The tale of an endemic shrimp's exceptional osmoregulation and the ancient Athalassic mangrove oasis.

The hyperarid mangrove in the Middle East is characterised by the absence of rivers or freshwater inputs and is one of the most extreme settings of this ecosystem on Earth. Endemic to Qatar's hyperarid mangroves, a Palaemon shrimp is uniquely confined to a sole mangrove site in the Arabian Gulf. Within these mangrove channels, we unveiled brine groundwater sources exceeding 70 ppt salinity, contrasting the local marine standard of 42 ppt. Concurrently, a mysid species typically linked to salt pans and groundwater coexists. Stable isotopic analysis implied the existence of a predator-prey dynamic between this mysid species and the studied shrimp. Then, investigating the endemic shrimp's adaptation to extreme salinity, we conducted osmolarity experiments and phylogenetic studies. Our findings demonstrate that this shrimp transitions from hypo- to hyper-osmoregulation, tolerating salinities from 18 to 68 ppt-an unprecedented osmoregulatory capacity among caridean shrimps. This speciation pattern likely arises from the species osmolarity adaptation, as suggested for other Palaemon congeners. Phylogenetic analysis of the studied Palaemon, along with the mangrove's geological history, suggests a profound evolutionary interplay between the ecosystem and the shrimp since the Eocene. This study proposes the hyperarid mangrove enclave as an Athalassic mangrove oasis-a distinctive, isolated ecosystem within the desert landscape.

Late rise in hemolymph osmolality in Macrobrachium acanthurus (diadromous freshwater shrimp) exposed to brackish water: Early reduction in branchial Na+/K+ pump activity but stable muscle HSP70 expression.

Some Macrobrachium shrimps (Caridea, Palaemonidae) are diadromous; freshwater adults are truly euryhaline, while larvae need saline water for development. Branchial Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) are involved in NaCl absorption in freshwater. This study aimed at verifying the time course of the osmoregulatory response of adult Macrobrachium acanthurus to high salinity brackish water (20‰), from the first 30min to 5days. The goal was to detect possible transition from hyper- to hyporegulation, the putative involvement of branchial NKA and CA, or the induction of muscular HSP70 expression. Hemolymph osmotic and ionic concentrations remained relatively stable and close to control levels until ~9h of exposure, but later increased consistently (~50%). A fast reduction in NKA activity (3-6h) was observed; these shrimps seem to shut off salt absorption already in the first hours. Later on, especially after 24h, hemolymph concentrations rise but HSP70 expression is not induced, possibly because constitutive levels are already sufficient to prevent protein damage. Time-dependent response mechanisms effective in high salinity brackish water, resulting in salt loading avoidance and suggestive of hyporegulation should be further investigated in decapods that evolutionary invaded freshwater.

Regulation of muscle hydration upon hypo- or hyper-osmotic shocks: differences related to invasion of the freshwater habitat by decapod crustaceans.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Lead hampers gill cell volume regulation in marine crabs: stronger effect in a weak osmoregulator than in an osmoconformer.

Hepatus pudibundus is a strictly marine osmoconformer crab, while Callinectes ornatus inhabits estuarine areas, behaving as a weak hyper-osmoregulator in diluted seawater. Osmoconformers are expected to have higher capacity for cell volume regulation, but gill cells of a regulator are expected to display ion transporters to a higher degree. The influence of lead nitrate (10 µM) on the ability of isolated gill cells from both species to volume regulate under isosmotic and hyposmotic conditions were here evaluated. Without lead, under a 25% hyposmotic shock, the gill cells of both species were quite capable of cell volume maintenance. Cells of C. ornatus, however, had a little swelling (5%) during the hyposmotic shock of greater intensity (50%), while cells of H. pudibundus were still capable of volume regulation. In the presence of lead, even under isosmoticity, the gill cells of both species showed about 10% volume reduction, indicating that lead promotes the loss of water by the cells. When lead was associated with 25% and 50% hyposmotic shock, C. ornatus cells lost more volume (15%), when compared to isosmotic conditions, while H. pudibundus cells showed volume regulation. We then analyzed the possible ways of action of lead on the mechanisms of cell volume regulation in the two species. Verapamil (100 µM) was used to inhibit Ca²âº channels, ouabain (100 µM) to inhibit Na⁺/K⁺-ATPase, and HgCl2 (100 µM) to inhibit aquaporins. Our results suggest that: (1) Ca²âº channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus, being the target of lead action in these cells; (2) aquaporins are much more relevant for water flux in H. pudibundus; and (3) the Na⁺/K⁺-ATPase is much more relevant for volume regulation in C. ornatus. Osmoregulators may be more susceptible to metal contamination than osmoconformers, especially in situations of reduced salinity, for two basic reasons: (1) lower capacity of volume regulation and (2) putative higher uptake of Pb²âº through ionic pathways that operate in salt absorption, such as, for example, the Na⁺/K⁺-ATPase.

Distinct patterns of water and osmolyte control between intertidal (Bunodosoma caissarum) and subtidal (Anemonia sargassensis) sea anemones.

Anemones are frequently found in rocky intertidal coasts. As they have highly permeable body surfaces, exposure to the air or to salinity variations inside tidal pools can represent intense osmotic and ionic challenges. The intertidal Bunodosoma caissarum has been compared with the subtidal Anemonia sargassensis concerning their response to air exposure or salinity changes. B. caissarum maintains tissue hydration through mucus production and dome-shape formation when challenged with air exposure or extreme salinities (fresh water or hypersaline seawater, 45 psu) for 1-2h. Upon exposure to mild osmotic shocks for 6h (hyposmotic: 25 psu, or hyperosmotic: 37 psu), B. caissarum was able to maintain its coelenteron fluid (CF) osmolality stable, but only in 25 psu. A. sargassensis CF osmolality followed the external medium in both salinities. Isolated cells of the pedal disc of B. caissarum showed full capacity for calcium-dependent regulatory volume decrease (RVD) upon 20% hyposmotic shock, at least partially involving the release of KCl via K(+)-Cl(-) cotransport, and also of organic osmolytes. Aquaporins (HgCl(2)-inhibited) likely participate in this process. Cells of A. sargassensis showed partial RVD, after 20 min. Cells from both species were not capable of regulatory volume increase upon hyperosmotic shock (20%). Whole organism and cellular mechanisms allow B. caissarum to live in the challenging intertidal habitat, frequently facing air exposure and seawater dilution.

Do osmoregulators have lower capacity of muscle water regulation than osmoconformers? A study on decapod crustaceans.

Decapod crustaceans occupy various aquatic habitats. In freshwater they are osmoregulators, while marine species are typically osmoconformers. Freshwater crustaceans are derived from marine ancestors. The hypothesis tested here was that osmoregulators, which can rely on salt transport by interface epithelia to prevent extracellular disturbance, would have a lower capacity of tissue water regulation when compared with osmoconformers. Four species of decapod crustaceans (the marine osmoconformer crab Hepatus pudibundus, and three osmoregulators of different habitats) have been exposed in vivo to a salinity challenge, for up to 24 hr. Osmoregulators were: the estuarine shrimp Palaemon pandaliformis, the diadromous freshwater shrimp Macrobrachium acanthurus, and the hololimnetic red crab Dilocarcinus pagei. H. pudibundus displayed hemolymph dilution already after 0.5 hr in 25 per thousand, reaching approximately 30% reduction in osmolality, but its muscle degree of hydration did not increase. To make the different in vivo salinity challenges directly comparable, the ratio between the maximum change in muscle hydration with respect to the control value measured for the species and the maximum change in hemolymph osmolality was calculated (x 1,000): H. pudibundus (25 per thousand, 8.1% kg H(2)O/mOsm x 10(3))>P. pandaliformis (2 per thousand, 9.2)>M. acanthurus (30 per thousand, 12.6)>P. pandaliformis (35 per thousand, 16.7)>D. pagei (7 per thousand, 60.4). Muscle slices submitted in vitro to a 30% osmotic challenge confirmed in vivo results. Thus, the estuarine/freshwater osmoregulators displayed a lower capacity to hold muscle tissue water than the marine osmoconformer, despite undergoing narrower variations in hemolymph osmolality.

Muscle water control in crustaceans and fishes as a function of habitat, osmoregulatory capacity, and degree of euryhalinity.

This study aimed at detecting possible patterns in the relationship between Anisosmotic Extracellular Regulation (AER) and Isosmotic Intracellular Regulation (IIR) in crustaceans and teleost fish from different habitats and evolutionary histories in fresh water (FW), thus different osmoregulatory capabilities, and degrees of euryhalinity. Crustaceans used were the hololimnetic FW Aegla schmitti, and Macrobrachium potiuna, the diadromous FW Macrobrachium acanthurus, the estuarine Palaemon pandaliformis and the marine Hepatus pudibundus; fishes used were the FW Corydoras ehrhardti, Mimagoniates microlepis, and Geophagus brasiliensis, and the marine-estuarine Diapterus auratus. The capacity for IIR was assessed in vitro following wet weight changes of isolated muscle slices incubated in anisosmotic saline (~50% change). M. potiuna was the crustacean with the highest capacity for IIR; the euryhaline perciforms G. brasiliensis and D. auratus displayed total capacity for IIR. It is proposed that a high capacity for IIR is required for invading a new habitat, but that it is later lost after a long time of evolution in a stable habitat, such as in the FW anomuran crab A. schmitti, and the Ostariophysian fishes C. ehrhardti and M. microlepis. More recent FW invaders such as the palaemonid shrimps (M. potiuna and M. acanthurus) and the cichlid G. brasiliensis are euryhaline and still display a high capacity for IIR.

Osmoregulation and tissue water regulation in the freshwater red crab Dilocarcinus pagei (Crustacea, Decapoda), and the effect of waterborne inorganic lead.

Inorganic lead has been measured in high concentrations in certain streams in Brazil. This study has evaluated the osmoregulatory effects of lead on the native freshwater red crab Dilocarcinus pagei. In order to probe its osmoregulatory and tissue volume regulatory capabilities and how it would be affected by lead, the crab has been submitted to individual and combined salt and chemical stresses (Pb2+). Male crabs were exposed for 10 days to either: (1) control (freshwater, FW), (2) brackish water of salinity 15 (BW), (3) inorganic lead in freshwater (FWPb), and (4) inorganic lead in brackish water (BWPb), 2.7 mgPb/L. In vivo, whole crabs lost weight transiently when exposed to Pb2+, both in FW and in BW. Haemolymph osmolality and ion concentrations increased and remained elevated upon exposure of crabs to BW, with or without Pb2+, showing a trend to hyper-conformation. In vitro, muscle weight decreased in isosmotic conditions upon exposure to Pb2+. Na+, Cl-, and ninhydrin positive substances (NPS) were increased in muscle exposed to hyperosmotic saline, well above what would be expected from simple efflux of water, suggesting a partial regulatory volume increase (RVI) capacity. This partial RVI involves the Na+, K+, 2Cl- -cotransporter and the Na+/H+ exchanger on Na+ and Cl- uptake, as judged from further decreases in muscle weight in the presence of the respective inhibitors. A breakdown of proteins into NPS seems to follow the uptake of inorganic ions. Pb2+ has affected water and ion movements in D. pagei both in the whole animal and in the isolated tissue. This study has highlighted the relevance of evaluating tissue volume regulation in aquatic animals confronted with metal polluted waters.