Intestinal FXYD12 and sodium-potassium ATPase: A comparative study on two euryhaline medakas in response to salinity changes.

FXYD proteins are the regulators of sodium-potassium ATPase (Na+/K+-ATPase, NKA). In teleosts, NKA is a primary driving force for the operation of many ion transport systems in the osmoregulatory organs (e.g. intestines). Hence, the purpose of this study was to determine the expression of FXYD proteins and NKA α-subunit in the intestines of two closely related medakas (Oryzias dancena and O. latipes), which came from different salinity habitats and have diverse osmoregulatory capabilities, to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA was the most predominant in the intestines of both medakas. The association of FXYD12 and NKA in the intestines of the two medaka species was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, the localization of FXYD12 and NKA was similar in the intestines of the two medaka species. However, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels), as well as NKA activity differed between the medakas. These results showed that FXYD12 may play a role in modulating NKA activity in the intestines of the two medakas following salinity changes in the maintenance of internal homeostasis. These findings contributed to knowledge of the expression and potential role of vertebrate FXYD12, the regulators of NKA, upon salinity challenge.

The ultrastructural characterization of mitochondria-rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus).

The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos) and the Mozambique tilapia (Oreochromis mossambicus), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae-free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)-rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt-rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)-acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron-dense, and were often vacuolated. The Mt in the PSCs of FW-acclimated milkfish and tilapia were larger and more electron-dense than those of their SW-acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo-osmotic environment. Conversely, the PSCs of tilapia were periodic acid-Schiff (PAS)-positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity-dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae-free and embedded pseudobranchs, whereas the PAS-positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol. 278:390-402, 2017. © 2017 Wiley Periodicals, Inc.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos.

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Comparison of Integrated Responses to Nonlethal and Lethal Hypothermal Stress in Milkfish (Chanos chanos): A Proteomics Study.

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills.

A Stenohaline Medaka, Oryzias woworae, Increases Expression of Gill Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-) Cotransporter 1 to Tolerate Osmotic Stress.

The present study aimed to evaluate the osmoregulatory mechanism of Daisy's medaka, O. woworae,as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of euryhaline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy's medaka belongs to the celebensis species group. The salinity tolerance of Daisy's medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy's medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, "Experimental (Exp.) 10‰ SW", was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy's medaka significantly increased. The protein abundance and distribution of branchial Na(+), K(+)-ATPase (NKA) and Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA αα-subunit protein-abundance was found in the 10‰ SW-acclimated fish. In gill cross-sections, more and larger NKA-immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the euryhaline medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

Different Modulatory Mechanisms of Renal FXYD12 for Na(+)-K(+)-ATPase between Two Closely Related Medakas upon Salinity Challenge.

Upon salinity challenge, the Na(+)-K(+)-ATPase (NKA) of fish kidney plays a crucial role in maintaining ion and water balance. Moreover, the FXYD protein family was found to be a regulator of NKA. Our preliminary results revealed that fxyd12 was highly expressed in the kidneys of the two closely related euryhaline medaka species (Oryzias dancena and O. latipes) from different natural habitats (brackish water and fresh water). In this study, we investigated the expression and association of renal FXYD12 and NKA α-subunit as well as potential functions of FXYD12 in the two medakas. These findings illustrated and compared the regulatory roles of FXYD12 for NKA in kidneys of the two medakas in response to salinity changes. In this study, at the mRNA and/or protein level, the expression patterns were similar for renal FXYD12 and NKA in the two medakas. However, different patterns of NKA activities and different interaction levels between FXYD12 and NKA were found in the kidneys of these two medakas. The results revealed that different strategies were used in the kidneys of the two medaka species upon salinity challenge. On the other hand, gene knockdown experiments demonstrated that the function of O. dancena FXYD12 allowed maintenance of a high level of NKA activity. The results of the present study indicated that the kidneys of the examined euryhaline medakas originating from brackish water and fresh water exhibited different modulatory mechanisms through which renal FXYD12 enhanced NKA activity to maintain internal homeostasis. Our findings broadened the knowledge of expression and functions of FXYD proteins, the modulators of NKA, in vertebrates.

FXYD11 mediated modulation of Na(+)/K(+)-ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments.

FXYD proteins regulate Na(+)/K(+)-ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka (Oryzias dancena) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka-α was elevated 48h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka-α mRNA expression and NKA activity increased significantly 12h and 1h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein-protein interaction between branchial NKA and FXYD11 increased acutely 12h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1h following post-exposure to SW, without the elevation of protein-protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts.

Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes.

Milkfish (Chanos chanos), an important marine aquaculture species in southern Taiwan, show considerable euryhalinity but have low tolerance to sudden drops in water temperatures in winter. Here, we used high throughput next-generation sequencing (NGS) to identify molecular and biological processes involved in the responses to environmental changes. Preliminary tests revealed that seawater (SW)-acclimated milkfish tolerated lower temperatures than the fresh water (FW)-acclimated group. Although FW- and SW-acclimated milkfish have different levels of tolerance for hypothermal stress, to date, the molecular physiological basis of this difference has not been elucidated. Here, we performed a next-generation sequence analysis of mRNAs from four groups of milkfish. We obtained 29669 unigenes with an average length of approximately 1936 base pairs. Gene ontology (GO) analysis was performed after gene annotation. A large number of genes for molecular regulation were identified through a transcriptomic comparison in a KEGG analysis. Basal metabolic pathways involved in hypothermal tolerance, such as glycolysis, fatty acid metabolism, amino acid catabolism and oxidative phosphorylation, were analyzed using PathVisio and Cytoscape software. Our results indicate that in response to hypothermal stress, genes for oxidative phosphorylation, e.g., succinate dehydrogenase, were more highly up-regulated in SW than FW fish. Moreover, SW and FW milkfish used different strategies when exposed to hypothermal stress: SW milkfish up-regulated oxidative phosphorylation and catabolism genes to produce more energy budget, whereas FW milkfish down-regulated genes related to basal metabolism to reduce energy loss.

Comparisons of two types of teleostean pseudobranchs, silver moony (Monodactylus argenteus) and tilapia (Oreochromis mossambicus), with salinity-dependent morphology and ion transporter expression.

There are essentially four different morphological types of pseudobranchs in teleosts, including lamellae-free, lamellae semi-free, covered, and embedded types. In the euryhaline silver moony (Monodactylus argenteus), the pseudobranch belongs to the lamellae semi-free type, which is characterized by one row of filaments on the opercular membrane and fusion on the buccal edge. The pseudobranchial epithelium of the moony contains two types of Na(+), K(+)-ATPase (NKA)-rich cells: chloride cells (CCs) and pseudobranch-type cells (PSCs). Our results revealed increased expression of NKA, the Na(+), K(+), 2Cl(-) cotransporter (NKCC), and the cystic fibrosis transmembrane conductance regulator (CFTR) for Cl(-) secretion and CCs profiles in the pseudobranchs of seawater (SW)-acclimated silver moonies, which indicates the potential role of pseudobranchs containing CCs in hypo-osmoregulation. In contrast, the pseudobranch of the Mozambique tilapia (Oreochromis mossambicus) belongs to the embedded type, which is covered by the connective tissues and only contains PSCs but not CCs. No sign of NKCC and CFTR-immunoreactivity (IR) was found in the pseudobranchs of SW and freshwater (FW) tilapia. However, higher NKA protein expression and larger sizes of NKA-IR PSCs were found in the pseudobranchs of FW-acclimated tilapia. Moreover, in the FW-acclimated moony, NKA-IR PSCs also exhibited higher numbers and larger sizes than in the SW individuals. Taken together, similar responses in low-salinity environments in different types of pseudobranchs indicated that the salinity-dependent morphologies of PSCs might be involved in critical functions for FW teleosts.

The inner opercular membrane of the euryhaline teleost: a useful surrogate model for comparisons of different characteristics of ionocytes between seawater- and freshwater-acclimated medaka.

The inner opercular membranes of the brackish medaka, Oryzias dancena, have numerous ionocytes, similar to the gill epithelia. By histological observation, this study demonstrated that it is possible to investigate the cellular morphology and function of ionocytes in the opercular membrane. The mitochondria-rich ionocytes in the opercular membranes were traced using rhodamine 123 and a cytochrome c oxidase IV antibody in vital and fixed situations, respectively. To validate different morphologies of seawater (SW)-type and freshwater (FW)-type ionocytes of the opercular membrane of euryhaline brackish medaka, a method of dual observation including immunofluorescence staining and subsequent scanning electron microscopy was used. The apical morphologies of SW- and FW-type ionocytes were hole and flat opening, respectively. In addition, the microvilli were found on the apical surface of the FW-type ionocytes. The SW-type ionocytes exhibited basolateral Na(+), K(+), 2Cl(-) cotransporter and the apical cystic fibrosis transmembrane conductance regulator. In contrast, in the apical region of FW-type ionocytes, Na(+), Cl(-) cotransporter and villin 1-like protein were expressed. In addition, histochemical staining of AgCl precipitation counterstained with a Na(+), K(+)-ATPase α-subunit antibody on the opercular membrane illustrated the role of Cl(-) secretion in the SW-type ionocytes of the brackish medaka. A combination of different observations in this study indicated that the opercular membrane could be a useful surrogate model for histological and functional studies on the epithelial ionocytes of fish gills.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways.

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 µM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways.

The lamellae-free-type pseudobranch of the euryhaline milkfish (Chanos chanos) is a Na(+), K(+)-ATPase-abundant organ involved in hypoosmoregulation.

In teleosts, the pseudobranch is hemibranchial, with a gill-like structure located near the first gill. We hypothesized that the pseudobranch of the milkfish might exhibit osmoregulatory ability similar to that of the gills. In this study, the obtained Na(+), K(+)-ATPase (NKA) activity and protein abundance profiles showed that these parameters were higher in the pseudobranchs of the seawater (SW)- than the freshwater (FW)-acclimated milkfish, opposite the situation in the gills. The pseudobranch of the milkfish contained two types of NKA-immunoreactive cells, chloride cells (CCs) and pseudobranch-type cells (PSCs). To further clarify the roles of CCs and PSCs in the pseudobranch, we investigated the distributions of two ion transporters: the Na(+), K(+), 2Cl(-) cotransporter (NKCC) and the cystic fibrosis transmembrane conductance regulator (CFTR). NKCC on the basolateral membrane and CFTR on the apical membrane were found only in pseudobranchial CCs of SW-acclimated individuals. Taken together, the results distinguished NKA-IR CCs and PSCs in the pseudobranch of milkfish using antibodies against NKCC and CFTR as markers. In addition, increases in the numbers and sizes of CCs as well as in NKA expression observed upon salinity challenge indicated the potential roles of pseudobranchs in hypo-osmoregulation in this euryhaline teleost.

Medaka villin 1-like protein (VILL) is associated with the formation of microvilli induced by decreasing salinities in the absorptive ionocytes.

INTRODUCTION: Villin 1 is an actin-regulatory protein involved in the formation of microvilli of mammalian enterocytes. The microvilli, finger-like protrusions, are more abundant on the apical surfaces of gill ionocytes in various freshwater (FW) teleosts than in seawater (SW) fishes. However, the plasticity in the mechanisms of microvillus formation in the gill ionocytes are poorly understood, and the actin-regulatory proteins involved in the formation of microvilli have not been identified in fishes. The present study used the euryhaline medaka (Oryzias dancena) as a model to explore the role of a homolog of villin 1 in the actin-organization of cellular morphologies induced by decreasing salinities. RESULTS: By ultrastructural observation, there are numerous actin filaments organized on the apical cortex of ion-absorptive ionocytes in the FW-acclimated medaka. From gills of the euryhaline medaka, we have identified the VILL sequence. The phylogenetic tree and functional domains suggest that VILL is the homolog of villin 1 in fishes. Immunofluorescence using a specific antibody revealed that VILL was specifically localized to the apical region of gill ionocytes along with microvilli in the FW medaka, but not in SW fish. The expression levels of Odvill mRNA and VILL protein were higher in the gills of the FW individuals than in the SW group and were induced when fish were transferred from SW to FW. A morpholino oligonucleotide for VILL knockdown eliminated the apical protrusions of ionocytes and pavement cells in the trunk epithelia of embryos. CONCLUSIONS: From a novel aspect of cytoskeletal functions, our findings highlighted the important role of VILL protein in the ionoregulation of aquatic vertebrates in response to different osmotic challenges. This study is the first to show that the expression of VILL is associated with the formation of microvilli in the absorptive ionocytes of a euryhaline fish. Loss-of-function experiments showed that the distribution of VILL may represent the molecular link between the cytoskeletal organization and cellular morphology of the absorptive ionocytes during hypoosmotic adaptation in aquatic vertebrates.

Expression profiles of branchial FXYD proteins in the brackish medaka Oryzias dancena: a potential saltwater fish model for studies of osmoregulation.

FXYD proteins are novel regulators of Na(+)-K(+)-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments.

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl(-) through the transporters including the Na(+), K(+)-ATPase (NKA), Na(+), K(+), 2Cl(-) cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl(-) secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (>1day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24h, the Cl(-) secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.

Effects of low environmental salinity on the cellular profiles and expression of Na+, K+-ATPase and Na+, K+, 2Cl- cotransporter 1 of branchial mitochondrion-rich cells in the juvenile marine fish Monodactylus argenteus.

The goal of this study was to determine the osmoregulatory ability of a juvenile marine fish, silver moony (Monodactylus argenteus), for the purpose of developing a new experimental species for ecophysiological research. In this study, M. argenteus was acclimated to freshwater (FW), brackish water (BW), or seawater (SW). The salinity tolerance of this euryhaline species was effective, and the fish survived well upon osmotic challenges. The largest apical surface of mitochondrion-rich cells was found in the FW individuals. Immunohistochemical staining revealed that Na(+), K(+)-ATPase immunoreactive (NKA-IR) cells were distributed in the interlamellar region of the gill filaments of the silver moony in all experimental groups. In addition to the filaments, NKA-IR cells were also found in the lamellae of the FW individuals. The number of NKA-IR cells in the gills of the FW individuals exceeded that of the BW and SW individuals. The NKA-IR cells of FW and SW individuals exhibited bigger size than that of BW fish. The NKA activities and protein expression of the NKA α-subunit in the gills of the FW individuals were significantly higher than in the BW and SW groups. Additionally, the relative amounts of Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were salinity-dependent in the gills. Immunofluorescent signals of NKCC1 were localized to the basolateral membrane of NKA-IR cells in all groups. In the gills of the FW individuals, however, some NKA-IR cells did not exhibit a basolateral NKCC1 signal. In conclusion, the present study illustrated the osmoregulatory mechanisms of this easy- and economic-to-rear marine teleost with euryhaline capacity and proved the silver moony to be a good experimental animal.

Salinity-dependent expression of the branchial Na+/K +/2Cl (-) cotransporter and Na+/K (+)-ATPase in the sailfin molly correlates with hypoosmoregulatory endurance.

In the branchial mitochondrion-rich (MR) cells of euryhaline teleosts, the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) is an important membrane protein that maintains the internal Cl(-) concentration, and the branchial Na(+)/K(+)-ATPase (NKA) is crucial for providing the driving force for many other ion-transporting systems. Hence this study used the sailfin molly (Poecilia latipinna), an introduced aquarium fish in Taiwan, to reveal that the potential roles of NKCC and NKA in sailfin molly were correlated to fish survival rates upon salinity challenge. Higher levels of branchial NKCC were found in seawater (SW)-acclimated sailfin molly compared to freshwater (FW)-acclimated individuals. Transfer of the sailfin molly from SW to FW revealed that the expression of the NKCC and NKA proteins in the gills was retained over 7 days in order to maintain hypoosmoregulatory endurance. Meanwhile, their survival rates after transfer to SW varied with the duration of FW-exposure and decreased significantly when the SW-acclimated individuals were acclimated to FW for 21 days. Double immunofluorescence staining showed that in SW-acclimated sailfin molly, NKCC signals were expressed on the basolateral membrane of MR cells, whereas in FW-acclimated molly, they were expressed on the apical membrane. This study illustrated the correlation between the gradual reductions in expression of branchial NKCC and NKA (i.e., the hypoosmoregulatory endurance) and decreasing survival rates after hyperosmotic challenge in sailfin molly.

The pharyngeal organ in the buccal cavity of the male Siamese fighting fish, Betta splendens, supplies mucus for building bubble nests.

The male Siamese fighting fish, Betta splendens, builds a bubble nest on the water surface to care for offspring during the reproductive period. To our knowledge, this study is the first to determine the composition of the bubble nest and to compare the pharyngeal organs of male and female Siamese fighting fish to determine the relationship between the pharyngeal organ and the ability to make bubble nests. Dot blots of the bubble nest probed with periodic acid-Schiff's (PAS) staining and Ponceau S solution revealed that the contents of the nest are glycoprotein rich. Dissection of the heads of Siamese fighting fish showed that the pharyngeal organ is located in the position through which inhaled air passes. The epithelial structure of the pharyngeal organ of the Siamese fighting fish, like that of other teleosts, has numerous wrinkles and papillae. Mucous goblet cells were observed on the epithelium of pharyngeal organs in male and female fish. The pharyngeal organ was found to be larger in male than in female fish. In addition, the epithelium of the pharyngeal organ in male fish has a greater number of mucous goblet cells than that in female fish. In Siamese fighting fish, this sexual dimorphism of the pharyngeal organ suggests that the male fish secretes more glycoprotein-rich mucus to build the bubble nest. Future work will focus on the type of mucous cells found in the epithelium of the pharyngeal organ that contributes to bubble formation and will determine the components of the mucus in the bubble nest.

Salinity-dependent expression of a Na+, K+, 2Cl- cotransporter in gills of the brackish medaka Oryzias dancena: a molecular correlate for hyposmoregulatory endurance.

This study used the brackish medaka (Oryzias dancena) to characterize Na+, K+, 2Cl- cotransporter (NKCC) expression from the genetic to cellular level in gills. Using RT-PCR to survey tissue distribution of nkcc1a, 1b, and 2, we report that gills of brackish medaka prominently express Odnkcc1a. The full-length cDNA of Odnkcc1a was cloned from gill tissue. In situ hybridization indicates that Odnkcc1a was localized to mitochondrion-rich (MR) cells. Higher mRNA levels of Odnkcc1a were found in gills from seawater (SW) and brackish water (BW) medaka when compared to freshwater (FW) fish. Furthermore, higher amounts of NKCC1a-like protein were detected by the monoclonal antibody in gills of SW and BW medaka compared to FW medaka. Double immunofluorescence staining revealed that NKCC1a-like protein colocalizes with Na+, K+-ATPase on the basolateral membrane of MR cells in BW and SW fish. In addition, transfer of brackish medaka from SW to FW revealed that expression of NKCC1a-like protein in gills was retained until 7days, which is a likely mechanism for maintaining hyposmoregulatory endurance. The study illustrates salinity-dependent expression of NKCC1a in branchial MR cells from brackish medaka and suggests a critical role for NKCC1a in hyposmoregulatory endurance of this fish.

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater.

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na(+)/K(+)-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA alpha-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and alpha-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell numberxcell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.