Two faces of one coin: Beneficial and deleterious effects of reactive oxygen species during short-term acclimation to hypo-osmotic stress in a decapod crab.

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.

Individual metabolism and behaviour as complementary endpoints to better understand mangrove crab community variations linked to wastewater inputs.

Mangrove forests are impacted by a large range of anthropogenic activities that challenge their functioning. For example, domestic wastewater (WW) discharges are known to increase vegetation growth but recent studies indicate that they have negative effects on benthic macrofauna, especially on mangrove crabs, these ecosystem engineers playing a key role on the functioning of the mangrove. In experimental areas regularly receiving WW at low tide (Mayotte Island, Indian Ocean), a drastic decrease in burrowing crab density has been reported. In this context, the individual behavioural and physiological responses of the fiddler crab Paraleptuca chlorophthalmus exposed to short-term (6 h) pulse of WW and ammonia-N (as a potential proxy of WW) were investigated. This species is one of the most sensitive to WW within the mangrove crab community. For the behavioural experiment, crabs could choose between the aquatic and aerial environment. Individual metabolic rate (O2 consumption) was monitored after 6 h of exposure in WW or ammonia-N. Aerobic and anaerobic metabolic markers (citrate synthase and lactate dehydrogenase activities, respectively) were also evaluated. Results indicate that crabs exposed to WW are more active and mobile than controls after 3 h. Crabs actively emersed from WW and reduced their activity and mobility after 6 h. A higher metabolic rate in WW occurred immediately (t = 0 h), 3 and 6 h after WW exposure, with also, a burst in aerobic bacterial consumption in WW, but no effect of ammonia-N. No effect of WW or ammonia-N was observed on enzymatic aerobic and anaerobic metabolic markers. Therefore, short-term pulses with domestic polluted wastewater trigger quick behavioural and metabolic responses that could be deleterious if prolonged. These results could contribute to the understanding of the community-scale changes observed in benthic macrofauna after several years of regular domestic pollution pulses.

Morphological and physiological traits of Mediterranean sticklebacks living in the Camargue wetland (Rhone river delta).

Three-spined sticklebacks (Gasterosteus aculeatus L.) living at the southern limit of the species distribution range could possess specific morphological and physiological traits that enable these fish to live at the threshold of their physiological capacities. Morphological analysis was carried out on samples of sticklebacks living in different saline habitats of the Camargue area (Rhone delta, northern Mediterranean coast) obtained from 1993 to 2017. Salinity acclimation capacities were also investigated using individuals from freshwater-low salinity drainage canals and from mesohaline-euryhaline lagoons. Fish were maintained in laboratory conditions at salinity values close to those of their respective habitats: low salinity (LS, 5‰) or seawater (SW, 30‰). Fish obtained from a mesohaline brackish water lagoon (BW, 15‰) were acclimated to SW or LS. Oxygen consumption rates and branchial Na+ /K+ -ATPase (NKA) activity (indicator of fish osmoregulatory capacity) were measured in these LS or SW control fish and in individuals subjected to abrupt SW or LS transfers. At all the studied locations, only the low-plated "leiurus" morphotype showed no spatial or temporal variations in their body morphology. Gill rakers were only longer and denser in fish sampled from the LS-freshwater (FW) drainage canals. All fish presented similar physiological capacities. Oxygen consumption rates were not influenced by salinity challenge except in SW fish transferred to LS immediately and 1 h after transfer. However, and as expected, gill NKA activity was salinity dependent. Sticklebacks of the Camargue area sampled from habitats with contrasted saline conditions are homogenously euryhaline, have low oxygen consumption rates and do not appear to experience significantly greater metabolic costs when challenged with salinity. However, an observed difference in gill raker length and density is most probably related to the nutritional condition of their habitat, indicating that individuals can rapidly acclimatize to different diets.

The effects of acute transfer to freshwater on ion transporters of the pharyngeal cavity in European seabass (Dicentrarchus labrax).

Gene expression of key ion transporters (the Na+/K+-ATPase NKA, the Na+, K+-2Cl- cotransporter NKCC1, and CFTR) in the gills, opercular inner epithelium, and pseudobranch of European seabass juveniles (Dicentrarchus labrax) were studied after acute transfer up to 4 days from seawater (SW) to freshwater (FW). The functional remodeling of these organs was also studied. Handling stress (SW to SW transfer) rapidly induced a transcript level decrease for the three ion transporters in the gills and operculum. NKA and CFTR relative expression level were stable, but in the pseudobranch, NKCC1 transcript levels increased (up to 2.4-fold). Transfer to FW induced even more organ-specific responses. In the gills, a 1.8-fold increase for NKA transcript levels occurs within 4 days post transfer with also a general decrease for CFTR and NKCC1. In the operculum, transcript levels are only slightly modified. In the pseudobranch, there is a transient NKCC1 increase followed by 0.6-fold decrease and 0.8-fold CFTR decrease. FW transfer also induced a density decrease for the opercular ionocytes and goblet cells. Therefore, gills and operculum display similar trends in SW-fish but have different responses in FW-transferred fish. Also, the pseudobranch presents contrasting response both in SW and in FW, most probably due to the high density of a cell type that is morphologically and functionally different compared to the typical gill-type ionocyte. This pseudobranch-type ionocyte could be involved in blood acid-base regulation masking a minor osmotic regulatory capacity of this organ compared to the gills.

Osmoregulation, bioenergetics and oxidative stress in coastal marine invertebrates: raising the questions for future research.

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.

Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

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.

Molecular characterization and expression of Na+/K+-ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer.

The Na+/K+-ATPase (NKA) is considered as the main pump involved in active ion transport. In the European sea bass, Dicentrarchus labrax, we found two genes encoding for the alpha 1 subunit isoforms (NKA α1a and NKA α1b). NKA α1a and NKA α1b isoform amino acid (aa) sequences were compared through phylogeny and regarding key functional motifs between salmonids and other acanthomorph species. Analysis of aa sequences of both isoforms revealed a high degree of conservation across teleosts. The expression pattern of both nka α1a and nka α1b was measured in the gill, kidney and posterior intestine of fish in seawater (SW) and transferred to fresh water (FW) at different exposure times. Nka α1a was more expressed than nka α1b whatever the condition and the tissue analyzed. After long-term salinity acclimation (2.5 years) either in FW or SW, transcript levels of nka α1a were higher in the kidney followed by the posterior intestine and the gill. Compared to SW conditions, expression of nka α1a in FW was significantly increased or decreased, respectively, in gill and posterior intestine. In contrast, branchial nka α1b was significantly decreased in FW-acclimated fish. Short-term FW acclimation seems to rapidly increase nka α1a transcript levels in the kidney unlike in gill tissues where different gene expression levels are detected only after long-term acclimation.

Salinity effects on osmoregulation and gill morphology in juvenile Persian sturgeon (Acipenser persicus).

The effect of abrupt and 5-day gradual salinity transfers from freshwater (FW) to 11 ‰ Caspian Sea brackish water (BW) was investigated in juvenile Persian sturgeon Acipenser persicus with three different weight groups: 1-2 g (1.62 ± 0.27 g), 2-3 g (2.55 ± 0.41 g) and 3-5 g (4.28 ± 0.76 g). Mortality rates, blood osmotic pressure, gill morphology and branchial Na+, K+-ATPase (NKA) activity were measured 4 and 10 days after abrupt transfer and 9 and 15 days after the initial gradual transfer (i.e. 4 and 10 days after reaching Caspian Sea salinity). Fish under 3 g could not survive increased salinity, and the blood osmotic pressure of the remaining surviving fish increased and remained elevated. However, heavier fish were able to survive and successfully acclimate, even to rapid salinity change with osmotic pressure reduced to Caspian Sea osmolality levels. At the gill level, the developmental increase in chloride cell volume and a higher NKA content most probably allow juveniles weighing more than 2 g to sharply increase NKA activity if the fish are transferred to BW. The rapid chloride cell proliferation occurring with increased salinity should strengthen this acclimation response. Therefore, a drastic physiological change occurs when fish weigh more than 2 g that allows migration to higher salinities. The triggering signal on chloride cells must be further investigated in order to optimize this functional step.

Salinity stress in the black-chinned tilapia Sarotherodon melanotheron.

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, O2 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 O2 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 O2 consumption both in normoxic and hypoxic conditions.

Population-specific responses to pollution exposure suggest local adaptation of invasive red swamp crayfish Procambarus clarkii along the Mediterranean French coastline.

Anthropogenic stressors can have an impact in a broad range of physiological processes and can be a major selective force leading to rapid evolution and local population adaptation. In this study, three populations of the invasive crayfish Procambarus clarkii were investigated. They are geographically separated for at least 20 years, and live in different abiotic environments: a freshwater inland lake (Salagou lake) with no major anthropogenic influence and two other coastal wetlands regularly polluted by pesticides along the Mediterranean coast (Camargue region and Bages-Sigean lagoon). Collected adults were genetically characterized using the mitochondrial COI gene and haplotype frequencies were analyzed for genetic variability within and between populations. Results revealed a higher genetic diversity for these invasive populations than any previous report in France, with more than seven different haplotypes in a single population. The contrasting genetic diversity between the Camargue and the other two populations suggest different times and sources of introduction. To identify differences in key physiological responses between these populations, individuals from each population were maintained in controlled conditions. Data on oxygen consumption rates indicate that the Salagou and Bages-Sigean populations possess a high inter-individual variability compared to the Camargue population. The low individual variability of oxygen consumption and low genetic diversity suggest a specific local adaptation for the Camargue population. Population-specific responses were identified when individuals were exposed to a pesticide cocktail containing azoxystrobin and oxadiazon at sublethal concentrations. The Salagou population was the only one with altered hydro-osmotic balance due to pollutant exposure and a change in protease activity in the hepatopancreas. These results revealed different phenotypic responses suggesting local adaptations at the population level.

Environmental stress responses in sympatric congeneric crustaceans: Explaining and predicting the context-dependencies of invader impacts.

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.

The hepatopancreas of the mangrove crab Neosarmatium africanum: a possible key to understanding the effects of wastewater exposure (Mayotte Island, Indian Ocean).

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 O2 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.

Are we neglecting earth while conquering space? Effects of aluminized solid rocket fuel combustion on the physiology of a tropical freshwater invertebrate.

Space launchers often use aluminized-solid fuel ("propergol") as propellant and its combustion releases tons of Al2O3 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) Al2O3 exposure (0.5 mg L-1) at normal pH (6.6); iii) decreased pH (4.5) (mimicking HCl release in the environment) with no Al2O3; or iv) Al2O3 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 Al2O3 and low pH caused the most impact: acetylcholinesterase and carboxylesterase activities decreased, indicating neurotoxicity and reduced detoxification capacity, respectively. Animal respiration was enhanced with Al2O3 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 Al2O3 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.

Wastewater bioremediation by mangrove ecosystems impacts crab ecophysiology: In-situ caging experiment.

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.

Description of the unusual digestive tract of Platax orbicularis and the potential impact of Tenacibaculum maritimum infection.

BACKGROUND: Ephippidae fish are characterized by a discoid shape with a very small visceral cavity. Among them Platax orbicularis has a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due to Tenacibaculum maritimum infection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ in P. orbicularis is still scarce. Therefore, the aims of this study are to characterize the GIT in non-infected P. orbicularis juveniles to then investigate the impact of T. maritimum on this multifunctional organ. METHODS: In the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged by T. maritimum were analysed and compared to the previously established GIT reference. RESULTS: The overlapped shape of the GIT of P. orbicularis is probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na+/K+ ATPase pump. An identification key allowing the characterization of the GIT sections using in toto histology is given. Secondly, individuals challenged with T. maritimum exhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.

Effects of domestic effluent discharges on mangrove crab physiology: Integrated energetic, osmoregulatory and redox balances of a key engineer species.

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 O2 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.

Salinity Variation in a Mangrove Ecosystem: A Physiological Investigation to Assess Potential Consequences of Salinity Disturbances on Mangrove Crabs.

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.

Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations.

In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 µg Cd2+/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na+/K+-ATPase and V-H+-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.

Salinity stress from the perspective of the energy-redox axis: Lessons from a marine intertidal flatworm.

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.