Ultrastructure and development of the gills in<Emphasis Type="Italic"> Rana dalmatina</Emphasis> (Amphibia,Anura)

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Ortons larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Recruitment and degeneration of mitochondrion-rich cells in the gills of Mozambique tilapia Oreochromis mossambicus during adaptation to a hyperosmotic environment

Cellular recruitment and degeneration of branchial mitochondrion-rich (MR) cells were examined in Mozambique tilapia transferred from hypoosmotic to hyperosmotic water. To examine apoptosis in the gills associated with salinity change, tilapia were directly transferred from freshwater to 70% seawater. The TUNEL assay showed that apoptotic cells in the gills were significantly increased at 1 day after transfer, which was supported by an electron-microscopic observation that gill MR cells underwent morphological changes characteristic of apoptosis such as an irregularly shaped electron-dense nucleus and distension of the tubular system. To further examine MR-cell recruitment, freshwater-acclimated tilapia were transferred either to freshwater or to 70% seawater after BrdU injection. Immunohistochemical detection of BrdU-labeled nuclei and Na(+)/K(+)-ATPase-rich MR cells allowed us to classify BrdU-labeled MR cells into two subtypes: a single MR cell and an MR-cell complex. Although newly generated single MR cells were observed similarly in both freshwater and 70% seawater-transferred fish, the density of MR-cell complexes was much higher in 70% seawater than in freshwater. Our findings indicated that transfer from hypoosmotic to hyperosmotic water enhanced apoptosis of freshwater-type MR cells, resulting in reduction in hyperosmoregulatory ability for freshwater adaptation, and stimulated the recruitment of MR-cell complexes to develop hypoosmoregulatory ability for seawater adaptation.     

Teleost chloride cell. II. Autoradiographic localization of gill Na,K- ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments

The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Oxygen, gills, and embryo behavior: mechanisms of adaptive plasticity in hatching

Many species alter the timing of hatching in response to egg or larval predators, pathogens, or physical risks. This plasticity depends on separation between the onset of hatching competence and physiological limits to embryonic development. I present a framework based on heterokairy to categorize developmental mechanisms and identify traits contributing to and limiting hatching plasticity, then apply it to a case of predator-induced hatching. Red-eyed treefrogs have arboreal eggs, and tadpoles fall into ponds upon hatching. Egg and tadpole predators select for earlier and later hatching, respectively. Embryos hatch up to 30% early in predator attacks, and later if undisturbed. They maintain large external gills throughout the plastic hatching period, delaying gill regression while development otherwise continues. Rapid gill regression occurs upon hatching. Prolonged embryonic development depends on external gills; inducing gill regression causes hatching. External hypoxia retards development, kills eggs, and induces hatching. Nonetheless, embryos develop synchronously and without hatching prematurely across a broad range of perivitelline PO₂, from 0.5–12.5 kPa. Embryos exploit spatial variation of PO₂ within eggs by positioning gills against patches of air-exposed surface. Respiratory plasticity and oxygen-sensitive behavior appear critical for the hatching plasticity that balances a predation risk trade-off across life stages.     

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60 S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.     

Biochemical characterization of isolated branchial mitochondria-rich cells of Oreochromis mossambicus acclimated to fresh water or hyperhaline sea water

Mitochondria-rich cells have been separated from other epithelial cells of tilapia (Oreochromis mossambicus) gills by density gradient centrifugation on Percoll. During centrifugation two main bands of cells formed. The viability of the cells in both bands was high (>90%). In one band, 45–47% of the total cell number was mitochondria-rich cells. The other band contained at least 80% pavement cells, representing the majority of other gill epithelial cell types. A comparison of the activities of four enzymes involved in major metabolic and ion regulatory functions was made between these two different fractions of cells. Furthermore, the separation of gill epithelial cells and determination of enzymatic activity was carried out in tilapia after the fish were acclimated to fresh water or hyperhaline sea water (60 mg·ml^-1 S) to gain an indication of the relative contribution of mitochondria-rich cells and pavement cells to both NaCl excretion and absorption. Regardless of acclimation salinity, the activities of Na⁺/K⁺-ATPase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were significantly higher in mitochondria-rich cells than in pavement cells. However, tilapia acclimated to hyperhaline sea water possessed significantly lower carbonic anhydrase activity in mitochondria-rich cells than in pavement cells. In contrast, no significant difference of carbonic anhydrase activity was observed between the two cell fractions in tilapia acclimated to fresh water.Abbreviations ATPase adenosine triphosphatase - CA carbonic anhydrase - DASPMI dimethylaminostyrylmethylpyridinium iodine - FW fresh-water - GIDH glutamate dehydrogenase - G6PDH glucose-6-phosphate dehydrogenase - HSW hyperhaline sea water (60 mg·ml^-1) - MR cells, mitochondria-rich cells - S salinity     

Do prostaglandins regulate external gill regression in anurans?

Although the endocrinological mechanism controlling regression of the internal, larval gills of anurans (frogs and toads) is well understood, the mechanism regulating loss of the external, embryonic gills is not known. Based on the homology of the mammalian ductus arteriosus with a portion of the amphibian branchial arches, and the regulation of blood flow in the mammalian ductus by prostaglandins of the E family (PGEs), we hypothesized that anuran external gill loss is also regulated by PGEs. To test this hypothesis, we topically applied both PGE2 and a synthetic analogue of PGE1, misoprostol, to embryos and young hatchlings of the red-eyed treefrog, Agalychnis callidryas. Both agents accelerated external gill regression. Furthermore, misoprostol overrode the inhibitory effect of hypoxia on gill regression in hatchlings and induced rapid loss of external gills in embryos, which normally maintain the gills until hatching. These observations support the hypothesis that PGEs regulate anuran external gill loss. The specific site of action for prostaglandins within the gills is not known; however, PGEs are secreted in the oral mucus of tadpoles, and this could be a natural topical source for these agents. PGEs offer a tool for manipulation of external gills and should facilitate tests of the physiological importance of these structures.     

Seasonal changes in sea-water tolerance of Arctic charr (Salvelinus alpinus)

Summary Groups of Arctic charr,Salvelinus alpinus, which had been acclimated to water with a salinity of 7 g·l^–1 and natural temperature and photoperiod, were exposed to water with different salinities and temperatures in June, September and February. At a salinity of 15 g·l^–1, plasma osmolality, plasma Na⁺, Cl^–, Mg²⁺ concentrations and the activity of gill Na-K-ATPase were stable, irrespective of temperature and season. In June, the charr were able to regulate blood plasma ionic levels within narrow limits when exposed to a salinity of 34 g·l^–1 (sea water) and a temperature of 8°C. The hypo-osmoregulatory capacity was less, but sufficient if the temperature was only 1°C during the seawater exposure. At the start of the experiment, the gill Na-K-ATPase activity was significantly higher in June than corresponding enzyme activities in September and February. Furthermore, an increase in gill Na-K-ATPase activity during the seawater exposure (8°C) was seen in June. Irrespective of ambient temperature and salinity, no fish died during the June experiments. In September and February, exposure to sea water produced marked increases in plasma osmolality and plasma ion concentrations. There were no changes in gill Na-K-ATPase activity. Consequently, the fish became dehydrated and were moribund after a short period of seawater exposure. Highest mortality was recorded when charr were exposed to winter sea conditions (34 g·l^–1 and 1°C) in February. The results indicate that an increase in daylength induce a hypo-osmoregulatory capacity in the Arctic charr during summer. In fall and winter, however, reduced daylength are accompanied by poor hypo-osmoregulatory capacity. This leads to high mortality as a result of increased electrolyte levels and dehydration.     

Adaptive branchial mechanisms in the sturgeon Acipenser naccarii during acclimation to saltwater

Variations of Na(+)/K(+)-ATPase activity and fatty-acid composition in the gills of the sturgeon Acipenser naccarii subjected to progressive acclimation to full seawater (35 ppt) were determined in relation to the hypo-osmoregulatory capacity of this species in the hyperosmotic medium. Blood samples were taken and gills arches were removed at intermediate salinity levels between 0 and 35 ppt and after 20 days at constant salinity (35 ppt). Plasma osmolality and Na(+)/K(+)-ATPase activity increased significantly with growing environmental salinity. Total saturated fatty acids (SFAs) decreased, while total polyunsaturated fatty acids (PUFAs) increased significantly with increasing salinity due mainly to changes in n-3 PUFAs (20:5n-3 and 22:6n-3). The n-3/n-6 ratio increased significantly during the acclimation process. The results show a direct relationship between salinity, increased gill Na(+)/K(+)-ATPase activity and ultrastructural changes of the gill chloride cells. Changes in the fatty-acid composition in gills of A. naccarii during progressive acclimation to full seawater suggest that variations of gill fatty acids may also have a role in osmoregulatory mechanisms.     

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.     

Rapid morphological oscillation of mitochondrion-rich cell in estuarine mudskipper following salinity changes

Morphological changes in the chloride cells or mitochondrion-rich (MR) cells in the skin under the pectoral fin of the estuarine mudskipper (Periophthalmus modestus) were examined in relation to intertidal salinity oscillation in river mouth. MR cells were distinguished between those in contact with the water (cells labeled with both mitochondrial probe DASPEI and Concanavalin-A, an apical surface marker of MR cells) and those that are not (DASPEI-positive only). After transfer of the fish from seawater to freshwater, no difference in the total MR cell density was observed, but the subpopulation of MR cells that are Concanavalin-A-positive decreased dramatically within 30 min. After 6 hr in freshwater, the fish were returned to seawater; the number of Con-A-positive MR cells increased to the initial levels rapidly. Thus, in seawater, mudskippers seem to open the apical crypts of the MR cells to secrete salt; in freshwater, they close the crypt of the MR cells tentatively, and tolerate hypotonicity until the rising tide. This unique response of chloride cells may also be seen in gills of other estuarine species.     

Ultrastructural features of chloride cells in the gill epithelium of the Atlantic salmon, Salmo salar, and their modifications during smoltification

To elucidate the ultrastructural modifications of the gill epithelium during smoltification, gills of the Atlantic salmon (Salmo salar) were examined by electron microscopy at three stages of this process, which were defined as follows: "parrs" were freshwater fish that had not yet started their transformation; "freshwater smolts" were freshwater fish that were ready to enter seawater; and "seawater smolts" were smolts that had been transferred from fresh water and maintained for 4 days in seawater (35%). In the gill epithelium of parrs, there were two types of chloride cells. The large chloride cells contained deeply stained mitochondria and numerous apical, irregular, dense, membrane-bound bodies that formed 77% of the chloride cell population and were distinguished easily from small chloride cells that have distinctly paler mitochondria and no dense bodies in their apical cytoplasm. In freshwater smolts, the large chloride cells formed 95% of the chloride-cell population. In contrast to the small chloride cells that were not modified, they almost doubled in size. Their tubular system developed extensively to form a tight network with regular meshes significantly smaller than those observed in parr chloride cells. Forty percent of the large chloride cells were associated with a new type of cell, the accessory cell, to which they were bound by shallow apical junctions. Half of these accessory cells were not seen to be in contact with the external medium. In seawater smolts, 80% of the large chloride cells were associated with accessory cells. Most accessory cells reached the external medium and sent numerous cytoplasmic interdigitations within the apical portion of the adjacent chloride cells. As a result, a section through the apical portion of the chloride cells and their associated accessory cells revealed a mosaic of interlocked cell processes bound together by an extended, shallow apical junction. It was concluded that the Atlantic salmon develops in fresh water most of the ultrastructural modifications of the gill epithelium which in most euryhaline fish are triggered by exposure to seawater. The effective transfer into seawater would act only as a final stimulus to achieve some adequacy between the freshwater smolt and its new environment.     

Teleost chloride cell. I. Response of pupfish Cyprinodon variegatus gill Na,K-ATPase and chloride cell fine structure to various high salinity environments

Certain euryhaline teleosts can tolerate media of very high salinity, i.e. greater than that of seawater itself. The osmotic gradient across the integument of these fish is very high and the key to their survival appears to be the enhanced ability of the gill to excrete excess NaCl. These fish provide an opportunity to study morphological and biochemical aspects of transepithelial salt secretion under conditions of vastly different transport rates. Since the cellular site of gill salt excretion is believed to be the "chloride cell" of the branchial epithelium and since the enzyme Na,K-ATPase has been implicated in salt transport in this and other secretory tissues, we have focused our attention on the differences in chloride cell structure and gill ATPase activity in the variegated pupfish Cyprinodon variegatus adapted to half-strength seawater (50% SW), seawater (100% SW), or double-stregth seawater (200% SW). The Na,K-ATPase activity in gill homogenates was 1.6 times greater in 100% SW. When 50% SW gills were compared to 100% SW gills, differences in chloride cell morphology were minimal. However, chloride cells from 200% SW displayed a marked hypertrophy and a striking increase in basal-lateral cell surface area. These results suggest that there are correlations among higher levels of osmotic stress, basal-lateral extensions of the cell surface, and the activity of the enzyme Na,K-ATPase.     

Cellular distributions of creatine kinase in branchia of euryhaline tilapia (Oreochromis mossambicus)

Although euryhalineteleosts can adapt to environmental fluctuation of salinity, theirenergy source for responding to changes in salinity and osmolarityremains unclear. This study examines the cellular localization ofcreatine kinase (CK) expression in branchia of tilapia(Oreochromis mossambicus). Western blot analysis ofmuscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted toseawater did not change. With the use of immunocytochemistry, three CKisoforms (MM, MB, and BB) were localized in mitochondria-rich (MR)cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To ourknowledge, this work presents the first evidence of CK expression in MRcells of tilapia gills, highlighting the potential role of CK inproviding energy for ion transport.

     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

Structural changes in the zonulae occludentes of the chloride cells of young adult lampreys following acclimation to seawater

Summary Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na⁺.     

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander,Hynobius nigrescens Stejneger

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na⁺, K⁺‐ATPase (NKA), vacuolar‐type H⁺‐ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43–65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria‐rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA‐, NKA‐ and ENaC‐, and VHA‐positive cells. VHA‐immunoreactive cells as well as NKA‐positive cells were observed during IY stages 43–65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid–base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium. J. Morphol., 2011. © 2011Wiley‐Liss, Inc.     

Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch

Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl(-)/HCO3(-) exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na(+)-K(+)-ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl(-)/HCO3(-) exchange in gills of Atlantic stingrays from both freshwater and marine environments.