Acute metabolic responses of two marine brachyuran crabs to dilute seawater: The aerobic cost of hyper regulation.

Hepatus pudibundus ("flecked box crab") is a stenohaline osmoconfomer, and restricted to marine habitats. Callinectes danae ("swimming crab Dana") lives in coastal/estuarine waters and is a weak hyper regulator. There is no consensus on which strategy is more expensive metabolically face salinity challenges: conformation with higher dependence on cell volume regulation, or hyper regulation, alleviating the need for intense cell volume regulation. Crabs were probed for their acute response to dilute seawater through exposures to salinities 35‰, 30‰, 25‰, and 20‰ for 2, 4, and 6 h. Hemolymph osmolality, lactate, and ions (chloride, sodium, magnesium, potassium) were assayed, as well as muscle water content. Water dissolved oxygen, ammonia, and pH levels were also measured. H. pudibundus conformed for osmolality and displayed increase in muscle hydration along the decrease in salinity down to 25‰, while C. danae efficiently maintained hemolymph osmo ionic stability, consumed more oxygen, acidified more the water, and released more ammonia. In 25‰, both species spent energy: H. pudibundus putatively controlling cell volume, and C. danae regulating hemolymph concentrations. In 20‰, H. pudibundus closed itself, avoiding the contact of the interface epithelia with the external environment and producing much lactate, whereas C. danae spent more energy (aerobic) in extracellular osmo ionic stability. Under these conditions, anisosmotic extracellular regulation (together with additional cell volume regulation) is more oxygen consuming than osmoconformation with a putatively more intense challenge to cell volume. The exposure to hyposalinity limits the occupation of estuarine environments by H. pudibundus in short and middle term.

Recognizing Salinity Threats in the Climate Crisis.

Climate change is causing habitat salinity to transform at unprecedented rates across the globe. While much of the research on climate change has focused on rapid shifts in temperature, far less attention has focused on the effects of changes in environmental salinity. Consequently, predictive studies on the physiological, evolutionary, and migratory responses of organisms and populations to the threats of salinity change are relatively lacking. This omission represents a major oversight, given that salinity is among the most important factors that define biogeographic boundaries in aquatic habitats. In this perspective, we briefly touch on responses of organisms and populations to rapid changes in salinity occurring on contemporary time scales. We then discuss factors that might confer resilience to certain taxa, enabling them to survive rapid salinity shifts. Next, we consider approaches for predicting how geographic distributions will shift in response to salinity change. Finally, we identify additional data that are needed to make better predictions in the future. Future studies on climate change should account for the multiple environmental factors that are rapidly changing, especially habitat salinity.

Osmoregulatory power influences tissue ionic composition after salinity acclimation in aquatic decapods.

Decapod crustaceans show variable degrees of euryhalinity and osmoregulatory capacity, by responding to salinity changes through anisosmotic extracellular regulation and/or cell volume regulation. Cell volume regulatory mechanisms involve exchange of inorganic ions between extra- and intra-cellular (tissue) compartments. Here, this interplay of inorganic ions between both compartments has been evaluated in four decapod species with distinct habitats and osmoregulatory strategies. The marine/estuarine species Litopenaeus vannamei (Lv) and Callinectes danae (Cd) were submitted to reduced salinity (15‰), after acclimation to 25 and 30‰, respectively. The freshwater Macrobrachium acanthurus (Ma) and Aegla schmitti (As) were submitted to increased salinity (25‰). The four species were salinity-challenged for both 5 and 10 days. Hemolymph osmolality, sodium, chloride, potassium, and magnesium were assayed. The same inorganic ions were quantified in muscle samples. Muscle hydration (MH) and ninhydrin-positive substances (NPS) were also determined. Lv showed slight hemolymph dilution, increased MH and no osmotically-relevant decreases in muscle osmolytes; Cd displayed hemolymph dilution, decreased muscular NaCl and stable MH; Ma showed hypo-regulation and steady MH, with no change in muscle ions; As conformed hemolymph sodium but hypo-regulated chloride, had stable MH and increased muscle NPS and ion levels. Hemolymph and muscle ions (especially chloride) of As were highly correlated (Pearson, +0.83). Significant exchanges between hemolymph and muscle ionic pools were more evident in the two species with comparatively less AER regulatory power, C. danae and A. schmitti. Our findings endorse that the interplay between extracellular and tissue ionic pools is especially detectable in euryhaline species with relatively lower osmoregulatory strength.

Distribution of Na+/K+-ATPase-immunoreactive ionocytes varies between two superorders of ray-finned fish: Ostariophysi and Acanthopterygii.

Ray-finned fishes of the superorder Ostariophysi are primarily freshwater (FW), and normally stenohaline. Differently, fishes of the superorder Acanthopterygii are essentially marine, and frequently euryhaline, with some secondary FW. Na+/K+-ATPase-immunoreactive ionocytes were localized in the branchial epithelia of 4 species of Ostariophysi and 3 of Acanthopterygii. The Ostariophysi grass carp (Ctenopharyngodon idella, Cypriniformes), twospot Astyanax (Astyanax bimaculatus) and piracanjuba (Brycon orbignyanus), Characiformes, and the jundiá (Rhamdia quelen, Siluriformes), all from FW, displayed ionocytes in the filament plus secondary lamellae (F + SL). In their turn, all the three species of Acanthopterygii showed immunoreactive ionocytes in the filaments only (F). They were the Nile tilapia (Oreochromis niloticus, Cichliformes) in FW, the dog snapper (Lutjanus jocu, Perciformes) in seawater (SW), and the green puffer (Sphoeroides greeleyi, Tetraodontiformes) in SW. Ionocytes normally extend their distribution to the secondary lamellae (F + SL) in Ostariophysi. In Acanthopterygii, we find more plasticity: ionocytes are more frequently restricted to the filament in SW, but also spread to SL in FW. It may be that the occurrence of ionocytes in SL is the ancestral condition, but some euryhaline acanthopterygians rely on the space of the SL for placement of additional ionocytes when in FW absorbing salt. Our study contributed to the identification of the pattern of ionocyte distribution in gills of Ostariophysi in respect to that of Acanthopterygii.

Biomarkers of homeostasis, allostasis, and allostatic overload in decapod crustaceans of distinct habitats and osmoregulatory strategies: an empirical approach.

The term "allostasis", meaning the assumption that homeostasis may not be as static as the term implies, has been vastly employed for mammals, and other vertebrates, for which the degree of internal stability is maximal, according to their higher complexity. We have here investigated how these states of homeostasis, allostasis, and allostatic overload could be diagnosed in decapod crustaceans, upon acute salinity challenges. Decapods of distinct lineages and habitats have been submitted to 3 salinity levels for 6 and 12 h. The first salinity was the habitat salinity (control), considered as the one that allows the homeostatic condition. The next salinity represented a mild challenge, that would potentially lead to allostasis, and the third salinity was intended to represent an overload, albeit not lethal. Species used were: the marine crab Hepatus pudibundus (Hp, osmoconformer, salinities 33, 25, and 20‰), the marine/estuarine swimming crab Callinectes danae (Cd, weak regulator, salinities 30, 20, and 10‰), and the diadromous freshwater prawn Macrobrachium acanthurus (Ma, strong regulator, salinities <0.5, 15, and 30‰). These 3 species follow a sequence of growing regulatory capacity (Hp

Euryhalinity of subtropical marine and estuarine polychaetes evaluated through carbonic anhydrase activity and cell volume regulation.

Polychaete worms are widespread and diverse in marine and estuarine habitats subject to varying salinity, in areas influenced by tides, demanding physiological adjustment for internal homeostasis. They are typically considered and reported to be osmoconformers, but they are not often studied for their osmoregulation. Here, three species of polychaete worms from distinct coastal habitats have been investigated: the spionid Scolelepis goodbody (intertidal in saline, exposed sandy beaches), the nereidid Laeonereis culveri (estuarine polyhaline), and the nephtyid Nephtys fluviatilis (estuarine oligohaline). The general objective here was to relate ecological aspects and physiology of the studied species. Constitutive whole body osmolality and carbonic anhydrase activity (CAA, relevant for osmoregulation, acid-base balance and respiration) have been assayed. In addition, cell volume regulatory capacity (from whole body cell dissociation) was challenged under hypoosmotic and hyperosmotic shocks (50% intensity), with respect to isosmotic control. S. googdbody and L. culveri, the two species from most saline environments (marine/estuarine), showed higher CAA than N. fluviatilis, which, in turn, displayed a hyperosmotic gradient to water of salinity 15. Cells from S. goodbody and L. culveri showed regulatory volume decrease upon swelling, with S. goodbody showing the largest volume increase. As in other more studied marine invertebrate groups, polychaetes also show variability in their osmoregulatory physiology, related to distinct saline challenges faced in their coastal habitats.

Benzocaine-induced stress in the euryhaline teleost, Centropomus parallelus and its implications for anesthesia protocols.

The use of anesthetic in fish farming is a traditional practice which aims to reduce the stress caused by transport and handling. However, anesthesia-induction protocols are commonly established and implemented without proper physiological/behavioral evaluation. Additionally, concentration and time of exposure to the anesthetic are often set without considering species-specific responses. The fat snook (Centropomus parallelus) is a fish with great potential for aquaculture. Given its remarkable euryhalinity, it can grow in fresh- or seawater. Most studies on fat snook anesthesia tested natural compounds (essential oils) instead of traditional anesthetics. However, the use of benzocaine is much more common in the commercial sector, as it is easy to obtain and of relatively low cost. The present study aimed at analyzing the effects benzocaine exposure on glucose and cortisol plasma levels (two traditional stress markers in teleost fish), as well as on plasma osmolality, chloride and magnesium, (indicators of osmo-ionic allostasis) in animals acclimated to different salinities. Results showed that while osmo-ionic allostasis was strictly maintained across the treatments, time of anesthesia had a strong positive relationship to plasma cortisol and glucose, regardless the salinity of exposure and acclimation. The results are discussed as they relate to anesthesia protocols and how stress response generated by time of anesthesia may challenge farming flexibility.

Ultrasonography as a promising methodology to indicate captured-induced abortion in viviparous elasmobranchs.

This study aimed to characterize morphological aspects related to abortion through a non-lethal approach in the shortnose guitarfish Zapteryx brevirostris, an endemic and threatened species commonly caught by artisanal fisheries. Two females with signs of abortion and one female exhibiting external signs of pregnancy were purchased alive at a fish market during the period when this species has developing embryos in southern Brazil. Scans were conducted using a portable ultrasound. Females with signs of abortion revealed an absence of embryos despite having a similar morphology of the uterus when compared to the pregnant female. Examination of the pregnant female revealed the presence of two embryos, measuring c. 100 mm, each detected through their midline. This study presents new data on uterine macromorphology following successive abortive events in a viviparous elasmobranch species and validates ultrasonography as a diagnostic tool for the species. Ultrasonography is an effective, non-lethal and less-invasive methodology that is recommended for use in future studies of abortion and other reproductive events in elasmobranchs.

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.

Evaluation of the water quality of the upper reaches of the main Southern Brazil river (Iguaçu river) through in situ exposure of the native siluriform Rhamdia quelen in cages.

Increase in industrial growth, urban and agricultural pollution, with consequent impacts on aquatic ecosystems are a major focus of research worldwide. Still, not many studies assess the impacts of contamination through in situ studies, using native species, also considering the influence of seasonality on their responses. This study aimed to evaluate the water quality of the basin of the Upper Iguaçu River, the main source of water supply to Curitiba, a major capital of Southern Brazil, and its Metropolitan area. Several biomarkers were evaluated after in situ exposure of the native catfish Rhamdia quelen inside cages for 7 days. Ten study sites were chosen along the basin, based on a diffuse gradient of contamination, corresponding to regions upstream, downstream, and within "great Curitiba". In each site, fish were exposed in Summer and Winter. The complex mixture of contaminants of this hydrographic basin generated mortality, and ion-, osmoregulatory and respiratory disturbances in the catfish as, for example, reduction of plasma osmolality and ionic concentrations, increased hematocrit levels and gill water content, altered branchial and renal activities of the enzyme carbonic anhydrase, as well as raised levels of plasma cortisol and glucose. Biomarkers were mostly altered in fish exposed in Great Curitiba and immediately downstream. There was a notable influence of season on the responses of the jundiá. A multivariate redundancy analysis revealed that the best environmental variables explained 30% of the variation in biomarkers after controlling for spatial autocorrelation. Thus, this approach and the chosen parameters can be satisfactorily used to evaluate contamination environments with complex mixtures of contaminants, in other urban basins as well.

Aquaporin in different moult stages of a freshwater decapod crustacean: Expression and participation in muscle hydration control.

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs.

Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na(+)/K(+)-ATPase and NKCC).

Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells.

Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker.

In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil.

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Ammonia excretion and expression of transport proteins in the gills and skin of the intertidal fish Lipophrys pholis.

Intertidal pools are intensely challenging environments, due to rapid and extreme fluctuations in water conditions during the tidal cycle. Emersion is another challenge intertidal fishes may face. Mechanisms of ammonia excretion and ion regulation were studied in the resident amphibious blennid Lipophrys pholis. The ammonia transporters Rhcg1 and Rhcg2 were cloned and characterized. Fish were challenged for 24h to 1) emersion, 2) fresh water (FW), and 3) high environmental ammonia (HEA; 1mM NH4Cl), or 4) ammonia loading (1.5µmol/g NH4HCO3). When air exposed, L. pholis maintained aquatic ammonia excretion rates (JAmm) while branchial Na(+)/K(+)-ATPase (NKA) activity increased, but no changes at the protein or mRNA levels of transporters were noted. In FW, JAmm decreased and osmotic problems were encountered. Skin NKA activity decreased, branchial Rhcg2, and skin Rhcg1 and Rhcg2 increased. Exposure to HEA only increased branchial Rhcg2 levels. Although internal ammonia loading only led to a modest non-significant increase in JAmm, skin NKA (activity and α-subunit), carbonic anhydrase protein levels, and branchial Rhcg1 levels increased. In summary, variable responses were observed involving both gill and skin but given the instability of its habitat, the constitutive expression of transporters is likely also of importance.

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.

Some euryhalinity may be more common than expected in marine elasmobranchs: the example of the South American skate Zapteryx brevirostris (Elasmobranchii, Rajiformes, Rhinobatidae).

Elasmobranchs are essentially marine, but ~15% of the species occur in brackish or freshwater. The Brazilian marine coastal skate Zapteryx brevirostris, non-reported in nearby estuaries, was submitted to 35, 25, 15, and 5 psu, for 6 or 12h (n=6). Plasma was assayed for osmolality, urea, and ions (Na(+), Cl(-), K(+), Mg(2+)). Muscle water content was determined, and the rectal gland, kidney and gills were removed for carbonic anhydrase (CA) and Na(+),K(+)-ATPase (NKA) activities. The skate survived to all treatments. Plasma osmolality and urea levels decreased respectively by 27% and 38% after 12h in 5 psu (with respect to levels when in seawater), but plasma Na(+), Cl(-), and Mg(2+) were well regulated. Plasma K(+) showed some conformation after 12h. Muscle hydration was maintained. Branchial CA and NKA did not respond to salinity. Rectal gland NKA decreased upon seawater dilution, while renal NKA increased. This skate was shown to be partially euryhaline. The analysis of plasma urea of elasmobranchs in brackish and freshwater versus salinity and time-allied to the widespread occurrence of some euryhalinity in the group-led us to revisit the hypothesis of a brackish water habitat for elasmobranch ancestors.

Direct relationship between osmotic and ionic conforming behavior and tissue water regulatory capacity in echinoids.

Echinoderms are considered marine osmoconforming invertebrates. However, many are intertidal or live next to estuaries, tolerating salinity changes and showing extracellular gradients to dilute seawater. Three species of echinoids - Lytechinus variegatus, which can occur next to estuarine areas, the rocky intertidal Echinometra lucunter, and the mostly subtidal Arbacia lixula - were submitted to a protocol of stepwise (rate of 2-3 psu/h) dilution, down to 15 psu, or concentration, up to 45 psu, of control seawater (35 psu). Coelomic fluid samples were obtained every hour. The seawater dilution experiment lasted 8h, while the seawater concentration experiment lasted 6h. Significant gradients (40-90% above value in 15 psu seawater) for osmolality, sodium, magnesium, and potassium were shown by L. variegatus and E. lucunter. A. lixula showed the smallest gradients, displaying the strongest conforming behavior. The esophagus of the three species was challenged in vitro with 20 and 50% osmotic shocks (hypo- and hyperosmotic). A. lixula, the most "conforming" species, showed the highest capacity to avoid swelling of its tissues upon the -50% hyposmotic shock, and was also the species less affected by salinity changes concerning the observation of spines and ambulacral feet movement in the whole-animal experiments. Thus, the most conforming species (A. lixula) displayed the highest capacity to regulate tissue water/volume, and was also the most euryhaline among the three studied species. In addition, tissues from all three species swelled much more than they shrank under osmotic shocks of same magnitude. This distinct trend to gain water, despite the capacity to hold some gradients upon seawater dilution, helps to explain why echinoderms cannot be fully estuarine, or ever enter fresh water.