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.

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.

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.

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.

Impact of ultraviolet-B radiation on planktonic fish larvae: alteration of the osmoregulatory function.

Coastal marine ecosystems are submitted to variations of several abiotic and biotic parameters, some of them related to global change. Among them the ultraviolet-B (UV-B) radiation (UVBR: 280-320 nm) may strongly impact planktonic fish larvae. The consequences of an increase of UVBR on the osmoregulatory function of Dicentrarchus labrax larvae have been investigated in this study. In young larvae of D. labrax, as in other teleosts, osmoregulation depends on tegumentary ion transporting cells, or ionocytes, mainly located on the skin of the trunk and of the yolk sac. As early D. labrax larvae passively drift in the top water column, ionocytes are exposed to solar radiation. The effect of UVBR on larval osmoregulation in seawater was evaluated through nanoosmometric measurements of the blood osmolality after exposure to different UV-B treatments. A loss of osmoregulatory capability occured in larvae after 2 days of low (50 µWcm(-2): 4 h L/20 h D) and medium (80 µWcm(-2): 4 h L/20 h D) UVBR exposure. Compared to control larvae kept in the darkness, a significant increase in blood osmolality, abnormal behavior and high mortalities were detected in larvae exposed to UVBR from 2 days on. At the cellular level, an important decrease in abundance of tegumentary ionocytes and mucous cells was observed after 2 days of exposure to UVBR. In the ionocytes, two major osmoeffectors were immunolocalized, the Na+/K(+)-ATPase and the Na+/K+/2Cl- cotransporter. Compared to controls, the fluorescent immunostaining was lower in UVBR-exposed larvae. We hypothesize that the impaired osmoregulation in UVBR-exposed larvae originates from the lower number of tegumentary ionocytes and mucous cells. This alteration of the osmoregulatory function could negatively impact the survival of young larvae at the surface water exposed to UVBR.

Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchus labrax: a candidate protein for osmosensing.

The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the TRP ion channels superfamily that has been proposed as a potential fish osmosensor in previous studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing the hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 min to 30 days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24 h to 30 d. In contrast, in the kidney, the TRPV4 expression decreased from 10 min to 7d of exposure to FW and then it increased at 30 d. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membranes in branchial lamellae, the cartilage of gills, the posterior pituitary gland and in the collecting ducts. Possible roles of TRPV4 are discussed.

Cellular and molecular osmoregulatory responses to cadmium exposure in Gammarus fossarum (Crustacea, Amphipoda).

Osmoregulation represents a reliable indicator of the physiological state of crustaceans. It is mainly effected in gills via Na(+)/K(+)-ATPase (NKA) providing the major driving force for ion transport. In the present study conducted in the freshwater amphipod Gammarus fossarum, the impact of an exposure to 15 µg Cd L(-1) for 3 and 7d was investigated on the haemolymph osmolality (HO), gill structure, NKA localization in gills and its relative expression. In Cd-exposed G. fossarum, mean HO significantly decreased compared to controls. In animals exposed for 3 and 7d, high inter-individual variations in HO values were noted, resulting in their separation into unimpacted, slightly impacted and impacted animals. In unimpacted individuals, gills retained their organization, showing a thicker gill epithelium than in controls; NKA fluorescence was continuously observed along the gill epithelium and was distributed on a broader area than in controls. In slightly impacted individuals, a thinner epithelium, a slight collapse of the gill and a lower NKA fluorescence were observed compared to unimpacted specimens. In impacted individuals, dramatic alterations of the gill structure, including hyperplasia and alteration of the pillars, resulting in the collapse of the gill and the disappearance of the haemolymphatic canals were observed, as well as very limited NKA fluorescence. Therefore, the degree of gill alteration and the intensity of NKA fluorescence observed in the different groups were correlated with their respective HO levels. The relative amount of the NKA α-subunit mRNA significantly increased in specimens exposed to Cd for 3d compared to controls, and then returned to control level after 7d. The relationships between the changes in HO values, NKA immunostaining and mRNA relative expression are discussed. These results confirm that HO represents a valuable biomarker to evaluate crustacean health, and they underline the interest to assess individual responses to contaminants.

Ontogeny of osmoregulation and salinity tolerance in the gilthead sea bream Sparus aurata.

The gilthead sea bream, Sparus aurata, is a euryhaline teleost that hatches in the open sea. The larvae drift to the coast and juveniles migrate into estuaries and lagoons where the salinity of the water may vary from brackish to hyper-saline. The ontogeny of osmoregulation in Sparus aurata was studied at successive stages, from day 1 (D1) post-hatch to the late juvenile stage (D300) after exposure to different salinities ranging from fresh water to 45.1 per thousand, at 18 degrees C. Survival ranged from between 5.1 and 39.1 per thousand at D3, and from 1.0 to 45.1 per thousand from D75. The fish were hyper-hypo-osmotic regulators at all studied stages. The acquisition of the full ability to hypo- and hyper-regulate occurred in four steps. The osmoregulatory capacity appeared age-dependent and reached its maximum level after D96, and the localization of ionocytes in the integument and gills occurred concurrently during development of the sea bream. However, the main site of osmoregulation shifted from the integument to the gills from D30 to D70, with a corresponding sharp increase in the osmoregulatory ability. Our results suggest that the early development of osmoregulatory ability, and thus of salinity tolerance in the sea bream may provide an advantageous flexibility for the timing of the migration between sea and estuaries and lagoons.

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods.

The tissue distribution and ontogeny of Na(+)/K(+)-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na(+)/K(+)-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na(+)/K(+)-ATPase. Na(+)/K(+)-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na(+)/K(+)-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na(+)/K(+)-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na(+)/K(+)-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 +/- 32.4 micromol g (FM) (-1) h(-1)) than in those of L. vulgaris (31.8 +/- 3.3 micromol g (FM) (-1) h(-1)). S. officinalis gills and pancreatic appendages achieved activities of 94.8 +/- 18.5 and 421.8 +/- 102.3 micromol(ATP) g (FM) (-1) h(-1), respectively. High concentrations of Na(+)/K(+)-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO(2)) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax.

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na(+)/K(+)-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na(+)/K(+)-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of juvenile Dicentrarchus labrax exposed to seawater and freshwater.

The European sea-bass, Dicentrarchus labrax is a euryhaline teleost whose high osmoregulatory abilities allow sea-lagoon migrations. In order to investigate the mechanism underlying the acclimation of juvenile fish to salinity, CFTR was studied in long-term (6 months) freshwater (FW)- and seawater (SW)-exposed fish, and in short-term (from day 0 to day 30) FW-exposed fish. Cellular and molecular approaches were combined to determine the functions of CFTR in the gills, posterior intestine and kidney. In the kidney, the expression of CFTR transcripts and protein is low. After a direct transfer from SW to FW, the CFTR mRNA is down-regulated in the gills within 1 day, followed by a protein decrease over 7 days. In the posterior intestine, first there is a protein decrease within one day and secondly at the mRNA level in 14 days. While in the gills the regulation is transcriptional, in the posterior intestine, there is first a post-transcriptional regulation followed by a transcriptional regulation after 14 days in FW. Over a long-term exposure, there is a transcriptional regulation in both organs. Coupled to other ion transports, CFTR contributes to ion regulation and thus to the adaptation of the European sea-bass to sea-lagoon transitions.