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.

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.

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.

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 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.

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.

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.