Mechanisms of Na+ uptake from freshwater habitats in animals.

Life in fresh water is osmotically and energetically challenging for living organisms, requiring increases in ion uptake from dilute environments. However, mechanisms of ion uptake from freshwater environments are still poorly understood and controversial, especially in arthropods, for which several hypothetical models have been proposed based on incomplete data. One compelling model involves the proton pump V-type H+ ATPase (VHA), which energizes the apical membrane, enabling the uptake of Na+ (and other cations) via an unknown Na+ transporter (referred to as the "Wieczorek Exchanger" in insects). What evidence exists for this model of ion uptake and what is this mystery exchanger or channel that cooperates with VHA? We present results from studies that explore this question in crustaceans, insects, and teleost fish. We argue that the Na+/H+ antiporter (NHA) is a likely candidate for the Wieczorek Exchanger in many crustaceans and insects; although, there is no evidence that this is the case for fish. NHA was discovered relatively recently in animals and its functions have not been well characterized. Teleost fish exhibit redundancy of Na+ uptake pathways at the gill level, performed by different ion transporter paralogs in diverse cell types, apparently enabling tolerance of low environmental salinity and various pH levels. We argue that much more research is needed on overall mechanisms of ion uptake from freshwater habitats, especially on NHA and other potential Wieczorek Exchangers. Such insights gained would contribute greatly to our general understanding of ionic regulation in diverse species across habitats.

Ontogenetical development of branchial chambers of Litopenaeus vannamei (Boone, 1931) and their involvement in osmoregulation: ionocytes and Na+/K+-ATPase.

Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither Na+/K+-ATPase expression, likely indicating that pleura, branchiostegites, or bud gills do not participate in osmoregulation. During early postlarval stages, the lack of Na+/K+-ATPase immunoreactivity of the ionocytes from the branchiostegites and epipodites suggests that they are immature ionocytes (ionocytes type I). It could be inferred from IIF and TEM results that epipodites and branchiostegites are involved in iono-osmoregulation from PL15, while gills and pleura do not participate in this function.

Ontogenetic Changes in Blood Osmolality During the Postembryonic Development of Zebrafish (Danio rerio).

The zebrafish Danio rerio is a teleost model species widely used in developmental genetics, biomedical studies, toxicology, and drug screening. Despite the interest of this species in research, little is known through indirect observations about its blood osmolality, which is a key parameter for diverse experiments. In this study, we directly measured blood osmolality using nano-osmometry at different stages of zebrafish postembryonic development. We found that blood osmolality is close to 240 mOsm·kg-1 in early larvae. It progressively increased to ∼270 mOsm·kg-1 during the larval development before reaching ∼300 mOsm·kg-1 after metamorphosis in juveniles and later in adults. These ontogenetic changes in blood osmolality illustrate the physiological changes in osmoregulation associated with postembryonic development, including metamorphosis. These values are of practical interest for adjusting the osmolality of fixatives and cell and tissue culture media for research using zebrafish as a model.

Physiological basis of interactive responses to temperature and salinity in coastal marine invertebrate: Implications for responses to warming.

Developing physiological mechanistic models to predict species' responses to climate-driven environmental variables remains a key endeavor in ecology. Such approaches are challenging, because they require linking physiological processes with fitness and contraction or expansion in species' distributions. We explore those links for coastal marine species, occurring in regions of freshwater influence (ROFIs) and exposed to changes in temperature and salinity. First, we evaluated the effect of temperature on hemolymph osmolality and on the expression of genes relevant for osmoregulation in larvae of the shore crab Carcinus maenas. We then discuss and develop a hypothetical model linking osmoregulation, fitness, and species expansion/contraction toward or away from ROFIs. In C. maenas, high temperature led to a threefold increase in the capacity to osmoregulate in the first and last larval stages (i.e., those more likely to experience low salinities). This result matched the known pattern of survival for larval stages where the negative effect of low salinity on survival is mitigated at high temperatures (abbreviated as TMLS). Because gene expression levels did not change at low salinity nor at high temperatures, we hypothesize that the increase in osmoregulatory capacity (OC) at high temperature should involve post-translational processes. Further analysis of data suggested that TMLS occurs in C. maenas larvae due to the combination of increased osmoregulation (a physiological mechanism) and a reduced developmental period (a phenological mechanisms) when exposed to high temperatures. Based on information from the literature, we propose a model for C. maenas and other coastal species showing the contribution of osmoregulation and phenological mechanisms toward changes in range distribution under coastal warming. In species where the OC increases with temperature (e.g., C. maenas larvae), osmoregulation should contribute toward expansion if temperature increases; by contrast in those species where osmoregulation is weaker at high temperature, the contribution should be toward range contraction.

Ontogeny of osmoregulation of the Asian shore crab Hemigrapsus sanguineus at an invaded site of Europe.

We studied the ontogeny of osmoregulation of the Asian shore crab Hemigrapsus sanguineus at an invaded area in the North Sea. H. sanguineus is native to Japan and China but has successfully invaded the Atlantic coast of North America and Europe. In the invaded areas, H. sanguineus is becoming a keystone species as driver of community structure and the adults compete with the shore crab Carcinus maenas. Strong osmoregulatory abilities may confer the potential to use and invade coastal areas already earlier in the life cycle. We reared larvae and first juveniles at 24°C in seawater from hatching to intermoult of each developmental stage (zoea I-V, megalopa, crab I). We exposed each stage to a range of salinities (0-39 ppt) for 24 h, and then we quantified haemolymph osmolality, using nano-osmometry. In addition, we quantified osmolality in field-collected adults after acclimation to the test salinities for 6 days. Larvae of H. sanguineus were able to hyper-osmoregulate at low salinities (15 and 20 ppt) over the complete larval development, although the capacity was reduced at the zoeal stage V; at higher salinities (25-39 ppt), all larval stages were osmoconformers. The capacity to slightly hypo-regulate at high salinity appeared in the first juvenile. Adults were able to hyper-osmoregulate at low salinities and hypo-regulate at concentrated seawater (39 ppt). H. sanguineus showed a strong capacity to osmoregulate as compared to its native competitor C. maenas, which only hyper-regulates at the first and last larval stages and does not hypo-regulate at the juvenile-adult stages. The capacity of H. sanguineus to osmoregulate over most of the life cycle should underpin the potential to invade empty niches in the coastal zone (characterized by low salinity and high temperatures). Osmoregulation abilities over the whole life cycle also constitute a strong competitive advantage over C. maenas.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

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.

The Legs Have It: In Situ Expression of Ion Transporters V-Type H(+)-ATPase and Na(+)/K(+)-ATPase in the Osmoregulatory Leg Organs of the Invading Copepod Eurytemora affinis.

The copepod Eurytemora affinis has an unusually broad salinity range, as some populations have recently invaded freshwater habitats independently from their ancestral saline habitats. Prior studies have shown evolutionary shifts in ion transporter activity during freshwater invasions and localization of ion transporters in newly discovered "Crusalis organs" in the swimming legs. The goals of this study were to localize and quantify expression of ion transport enzymes V-type H(+)-ATPase (VHA) and Na(+)/K(+)-ATPase (NKA) in the swimming legs of E. affinis and determine the degree of involvement of each leg in ionic regulation. We confirmed the presence of two distinct types of ionocytes in the Crusalis organs. Both cell types expressed VHA and NKA, and in the freshwater population the location of VHA and NKA in ionocytes was, respectively, apical and basal. Quantification of in situ expression of NKA and VHA established the predominance of swimming leg pairs 3 and 4 in ion transport in both saline and freshwater populations. Increases in VHA expression in swimming legs 3 and 4 of the freshwater population (in fresh water) relative to the saline population (at 15 PSU) arose from an increase in the abundance of VHA per cell rather than an increase in the number of ionocytes. This result suggests a simple mechanism for increasing ion uptake in fresh water. In contrast, the decline in NKA expression in the freshwater population arose from a decrease in ionocyte area in legs 4, likely resulting from decreases in number or size of ionocytes containing NKA. Such results provide insights into mechanisms of ionic regulation for this species, with added insights into evolutionary mechanisms underlying physiological adaptation during habitat invasions.

Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes.

In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

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.

Differential distribution of V-type H(+)-ATPase and Na (+)/K (+)-ATPase in the branchial chamber of the palaemonid shrimp Macrobrachium amazonicum.

V-H(+)-ATPase and Na(+)/K(+)-ATPase were localized in the gills and branchiostegites of M. amazonicum and the effects of salinity on the branchial chamber ultrastructure and on the localization of transporters were investigated. Gills present septal and pillar cells. In freshwater (FW), the apical surface of pillar cells is amplified by extensive evaginations associated with mitochondria. V-H(+)-ATPase immunofluorescence was localized in the membranes of the apical evaginations and in clustered subapical areas of pillar cells, suggesting labeling of intracellular vesicle membranes. Na(+)/K(+)-ATPase labeling was restricted to the septal cells. No difference in immunostaining was recorded for both proteins according to salinity (FW vs. 25 PSU). In the branchiostegite, both V-H(+)-ATPase and Na(+)/K(+)-ATPase immunofluorescence were localized in the same cells of the internal epithelium. Immunogold revealed that V-H(+)-ATPase was localized in apical evaginations and in electron-dense areas throughout the inner epithelium, while Na(+)/K(+)-ATPase occurred densely along the basal infoldings of the cytoplasmic membrane. Our results suggest that morphologically different cell types within the gill lamellae may also be functionally specialized. We propose that, in FW, pillar cells expressing V-H(+)-ATPase absorb ions (Cl(-), Na(+)) that are transported either directly to the hemolymph space or through a junctional complex to the septal cells, which may be responsible for active Na(+) delivery to the hemolymph through Na(+)/K(+)-ATPase. This suggests a functional link between septal and pillar cells in osmoregulation. When shrimps are transferred to FW, gill and branchiostegite epithelia undergo ultrastructural changes, most probably resulting from their involvement in osmoregulatory processes.

Without gills: localization of osmoregulatory function in the copepod Eurytemora affinis.

The Pancrustacea, which include crustaceans and hexapods, have successfully colonized marine, freshwater, and terrestrial habitats. While members of the class Malacostraca (e.g., crabs, shrimp) often display immense osmoregulatory capacities, more basally branching crustaceans (e.g., copepods, branchiopods) tend to possess less-specialized osmoregulatory structures that have been poorly characterized. Remarkably, some of these more basal taxa have also colonized diverse habitats. For instance, the copepod Eurytemora affinis has recently invaded freshwater habitats multiple times independently but lack obvious osmoregulatory structures. To explore localization of ion exchange, we performed silver staining, immunohistochemical staining, and transmission electron microscopy. Our results revealed localization of ion transport within the maxillary glands and on four pairs of swimming legs. Silver staining revealed ion exchange at the maxillary pores and on the endopods and exopods of swimming legs P1 through P4. Immunohistochemical assays localized ion transport enzymes V-type H(+)-ATPase and Na(+)/K(+)-ATPase in the maxillary glands and swimming legs as well. Finally, transmission electron microscopy identified specialized ionocytes within these anatomical regions. These investigations uncovered novel osmoregulatory structures at the swimming legs, which we designate the "Crusalis organs." Our findings identified specific tissues specialized for ion transport, potentially enabling this small crustacean to rapidly transition into freshwater habitats.

Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs.

The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na(+)/K(+)-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.

The ClC-3 chloride channel and osmoregulation in the European sea bass, Dicentrarchus labrax.

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.

Individual and combined effects of copper and parasitism on osmoregulation in the European eel Anguilla anguilla.

The European eel (Anguilla anguilla), a catadromous species, breeds in the sea and migrates to estuarine, lagoon or freshwater habitats for growth and development. Yellow eels, exposed to low or fluctuating salinities, are also exposed to multiple other stressors as pollution, over-fishing and parasitism, which contribute to the dramatic decrease of eel populations in several European countries. The objective of this study was to evaluate the single and combined effects of waterborne copper and experimental infestation of eels with the nematode Anguillicoloides crassus after a salinity challenge from nearly isotonic (18ppt) to hypo- (5ppt) and hypertonic (29ppt) conditions, in order to investigate the osmoregulatory capacity of eels exposed to these stressors. In a nearly isotonic condition (18ppt), blood osmolality remained constant over the 6 weeks contamination to Cu(2+) and Anguillicoloides crassus. In fish exposed to a salinity challenge of 29ppt for 2 weeks, no significant effect was recorded in blood osmolality, Na(+)/K(+)-ATPase (NKA) activity, Na(+) and Cl(-) concentrations. After 2 weeks at 5ppt however, a significant blood osmolality decrease was detected in fish exposed to Anguillicoloides crassus infestation with or without Cu(2+) addition. This decrease may originate from lower Cl(-) levels measured in eels exposed to both stressors. Blood Na(+) levels remained relatively stable in all tested animals, but gill NKA activities were lower in eels exposed to combined stress. No apparent branchial lesions were detected following the different treatments and immunolocalization of NKA revealed well-differentiated ionocytes. Thus, the 5ppt challenge in eels exposed to copper and Anguillicoloides crassus seems to clearly enhance iono/osmoregulatory disturbances. Funded by ANR CES/CIEL 2008-12.

Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

Osmoregulatory strategies in natural populations of the black-chinned tilapia Sarotherodon melanotheron exposed to extreme salinities in West African estuaries.

The effect of salinity was studied in natural populations of the black-chinned tilapia (Sarotherodon melanotheron) from West Africa. This euryhaline species colonizes nearly all coastal environments from bays to lagoons characterized by salinities ranging from fresh water to hypersaline water over 100 ‰. Individuals were sampled during the dry season at several locations characterized by different levels of salinity (3-102 ‰). Their osmotic status and their gills were analyzed. The branchial mitochondria-rich cells (MRC), localized at the basis of the filaments and along the lamellae in fish taken from the saline stations, showed a wide plasticity with significant differences in their number and size. The most striking results were a significant larger area (≈3x) and a higher number (≈55x) of MRC at high salinity (102 ‰) compared to low salinity (3 ‰). The major ion transporters and channels were localized by immunocytochemistry and different expression patterns have been recorded between stations. Despite an increased Na⁺/K⁺-ATPase (NKA) α-subunit expression and NKA activity, pointing to an increased monovalent ion excretion, a severe osmotic imbalance was recorded in animals living in hypersaline environments.

Impact of environmental DDT concentrations on gill adaptation to increased salinity in the tilapia Sarotherodon melanotheron.

Estuaries of tropical developing countries suffering from severe droughts induced by climate change are habitats to fish, which face drastic salinity variations and the contact with pollutants. The Western Africa tilapia Sarotherodon melanotheron is highly resistant to hypersalinity, but the effect of human-released xenobiotics on its adaptation is barely known. Controlled experiments were conducted to observe S. melanotheron gill adaptation to abrupt salinity variations in the presence of waterborne DDT, at concentrations detected in their natural habitat. The gills appeared as an important site of DDT conversion to DDD and/or depuration. A 12-days DDT exposure resulted in decreased gill epithelium thickness at all salinities (from fresh- to hypersaline-water), and the structure of gills from freshwater fish was particularly altered, relative to controls. No unbalance in tilapia blood osmolality was observed following DDT exposure, which however caused a decrease in branchial Na(+)-K(+)-ATPase (NKA) activity. Gill cellular NKA expression was reduced in salt-water, together with the expression of the CFTR chloride channel in hypersaline water. Although S. melanotheron seems very resistant (especially in seawater) to short-term waterborne DDT contamination, the resulting alterations of the gill tissue, cells and enzymes might affect longer term respiration, toxicant depuration and/or osmoregulation in highly fluctuating salinities.

Rapid evolution of body fluid regulation following independent invasions into freshwater habitats.

Colonizations from marine to freshwater environments constitute among the most dramatic evolutionary transitions in the history of life. Colonizing dilute environments poses great challenges for acquiring essential ions against steep concentration gradients. This study explored the evolution of body fluid regulation following freshwater invasions by the copepod Eurytemora affinis. The goals of this study were to determine (1) whether invasions from saline to freshwater habitats were accompanied by evolutionary shifts in body fluid regulation (hemolymph osmolality) and (2) whether parallel shifts occurred during independent invasions. We measured hemolymph osmolality for ancestral saline and freshwater invading populations reared across a range of common-garden salinities (0.2-25 PSU). Our results revealed the evolution of increased hemolymph osmolality (by 16-31%) at lower salinities in freshwater populations of E. affinis relative to their saline ancestors. Moreover, we observed the same evolutionary shifts across two independent freshwater invasions. Such increases in hemolymph osmolality are consistent with evidence of increased ion uptake in freshwater populations at low salinity, found in a previous study, and are likely to entail increased energetic costs upon invading freshwater habitats. Our findings are consistent with the evolution of increased physiological regulation accompanying transitions into stressful environments.