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Background: Homeostasis of ions and water is important for the maintenance of cellular functions. The regulation of the homeostasis is particularly important in euryhaline fish that migrate between freshwater (FW) and seawater (SW) environments. The fish gill, the major tissue that forms an interface separating the extracellular fluids and external water environment, has an effective transport system to maintain and regulate a constant body osmolality. In fish gills, the two major epithelial cells, pavement cells (PVCs) and mitochondria-rich cells (MRCs), are known to play key and complementary roles in ion transport at the interface. Discovering the robust mechanisms underlying the two cell types' response to osmotic stress would benefit our understanding of the fundamental mechanism allowing PVCs and MRCs to handle osmotic stress. Owing to the limited genomic data available on estuarine species, existing knowledge in this area is slim. In this study, transcriptome analyses were conducted using PVCs and MRCs isolated from Japanese eels adapted to FW or SW environments to provide a genome-wide molecular study to unravel the fundamental processes at work., Results: The study identified more than 12,000 transcripts in the gill cells. Interestingly, remarkable differential expressed genes (DEGs) were identified in PVCs (970 transcripts) instead of MRCs (400 transcripts) in gills of fish adapted to FW or SW. Since PVCs cover more than 90 % of the gill epithelial surface, the greater change in gene expression patterns in PVCs in response to external osmolality is anticipated. In the integrity pathway analysis, 19 common biological functions were identified in PVCs and MRCs. In the enriched signaling pathways analysis, most pathways differed between PVCs and MRCs; 14 enriched pathways were identified in PVCs and 12 in MRCs. The results suggest that the osmoregulatory responses in PVCs and MRCs are cell-type specific, which supports the complementary functions of the cells in osmoregulation., Conclusions: This is the first study to provide transcriptomic analysis of PVCs and MRCs in gills of eels adapted to FW or SW environments. It describes the cell-type specific transcriptomic network in different tonicity. The findings consolidate the known osmoregulatory pathways and provide molecular insight in osmoregulation. The presented data will be useful for researchers to select their targets for further studies.

Osmoregulation in fish has been a classical research topic for several decades. Salmon and eels are the widely used model animals because of their wide distribution in different geographical locations and spawning migration between fresh- and salt-water habitats. Numerous fish osmoregulatory hormones and ion transporters were identified for their essential roles in acclimation and adaptation to waters of different salinities. Because of the lack of a genomic database, the scope of most studies, however, is very limited. Recently, our group reported the first high-throughput transcriptomic and proteomic studies to identify hyperosmotic-responsive genes/proteins in gills of Japanese eels. In this study, we aimed to decipher changes in hypo-osmotic-responsive proteins in fish acclimating from seawater (SW) to freshwater (FW) conditions. We collected gill samples from SW-adapted and SW-to-FW-acclimating fish. The respective gill proteins were extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) and analyzed using a high-resolution mass spectrometer. In the short-term transfer from SW to FW, 51 hypo-responsive proteins were detected, and 24 unique hypo-osmotic-responsive proteins were identified (15 up-regulated and nine down-regulated proteins). Our data support the use of an omics approach to facilitate the application of functional genomics in non-model organisms., Biological Significance: By combining transcriptomic and proteomic approaches, the study has provided the most comprehensive, targeted investigation of eel gill hypo-osmotic responsive proteins that provides molecular insights of osmoregulation mechanisms in a non-model organism, eel. This article is part of a Special Issue entitled: Proteomics of non-model organisms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Osmoregulation is critical for the survival of fishes that migrate between freshwater (FW) and seawater (SW). The eel, as a catadromous fish, has been studied for decades to reveal the mechanisms of osmoregulation. These studies, however, have been limited by the lack of a genomic database to decipher the mechanism of osmoregulation at a molecular level. In this study, using high-throughput transcriptomic and proteomic technologies, we have provided the first genome-wide study to identify hyperosmotic responsive proteins in the gills of the Japanese eel. Deep sequencing using the 454 platform produced over 660,000 reads with a mean length of 385 bp. For the proteomic study, we collected gill samples from three different treatment groups of fish that had fully adapted to FW/SW or were transferred from FW to SW for 6h. The respective group of gill proteins were extracted and labeled using an isobaric tag for relative and absolute quantitation (iTRAQ) using LTQ-Orbitrap, a high resolution mass spectrometer. Among the 1519 proteins identified from the gill samples, 96 proteins were differentially expressed between FW and SW adapted fish. Nineteen hyperosmotic responsive proteins were detected (10 up-regulated and 9 down-regulated proteins) after 6h post FW to SW transfer., Biological Significance: The study has provided the most comprehensive, targeted investigation of eel gill proteins to date, and shown the powerfulness of combining transcriptomic and proteomic approaches to provide molecular insights of osmoregulation mechanisms in a non-model organism, eel., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Eukaryotic cells undergo rapid regulatory processes to maintain cellular homeostasis upon osmotic stress. In fishes, gill epithelial cells play main roles in these processes. Although osmoregulatory functions of fish gills have been well studied, little is known about the underlying mechanisms, particularly the hypertonic-induced signalling pathways during osmotic stress. This study reports for the first time on the osmo-sensing signal cascade that related to the medaka osmotic stress transcription factor 1 (Ostf1), a hypertonic induced immediate early gene, under hypertonic stress. Quantitative real-time PCR showed the rapid increase of Ostf1 in gill after transfer of medaka from fresh water to 50% seawater; particularly Ostf1b whose mRNA expression increased to 4 folds at 0.5h and reached to 10 folds at 6h after the transfer. The in vivo knockdown of Ostf1b profoundly inhibited SEK and JNK phosphorylation, but not p38 and ERK phosphorylation in the medaka gill tissue. To further investigate the possible role of Ostf1b in the JNK pathway, Ostf1b was ectopically expressed in HEK293 cells. Results indicated that Ostf1b is a downstream target of SEK and JNK and exerts a positive feedback loop on the JNK signalling pathway via activation of GCK and/or MLK3 proteins. Additionally, MAPK inhibitors experiments suggested that activation of the JNK pathway by hypertonicity is involved in the maintenance of Ostf1b stability, which in turn provides continuous stimulation of GCK for JNK phosphorylation. Lastly, changes in transcription levels of different water/ion transporters were found in knockdown or ecoptic over-expression of Ostf1b in medaka gills and human embryonic kidney cells, suggesting the role of Ostf1b in modulation of critical water channel/ion transporters during osmotic stress., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake., (Copyright © 2011 Wiley-Liss, Inc.)

It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l(-1)) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+ -ATPase, Na+/K+/2Cl- cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, alpha and beta subunits of Na+/K+ -ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

Fish gills are the major osmoregulatory tissue that contact the external water environment and have developed an effective osmoregulatory mechanism to maintain cellular function. Marine medaka (Oryzias melastigma) has the ability to live in both seawater and fresh water environments. The present study performed a seawater (SW) to 50% seawater (SFW) transfer, and the gill samples were used for comparative transcriptomic analysis to study the alteration of hypo-osmotic stress on immune responsive genes in this model organism. The result identified 518 differentiated expressed genes (DEGs) after the SW to SFW transfer. Various pathways such as p53 signaling, forkhead box O signaling, and the cell cycle were enriched. Moreover, the immune system was highlighted as one of the top altered biological processes in the enrichment analysis. Various cytokines, chemokines, and inflammatory genes that participate in the IL-17 signaling pathway were suppressed after the SW to SFW transfer. On the other hand, some immunoglobulin-related genes were up-regulated. The results were further validated by real-time qPCR. Taken together, our study provides additional gill transcriptome information in marine medaka; it also supports the notion that osmotic stress could influence the immune responses in fish gills. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was applied to the laboratory medaka to understand how the osmotic gradient could influence the composition of the gill microbiota communities. The data suggested that the shift of the gill microbiota community has relied on the first sense of osmolality differences, and such changes were accomplished by the enriched osmosensing and metabolic pathways. The fish gill is the first tissue that is exposed to the external media and undergoes continuous osmotic challenges. Recently, our group published an article entitled "Integrated Omics Approaches Revealed the Osmotic Stress-Responsive Genes and Microbiota in Gill of Marine Medaka" in the journal mSystems (e0004722, 2022), and suggested the possible host-bacterium interaction in the fish gill during osmotic stress. The previous study was performed by the progressive fresh water transfer (i.e., seawater to fresh water transfer via 50% seawater (FW)). Our group hypothesized that osmotic gradient could be a factor that determines the microbiota communities in the gill. The current 16S rRNA metagenomic sequencing study found that the direct transfer (i.e., seawater to fresh water (FWd)) could result in different gill microbiota communities in the same fresh water endpoints. Pseduomonas was the dominant bacteria (more than 55%) in the FWd gill. The Kyoto Encyclopedia of Genes and Genomes and MetaCyc analysis further suggested that the FWd group had enhanced osmosensing pathways, such as the ATP-binding cassette transporters, taurine degradation, and energy-related tricarboxylic acid metabolism compared to the FW group. [ABSTRACT FROM AUTHOR]