Shift of Chloride Cell Distribution during Early Life Stages in Seawater-Adapted Killifish, Fundulus heteroclitus

The shift of chloride cell distribution was investigated during early life stages of seawater-adapted killifish (Fundulus heteroclitus). Chloride cells were detected by immunocytochemistry with an an-tiserum specific for Na(+), K(+)-ATPase in whole-mount preparations and paraffin sections. Chloride cells first appeared in the yolk-sac membrane in the early embryonic stage, followed by their appearance in the body skin in the late embryonic stage. Immunoreactive chloride cells in the yolk-sac membrane and body skin often formed multicellular complexes, as evidenced by the presence of more than one nucleus. The principal site for chloride cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gill and opercular membrane in larval and later developmental stages. Our observations suggest that killifish embryos and newly-hatched larvae could maintain their ion balance through chloride cells present in the yolk-sac membrane and body skin until branchial and opercular chloride cells become functional.     

Developmental Sequence of Chloride Cells in the Body Skin and Gills of Japanese Flounder (Paralichthys olivaceus) Larvae

The developmental sequence of chloride cells was examined in both the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae by whole-mount immunocytochemistry using an antiserum specific for Na(+),K(+)-ATPase. In premetamorphic larvae at 0 and 4 days after hatching (days 0 and 4), immunoreactive chloride cells were distributed only in the yolk-sac membrane and body skin. Premetamorphic larvae at days 8-18 possessed both cutaneous and branchial chloride cells. Large chloride cells in the skin of premetamorphic larvae often formed multicellular complexes, suggestive of their ion-secreting function. Cutaneous chloride cells decreased in size and density at the beginning of metamorphosis (days 21 and 24), and disappeared at the metamorphic climax (days 28 and 33). In contrast, branchial chloride cells first appeared at day 8, and increased during metamorphosis. These results indicate that the site for ion secretion in seawater may shift from cutaneous to branchial chloride cells during metamorphosis. The appearance of branchial chloride cells before the differentiation of gill lamellae suggests that the primary function of the gills during the early development is ion regulation rather than gas exchanges.     

Immunolocalization of Vacuolar-Type H(+)-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H(+)-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na(+) in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na(+) uptake in exchange for H(+) secretion through V-ATPase in FW-adapted tilapia during early life stages.     

Distribution of chloride cells in teleost larvae

Distribution and density of the chloride cells in the newly hatched larvae of teleosts vary depending on species and environmental salinity at hatching. In the euryhaline freshwater ayu (Plecoglossus altivelis), chloride cells are concentrated in the skin posterior to the pectoral fins and gradually decrease in number toward the head and tail. In the stenohaline sea water flounder (Kareius bicoloratus), most chloride cells are localized at the inner membrane of gill chambers and in the skin near the openings of gill chambers, but only a few cells appear in the skin of the yolk sac. In the stenohaline freshwater carp (Cyprinus carpio), only a few small chloride cells are scattered in the body skin. The density and abundance of chloride cells appears to be correlated with the different requirements for osmoregulation in teleost larvae.     

Gross morphology and surface ultrastructure of the gills of Odontesthes argentinensis (Actinopterygii, Atherinopsidae) from a Southwestern Atlantic coastal lagoon

Odontesthes argentinensis was collected from Mar Chiquita Coastal Lagoon, the Southernmost coastal Atlantic Lagoon of Argentina. The morphology of the gills was analyzed by scanning electron microscopy. The morphology of the superficial structures of the gill filaments and pharyngeal region of the gill arch was discussed and related to their functional aspects. The gills arches are structurally similar to those of other teleosts and bring out the osmoregulatory capacity of this species. The epithelium that covers the surface of the filaments and the pharyngeal region of the gill arch is formed by polygonal pavement cells with conspicuous microridges. These folds in the membrane are not denoted in the epithelium of the respiratory lamellae. Apical crypts of chloride cells are present on the afferent and interlamellar filament surfaces, but are absent elsewhere on the gill arch. The highest density of mucous cells is observed into the gill filament and the pharyngeal region which indicates the existence of a protective strategy of the respiratory lamellae and the pharynx. The epithelium of the gill arches and the rakers is studded with spines. There are taste buds along the whole pharyngeal region that may be associated with their participation in tasting at this zone.     

Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost Oreochromis mossambicus

The purpose of this study is to provide biochemical evidence for the functions of the mitochondria-rich cell (MR cell) in the yolk-sac epithelium of the developing larvae of tilapia Oreochromis mossambicus. Western blotting with the antibody (6F) raised against avian Na-K-ATPase alpha1 subunit demonstrated the presence of Na-K-ATPase in yolk-sac epithelium of tilapia larvae and about 1. 46-fold more of the enzyme in seawater larvae than in freshwater ones. The yolk-sac MR cells were immunoreacted to the antibody (alpha5) against the alpha subunit of avian Na-K-ATPase and were double-labeled with anthroylouabain and dimethylaminostyrylethyl-pyridiniumiodine, suggesting the existence and activity of Na-K-ATPase in these cells. Binding of 3H-ouabain in the yolk sac of seawater larvae was much higher than in that of freshwater larvae (4.183+/-0.143 pmol/mg protein versus 1.610+/-0. 060 pmol/mg protein or 0.0508+/-0.0053 pmol/yolk sac versus 0. 0188+/-0.0073 pmol/yolk sac). These biochemical results are further evidence that yolk-sac MR cells are responsible for a major role in the osmoregulatory mechanism of early developmental stages before the function of gills is fully developed.     

Ontogenic change in tissue osmolality and developmental sequence of mitochondria-rich cells in Mozambique tilapia developing in freshwater

We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na⁺/K⁺-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day − 3), followed by their appearance in the body skin on day − 2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days − 1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages.     

Placentation in the opossum, Didelphis virginiana

For the first 9 days of gestation, opossum embryos float in uterine secretions, separated from maternal tissues by a shell membrane. Each embryo is part of the wall of its hollow embryonic sphere. By the 10th day of development, the embryo becomes enveloped by both the amnion and yolk-sac. The yolk-sac consists of vascular and non-vascular portions and, together with the surrounding trophectoderm (trophoblast), forms the yolk-sac placenta of the opossum: the allantois does not contribute to formation of the placenta. The vascular portion of the yolk-sac placenta establishes an intimate relationship with the uterine epithelium soon after loss of the shell membrane. The yolk-sac placenta is non-invasive. Cells of the trophoblast exhibit numerous microvilli, an apical endocytic complex and the lateral and basal cell membrane are elaborately folded. These features suggest a cell that is active in the transport of materials. Junctional complexes between cells of the trophoblast and uterine epithelium were not observed. The uterine epithelium changes from ciliated pseudostratified columnar with few infoldings of lateral and basal cell membranes, to non-ciliated simple columnar in which these membranes show elaborate infoldings. The cells show numerous inclusions and mitochondria are polarized to the basal half of the cell. These features suggest a cell that also is active in the transport of materials.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.     

Immunocytochemical distribution of cytochrome P4501A (CYP1A) in developing gilthead seabream, Sparus aurata

CYP1A is a major inducible enzyme in the metabolism of xenobiotic substrates. In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A) during the period of endogenous nutrition (from hatching until day 4) in developing gilthead seabream, Sparus aurata larvae. For this purpose, a polyclonal antiserum (BN-1, Biosense Laboratories) directed against conserved piscine CYP1A sequences was used on paraffin-embedded sections from seabream larvae. From hatching onward, CYP1A immunoreactivity was observed in the following tissues and cells: syncytial, oil-globule envelopes and matrix of the yolk-sac, kidney (epithelia of renal tubules), cardiac muscle cells, skin epidermal cells, troncal musculature, enterocytes of different intestinal regions, goblet cells of the bucco-pharyngeal region, gill epithelial cells and the endothelia of the vascular system of various tissues (especially from liver and brain). Moreover, eye (retina), olfactory epithelium and some positive nerve fibers located in the proximity of the olfactory bulbs and running ventrally toward the posterior brain were strongly CYP1A immunoreactive. In general, the intensity of immunostaining increased with larval development.     

Changes in chloride cell distribution during early larval stages of Clupea harengus

A non-uniform distribution of cutaneous chloride cells was found in the early, pre-feeding larval stages of herring Clupea harengus . Chloride cells on the head, yolk-sac and trunk regions were unevenly distributed, whereas more densely packed chloride cells were observed in the pericardial and prebranchial regions. The pattern of chloride cell distribution changed during development and two distinct changes are described. The density of choride cells on the ventral trunk increased substantially during the period of yolk absorption, presumably due to contraction of the yolk sac and selective retention of yolk-sac chloride cells. Also during this period the cells on the lateral body wall increased in number and became distributed in segmental bands overlying the myosepta. Most chloride cells were found in association with the haemocoel or primordial blood vessels. Superficial segmental blood vessels were not found in the early larva, but the segmental bands of chloride cells overlay nerve tracts in the myosepta which were tentatively identified as the focal innervation of myotomes. It is concluded that both the circulatory system and the peripheral nervous system may play a role in determining chloride cell distribution in early larvae.     

Thyroid status alters gill ionic metabolism and chloride cell morphology as evidenced by scanning electron microscopy in a teleost Anabas testudineus (Bloch): short and long term in vivo study

Gill is the main organ of osmotic regulation in teleosts and chloride cells are the sites of ion transport across gill epithelium. Thyroid hormones are implicated in the regulation of osmotic balance in teleosts also. Treatment with 6-propyl thiouracil (6-PTU) inhibited the membrane bound enzyme Na+K+ ATPase in the gill while triiodothyronine (T3) injection stimulated it in a short-term in vivo study in the teleost Anabas testudineus. Na+, K+ and Ca2+ ions were also decreased in the 6-PTU treated fish and the T3 treatment increased their concentrations in the gill lamellae. The gill morphology also changed according to the thyroid status in the long term study. 6-PTU treatment altered the typical serrated morphology of the gill lamellae, while the T3 treatment reversed it. T3 injection increased the density of pavement and chloride cells as evidenced by scanning electron microscopy. The results demonstrate that physiological status of the thyroid influences gill Na+ pump activity and chloride cell morphological changes. Further, the study suggests a regulatory role of T3 on gill ions (Na+, K+ and Ca2+), Na+K+ and Ca2+ ATPase activity and the different gill cell types in A. testudineus.     

Morphological, histochemical and immunohistochemical study of the gill epithelium in the abyssal teleost fish Coelorhynchus coelorhynchus

Histochemical and immunohistochemical study was carried out on nitrinergic innervation and neuroendocrine system in the gill epithelium of the abyssal fish Coelorhynchus coelorhynchus. The results showed that nNOS-positive nerve fibers, originating from the branchial arch were present in the subepithelial tissue of branchial primary filament. nNOS-positive neuroendocrine cells were also present in the primary filaments and secondary lamellae. Numerous mucous cells in the gill epithelium were AB/PAS-positive, while sialic acid was absent as confirmed by neuraminidase reaction and WGA lectin histochemistry. The mucus compounds in abyssal teleost fish are different from those found in pelagic species, being related to their living conditions. In abyssal species, greater numbers of chloride and neuroendocrine cells are involved in the movement of water and electrolytes. Neuroendocrine cells possess oxygen receptors which mediate the cardiovascular and ventilatory response to oxygen deficiency, as reported in teleost species. Besides, NO contributes through nervous stimulation to the regulation of vascular tone and blood circulation in the gill.     

Histomorphology of the early yolk-sac larvae of the Atlantic halibut (Hippoglossus hippoglossus L.)—an indication of the timing of functionality

Halibut larvae hatch at a very immature stage, and the duration of the yolk-sac period is very long (up to 50 days). This paper describes the histomorphological development of organs of the yolk-sac larvae (6° C) by use of light and electron microscopy. Rudimentary branchial cavities were open from 2 days after hatching. Kidneys seemed functional 16 days after hatching and onwards, and primitive lamellae on the gill arches were beginning to form at this age. Pancreatic zymogen granules were first observed 20daysafter hatching. The liver was segmented into lobes between 20 and 23 days after hatching, and the gall bladder seemed functional from day 23. The hindgut became extensively folded from day 26, and branchial capillaries were first observed at this stage. The larvae were able to catch food particles 24 days after hatching. Judging from ultrastructural observations, it seemed that halibut larvae were able to digest food particles between day 24 and 26 after hatching (around 150 daydegrees and 50% yolk absorption).     

Identification of respiratory and ion-transporting epithelia in the phyllosoma larvae of the slipper lobster Scyllarus arctus

Phyllosoma larvae of the Palinura lack a branchial cavity and gills. In the phyllosoma, gas and ion exchanges that occur at the level of the gill in the adult must occur in other parts of the body or through the entire body. The objective of this study was to localize epithelia bordering the body of the phyllosoma larvae that had features comparable to those of the gill epithelia of adult decapods. The first phyllosoma instar of the small Mediterranean slipper lobster Scyllarus arctus was studied. First, we used a silver nitrate staining method to identify parts of the body with high ionic permeability. Confocal laser scanning microscopy with a fluorescent vital stain for mitochondria, dimethylaminostyrylmethylpyridiniumiodine (DASPMI), was then used to localize cells with a high density of mitochondria. Next, an ultrastructural study of selected epithelia was carried out. A thick (5 microns) mitochondria-rich epithelium covers the ventral side of the cephalic shield; its cells are characterized by the presence of well-developed apical infoldings adjacent to the cuticle. This part of the body has a high ionic permeability as indicated by a positive silver nitrate staining. The ventral mitochondria-rich epithelium might be involved in active ion transport. The rest of the body, particularly the dorsal side of the shield and the appendages, shows a lower ionic permeability (no positive silver nitrate staining) and is limited by a thin (1 micron) epithelium with low numbers of mitochondria. This epithelium exhibits features of a typical respiratory epithelium.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na(+) and Cl(-) transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na(+) and Cl(-) gradients were measured at the yolk-sac surface, indicating secretions of Na(+) and Cl(-) from the yolk-sac skin. With Na(+) pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ～60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na(+) and Cl(-) were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl(-) flux, but not Na(+) flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na(+) and Cl(-) secretion. Following freshwater (FW) to SW transfer, Na(+) and Cl(-) secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na(+) and Cl(-) secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na(+) and Cl(-) secretions declined dramatically after SW to FW transfer and Na(+)/Cl(-) uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na(+) excretion and MRCs possess a functional plasticity in changing from a Na(+)/Cl(-)-secreting cell to a Na(+)/Cl(-)-absorbing cell.     

Surface ultrastructural changes in the gills of sockeye salmon (teleostei: Oncorhynchus nerka) during seawater transfer: Comparison of successful and unsuccessful seawater adaptation

Sockeye salmon were transferred rapidly from freshwater to seawater and the changes in gill morphology, in particular the distribution and sizes of chloride and mucous cells on the afferent filamental surface examined. Salmon that successfully adapted to seawater were compared with salmon that did not adapt to seawater and died as a consequence of osmoregulatory failure. The number of mucus cells (density), determined from scanning electron microscopy, increased significantly after seawater challenge. A greater increase in mucus cell density occurred in the salmon that failed to adapt to seawater. Light microscopy of transverse sections of gills detected no difference in mucus cell numbers after seawater challenge. It is proposed that mucus cells that lie just beneath the gill epithelium are activated in response to the seawater challenge, and migrate and open onto the epithelium. Freshwater-adapted salmon that had low densities of chloride cells prior to the seawater challenge failed to adapt, whereas salmon that had high densities of chloride cells adapted successfully to seawater. In the latter, the density of chloride cells on the afferent surface decreased after 30 days in seawater. The apical surface of the chloride cells of freshwater-adapted sockeye were either smooth or covered with microvilli. A greater proportion of microvilli-covered chloride cells occurred in the freshwater-adapted salmon that subsequently adapted to seawater.     

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.     

The surface epithelium of teleostean fish gills. Cellular and junctional adaptations of the chloride cell in relation to salt adaptation

Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy. In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions. There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells. Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea water-adapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment. Based on these observations we discuss how solutes could transfer across the epithelium.