). Staining with ROS-sensitive dyes - DHE for detection of superoxide anion (O2•(-)) formation and C-H DFFDA for other ROS species aside from O2•(-) (H2O2, HOO• and ONOO) - showed increased ROS formation following anoxia-reoxygenation treatment. Animals exposed to hyperoxic, normoxic and anoxic treatments displayed no significant differences in O2•(-) formation, whereas mitochondrial ROS formation as detected by C-H2DFFDA was higher after hyperoxic exposure and lowest under near-anoxia conditions compared with the normoxic control group. Macrostomum lignano seems to be a species that is tolerant of a wide range of oxygen concentrations (being able to maintain aerobic metabolism from extremely low PO2 up to hyperoxic conditions), which is an essential prerequisite for successfully dealing with the drastic environmental oxygen variations that occur within intertidal sediments.

Patella ferruginea Gmelin, 1791 is an endangered marine gastropod endemic to the Western Mediterranean. Its range is restricted to the Sardinian-Corsican region (SCR), North Africa, a few scattered sites in Southern Spain, and Sicily. Inter-simple sequence repeat (ISSR) markers and three different mitochondrial DNA (mtDNA) regions, Cytochrome c Oxidase subunit I, 12S (small-subunit ribosomal RNA gene) and 16S (large-subunit ribosomal RNA gene), were used to investigate the presence of genetic population structuring. The mtDNA sequences showed very low levels of genetic differentiation. Conversely, ISSRs showed the presence of two main genetic groups, corresponding to Spain, North Africa and Sicily and the SCR. The SCR was further split into two subgroups. The ISSR results suggest that, on a regional scale, the genetic structure of P. ferruginea is mainly determined by the restriction of gene flow by dispersal barriers. On a more local scale human harvesting may play a crucial role in population structuring by increasing the effect of genetic drift.

Many neurotransmitters, such as γ-amino butyric acid (GABA), can act as chemical cues influencing settlement and metamorphosis in benthic marine invertebrates. This effect has been described especially in mollusks, such as mussels, clams, or haliotids. This study describes the first record of the effect of GABA on patellogastropod populations. Special attention was paid to the effect of the compound on recruitment processes. The experiment was carried out using 10 × 10 cm artificial limestone plates that were drilled into intertidal rocks at different inclinations, and periodically treated with a 1 mM GABA solution. A total of five limpet species was considered (four patellid limpet species and the pulmonate Siphonaria pectinata). Each individual recorded on the plates as well as within a 20 × 20 cm quadrant was measured, identified to species level, and its straight-line distance to the application point was registered. Treated surfaces were the first to possess both adults and recruits. Individuals also were found in a higher number around GABA-treated plates than around controls. The results indicated that the compound may not only enhance recruitment, but also might accelerate it. Recruits were located at higher distances from GABA treated plates than from control surfaces. This supports the hypothesis that this is the life history stage most sensitive to the compound. The behavior shown by the individuals belonging to the two endangered patellid species present in the study area (Patella ferruginea and Cymbula nigra) also were analyzed. For the former, the results indicated that the use of GABA may have similar effects on recruitment similar to the presence of adult conspecifics. This is the first report of the effect of GABA on patellid limpet recruitment and population dynamics. Conservational implications of the results are discussed.