Catherine Lorin-Nebel

The Na + /K + /2Cl - cotransporter in the sea bass Dicentrarchus labrax during ontogeny: involvement in osmoregulation

SUMMARY This study combines a cellular and molecular analysis of the Na+/K+/2Cl- cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1-4 days), medium- (7-21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na+/K+-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of Dicentrarchus labrax during ontogeny

The expression and localization of the cystic fibrosis transmembrane conductance regulator (CFTR) were determined in four osmoregulatory tissues during the ontogeny of the sea‐bass Dicentrarchus labrax acclimated to fresh water and sea water. At hatch in sea water, immunolocalization showed an apical CFTR in the digestive tract and integumental ionocytes. During the ontogeny, although CFTR was consistently detected in the digestive tract, it shifted from the integument to the gills. In fresh water, CFTR was not present in the integument and the gills, suggesting the absence of chloride secretion. In the kidney, the CFTR expression was brief from D4 to D35, prior to the larva–juvenile transition. CFTR was apical in the renal tubules, suggesting a chloride secretion at both salinities, and it was basolateral only in sea water in the collecting ducts, suggesting chloride absorption. In the posterior intestine, CFTR was located differently from D4 depending on salinity. In sea water, the basolateral CFTR may facilitate ionic absorption, perhaps in relation to water uptake. In fresh water, CFTR was apical in the gut, suggesting chloride secretion. Increased osmoregulatory ability was acquired just before metamorphosis, which is followed by the sea‐lagoon migration.

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.

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.

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.

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.

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.

Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes

European sea bass Dicentrarchus labrax undertake seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. Their ability to cope with these unstable habitats is undeniable, but it is still not clear how and to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18°C (temperate) or 24°C (warm) for 2weeks and then transferred to fresh water (FW) or SW at the respective temperature. Transfer to FW for two weeks resulted in decreased blood osmolalities and plasma Cl- at both temperatures. In FW warm conditions, plasma Na+ was ~15% lower and Cl- was ~32% higher than in the temperate-water group. Branchial Na+/K+-ATPase (NKA) activity measured at the acclimation temperature (Vapparent) did not change according to the conditions. Branchial Na+/K+-ATPase activity measured at 37°C (Vmax) was lower in warm conditions and increased in FW compared to SW conditions whatever the considered temperature. Mitochondrion-rich cell (MRC) density increased in FW, notably due to the appearance of lamellar MRCs, but this increase was less pronounced in warm conditions where MRCs size was lower. In SW warm conditions, pavement cell apical microridges are less developed than in other conditions. Overall gill morphometrical parameters (filament thickness, lamellar length and width) differ between fish that have been pre-acclimated to different temperatures. This study shows that a thermal change affects gill plasticity affecting whole-organism ion balance two weeks after salinity transfer.

Dynamic expression of vasotocin and isotocin receptor genes in the marbled eel (Anguilla marmorata) following osmotic challenges

To examine the physiological roles of arginine vasotocin receptor (AVTR) and isotocin receptor (ITR) in osmoregulation of a euryhaline teleost, the marbled eel (Anguilla marmorata), three different genes coding for AVTRV1a2, AVTRV2 and ITR were cloned by screening an A. marmorata cDNA library. These receptors were expressed differentially and ubiquitously in the eight tissues we examined. The changes in mRNA expression levels of AVTRV1a2, AVTRV2, and ITR were assessed in a time-course study following salinity transfer from fresh water (FW, 0‰) to fresh water (FW, 0‰), brackish water (BW, 10‰) or saline water (SW, 25‰). When eels were transferred to BW, mRNA levels underwent an adaptive period, from 0 to 24 h, and a chronic regulatory period, starting at 24 h after transfer. In the adaptive period, the relative mRNA expression of AVTRV1a2, AVTRV2, and ITR increased in BW. But after this adaptive period, the mRNA levels of the three genes were significantly decreased compared to FW (control group, 0 h). The mRNA expression levels of AVTRV1a2, AVTRV2 and ITR were low in SW. The protein level of AVTRV1a2, a key protein in the brain, was also investigated and found to be consistent with mRNA results. Our results indicated that the nonapeptide receptor system may play a role in the acute stress response induced by hyper-osmotic challenge in marbled eels.