The effects of acute transfer to freshwater on ion transporters of the pharyngeal cavity in European seabass (Dicentrarchus labrax).

Gene expression of key ion transporters (the Na+/K+-ATPase NKA, the Na+, K+-2Cl- cotransporter NKCC1, and CFTR) in the gills, opercular inner epithelium, and pseudobranch of European seabass juveniles (Dicentrarchus labrax) were studied after acute transfer up to 4 days from seawater (SW) to freshwater (FW). The functional remodeling of these organs was also studied. Handling stress (SW to SW transfer) rapidly induced a transcript level decrease for the three ion transporters in the gills and operculum. NKA and CFTR relative expression level were stable, but in the pseudobranch, NKCC1 transcript levels increased (up to 2.4-fold). Transfer to FW induced even more organ-specific responses. In the gills, a 1.8-fold increase for NKA transcript levels occurs within 4 days post transfer with also a general decrease for CFTR and NKCC1. In the operculum, transcript levels are only slightly modified. In the pseudobranch, there is a transient NKCC1 increase followed by 0.6-fold decrease and 0.8-fold CFTR decrease. FW transfer also induced a density decrease for the opercular ionocytes and goblet cells. Therefore, gills and operculum display similar trends in SW-fish but have different responses in FW-transferred fish. Also, the pseudobranch presents contrasting response both in SW and in FW, most probably due to the high density of a cell type that is morphologically and functionally different compared to the typical gill-type ionocyte. This pseudobranch-type ionocyte could be involved in blood acid-base regulation masking a minor osmotic regulatory capacity of this organ compared to the gills.

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.

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.

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.

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality.

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na(+) /K(+) -ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na(+) /K(+) /2Cl⁻ cotransporters (NKCCs) and the chloride channel "cystic fibrosis transmembrane regulator" (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.

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.

Digestive tract ontogeny of Dicentrarchus labrax: implication in osmoregulation.

The ontogeny of the digestive tract (DT) and of Na(+)/K(+)-ATPase localization was investigated during the early postembryonic development (from yolk sac larva to juvenile) of the euryhaline teleost Dicentrarchus labrax reared at two salinities: seawater and diluted seawater. Histology, electron microscopy and immunocytochemistry were used to determine the presence and differentiation of ion transporting cells. At hatching, the DT is an undifferentiated straight tube over the yolk sac. At the mouth opening (day 5), it comprises six segments: buccopharynx, esophagus, stomach, anterior intestine, posterior intestine and rectum, well differentiated at the juvenile stage (day 72). The enterocytes displayed ultrastructural features similar to those of mitochondria-rich cells known to be involved in active ion transport. At hatching, ion transporting cells lining the intestine and the rectum exhibited a Na(+)/K(+)-ATPase activity which increased mainly after the larva/juvenile (20 mm) metamorphic transition. The immunofluorescence intensity was dependent upon the stage of development of the gut as well as on the histological configuration of the analyzed segment. The appearance and distribution of enteric ionocytes and the implication of the DT in osmoregulation are discussed.

Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system.

The effects of long-term freshwater acclimatization were investigated in juvenile sea-bass Dicentrarchus labrax to determine whether all sea-bass juveniles are able to live in freshwater and to investigate the physiological basis of a successful adaptation to freshwater. This study particularly focused on the ability of sea-bass to maintain their hydromineral balance in freshwater and on their ion (re)absorbing abilities through the gills and kidneys. Two different responses were recorded after a long-term freshwater acclimatization. (1) Successfully adapted sea-bass displayed standard behavior; their blood osmolality was maintained almost constant after the freshwater challenge, attesting to their efficient hyperosmoregulation. Their branchial and renal Na+/K+-ATPase abundance and activity were high compared to seawater fish due to a high number of branchial ionocytes and to the involvement of the urinary system in active ion reabsorption, producing hypotonic urine. (2) Sea-bass that had not successfully adapted to freshwater were recognized by abnormal schooling behavior. Their blood osmolality was low (30% lower than in the successfully adapted sea-bass), which is a sign of acute osmoregulatory failure. High branchial Na+/K+-ATPase abundance and activity compared to successfully adapted fish were coupled to a proliferation of gill chloride cells, whose ultrastructure did not display pathological signs. The large surface used by the gill chloride cells might negatively interfere with respiratory gas exchanges. In their urinary system, enzyme abundance and activity were low, in accordance with the observed lower density of the kidney tubules. Urine was isotonic to blood in unsuccessfully adapted fish, ruling out any participation of the kidney in hyperosmoregulation. The kidney failure seems to generate a compensatory ion absorption through increased gill activity, but net ion loss through urine seems higher than ion absorption by the gills, leading to lower hyper-osmoregulatory performance and to death.

Osmoregulation, immunolocalization of Na+/K+-ATPase, and ultrastructure of branchial epithelia in the developing brown shrimp, Crangon crangon (Decapoda, Caridea).

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+-ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0 per thousand to 32.2 per thousand, salinity tolerance was generally high (survival rates: 70%-100%) in all developmental stages, but it decreased in media <10.2 per thousand. Zoeal stages and decapodids slightly hyperregulated at 17.0 per thousand and osmoconformed in media > or =25.5 per thousand. At 10.2 per thousand, these stages showed high mortality, and only juveniles survived at 5.3 per thousand. Juveniles hyperregulated at 10.2 per thousand and 17.0 per thousand, osmoconformed at 25.5 per thousand, and hyporegulated in media > or =32.2 per thousand. Large juveniles hyperregulated also at 5.3 per thousand. Expression of the Na+/K+-ATPase and ion-transporting cells was located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion-transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+-ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyperosmoconforming decapodid to an effectively hyper-/hyporegulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion-transporting cells and the expression of Na+/K+-ATPase.

Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk X-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus.

We studied the ontogeny of the eyestalk structure and of the L-CHH and d-Phe3-CHH synthesis in the X-organ/sinus gland (XO/SG) complex by light microscopy and immunocytochemistry in the freshwater crustacean Astacus leptodactylus. The optic ganglia start to differentiate in embryos at EI 190 microm (EI: eye index; close to 410 microm at hatching). At EI 270 microm, the three medullae (externa, interna, and terminalis) and the lamina ganglionaris are present and are organized as in the adult eyestalk. The L-CHH was localized in perikarya of neuroendocrine cells, in their tracts, and in SG from the metanauplius stage to the adult. The d-Phe3-CHH was visualized in XO perikarya, in their tracts and in SG of embryos from EI 350 microm and in all later studied stages. Co-localization of both CHH stereoisomers always occurred in the d-Phe3-CHH-producing cells. These results show that the synthesis of CHH enantiomers starts during the embryonic life in A. leptodactylus, and that the d-isomer is synthesized later than its L-counterpart. We discuss the post-translational isomerization as a way to generate hormonal diversity and the putative relation between d-Phe3-CHH synthesis and the ability to osmoregulate, occurring late during the embryonic life of Astacus leptodactylus.

Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus.

This study investigates the involvement of eyestalk neuroendocrine factors on osmoregulation in the crayfish Astacus leptodactylus maintained in freshwater. Eyestalk removal was followed by a significant decrease in hemolymph osmolality and Na(+) concentration and by a 50% increase in mass after one molting cycle. Several neurohormones have been isolated from the sinus gland through high-performance liquid chromatography (HPLC), and different crustacean hyperglycemic hormone (CHH)-related peptides, including stereoisomers (L-CHH and D-Phe(3) CHH), have been identified by direct enzyme-linked immunosorbent assay (ELISA). A glucose quantification bioassay demonstrated a strong hyperglycemic activity following injection of the immunoreactive chromatographic fractions and showed that the D-Phe(3) CHH was the most efficient. Destalked crayfish were then injected with purified CHH HPLC fractions. The D-Phe(3) CHH fraction significantly increased the hemolymph osmolality and Na(+) content 24 h after injection. Two other CHH-related peptides caused a smaller increase in Na(+) concentration. No significant variation was observed in hemolymph Cl(-) concentration following injection of any of the CHH isoforms. These results constitute the first observation of the effects of a CHH isoform, specifically the D-Phe(3) CHH, on osmoregulatory parameters in a freshwater crustacean. The effects of eyestalk ablation and CHH injection on osmoregulation and the identification of different CHH-related peptides and isoforms in crustaceans are discussed.