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Functional dynamics of claudin expression in Japanese medaka (Oryzias latipes): Response to environmental salinity
Abstract
Salinity regulation of 13 claudin paralogs was investigated in osmoregulatory organs of euryhaline Japanese medaka. They were identified by blast-search in the medaka genome database based on representation in osmoregulatory organs of other teleosts. Our hypothesis was that, because of their sequence similarities to mammalian orthologs previously characterized as barrier- and ion-selective channel-forming proteins, these paralogs would respond to salinity according to expected modulation of osmoregulatory function. Cldn10c, -10d, -10e, -10f, -27a, -28a, -28b and -30c had 4- to 100-fold higher expression in gill than other examined organs. Two splice variants of cldn10b were predominantly expressed in kidney, while cldn15a and -b and -25 were found mainly in intestine. In gills, cldn27a, -28a, -28b and -30c did not change between fresh water (FW) and seawater (SW)-acclimated fish, while cldn10c, -10d, -10e, -10f were most abundant in SW. Short-term SW transfer induced up-regulation of cldn10 gill paralogs after 1 day, decrease in cldn28b and no difference for cldn27a, -28a and -30c. The reverse pattern was observed after FW transfer of SW medaka. Intestinal cldn15a and -25 did not differ between FW and SW fish. However, cldn15b was 10-fold higher in FW than SW, suggesting a role in functional modulation of the intestine related to water and salt transport. In kidney, cldn10bs were elevated in SW fish, suggesting a role in paracellular ion transport in the marine nephron. Based on in silico analysis, most gill Cldn10s were predicted to form cation pores, whereas Cldn27a, 28a, 28b and 30c may increase epithelial resistance.
Copyright © 2015. Published by Elsevier Inc.
... In mice, CLDN15-mediated Na + back-flux into the intestinal lumen is essential for active glucose absorption through the Na + /glucose cotransporter, safeguarding monosaccharide uptake [30]. In fishes, Cldn15a paralogs have been found to be expressed specifically in the gastrointestinal (GI) tract (salmon [31,32], zebrafish [33], medaka [34]). In medaka, we previously identified an additional new paralog, Cldn15b, which is also primarily expressed in the intestine at levels several orders of magnitude higher than any other examined organs [34]. ...
... In fishes, Cldn15a paralogs have been found to be expressed specifically in the gastrointestinal (GI) tract (salmon [31,32], zebrafish [33], medaka [34]). In medaka, we previously identified an additional new paralog, Cldn15b, which is also primarily expressed in the intestine at levels several orders of magnitude higher than any other examined organs [34]. ...
... The time-course experiment showed that cldn15b was not significant affected by SW before 24 h and 168 h while the inhibitory effect on the two aqps was apparent at the 6 h and 24 h mark but not significant at the 168 h time point. Thus, while the dynamics of regulation are not straightforward, the overall inhibitory effect of SW on cldn15b, aqp1a, and aqp8ab observed previously was confirmed [8,34]. ...
When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two cldn15 paralogs, cldn15a and cldn15b, have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of cldn15a, while cldn15b was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.
... In Atlantic salmon (Salmo salar), spotted green puffer fish (Tetraodon nigroviridis) and Japanese medaka (Oryzias latipes), SW-acclimation entails the elevated expression of branchial cldn10c, cldn10d and cldn10e (Tipsmark et al., 2008a;Bui and Kelly, 2014;Bossus et al., 2015). Based on the amino acid sequences of their first extracellular loops, these paralogs could be involved in forming the cation-selective pathway associated with Na + extrusion (Günzel et al., 2009;Bossus et al., 2015). ...
... In Atlantic salmon (Salmo salar), spotted green puffer fish (Tetraodon nigroviridis) and Japanese medaka (Oryzias latipes), SW-acclimation entails the elevated expression of branchial cldn10c, cldn10d and cldn10e (Tipsmark et al., 2008a;Bui and Kelly, 2014;Bossus et al., 2015). Based on the amino acid sequences of their first extracellular loops, these paralogs could be involved in forming the cation-selective pathway associated with Na + extrusion (Günzel et al., 2009;Bossus et al., 2015). Mammalian Cldn4 shares a common ancestor with 13 teleost Cldns (Baltzegar et al., 2013), some of which (Cldn28a and Cldn30) have salinity-dependent expression in the gill of Atlantic salmon and Mozambique tilapia. ...
... In both species, cldn28a and cldn30 levels are elevated in FW-acclimated animals (Tipsmark et al., 2008a(Tipsmark et al., , 2008b. Cldn30 is directly involved in the tightening of fish epithelia (Engelund et al., 2012;Kwong and Perry, 2013), while the sequence of Cldn28a suggests a similar capacity as its barrier-forming mammalian homologs (Hou et al., 2013;Bossus et al., 2015). Cortisol and prolactin directly decrease the permeability of primary gill cultures of trout epithelia Zhou et al., 2003). ...
In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin.
... In addition to the structure of TJs, the expression of different TJ proteins, including claudins, occludins, zona occludens (ZO) and tricellulins, are regulated in different ways in response to ambient ion content and contribute to changes in gill permeability (Chasiotis, Kolosov, Bui, et al., 2012;Kolosov et al., 2013). For example, paralogs of Claudin 3, 27, 28 and 30 have been identified as putative 'FW claudins' in a number of euryhaline species (Bossus et al., 2015;Duffy et al., 2011;Tipsmark, Baltzegar, et al., 2008;Tipsmark, Luckenbach, et al., 2008;Tipsmark et al., 2016), although there may be exceptions to this generalisation (Bagherie-Lachidan et al., 2009). The TJ protein occludin is also a barrier-forming protein that is important in hyperregulating FW fishes (Chasiotis et al., 2009(Chasiotis et al., , 2012b. ...
... Marine fishes rely on the expression of TJs that form a cation-selective pore that allows for the secretion of Na + from the blood to the surrounding SW environment (Section 6.1.1). Several studies have demonstrated that the expression of paralogs of Claudin 10 is increased in the gills of euryhaline fishes following transfer from FW to SW (Bossus et al., 2015;Marshall et al., 2018;Tipsmark et al., 2016). Additionally, electrophysiological and molecular evidence supports the presence of a Na + and/or K + -selective pore in SW-acclimated killifish that is likely related to the expression of Claudin 10 paralogs (Marshall et al., 2018). ...
Fish aquaculture facilities strive to maintain abiotic conditions that optimise somatic growth. Two physiological systems that are critically linked to environmental abiotic factors are ion and acid‐base regulation. Given the detrimental effects of ionic and acid‐base imbalances coupled with the potential energetic costs of ion and acid‐base regulation, it is important to understand how changes in abiotic variables such as salinity, temperature and pH alter ionoregulatory and acid‐base physiology. The gill is the predominant site of ion and acid‐base regulation, two linked processes that are vital to the health of fishes. Most species maintain ion levels in the blood plasma within a narrow range, independent of external ion concentrations. In freshwater (FW), fishes must actively take up ions from their dilute environment to counteract passive ion losses, while in seawater (SW), ion excretion serves to counteract passive salt gain. Ion uptake (FW) and excretion (SW) are coordinated by different subtypes of ion‐transporting cells (ionocytes) expressed in the gill epithelium. In this review, we discuss the function of FW and SW ionocytes in the movement of ions across the gill epithelium and address other features of the gill that are integral to ionoregulation. We also discuss the mechanisms of acid‐base regulation by the gill, which is intimately related to ion regulation because it is coordinated by Na+‐ and Cl−‐linked fluxes of acid (H+) and base (HCO3‐) equivalents. These fundamental physiological principles are integral in understanding how fishes in aquaculture respond to relevant disturbances that may occur under culture conditions.
... For instances, claudin-15a, claudin-b and claudin-c were highly expressed in the middle intestine of grass carp (Xu et al. 2014), consistent with the results of zebrafish and Atlantic salmon (Tipsmark et al. 2010;Kumai et al. 2011). Also cldn15a, cldn15b and cladn25 are also found mainly in the gut of medaka (Oryzias latipes) (Bossus et al. 2015). In cultured intestinal epithelial cells, inhibition of the expression of claudin-3 results in an increase in paracellular permeability (McLaughlin et al. 2004). ...
... Furthermore, kidney cells from spotted scat can tolerate hypertonic stress (Gui et al. 2016). Previous studies have indicated that some claudin 10b variants were mainly expressed in the kidney of fish, like zebrafish (Danio rerio), fugu, and medaka (Loh et al. 2004;Baltzegar et al. 2013;Bossus et al. 2015). After seawater transfer, expression levels of the claudin 3a, claudin 3b, and claudin 3c were increased in the salmon kidney (Tipsmark et al. 2012); this result suggested that renal expression of these claudin genes is important to paracellular ion selection. ...
Claudins are a family of integrated membrane-bound proteins involving in paracellular tightness, barrier forming, ion permeability, and substrate selection at tight junctions of chordate epithelial and endothelial cells. Here, 39 putative claudin genes were identified in the Pungitius sinensis based on the high throughput RNA-seq. Conservative motif distribution in each group suggested functional relevance. Divergence of duplicated genes implied the species’ adaptation to the environment. In addition, selective pressure analyses identified one site, which may accelerate functional divergence in this protein family. Pesticides cause environmental pollution and have a serious impact on aquatic organisms when entering the water. The expression pattern of most claudin genes was affected by organophosphorus pesticide, indicating that they may be involved in the immune regulation of organisms and the detoxification of xenobiotics. Protein–protein network analyses also exhibited 439 interactions, which implied the functional diversity. It will provide some references for the functional study on claudin genes.
... In addition to a high density of basolateral Na + ,K + -ATPase (Nka), ionocytes are equipped with specific apical and basolateral ion transport proteins that work together in ion absorption and excretion. One FW-type ionocyte Japanese medaka has previously been used as a model organism for studying ionic and osmotic regulation in a euryhaline teleost [3,29,[32][33][34][35], and aquaporin tissue distribution was characterized in a previous study with a focus on the dynamics in the intestine [33]. The current study aims to answer three main questions: (i) in which cell types do we find the two aquaporin paralogs, AQP1 and AQP3, expressed in the gill of medaka?; (ii) how are branchial AQP1 and AQP3 mRNA and protein expression and localization affected by environmental salinity?; and (iii) are PRL and cortisol involved in endocrine control of AQP expression? ...
... In individual experiments, 300 or 500 ng of total RNA was used for cDNA synthesis using the Applied Biosystems High Capacity cDNA Reverse Transcription kit (Thermo Fisher). qPCR primers were previously validated and published in Bossus et al. [34] and Madsen et al. [33]. Elongation Factor 1 alpha (EF1a), beta actin (bact), and ribosomal protein L7 (RPL7) were analyzed as normalization genes in all experiments. ...
Aquaporins (AQPs) facilitate transmembrane water and solute transport, and in addition to contributing to transepithelial water transport, they safeguard cell volume homeostasis. This study examined the expression and localization of AQP1 and AQP3 in the gills of Japanese medaka (Oryzias latipes) in response to osmotic challenges and osmoregulatory hormones, cortisol, and prolactin (PRL). AQP3 mRNA was inversely regulated in response to salinity with high levels in ion-poor water (IPW), intermediate levels in freshwater (FW), and low levels in seawater (SW). AQP3 protein levels decreased upon SW acclimation. By comparison, AQP1 expression was unaffected by salinity. In ex vivo gill incubation experiments, AQP3 mRNA was stimulated by PRL in a time- and dose-dependent manner but was unaffected by cortisol. In contrast, AQP1 was unaffected by both PRL and cortisol. Confocal microscopy revealed that AQP3 was abundant in the periphery of gill filament epithelial cells and co-localized at low intensity with Na+,K+-ATPase in ionocytes. AQP1 was present at a very low intensity in most filament epithelial cells and red blood cells. No epithelial cells in the gill lamellae showed immunoreactivity to AQP3 or AQP1. We suggest that both AQPs contribute to cellular volume regulation in the gill epithelium and that AQP3 is particularly important under hypo-osmotic conditions, while expression of AQP1 is constitutive.
... ionocytes) that are involved in salt secretion in SW (Bui et al., 2010;Bui and Kelly, 2014;Kolosov et al., 2014); (3) select Cldn-10 isoforms in gill ionocytes exhibit alterations in subcellular distribution when a euryhaline fish is acclimated to SW versus FW (Bui and Kelly, 2014); (4) select Cldn-10 isoforms in the gill exhibit increased mRNA and/or protein abundance in euryhaline fishes under conditions where the gill epithelium becomes salt secreting (i.e. following transfer from FW to SW) (Tipsmark et al., 2008;Bui et al., 2010;Kelly, 2014, 2015;Bossus et al., 2015); and (5) salmonid cldn-10e responded to endocrine factors that direct the functional re-organization of the gill epithelium following salinity change (Tipsmark et al., 2009;Trubitt et al., 2015). Despite this growing evidence that select Cldn-10 isoforms underlie branchial salt secretion, no study has exclusively focused on elucidating a role for Cldn-10 isoforms in the gill of a euryhaline fish, combining the molecular physiology of Cldn-10 salinity regulation with measured cation selectivity across branchial tissue in SW and hypersaline SW. ...
... abundance in the gills of SW-versus FW-acclimated animals (Bui et al., 2010;Bui and Kelly, 2014;Bossus et al., 2015). In addition, transcript abundance of cldn-10e has also been reported to increase in the gill of SW-acclimated Atlantic salmon (Tipsmark et al., 2008). ...
To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from fresh water (FW) to seawater (SW, 35 ‰) and from SW to hypersaline SW (2SW, 60 ‰) in a time course with transfer control groups (FW to FW and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and -10e by 2-fold, whilst cldn-10c and -10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d, and transiently altered cldn-10e abundance, but increased cldn-10c and -10f by 4-fold. This was coincident with an increased number of single-stranded junctions (observed by TEM). For both salinity transfers; (1) cldn-10e mRNA was acutely responsive (i.e. after 24 h), (2) other responsive cldn-10 isoforms increased later (3-7 days), and (3) cystic fibrosis transmembrane conductance regulator (cftr) mRNA was elevated in accordance with established changes in transcellular Cl− movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and -10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na+>K+>Rb+∼Cs+>Li+. Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes.
... Previous studies have provided evidence identifying the ocln gene as an osmoregulation-related gene in Acipenser baerii [51], while the cldn19 gene played significant roles in osmoregulatory physiology of Petromyzon marinus [52]. Moreover, the PSG cldn10, which predominantly expressed in osmoregulatory organs of teleost fishes [38,53], was found have two specific AA replacements in the two Pseudaspius species (Additional file 2: Figure S6A). In addition, osmotic regulation affected the expression of many SLCs (solute carriers), such as slc15a2, slc6a8, slc12a1, and slc2a3 [24,[54][55][56]. ...
Background
Cyprinidae, the largest fish family, encompasses approximately 367 genera and 3006 species. While they exhibit remarkable adaptability to diverse aquatic environments, it is exceptionally rare to find them in seawater, with the Far Eastern daces being of few exceptions. Therefore, the Far Eastern daces serve as a valuable model for studying the genetic mechanisms underlying seawater adaptation in Cyprinidae.
Results
Here, we sequenced the chromosome-level genomes of two Far Eastern daces (Pseudaspius brandtii and P. hakonensis), the two known cyprinid fishes found in seawater, and performed comparative genomic analyses to investigate their genetic mechanism of seawater adaptation. Demographic history reconstruction of the two species reveals that their population dynamics are correlated with the glacial-interglacial cycles and sea level changes. Genomic analyses identified Pseudaspius-specific genetic innovations related to seawater adaptation, including positively selected genes, rapidly evolving genes, and conserved non-coding elements (CNEs). Functional assays of Pseudaspius-specific variants of the prolactin (prl) gene showed enhanced cell adaptation to greater osmolarity. Functional assays of Pseudaspius specific CNEs near atg7 and usp45 genes suggest that they exhibit higher promoter activity and significantly induced at high osmolarity.
Conclusions
Our results reveal the genome-wide evidence for the evolutionary adaptation of cyprinid fishes to seawater, offering valuable insights into the molecular mechanisms supporting the survival of migratory fish in marine environments. These findings are significant as they contribute to our understanding of how cyprinid fishes navigate and thrive in diverse aquatic habitats, providing useful implications for the conservation and management of marine ecosystems.
... Cluster C5 and C13 cells were classified as pavement cells owing to their enrichment for claudin 4 (cldn4) and several other genes encoding claudin proteins which are important for establishing tight junctions, including cldn23.1, cldn8, claudin-23-like (LOC100692699), and claudin-7-A (LOC100710159) [42]. The gene evpl encoding envoplakin is involved in the formation of the cornified envelope and was specifically expressed in pavement cells of fish gills (Fig. 1D) [43], further supporting our annotation. ...
... In fish skin, claudin expression has been shown to vary in response to environmental salinity, with the expression of certain claudin-10 isoforms increasing following SW acclimation in several species (Bui and Kelly, 2014). This is consistent with studies of the gill of several teleost species, where SW transfer also increases the expression of claudin-10 isoforms (Tipsmark et al., 2008a, Tipsmark et al., 2008b, Tipsmark et al., 2016Bossus et al., 2015) and with the opercular skin of Fundulus heteroclitus (Marshall et al., 2018;Chen et al., 2021). Certain claudin-10 isoforms are thought to provide the paracellular pathway with cation selective pores, which facilitate ion efflux in SW (Marshall et al., 2018). ...
The mechanisms underpinning ionic transport and barrier function have been relatively well characterised in amphibians and fish. In teleost fish, these processes have mostly been characterised in the gill and intestine. In contrast, these processes remain much less clear for the trunk skin of fish. In this study, we measured barrier function and active transport in the trunk skin of the rainbow trout, using the Ussing chamber technique. The effects of epithelial damage, skin region, salinity, and pharmacological inhibition were tested. Skin barrier function decreased significantly after the infliction of a superficial wound through the removal of scales. Wound healing was already underway after 3 h and, after 24 h, there was no significant difference in barrier function towards ions between the wounded and control skin. In relation to salinity, skin permeability decreased drastically following exposure to freshwater, and increased following exposure to seawater. Changes in epithelial permeability were accompanied by salinity-dependent changes in transepithelial potential and short-circuit current. The results of this study support the idea that barrier function in rainbow trout trunk skin is regulated by tight junctions that rapidly respond to changes in salinity. The changes in transepithelial permeability and short circuit current also suggest the presence of an active transport component. Immunostaining and selective inhibition suggest that one active transport component is an apical V-ATPase. However, further research is required to determine the exact role of this transporter in the context of the trunk skin.
... In the last few years, marine medaka has been used to study the environmental marine pollution (13,14) due to its seawater habitat and well-known genome (15,16). However, most of the salinity works in medaka species were performed in the Japanese medaka (Oryzias latipes) (10,(17)(18)(19)(20), which lives in fresh water. The current study applied RNA sequencing to provide an overview of the hypotonic osmoregulatory mechanism in gill of marine medaka. ...
Aquatic fishes face osmotic stress continuously, and the gill is the first tissue that senses and responds to the external osmotic challenges. However, the understandings of how the gill microbiota could respond to osmotic stress and their potential host-bacterium relationships are limited. The objectives of the current study are to identify the hypotonic responsive genes in the gill cells and profile the gill microbiota communities after fresh water transfer experiment via transcriptome sequencing and 16S rRNA gene sequencing. Transcriptome sequencing identified 1,034 differentially expressed genes (DEGs), such as aquaporin and sodium potassium chloride cotransporter, after the fresh water transfer. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further highlighted the steroid biosynthesis and glycosaminoglycan biosynthesis pathways in the gill. Moreover, the 16S rRNA gene sequencing identified Vibrio as the dominant bacterium in the seawater, which changed to Pseudomonas and Cetobacterium after the fresh water transfer. The alpha diversity analysis suggested that the gill bacterial diversity was lower in the fresh water transferred group. The KEGG and MetaCyc analysis further predicted the alteration of the glycosaminoglycan and chitin metabolisms in the gill bacteria. Collectively, the common glycosaminoglycan and chitin pathways in both the gill cells and gill microbiota suggest the host-bacterium interaction in gill facilitates the fresh water acclimation.
IMPORTANCE This is the first study using the transcriptome and 16S rRNA gene sequencing to report the hypotonic responsive genes in gill cells and the compositions of gill microbiota in marine medaka. The overlapped glycosaminoglycan- and chitin-related pathways suggest host-bacterium interaction in fish gill during osmotic stress.
... The downregulation of OCLN, CLND10 and CLDN7 was presumably related to the rearrangement of gill in G. przewalskii in FW and changed permeability. Medaka acclimated to seawater had a higher transcriptional level of CLDN10 than that in FW, which played a key role in gill plasticity (Bossus et al., 2015). The loosened tight junction also indicated the potential variation in the permeability at low salinity. ...
Gymnocypris przewalskii is a native cyprinid fish that dwells in the Lake Qinghai with salinity of 12–13‰. It migrates annually to the freshwater rivers for spawning, experiencing the significant changes in salinity. In the present study, we performed the physiological, morphological and transcriptomic analyses to understand the osmoregulation in G. przewalskii. The physiological assay showed that the osmotic pressure of G. przewalskii was almost isosmotic to the brackish lake water. The low salinity reduced its ionic concentrations and osmotic pressure. The plasticity of gill microstructure was linked to the salinity variations, including the presence of mucus and intact tight junctions in brackish water and the development of the mitochondria-rich cells and the loosened tight junctions in freshwater. RNA-seq analysis identified 1926 differentially expressed genes, including 710 and 1216 down- and up-regulated genes in freshwater, which were enriched in ion transport, cell-cell adhesion, and mucus secretion. Genes in ion uptake were activated in low salinity, and mucus pathways and tight junction showed the higher transcription in brackish water. The isosmoticity between the body fluid and the environment suggested G. przewalskii was in the metabolic-saving condition in the brackish water. The decreased salinity disrupted this balance, which activated the ion uptake in freshwater to maintain osmotic homeostasis. The gill remodeling was involved in this process through the development of the mitochondria-rich cells to enhance ion uptake. The current finding provided insights into the potential mechanisms of G. przewalskii to cope with salinity alteration.
... The barrier-forming CLDN3 genes were duplicated and are expressed in different intestinal segments of the euryhaline pufferfish, Tetraodon nigroviridis; in addition, the expression levels decrease after transfer to SW [33]. The cldn15a, cldn15b and cldn25 are expressed in the medaka intestine, and cldn15b expression is profoundly downregulated in SW [21]. As the aqp genes are downregulated in the medaka intestine after SW transfer [139], the decreased cldn15 expression in SW may be related to increased water permeability of the paracellular route (see 4.2.1). ...
Adaptation to a hypertonic marine environment is one of the major topics in animal physiology research. Marine teleosts lose water osmotically from the gills and compensate for this loss by drinking surrounding seawater and absorbing water from the intestine. This situation is in contrast to that in mammals, which experience a net osmotic loss of water after drinking seawater. Water absorption in fishes is made possible by (1) removal of monovalent ions (desalinization) by the esophagus, (2) removal of divalent ions as carbonate (Mg/CaCO 3 ) precipitates promoted by HCO 3 ⁻ secretion, and (3) facilitation of NaCl and water absorption from diluted seawater by the intestine using a suite of unique transporters. As a result, 70–85% of ingested seawater is absorbed during its passage through the digestive tract. Thus, the digestive tract is an essential organ for marine teleost survival in the hypertonic seawater environment. The eel is a species that has been frequently used for osmoregulation research in laboratories worldwide. The eel possesses many advantages as an experimental animal for osmoregulation studies, one of which is its outstanding euryhalinity, which enables researchers to examine changes in the structure and function of the digestive tract after direct transfer from freshwater to seawater. In recent years, the molecular mechanisms of ion and water transport across epithelial cells (the transcellular route) and through tight junctions (the paracellular route) have been elucidated for the esophagus and intestine. Thanks to the rapid progress in analytical methods for genome databases on teleosts, including the eel, the molecular identities of transporters, channels, pumps and junctional proteins have been clarified at the isoform level. As 10 y have passed since the previous reviews on this subject, it seems relevant and timely to summarize recent progress in research on the molecular mechanisms of water and ion transport in the digestive tract in eels and to compare the mechanisms with those of other teleosts and mammals from comparative and evolutionary viewpoints. We also propose future directions for this research field to achieve integrative understanding of the role of the digestive tract in adaptation to seawater with regard to pathways/mechanisms including the paracellular route, divalent ion absorption, metabolon formation and cellular trafficking of transporters. Notably, some of these have already attracted practical attention in laboratories.
... Such investigations have revealed the importance of branchial NKA isoform switching for salinity acclimation (Bystriansky et al., 2006;Nilsen et al., 2007;Richards et al., 2003;Tipsmark et al., 2011;Urbina et al., 2013), and for acclimation to ion-poor conditions (Esbaugh et al., 2019). In addition, numerous studies have employed qRT-PCR to characterize the effects of ionoregulatory challenges (e.g., altered salinity, acid exposure, ion-poor conditions) on the transcript abundance of genes encoding iontransporting proteins Breves et al., 2020;Lema et al., 2018;Masroor et al., 2019;Scott et al., 2004aScott et al., , 2005Tipsmark et al., 2008b;Wu et al., 2010;Yan et al., 2007), tight junction proteins such as claudins and occludins (Bossus et al., 2015;Chasiotis et al., 2012;Kolosov et al., 2020;Marshall et al., 2018;Tipsmark et al., 2008a), and neuroendocrine regulators (Breves et al., 2013(Breves et al., , 2010Kwong and Perry, 2015;Lin et al., 2014Lin et al., , 2012bNilsen et al., 2008). ...
The mechanisms underlying ionoregulation in fishes have been studied for nearly a century, and reductionist methods have been applied at all levels of biological organization in this field of research. The complex nature of ionoregulatory systems in fishes makes them ideally suited to reductionist methods and our collective understanding has been dramatically shaped by their use. This review provides an overview of the broad suite of techniques used to elucidate ionoregulatory mechanisms in fishes, from the whole-animal level down to the gene, discussing some of the advantages and disadvantages of these methods. We provide a roadmap for understanding and appreciating the work that has formed the current models of organismal, endocrine, cellular, molecular, and genetic regulation of ion balance in fishes and highlight the contribution that reductionist techniques have made to some of the fundamental leaps forward in the field throughout its history.
... In this study, blood chloride levels were decreased in FW-intolerant compared to FW-tolerant fish, but this observation was not made consistently over the study (shown in experiment 1 performed on 8-month-old fishes, Fig. 3d, FW t vs FW i ). A possible chloride loss could be the cause of decreased chloride levels and may indicate leakiness of the gill epithelium, e.g., less tight junction-related proteins might be expressed (Chasiotis et al. 2012;Bui and Kelly 2014;Bossus et al. 2015). This could also be linked to decreased chloride uptake at the gill and/or renal levels. ...
... Claudin-10 mRNA expression levels have been related ammonia exposure (Connon, Deanovic, Fritsch, D'Abronzo, & Werner, 2011), rearing density (Sveen et al., 2016), and salinity (Bossus, Madsen, & Tipsmark, 2015;Kolosov, Bui, Chasiotis, & Kelly, 2013;Marshall et al., 2018) in fishes. Spatial variation in cell membrane proteins is consistent with environmental differences between basins in chemicals such as sodium, chloride, and phosphorous (Environment Canada, 2011), although the biological impacts of these spatial chemical differences are unknown. ...
RNA sequencing is an effective approach for studying aquatic species yielding both physiological and genomic data. However, its population genetic applications are not well‐characterized. We investigate this possible role for RNA sequencing for population genomics in Lake Winnipeg, Manitoba, Canada, walleye (Sander vitreus ). Lake Winnipeg walleye represent the largest component of the second‐largest freshwater fishery in Canada. In the present study, large female walleye were sampled via nonlethal gill biopsy over two years at three spawning sites representing a latitudinal gradient in the lake. Genetic variation from sequenced mRNA was analyzed for neutral and adaptive markers to investigate population structure and possible adaptive variation. We find low population divergence (F ST = 0.0095), possible northward gene flow, and outlier loci that vary latitudinally in transcripts associated with cell membrane proteins and cytoskeletal function. These results indicate that Lake Winnipeg walleye may be effectively managed as a single demographically connected metapopulation with contributing subpopulations and suggest genomic differences possibly underlying observed phenotypic differences. Despite its high cost relative to other genotyping methods, RNA sequencing data can yield physiological in addition to genetic information discussed here. We therefore argue that it is useful for addressing diverse molecular questions in the conservation of freshwater species.
... In this study, blood chloride levels were decreased in FW-intolerant compared to FW-tolerant fish, but this observation was not made consistently over the study (shown in experiment 1 performed on 8-month-old fishes, Fig. 3d, FW t vs FW i ). A possible chloride loss could be the cause of decreased chloride levels and may indicate leakiness of the gill epithelium, e.g., less tight junction-related proteins might be expressed (Chasiotis et al. 2012;Bui and Kelly 2014;Bossus et al. 2015). This could also be linked to decreased chloride uptake at the gill and/or renal levels. ...
In teleosts, the regulation of hydromineral balance has a direct impact on several physiological functions, biochemical processes, and can influence behaviour, distribution and survival. As European sea bass Dicentrarchus labrax undertake seasonal migrations from seawater (SW) to brackish, estuarine and fresh water (FW) in their habitat, this study investigates their capacity to tolerate fresh water and explores intraspecific variations in physiological responses. Juvenile D. labrax were transferred from SW to FW at various ages. Freshwater-tolerant and non-tolerant phenotypes were discriminated according to behavioural and morphological characteristics. About 30% of the fish exposed to FW were identified as freshwater intolerant following FW challenges performed at different ages. Interestingly, intolerant fish exhibited the same phenotypic traits: erratic swimming, lower speed, isolation from the shoal and darker colour. Freshwater-intolerant fish were also characterised by a significant lower blood osmolality compared to tolerant fish, and significantly lower Na⁺/K⁺-ATPase α1a expression in the posterior kidney. An imbalance in ion regulatory mechanisms was further confirmed by a blood Na⁺/Cl⁻ ratio imbalance observed in some freshwater-intolerant fish. The analysis of glucocorticoid and mineralocorticoid receptor expression levels in gills and kidney revealed significant differences between freshwater-intolerant and -tolerant fish in both organs, suggesting differential stress-related responses. This study clearly shows an intraspecific difference in the responses following FW transfer with a decreased renal ion uptake capacity as a major cause for freshwater intolerance.
... In Atlantic salmon, claudin 10 transcription in gill increased during smoltification and salt-water acclimation, suggesting that claudin 10 is involved in osmoregulation (Tipsmark et al., 2008). This is also true for euryhaline Japanese medaka (Oryzias latipes), where claudin 10 has been suggested to be involved in osmoregulation in gills and kidney (Bossus et al., 2015). Cortisol treatment of cultured gill epithelia from puffer fish (Tetraodon nigroviridis) dosedependently altered transcription of selected claudins (Bui et al., 2010). ...
For the full thesis please visit: http://bora.uib.no/handle/1956/19024.
The work described in this thesis focuses on the response mechanisms in the skin of post-smolt Atlantic salmon (Salmo salar L.) under the following challenging conditions:
•i. The effect of high biomass and low specific water flow on skin
integrity (paper I)
•ii. The effect of high biomass followed by acute challenge test on mucin transcription in skin (paper II)
•iii. General wound healing mechanisms in Atlantic salmon skin (paper III) and the effect of high biomass on wound healing (paper IV)
•iv. The effect of handling stress on mucin transcription (paper II)
High biomass is predicted to provide greater profitability in the production of fish in closed systems. Similarly, low water flow can reduce costs associated with water treatment and water supply. In paper I, these hypotheses were challenged. Five fish densities (25, 50, 75, 100 and 125 kg/m3) and four levels of specific water flow (0.2, 0.3, 0.4 and 0.5 l /kg fish/min) were established to detect effects on salmon skin. After eight weeks, both high biomass (≥ 100 kg/m3) and low specific water flow, (0.2-0.3 l /kg/min) led to activated stress and immune responses in the skin of post-smolt, including increased transcription of mucin-like genes.
The main constituency of the mucus layer are big gel-forming proteins called mucins. In paper II, seven gel-forming mucins, including two mucin2 and five mucin5 variants, were identified based on a computational pipeline consisting of annotation, transcription, domain structure and phylogenetic analysis. Mucin2 was predominantly expressed in the intestine, while the mucin5 family was expressed in many organs, including skin and gills. In order to investigate transcriptional regulation of mucins during stress conditions, two controlled experiments were conducted. In the first experiment, handling stress induced mucin transcription in the gill, while transcription decreased in the skin and intestine. In the second experiment, long term intensive rearing conditions interrupted by additional acute challenge test led to increased transcription of mucin genes in the skin at one, seven and fourteen days post-challenge.
Farmed fish are particularly vulnerable to skin damage which may occur after handling, confinement, infections and more. The same procedures may trigger stress responses in the fish. Thus, the goal of paper III was to describe the general cutaneous wound healing mechanisms in post-smolts while paper IV focused on the effects of chronic stress on wound healing. Two experimental groups were created where low fish density was used as control (x̄ = 14 kg/m3) and high fish density (x̄ = 126 kg/m3) as chronic stress factor. Deep cutaneous wounds were inflicted on the flank of the fish by using a 5mm punch biopsy tool and the following healing process was documented with seven sampling points over a period of 53 days. The results showed that the healing process was dominated by an early acute inflammatory phase and a later regenerative stage with connective tissue formation and wound contraction. Both the histological analyses and the transcriptional responses fully supported these findings. Stress changed several steps in the healing process, including re-epithelialization, the mucus response, scale mineralization, pigmentation, formation of fibrous tissue and wound contraction, as demonstrated by histology and picture analysis. The transcriptional analyses showed that the inflammatory response was enhanced by chronic stress, while several genes involved in tissue repair were downregulated. These alterations may be used as key check points in future studies when evaluating the effect of a given treatment on cutaneous wound healing.
In conclusion, the challenging conditions applied in this thesis in general enhanced the inflammatory reactions or altered the mucus response in the skin of post-smolt Atlantic salmon. Molecular analyses on intact skin and wound samples can thus be used to detect adverse effects of the aquatic rearing environment, including effects that cannot be detected by traditional welfare analyses.
... In the intestine of Atlantic salmon (Salmo salar), claudin-15 could enhance the Na + movement by increasing paracellular permeability [100]. In Japanese medaka (Oryzias latipes) intestine, claudin-15a expression were much higher than claudin-15b [101]. Thus, DON up-regulated claudin-15a (rather than claudin-15b) gene expression, which might be a feedback mechanism for increasing Na + transport capacity in the fish intestine. ...
... The candidate gene rgs9bp is a regulator of G-protein signaling, which involves in bone metabolism in catfish 65 . Whereas, cldn10a and cldn10b are associated with salinity regulation in Japanese medaka (Oryzias latipes) 66 . It is unexpected that no feed efficiency-associated candidate genes were detected in other QTL intervals or nearby regions because the markers within these regions failed to be mapped to current assemblied genome of crucian carp. ...
QTL is a chromosomal region including single gene or gene clusters that determine a quantitative trait. While feed efficiency is highly important in aquaculture fish, little genetic and genomic progresses have been made for this trait. In this study, we constructed a high-resolution genetic linkage map in a full-sib F1 family of crucian carp (Carassius auratus) consisting of 113 progenies with 8,460 SNP markers assigning onto 50 linkage groups (LGs). This genetic map spanned 4,047.824 cM (0.478 cM/marker) and covered 98.76% of the crucian carp genome. 35 chromosome-wide QTL affecting feed conversion efficiency (FCE, 8 QTL), relative growth rate (RGR, 9 QTL), average daily gain (ADG, 13 QTL) and average daily feed intake (ADFI, 5 QTL) were detected on 14 LGs, explaining 14.0–20.9% of the phenotypic variations. In LGs of LG16, LG25, LG36 and LG49, several QTL affecting different traits clustered together at the identical or close regions of the same linkage group. Seven candidate genes, whose biological functions may involve in the energy metabolism, digestion, biosynthesis and signal transduction, were identified from these QTL intervals by comparative genomics analysis. These results provide a basis for elucidating genetic mechanism of feed efficiency and potential marker-assisted selection in crucian carp.
... In addition to the transcellular pathway, water movement across epithelia is regulated intercellularly (between cells) by tight junction proteins, such as CLDN in teleosts (29). Teleosts have more than 50 CLDN genes (41), of which cldn15a,b and cldn25 are predominantly expressed in the intestine of medaka (8) and Atlantic salmon (45). Nine CLDN genes were expressed significantly in the eel esophagus, as detected by RNA-seq, of which cldn15a was most abundant, and its expression was profoundly upregulated after SW acclimation (Fig. 4M). ...
... Genome scanning of oceanic and FW stickleback has identified many osmoregulatory genes under strong directional selection between these environments, including atp1a1 (NKA), aqp3 (aquaporin 3; a water channel), and kir2.2 (a potassium channel) DeFaveri et al. 2011). Because performance differed in FW, researchers should consider examining the expression or abundance of transporters associated with ion uptake (e.g., NHE, VHA, NCC; Evans 2011), as well as tight junction and mucous proteins (mucins), which help reduce diffusive ion loss across the gill epithelium (Jones et al. 2012a;Bossus et al. 2015). ...
Maintaining ion balance in environments of changing salinity is one of the greatest physiological challenges facing aquatic organisms and by comparing populations inhabiting different salinity regimes, we can learn how physiological plasticity evolves in response to local osmotic stress. I characterized the evolution of osmoregulatory responses in representative marine, anadromous, and freshwater (FW) populations of Threespine Stickleback (Gasterosteus aculeatus) by comparing survival and physiological measures in F1-generation fish following salinity challenge. Juveniles from a population landlocked for ~10,000 years displayed ontogenetically-delayed seawater (SW) tolerance, a lower maximum salinity threshold, and did not upregulate the Na+/K+-ATPase (NKA) ion transporter as much as marine counterparts (Chapter 1). Stickleback also responded to salinity stress by remodeling their gill epithelium: I observed a higher density of ionoregulatory cells when juveniles were subjected to both low and high salinities, and the latter treatment induced strong upregulation of ion secretory cells (Chapter 2). Finally, I examined the speed at which osmoregulatory plasticity evolves by comparing halotolerance between an anadromous population and descendants that had been FW-restricted for only two generations (Chapter 3). The lake-introduced group had improved survival in FW, but also retained SW tolerance and had similar increases in gill NKA activity, gill Na+/K+/2Cl- cotransporter abundance, and organic osmolytes in SW. Overall, the differentiated responses to salinity I observed among stickleback populations indicate that osmoregulation has evolved in a manner consistent with local adaptation and following FW invasions, positive selection on FW tolerance acts more rapidly than relaxed selection on SW tolerance.
... Another candidate gene cldn10 encodes a component of tight junctions. Seawater transfer induced elevation of cldn10 expression in the gills of medaka (Bossus et al. 2015). It is likely that differential expression of cldn10 can contribute to divergence in salinity tolerance between ecotypes. ...
Adaptation to different salinities can drive and maintain divergence between populations of aquatic organisms. Anadromous and stream ecotypes of threespine stickleback (Gasterosteus aculeatus) are an excellent model to explore the genetic mechanisms underlying osmoregulation divergence. Using a parapatric pair of anadromous and stream stickleback ecotypes, we employed an integrated genomic approach to identify candidate genes important for adaptation to different salinity environments. Quantitative trait loci (QTL) mapping of plasma sodium concentrations under a seawater challenge experiment identified a significant QTL on chromosome 16. To identify candidate genes within this QTL, we first conducted RNA-seq and microarray analysis on gill tissue to find ecotypic differences in gene expression that were associated with plasma Na+ levels. This resulted in identification of ten candidate genes. Quantitative PCR analysis on gill tissue of additional Japanese stickleback populations revealed that the majority of the candidate genes showed parallel divergence in expression levels. Secondly, we conducted whole genome sequencing and found five genes that are predicted to have functionally important amino acid substitutions. Finally, we conducted genome scan analysis and found that eight of these candidate genes were located in genomic islands of high differentiation, suggesting that they may be under divergent selection. The candidate genes included those involved in ATP synthesis and hormonal signaling, whose expression or amino acid changes may underlie variation in salinity tolerance. Further functional molecular analysis of these genes will reveal the causative genetic and genomic changes underlying divergent adaptation. This article is protected by copyright. All rights reserved.
... Because performance differed in fresh water, researchers should consider examining the expression or abundance of transporters associated with ion uptake [e.g. NHE, VHA, NCC (Evans, 2011)], as well as tight junction and mucous proteins (mucins), which help reduce diffusive ion loss across the gill epithelium (Jones et al., 2012a;Bossus et al., 2015). These proteins may differ among salinity-divergent stickleback populations with respect to their gene expression, abundance, or isoform variants. ...
Background: Post-Pleistocene diversification of threespine stickleback in fresh water offers a valuable opportunity to study how changes in environmental salinity shape physiological evolution in fish. In Alaska, the presence of both ancestral oceanic populations and derived landlocked populations, including recent lake introductions, allows us to examine rates and direction of evolution of osmoregulation following halohabitat transition. Hypotheses: Strong selection for enhanced freshwater tolerance will improve survival of recently lake-introduced stickleback in ion-poor conditions compared with their oceanic ancestors. Trade-offs between osmoregulation in fresh water and seawater will allow members of the ancestral population to survive better in response to seawater challenge, as mediated by upregulating salt-secreting transporters in the gill. Poorer hypo-osmoregulatory performance of derived fish will be marked by higher levels of taurine and other organic osmolytes. Methods: We reared clutches at a common salinity from an anadromous and a descendant population, Scout Lake, which has been landlocked for only two generations. We challenged 6-week-old juveniles with extreme low and high salinity treatments and sampled fish over 10 days to investigate putative molecular mechanisms underlying differences in halotolerance. We measured whole-body organic osmolyte content as well as gill Na⁺/K⁺-ATPase (NKA) activity and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) protein abundance. Other juveniles from these populations and also from Cheney Lake, a fourth-generation landlocked descendant, were gradually salt-acclimated to determine maximum halotolerance limits. Results: Scout Lake stickleback exhibited 67% higher survival in fresh water than the ancestral anadromous population, but individuals from both groups exhibited similar seawater tolerance. Likewise, the gradual salinity threshold for each population was equivalent (71 ppt). Gill NKA activity and NKCC abundance were both higher in seawater-challenged fish, but did not differ between populations. Sticklebacks from both populations responded to acute salinity stress by transiently increasing osmolyte levels in seawater and decreasing them in fresh water. Conclusion: Enhanced freshwater tolerance has evolved rapidly in recently landlocked stickleback compared with their anadromous ancestors (0.569 haldanes), but the former have retained ancestral seawater-osmoregulatory function.
... In Atlantic salmon, claudin 10 transcription in gill increased during smoltification and salt-water acclimation, suggesting that claudin 10 is involved in osmoregulation (Tipsmark et al., 2008). This is also true for euryhaline Japanese medaka (Oryzias latipes), where claudin 10 has been suggested to be involved in osmoregulation in gills and kidney (Bossus et al., 2015). Cortisol treatment of cultured gill epithelia from puffer fish (Tetraodon nigroviridis) dosedependently altered transcription of selected claudins (Bui et al., 2010). ...
Prolonged production of Atlantic salmon (Salmo salar) post-smolts in closed-containment systems has prompted research into biological requirements under higher production intensities. This study examined the effect of fish density and specific water flow on skin health in post post-smolts particularly focusing on epithelial cell morphology and gene expression.
... Japanese medaka have been used for a while as a model for understanding ionoregulatory function in euryhaline fishes (Bossus et al. 2015;Hsu et al. 2014;Takei 2002, 2003;Madsen et al. 2014;Sakamoto et al. 2001). This FW species is native to marshes, ponds and irrigation canals of rice fields in Japan, Korea and China (Takehana et al. 2003) and is capable of adapting to a wide range of salinities (Haruta et al. 1991;Inoue and Takei 2002;Miyamoto et al. 1986;Shen et al. 2011). ...
Some euryhaline teleosts exhibit a switch in gill Na+/K+-ATPase (Nka) α isoform when moving between fresh water (FW) and seawater (SW). The present study tested the hypothesis that a similar mechanism is present in Japanese medaka and whether salinity affects ouabain, Mg2+, Na+ and K+ affinity of the gill enzyme. Phylogenetic analysis classified six separate medaka Nka α isoforms (α1a, α1b, α1c, α2, α3a and α3b). Medaka acclimated long-term (>30 days) to either FW or SW had similar gill expression of α1c, α2, α3a and α3b, while both α1a and α1b were elevated in SW. Since a potential isoform shift may rely on early changes in transcript abundance, we conducted two short-term (1–3 days) salinity transfer experiments. FW to SW acclimation induced an elevation of α1b and α1a after 1 and 3 days. SW to FW acclimation reduced α1b after 3 days with no other α isoforms affected. To verify that the responses were typical, additional transport proteins were examined. Gill ncc and nhe3 expression were elevated in FW, while cftr and nkcc1a were up-regulated in SW. This is in accordance with putative roles in ion-uptake and secretion. SW-acclimated medaka had higher gill Nka V
max and lower apparent K
m for Na+ compared to FW fish, while apparent affinities for K+, Mg2+ and ouabain were unchanged. The present study showed that the Japanese medaka does not exhibit a salinity-induced α isoform switch and therefore suggests that Na+ affinity changes involve altered posttranslational modification or intermolecular interactions.
The endocrine system is an essential regulator of the osmoregulatory organs that enable euryhaline fishes to maintain hydromineral balance in a broad range of environmental salinities. Because branchial ionocytes are the primary site for the active exchange of Na⁺, Cl⁻, and Ca²⁺ with the external environment, their functional regulation is inextricably linked with adaptive responses to changes in salinity. Here, we review the molecular-level processes that connect osmoregulatory hormones with branchial ion transport. We focus on how factors such as prolactin, growth hormone, cortisol, and insulin-like growth-factors operate through their cognate receptors to direct the expression of specific ion transporters/channels, Na⁺/K⁺-ATPases, tight-junction proteins, and aquaporins in ion-absorptive (freshwater-type) and ion-secretory (seawater-type) ionocytes. While these connections have historically been deduced in teleost models, more recently, increased attention has been given to understanding the nature of these connections in basal lineages. We conclude our review by proposing areas for future investigation that aim to fill gaps in the collective understanding of how hormonal signaling underlies ionocyte-based processes.
Claudin (Cldn) -10 tight junction (TJ) proteins are hypothesized to form the paracellular Na+ secretion pathway of hyposmoregulating mummichog (Fundulus heteroclitus) branchial epithelia. Organ-specific expression profiles showed that only branchial organs (the gill and opercular epithelium, OE) exhibited abundant cldn-10 paralog transcripts, which typically increased following sea water (SW) to hypersaline (2SW) challenge. Post-translational properties, protein abundance, and ionocyte localization of Cldn-10c, were then examined in gill and OE. Western blot analysis revealed two Cldn-10c immunoreactive bands in the mummichog gill and OE at ∼29 kDa and ∼40 kDa. The heavier protein could be eliminated by glycosidase treatment, demonstrating the novel presence of a glycosylated Cldn-10c. Protein abundance of Cldn-10c increased in gill and OE of 2SW-exposed fish. Cldn-10c localized to the sides of gill and OE ionocyte apical crypts and partially colocalized with cystic fibrosis transmembrane conductance regulator and F-actin, consistent with TJ complex localization. Cldn-10c immunofluorescent intensity increased but localization was unaltered by 2SW conditions. In support of our hypothesis, cldn-10/Cldn-10 TJ protein dynamics in gill and OE of mummichogs and TJ localization are functionally consistent with the creation and maintenance of salinity-responsive, cation-selective pores that facilitate Na+ secretion in hyperosmotic environments.
Given the limited data available for estuarine/marine fish species and potential risk of being exposed to the herbicide atrazine, additional toxicity data regarding sensitive life-stages are needed. As such, this work sought to characterize: 1) the acute larval toxicity, and 2) early life-stage toxicity of technical atrazine in the model marine species sheepshead minnow (Cyprinodon variegatus). Atrazine was observed to be slightly to moderately toxic towards C. variegatus under acute conditions (as per U.S. EPA 2017 criteria). After 96 h exposure, mortality rates of 5%, 15%, 35%, and 90% were observed among fish exposed to atrazine at 4.6, 7.6, 13, and 22 mg a.i./L, respectively. Sub-lethal effects were observed among surviving fish exposed to > 3.2 mg a.i/L. The 96 h LC50 was 13 mg a.i./L and the NOEC was 3.2 mg a.i./L. In the 33 d early-life stage test, mean embryo survival rates in 0.15, 0.30, 0.57, 1.1, and 2.2 mg a.i./L treatments ranged from 71% to 79% and were not different from survival in the control (78%). Following 28 d post-hatch exposure (Day 33), mean larval survival ranged from 98% to 100% in all treatments and the control. Larval length and wet weight were the most sensitive indicators of the toxicity of atrazine to early life-stage sheepshead minnow. The NOEC for growth was 1.1 mg a.i./L and the LOEC was 2.2 mg a.i./L. Based on these, the MATC for atrazine to sheepshead minnow embryos and larvae was estimated to be 1.6 mg a.i./L. These results were consistent with previous investigations in sheepshead minnow and other marine fish species. Based on the results, atrazine would not be expected to pose unacceptable risks for sheepshead minnow early life-stages at environmentally relevant concentrations.
Euryhaline teleost kidneys undergo a major functional switch from being filtratory in fresh water (FW) to being predominantly secretory in seawater (SW) conditions. The transition involves both vascular and tubular effects. There is consensus that glomerular filtration rate is greatly reduced upon exposure to hyperosmotic conditions. Yet, regulation at the tubular level has only been examined sporadically in a few different species. This study aimed to obtain a broader understanding of transcriptional regulation in proximal vs. distal tubular segments during osmotic transitions. Proximal and distal tubule cells were dissected separately by laser capture micro-dissection, RNA was extracted, and relative mRNA expression level of >30 targets involved in solute and water transport were quantified by qPCR in relation to segment type in fish acclimated to FW or SW. The gene categories were aquaporins, solute transporters, fxyd proteins and tight junction proteins. aqp8bb1, aqp10b1, nhe3, sglt1, slc41a1, cnnm3, fxyd12a, cldn3b, cldn10b, cldn15a and cldn12 were expressed at higher level in proximal compared to distal tubules. aqp1aa, aqp1ab, nka-a1a, nka-a1b, nkcc1a, nkcc2, ncc, clc-k, slc26a6C, sglt2, fxyd2, cldn3a and occln were expressed at higher level in distal compared to proximal tubules. Expression of aqp1aa, aqp3a1, aqp10b1, ncc, nhe3, cftr, sglt1, slc41a1, fxyd12a, cldn3a, cldn3b, cldn3c, cldn10b, cldn10e, cldn28a and cldn30c were higher in SW- than in FW-acclimated salmon, whereas the opposite was the case for aqp1ab, slc26a6C and fxyd2. The data shows distinct segmental distribution of transport genes and a significant regulation of tubular transcripts when kidney function is modulated during salinity transitions.
Claudins are tight junction proteins mostly appreciated in their function of paracellular barrier-formation. Compared to a virtual absence of any tight junctions, their paracellular sealing role certainly stands out. Yet, it was recognized immediately after the discovery of the first claudins, that some members of the claudin protein family were able to convey size and charge selectivity to the paracellular pathway. Thus, paracellular permeability can be fine-tuned according to the physiological needs of a tissue by inserting these channel-forming claudins into tight junction strands. Precise permeability adjustment is further suggested by the presence of numerous isoforms of channel-forming claudins (claudin-10b-, -15-, -16-like isoforms) in various vertebrate taxa. Moreover, their expression and localization are controlled by multiple transcriptional and posttranslational mechanisms. Consequently, mutation or dysregulation of channel-forming claudins can cause severe diseases. The present review therefore aims at providing an up-to-date report of the current research on these aspects of channel-forming claudins and their possible implications on future developments.
Deoxynivalenol (DON) is a common mycotoxin existed in animal feed, and lead to significant economic loss due to its negative impacts on animal growth performance and animal health. The gill is a primary mucosal immune organ in teleosts, and the structural integrity of the gill is closely relevant with fish healthy growth. Hence, this study assessed the influences of DON on the gill structural integrity of juvenile grass carp, Ctenopharyngodan idella (initial average weight 12.17 ± 0.01 g), when offered with six different diets which contained various content of DON (27, 318, 636, 922, 1243 and 1515 μg/kg diet) for 60 days. Our research firstly systematically elaborated that DON caused histopathological lesions, oxidative injury, reduction of antioxidant ability, apoptosis as well as damages of tight junctions in fish gills. Comparing these data to the control, we found that DON at dose of more than 318 μg/kg diet led to oxidative injury, apoptosis and disruption of tight junctions in fish gill, which were likely to be relevant with Nrf2, JNK and MLCK signalling pathways, respectively. It was worth noting that DON was not found to affect the gene expressions of Keap1b (rather than Keap1a), claudin-b, claudin-3c and claudin-15b (not claudin-15a) in fish gills. Furthermore, based on MDA and T-AOC activities in the gill, the maximum permissible levels of DON were evaluated to be 375.60 as well as 412.91 μg/kg diet in grass carp, respectively.
Erucic acid (EA), an anti-nutritional factor naturally present in rapeseed meal, could inhibit animal growth. While most studies about the detrimental effect of EA have been restricted mainly to terrestrial animal, and the information on aquatic animal is lacking. This study focused on investigating the impact of EA on growth performance, intestinal oxidative damage, cell apoptosis and dysfunction of cell-cell tight junction of on-growing grass carp (Ctenopharyngodon idella) (129.17 ± 0.19 g) which were offered with six diets containing graded levels of EA (0.00, 0.29, 0.60, 0.88, 1.21 and 1.50% diet) for 60 days. We were the first to find that EA inhibited growth and disrupted intestinal structural integrity of on-growing grass carp involved in: (1) reducing feed utilization and decreasing intestinal growth of fish; (2) causing intestinal hyperemia and hyperplasia of intestine villi of fish; (3) inducing oxidative damage, cell apoptosis and dysfunction of cell-cell tight junction associated with Nrf2, p38MAPK and MLCK signaling pathways in the intestine of fish, respectively. Interestingly, EA did not affect the activity and mRNA level of CuZnSOD and the mRNA levels of GSTP2, Keap1b, JNK, Bcl-2 (only in PI and DI), caspase-3 (only in DI), claudin-12 and -15b of fish intestine. Finally, broken-line regression analysis demonstrated that the maximum tolerance levels of EA for on-growing grass carp (129.17–471.18 g) for 60 days based on PWG (percent weight gain), MDA in PI (proximal intestine), ROS in MI (mid intestine) and PC in DI (distal intestine) were estimated to be 0.64%, 0.48%, 0.48% and 0.53% diet, respectively.
In this study, we investigated the effects of dietary myo-inositol on the growth and intestinal physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (221.83 ± 0.84 g) were fed six diets containing graded levels of myo-inositol (27.0, 137.9, 286.8, 438.6, 587.7 and 737.3 mg/kg) for 10 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that compared with optimal myo-inositol levels, myo-inositol deficiency (27.0 mg/kg diet): (1) decreased glutathione (GSH) contents and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes [not glutathione-S-transferase (gst) p1 and gstp2] and NF-E2-related factor 2 (nrf2), whereas up-regulated the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents, and the mRNA levels of Kelch-like-ECH-associated protein 1 (keap1) in three intestinal segments of young grass carp (P < 0.05). (2) Up-regulated cysteinyl aspartic acid-protease (caspase)-2, -3, -7, -8, -9, apoptotic protease activating factor-1 (apaf-1), Bcl2-associated X protein (bax), fas ligand (fasl), gen-activated protein kinase (p38mapk) and c-Jun N-terminal protein kinase (jnk) mRNA levels, whereas down-regulated B-cell lymphoma-2 (bcl-2), inhibitor of apoptosis proteins (iap) and myeloid cell leukemia-1 (mcl-1) mRNA levels in three intestinal segments of young grass carp (P < 0.05). (3) Down-regulated mRNA levels of cell cycle proteins cyclin b, cyclin d, cyclin e and E2F transcription factor 4 (e2f4) in three intestinal segments of young grass carp (P < 0.05). (4) Down-regulated the mRNA levels of zonula occludens (zo) 1, zo-2, occludin, claudin-b, -c, -f, -3c, -7a, -7b as well as -11, and up-regulated the mRNA levels of claudin-12, -15a (not -15b) and myosin light chain kinase (mlck) in three intestinal segments of young grass carp (P < 0.05). All above data indicated that dietary myo-inositol deficiency could damage physical barrier function in three intestinal segments of fish. Finally, the myo-inositol requirements based on the percent weight gain (PWG), reactive oxygen species (ROS) contents in the proximal intestine (PI), relative mRNA levels of caspase-2 (PI), cyclin b (MI) as well as claudin-b (PI) were estimated to be 276.7, 304.1, 327.9, 416.7 and 313.2 mg/kg diet, respectively.
This study evaluates the effects of dietary phosphorus on the growth, immune function and structural integrity (head kidney, spleen and skin) of young grass carp (Ctenopharyngodon idella) that were fed graded levels of available phosphorus (0.95-8.75 g/kg diet). Results indicated that phosphorus deficiency decreased the growth performance of young grass carp. In addition, the results first demonstrated that compared with the optimal phosphorus level, phosphorus deficiency depressed the lysozyme (LZ) and acid phosphatase (ACP) activities and the complement 3 (C3), C4 and immunoglobulin M (IgM) contents, and down-regulated the mRNA levels of antimicrobial peptides, anti-inflammatory cytokines, inhibitor of κBα (IκBα) and target of rapamycin (TOR), whereas it up-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) p65 and NF-κB p52 mRNA levels to decrease fish head kidney and spleen immune functions. Moreover, phosphorus deficiency up-regulated the mRNA levels of Kelch-like-ECH-associated protein 1a (Keap1a), Fas ligand (FasL), apoptotic protease activating factor-1 (Apaf-1), Bcl-2 associated X protein (Bax), caspase -2, -3, -7, -8 and -9, p38 mitogen-activated protein kinase (MAPK) and myosin light chain kinase (MLCK), whereas it depressed the glutathione (GSH) contents and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes, NF-E2-related factor 2 (Nrf2), B-cell lymphoma protein-2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and tight junction complexes to attenuate fish head kidney and spleen structural integrity. In addition, phosphorus deficiency increased skin hemorrhage and lesions morbidity. Finally, based on the percent weight gain (PWG) and the ability to combat skin hemorrhage and lesions, the dietary available phosphorus requirements for young grass carp (254.56-898.23 g) were estimated to be 4.10 and 4.13 g/kg diet, respectively. In summary, phosphorus deficiency decreases the growth performance, and impairs immune function and structural integrity in the head kidney, spleen and skin of young grass carp.
Several gill claudin (Cldn) tight junction proteins in Japanese medaka are regulated by salinity (cldn10 paralogs and cldn28b), while others are constitutively expressed (cldn27a, cldn28a and cldn30c). The role of the endocrine system in this regulation has yet to be understood. The in vitro effects of cortisol and prolactin on cldn expression in gill explant cultures were investigated in medaka. ncc2b and cftr were used as markers of specific ionocytes associated with freshwater- and seawater-acclimation, respectively. Concentration-response experiments were performed by overnight incubation with 0, 0.1, 1 and 10 ?g mL(-1) cortisol or 0, 0.01, 0.1 and 1 ?g mL(-1) ovine prolactin. Cortisol significantly up-regulated cftr, ncc2b, cldn10 paralogs, cldn27a and cldn30c from 1.2- to 5-fold control levels at 10 ?g mL(-1). Cortisol had no effect on cldn28a and cldn28b. Prolactin had a concentration-dependent effect, decreasing expression of cftr (1 ?g mL(-1), 2.2-fold) while increasing ncc2b (from 0.1 ?g mL(-1), 6-7-fold). Prolactin up-regulated expression of 3 cldns: cldn28b (0.1 and 1 ?g mL(-1)), cldn10c and cldn10f (1 ?g mL(-1)), with up to 2-, 2.5- and 2-fold of control level, respectively. A combination experiment with both hormones showed that they act in synergy on cldn28b and have an additive effect on cftr, ncc2b, cldn10c and cldn10f. Our results showed that cortisol and prolactin are essential to maintain the expression of specific branchial claudins. This work also provides evidence that both hormones act directly on gill of medaka to modulate determinants of paracellular ion movement.
This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg−1 diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis morbidity of young grass carp were estimated to be 286.82 g kg−1 diet (250.66 g digestible protein kg−1 diet) and 292.10 g kg−1 diet (255.47 g digestible protein kg−1 diet), respectively.
Expression profiles of claudin-6, -10d, and -10e in the euryhaline teleost fish Tetraodon nigroviridis revealed claudin-6 in brain, eye, gill, and skin tissue, while claudin-10d and -10e were found in brain, gill and skin only. In fishes, the gill and skin are important tissue barriers that interface directly with surrounding water, but these organs generally function differently in osmoregulation. Therefore, roles for gill and skin claudin-6, -10d, and -10e in the osmoregulatory strategies of Tetraodon were investigated. In the gill epithelium claudin-6, -10d, and -10e co-localized with Na(+)-K(+)-ATPase immunoreactive (NKA-ir) ionocytes and differences in sub-cellular localization could be observed in hypo- (freshwater, FW) versus hyperosmotic (seawater, SW) environments. Claudin-10d and -10e abundance increased in the gills of fish acclimated to SW versus FW while claudin-6 abundance decreased in SW. Taken together with our knowledge of claudin-6 and -10 function in other vertebrates, data support the idea that in SW-acclimated Tetraodon, these claudins are abundant in gill ionocytes where they contribute to the formation of a Na(+) shunt and 'leaky' epithelium, both of which are characteristic of salt secreting SW fish gills. Skin claudin-10d and -10e abundance also increased in fish acclimated to SW versus those in FW, but so did claudin-6. In skin, claudin-6 was found to co-localize with NKA-ir cells, but claudin-10d and -10e did not. This study provides direct evidence that the gill epithelium contains salinity responsive tight junction proteins that are abundant primarily in ionocytes. These same proteins also appear to play a role in the osmoregulatory physiology of the epidermis.
In vertebrates, tight junction (TJ) proteins play an important role in epithelium formation and development, the maintenance of tissue integrity and regulating TJ permeability. In this study, primary cultured model gill epithelia composed of pavement cells (PVCs) were used to examine TJ protein transcript abundance during the development of epithelium confluence and epithelium resistive properties. Differences in TJ protein expression patterns and transcript abundance between gill models composed of PVCs and models composed of PVCs and mitochondrion-rich cells (MRCs) were also examined. Marked alterations in TJ protein transcript abundance were observed during the formation of confluence in flask cultured model gill epithelia. In contrast, during the formation of tissue resistance in insert cultured epithelia (i.e. epithelia cultured on a permeable substrate), changes in TJ protein mRNA abundance were conservative, despite paracellular marker flux decreasing by orders of magnitude. In both cases significant changes in claudin-8b,-8d, -27b, -28b and -32a transcript abundance were observed, suggesting that temporal alterations in the abundance of these genes are important endpoints of model gill epithelium integrity. When MRCs were present in cultured gill models, the mRNA abundance of several TJ proteins significantly altered and claudin-10c, -10d and -33b were only detected in preparations possessing MRCs. These data provide insight into the role of select TJ proteins in the formation and development of gill epithelia and the maintenance of gill barrier properties. In addition, observations reveal a heterogeneous distribution of claudin TJ proteins in the gill epithelial cells of rainbow trout.
In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr⁶⁷ mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na⁺ permeability, without affecting the Cl⁻ permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl⁻ permeability. The Y67F mutant restored the Na⁺ permeability, Cl⁻ permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore.
Background: Claudin pore domain contains a highly conserved aromatic residue.
Results: Cation selectivity of claudin-2 and claudin-10b was impaired by substitution of residues lacking an aromatic group.
Conclusion: The aromatic residue confers cation selectivity by cation-π interaction and restricting the pore diameter.
Significance: This advances our understanding of the paracellular ion selectivity mechanism.
The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac. Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression. Additionally, Na(+) efflux was substantially increased in the claudin-b morphants which contributed to a significant reduction in whole-body Na(+) levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na(+). Nevertheless, the elevated loss of Na(+) in the morphants was compensated by an increase in Na(+) uptake. Notably, we observed that the increased Na(+) uptake in the morphants was attenuated in the presence of the selective Na(+)/Cl(-)-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl(-)-free water. These results suggested that the increased Na(+) uptake in the morphants was at least in part mediated by NCC. Furthermore, treatment with an H(+)-ATPase inhibitor bafilomycin A1 was found to reduce Na(+) uptake in the morphants, suggesting that H(+)-ATPase activity was essential to provide a driving force for Na(+) uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na(+) handling in zebrafish.
Claudins are tight junction membrane proteins that regulate paracellular permeability of renal epithelia to small ions, solutes, and water. Claudins interact within the cell membrane and between neighboring cells to form tight junction strands and constitute both the paracellular barrier and the pore. The first extracellular domain of claudins is thought to be the pore-lining domain and contains the determinants of charge selectivity. Multiple claudins are expressed in different nephron segments; such differential expression likely determines the permeability properties of each segment. Recent evidence has identified claudin-2 as constituting the cation-reabsorptive pathway in the proximal tubule; claudin-14, -16, and -19 as forming a complex that regulates calcium transport in the thick ascending limb of the loop of Henle; and claudin-4, -7, and -8 as determinants of collecting duct chloride permeability. Mutations in claudin-16 and -19 cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. The roles of other claudins in kidney diseases remain to be fully elucidated. Expected final online publication date for the Annual Review of Physiology Volume 75 is February 10, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The anadromous salmonid life cycle includes both fresh water (FW) and seawater (SW) stages. The parr-smolt transformation (smoltification) pre-adapt the fish to SW while still in FW. The osmoregulatory organs change their mode of action from a role of preventing water inflow in FW, to absorb ions to replace water lost by osmosis in SW. During smoltification, the drinking rate increases, in the intestine the ion and fluid transport increases and is further elevated after SW entry. In SW, the intestine absorbs ions to create an inwardly directed water flow which is accomplished by increased Na(+), K(+)-ATPase (NKA) activity in the basolateral membrane, driving ion absorption via ion channels and/or co-transporters. This review will aim at discussing the expression patterns of the ion transporting proteins involved in intestinal fluid absorption in the FW stage, during smoltification and after SW entry. Of equal importance for intestinal fluid absorption as the active absorption of ions is the permeability of the epithelium to ions and water. During the smoltification the increase in NKA activity and water uptake in SW is accompanied by decreased paracellular permeability suggesting a redirection of the fluid movement from a paracellular route in FW, to a transcellular route in SW. Increased transcellular fluid absorption could be achieved by incorporation of aquaporins (AQPs) into the enterocyte membranes and/or by a change in fatty acid profile of the enterocyte lipid bilayer. An increased incorporation of unsaturated fatty acids into the membrane phospholipids will increase water permeability by enhancing the fluidity of the membrane. A second aim of the present review is therefore to discuss the presence and regulation of expression of AQPs in the enterocyte membrane as well as to discuss the profile of fatty acids present in the membrane phospholipids during different stages of the salmonid lifecycle.
The tilapia, Oreochromis mossambicus, is a truly euryhaline species in that it lives, grows, and reproduces in freshwater as well as in full-strength seawater. The gills, intestine, and kidneys show ionoregulatory adaptations fundamental for the calcium balance of this fish in these vastly different ionic media. This review focuses on calcium flows in these ionoregulatory organs and the changes that occur in the Cal²⁺-transporting mechanisms in the basolateral plasma membrane compartment of the cells that make up their ion-transporting epithelia. Influx of Ca²⁺ via the gills is comparable in freshwater and seawater; also, the Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger in branchial epithelial plasma membranes have comparable activities in fish adapted to freshwater and in those adapted to seawater. Ussing chamber experiments with isolated opercular membranes (a flat epithelium with chloride cells) suggest that chloride-cell-mediated, inward Ca²⁺ transport is largely dependent on Na⁺-dependent mechanisms. The Ca²⁺-ATPase is thought to play a "housekeeping" role in cellular Ca²⁺ regulation. Intestinal epithelial Ca²⁺ flux is lower in seawater fish than in freshwater fish, and this may reflect adaptation to the imminent overload of calcium in seawater, where for the uptake of water the fish drinks a 10-millimolar Ca solution. Intestinal Ca²⁺ transport is fully dependent on serosal Na⁺. Accordingly, a powerful Na⁺/Ca²⁺ exchanger operates in the basolateral plasma membrane of the enterocyte, and in particular the capacity of this transporter decreases in seawaterfish. The kidney of freshwater fishes produces a typical dilute and hypocalcic urine; in seawater, urine production decreases and the urine calcium concentration is always higher than that of the plasma. Exchange activity of Na⁺ and Ca²⁺ is undetectably low or absent in renal tissue plasma membranes. However, a high-afinity, high-capacity Ca²⁺-ATPase activity appears to correlate with Ca²⁺ reabsorption, as its activity significantly decreases after transfer of the fish to seawater. It thus appears that ATP- and Na⁺-dependent Ca²⁺ pumps are differentially expressed in gills, intestine, and kidney. Their activity may explain an ATP- or Na⁺-dependence of Ca²⁺ flow in the pertinent epithelium.
Paracellular permeability characteristics of the fish gill epithelium are broadly accepted to play a key role in piscine salt and water balance. This is typically associated with differences between gill epithelia of teleost fishes residing in seawater versus those in freshwater. In the former, the gill is 'leaky' to facilitate Na(+) secretion and in the latter, the gill is 'tight' to limit passive ion loss. However, studies in freshwater fishes also suggest that varying epithelial 'tightness' can impact ionoregulatory homeostasis. Paracellular permeability of vertebrate epithelia is largely controlled by the tight junction (TJ) complex, and the fish gill is no exception. In turn, the TJ complex is composed of TJ proteins, the abundance and properties of which determine the magnitude of paracellular solute movement. This review provides consolidated information on TJs in fish gills and summarizes recent progress in research that seeks to understand the molecular composition of fish gill TJ complexes and what environmental and systemic factors influence those components.
The intestine is one of the major osmoregulatory organs in fish. During the salmon parr–smolt transformation, the intestine must change its functions from the freshwater (FW) role of preventing water inflow, to the seawater (SW) role of actively absorbing ions and water.This development can be assessed as an increased intestinal fluid transport (Jv) during the parr–smolt transformation. The developmental changes taking place during parr–smolt transformation are governed by a number of endocrine systems, of which cortisol is the main stimulator of Jv. In order to further elucidate the mechanisms behind the elevation of Jv during parr–smolt transformation, juvenile Atlantic salmon were followed during natural (1+age) as well as photoperiod-induced (0+age) smoltification. Plasma cortisol levels, gill and intestinal Na+,K+-ATPase activity, Jv (only during natural smoltification) and intestinal paracellular permeability were measured. In natural smolting as well as in photoperiod-induced smolting, normal patterns of plasma cortisol levels and gill Na+,K+-ATPase activity, with clearly defined, transient peaks were obtained. When fish were transferred to SW, a second elevation in plasma cortisol levels and gill Na+,K+-ATPase activity occurred, whereas Jv remained at similar levels as in FW fish. As to the mechanisms behind the increased Jv during parr–smolt transformation, the intestinal Na+,K+-ATPase activity increases in the anterior intestine and the paracellular permeability, as judged by transepithelial resistance (TER), appears to decrease in the posterior intestine. These events correspond with the increase in Jv seen during this developmental stage. Furthermore, the increase in the physiological parameters follows the changes in plasma cortisol levels, shifted by a couple of weeks. When the fish were transferred to SW, a further increase in Na+,K+-ATPase activity was apparent in both anterior and posterior intestine and the paracellular permeability decreases. To summarize, the increased Jv seen during the parr–smolt transformation of Atlantic salmon may be due to an increase in the paracellular water flow of the posterior intestine. When the fish enter SW, the water flow appears to be directed from the paracellular pathway towards a more transcellular route with increased intestinal Na+,K+-ATPase activity as the main driving force.
The Salton Sea is a shallow (mean depth = 8 m; maximum depth = 15 m), saline (41-45 g l-1), intermittently mixing, 57 km long, 980 km2 lake located in the arid southwestern United States. The Sea is a wind driven system, with predominant winds paralleling the long axis of the lake, being strongest in spring and weakest in summer and fall. The Sea mixed daily or nearly daily between September and January. During this cooling period, moderate to high levels of dissolved oxygen (3-11 mg l-1) were found throughout the water column. Mean water column temperature ranged from a minimum of 13-14°C in early January to a maximum of 30-34°C in July-September. During most of this warming period, the Sea was thermally stratified but subject to periodic wind driven mixing events. Winds were stronger in spring 1998 than in 1997 or 1999, causing more rapid heating of the lake that year and also delaying onset of anoxic conditions in bottom waters. During summer months, mid-lake surface waters were sometimes supersatured with oxygen, and bottom waters were hypoxic or anoxic with sulfide concentrations > 5 mg l-1. Oxic conditions (> 1 mg O2 l-1) often extended a few meters deeper nearshore than they did well offshore as a consequence of greater mixing nearshore. Mixing events in late summer deoxygenated the entire water column for a period of days. Consumption of oxygen by sulfide oxidation likely was the principal mechanism for these deoxygenation events. Sulfide concentrations in surface waters were 0.5-1 mg l-1 approximately 3 days after one mixing event in mid-August 1999. These mixing events were associated with population crashes of phytoplankters and zooplankters and with large fish kills. In the southern basin, freshwater inflows tended to move out over the surface of the Sea mixing with saline lake water as a function of wind conditions. Salinity gradients often contributed more to water column stability than did thermal gradients in the southeasternmost portion of the lake.
In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.
Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.
Intestinal water absorption is greatly enhanced in salmonids upon acclimation from freshwater (FW) to seawater (SW); however, the molecular mechanism for water transport is unknown. We conducted a pharmacological characterization of water absorption in the rainbow trout intestine along with an investigation of the distribution and cellular localization of 3 aquaporins (Aqp1aa, -1ab and -8ab) in pyloric caeca, middle (M) and posterior (P) intestine of the Atlantic salmon. In vitro iso-osmotic water absorption (Jv) was higher in SW than FW-trout and was inhibited by (mmol L-1): 0.1 KCN (41%), 0.1 ouabain (72%) and 0.1 bumetanide (82%) suggesting that active transport, Na+,K+-ATPase and Na+,K+,2Cl--cotransport are involved in establishing the driving gradient for water transport. Jv was also inhibited by 1 mmol L-1 HgCl2, serosally (23% in M and 44% in P), mucosally (27% in M) or both (61% in M and 58% in P), suggesting involvement of both apical and basolateral aquaporins in water transport. The inhibition was antagonized by 5 mmol L-1 mercaptoethanol. By comparison, 10 mmol L-1 mucosal tetraethylammonium, an inhibitor of certain aquaporins, inhibited Jv by 20%. In the presence of glucose, mucosal addition of phloridzin inhibited water transport by 20%, suggesting that water transport is partially linked to the Na+-glucose cotransporter. Using polyclonal antibodies against salmon Aqp1aa, -1ab and -8ab, we detected Aqp1aa, and -1ab immunoreactivity in the brush border and sub-apical region of enterocytes in all intestinal segments. The Aqp8ab antibody showed a particularly strong immunoreaction in the brush border and sub-apical region of enterocytes throughout the intestine and also stained lateral membranes and peri-nuclear regions though at lower intensity. The present localization of 3 aquaporins in both apical and lateral membranes of salmonid enterocytes facilitates a model for transcellular water transport in the intestine of SW-acclimated salmonids.
WATER BALANCE IN TELEOST FISH IS MAINTAINED WITH CONTRIBUTIONS FROM THE MAJOR OSMOREGULATORY ORGANS: intestine, gills, and kidney. Overall water fluxes have been studied in all of these organs but not until recently has it become possible to approach the mechanisms of water transport at the molecular level. This mini-review addresses the role of the kidney in osmoregulation with special emphasis on euryhaline teleosts. After a short review of current knowledge of renal functional morphology and regulation, we turn the focus to recent molecular investigations of the role of aquaporins in water and solute transport in the teleost kidney. We conclude that there is much to be achieved in understanding water transport and its regulation in the teleost kidney and that effort should be put into systematic mapping of aquaporins to their tubular as well as cellular localization.
The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.
Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na(+) gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na(+)-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.
In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H(+), Na(+), and NH(4)(+) gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na(+) uptake and NH(3)/NH(4)(+) excretion were detected at most mitochondrion-rich cells (MRCs). H(+) probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 muM) blocked 35% of the NH(3)/NH(4)(+) secretion and 54% of the Na(+) uptake, suggesting that the Na(+)/H(+) exchanger (NHE) is involved in Na(+) and NH(3)/NH(4)(+) transport. Low-Na(+) water (<0.001 mM) or high-NH(4)(+) (5 mM) acclimation simultaneously increased Na(+) uptake and NH(3)/NH(4)(+) excretion but decreased or even reversed the H(+) gradient at the skin and MRCs. The correlation between NH(4)(+) production and H(+) consumption at the skin surface suggests that MRCs excrete nonionic NH(3) (base) by an acid-trapping mechanism. Raising the external NH(4)(+) significantly blocked NH(3)/NH(4)(+) excretion and Na(+) uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH(3)/NH(4)(+) excretion and Na(+) uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na(+)/H(+) exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na(+) acclimation. This study suggests a novel Na(+)/NH(4)(+) exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H(+) gradients across apical membranes of MRCs.
The tight junction protein claudin-10 is known to exist in two isoforms, resulting from two alternative exons, 1a and 1b (Cldn10a, Cldn10b). Here, we identified and characterized another four claudin-10 splice variants in mouse and human. One (Cldn10a_v1) results from an alternative splice donor site, causing a deletion of the last 57 nucleotides of exon 1a. For each of these three variants one further splice variant was identified (Cldn10a_v2, Cldn10a_v3, Cldn10b_v1), lacking exon 4. When transfected into MDCK cells, Cldn10a, Cldn10a_v1 and Cldn10b were inserted into the tight junction, whereas isoforms of splice variants lacking exon 4 were retained in the endoplasmic reticulum. Cldn10a transfection into MDCK cells confirmed the previously described increase in paracellular anion permeability. Cldn10a_v1 transfection had no direct effect, but modulated Cldn10a-induced organic anion permeability. At variance with previous reports in MDCK-II cells, transfection of high-resistance MDCK-C7 cells with Cldn10b dramatically decreased transepithelial resistance, increased cation permeability, and changed monovalent cation selectivity from Eisenman sequence IV to X, indicating the presence of a high field-strength binding site that almost completely removes the hydration shell of the permeating cations. The extent of all these effects strongly depended on the endogenous claudins of the transfected cells.
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.
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 junction 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 fudamental 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.
Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1-based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.
Changes in protein and mRNA expression of Na(+),K(+)-ATPase in gills and pyloric caeca of brown trout were investigated on a detailed time course after transfer from freshwater to 25 ppt seawater (SW). A transient deflection in plasma osmolality and muscle water content lasting from 4 h until day 3 was followed by restoration of hydromineral balance from day 5 onward. Gills and pyloric caeca responded to SW transfer by increasing Na(+),K(+)-ATPase activity from days 5 and 3, respectively, onward. In both tissues, this response was preceded by an increase in alpha-subunit Na(+), K(+)-ATPase mRNA as early as 12 h posttransfer. The similarity of the response in these two organs suggests that they both play significant physiological roles in restoring hydromineral balance after abrupt increase in salinity. Further, SW transfer induced a slight, though significant, increase in primary gill filament Na(+), K(+)-ATPase immunoreactive (NKIR) cell abundance. This was paralleled by a marked (50%) decrease in secondary lamellar NKIR cell abundance after less than 1 d in SW. Thus, SW acclimation in brown trout is characterised by a lasting decrease in overall NKIR cell abundance in the gill. We propose that SW transfer stimulates Na(+),K(+)-ATPase enzymatic activity within individual chloride cells long before (<1 d) it becomes apparent in measurements of whole-gill homogenate enzymatic activity. This is supported by the early stabilisation (12 h) of hydromineral balance.
This chapter analyzes the role of kidney in some marine creatures. In freshwater forms, the kidney functions largely as a water excretory device. With rare exceptions, this is accomplished by filtration at the renal glomerulus and implies the presence of suitable cellular components to conserve filtered ions and excrete dilute urine. In marine teleost forms, the kidney functions chiefly as an excretory device for magnesium and sulfate ions. In glomerular marine forms, associated machinery must be present to conserve water, monovalent ions, and other filtered plasma constituents. The cartilaginous sharks, skates, and rays, because they are hyperosmotic in a marine environment, have combined the principal functions of the kidneys of both freshwater and marine bony fishes. The chapter assembles all available information concerning the structure and function of the fish kidney and provides a unifying synthesis for the understanding of this organ's role in body fluid regulation, of specific nephron function in fishes, and of the evolutionary significance of the regions of the nephron in fishes and higher vertebrates.
The physiological mechanisms required for fish to live in freshwater and seawater are well described for some species and are the same mechanisms exploited in amphihaline species that migrate between freshwater and seawater. Many fish not only tolerate, but can acclimate and adapt to conditions outside conventional freshwater and seawater conditions, specifically salinities greater than seawater, alkaline waters (up to pH 10), and ion-poor waters. These environments exist around the globe and in many instances can support recreational and commercial fisheries. This chapter will describe the chemical characteristics of these water types, the physiological challenges associated with living in these extreme environments, and the physiological solutions that permit fish not only to survive, but in some cases to thrive, in hypersaline, alkaline, and ion-poor waters.
Aquatic habitats are remarkably diverse with respect to the chemical and physical properties of the water. For example, salinity may vary between full-strength seawater and near distilled water while pH may differ by as much as 6.0 pH units. Furthermore, aquatic environments, especially the freshwater ecosystems, are typically unstable and characterized by marked natural fluctuations of temperature, pH, oxygen, carbon dioxide, and dissolved ions. The ability of fish to inhabit these diverse and oscillating environments arises from a variety of adaptive physiological mechanisms. Owing to the location of the gill between the external and internal environments and its crucial role in gas transfer, acid-base balance, and ionic regulation, adaptive changes in branchial function are especially important. In many instances, the compensatory adjustments of gill function originate from profound morphological changes. Generally, the morphological adjustments to environmental changes are considered to be adaptive, although in certain instances gill function may actually be impaired. Moreover, owing to the multi-functional nature of the gill, morphological amelioration of a particular physiological function might compromise another.
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.
We investigated the salinity dependent expression dynamics of 7 aquaporin paralogs (aqp1a, -3a, -7, -8ab, -10a, -10b and -11a) in several tissues of euryhaline Japanese medaka (Oryzias latipes). All paralogs except aqp7 and -10a had a broad tissue distribution and several were affected by salinity in both osmoregulatory and non-osmoregulatory tissues. In the intestine, aqp1a, -7, -8ab and -10a decreased upon seawater (SW)-acclimation, both when comparing long-term acclimated fish and during 1-3 days of the transition period (freshwater (FW)-to-SW and SW-to-FW). In the gill, aqp3a was lower and aqp10a higher in SW than in FW. In the kidney no aqps were affected by salinity. In the skin, aqp1a and -3a were lower in SW than in FW. In the liver aqp8ab and -10a were lower in SW than in FW. Further, 6 Na(+),K(+)-ATPase α-subunit isoform transcripts were analyzed in the intestine but showed no consistent response to salinity, suggesting that water transport is not regulated at this level. In contrast, mRNA of the Na(+),K(+), 2Cl(-)-cotransporter type-2 showed a strong increase in the intestine in SW compared to FW fish. Using custom made antibodies, we localized Aqp1a, -8ab and -10a proteins in the apical region of enterocytes of FW fish. Apical staining intensity strongly decreased, vanished or moved to sub apical regions, when fish were acclimated to SW, supporting the lower mRNA expression in SW. Western blots confirmed the decrease in Aqp1a and -10a protein in SW. The strong decrease in aquaporin expression in the intestine of SW fish is surprising and challenges the paradigm for transepithelial intestinal water absorption in SW fishes.
The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na(+)/H(+) exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC), Na(+)-K(+)-ATPase (NKA) and anion exchanger (AE); (2) Na(+)-Cl(-) cotransporter (NCC) cells with apical NCC and basolateral H(+)-ATPase; and (3) epithelial Ca(2+) channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.
Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions.
Euryhaline fishes live in a wide salinity range from freshwater to seawater and hypersaline environments. Euryhaline fishes such as salmon, eels, and tilapia are economically important in worldwide fisheries and aquaculture. This chapter summarizes mechanisms used by hagfishes, lampreys, elasmobranchs, and teleosts to maintain ionic and osmotic homeostasis in changing environmental salinity. Hyperosmoregulation in dilute environments uses gills for active ion uptake, intestine for dietary ion uptake, and kidneys for renal uptake. Extrarenal NaCl uptake occurs via high-affinity uptake mechanisms or low-affinity Na+/Cl- cotransport. In seawater, hypoosmoregulation involves reflexive drinking, intestinal absorption of salts and water, NaCl secretion at the gills, and renal ion excretion. Na+ and Cl- excretion occurs through Na+/K+-ATPase, the Na+/K+/2Cl- cotransporter, and apical cystic fibrosis transmembrane conductance regulator. These mechanisms are magnified in hypersaline conditions. Active extrarenal salt transport takes place through mitochondrion-rich ionocytes in skin and gill epithelia. The mechanisms of ion and water transport are summarized, with models presented for seawater, marine, and euryhaline conditions.
The short-term osmoregulatory capacity of sea bass (Dicentrarchus labrax) larvae and juveniles at consecutive developmental stages was evaluated by an analysis of drinking rates and whole-body and branchial Na+/K+ ATPase activity. The effect of acute salinity stress on those parameters was assessed from hatching to the juvenile stage. Drinking rate normalised to body weight (DRbw) decreased throughout postembryonic development. DRbw was not affected by salinity in yolk sac larvae. However, significant differences were observed in older larvae and in juveniles submitted to hypo- and hyperosmotic challenges. Post yolk sac larvae and juveniles always drink, but at a lower rate in hypoosmotic media compared to hyperosmotic media. A correlation was established between DRbw and blood osmolality during the postembryonic development of the sea bass. Na+/K+ ATPase specific and total activity in whole larval homogenates decreased from hatching to the 5-mm yolk sac larva stage and increased in 10-mm stage larvae. The enzyme specific activity in the gills, from post yolk sac larvae to juveniles, decreased whereas the total activity increased during the same period. Developing sea bass have a better capacity to cope with hypoosmotic than hyperosmotic media.
We examined the hypoosmoregulatory ability of a model fish, medaka (Oryzias latipes), in relation to the gill-chloride cells or mitochondrion-rich (MR) cells, and to cortisol. When the medaka were transferred from freshwater (FW) to 30‰ seawater (SW), muscle water content decreased by 8% after 2 h and normalized within 1 week. Size and density of MR cells in the gill filament increased after 1–2 weeks in SW. Immersion of medaka in FW containing cortisol (10 μg/ml) for 1–2 weeks also doubled the number of MR cells, and abolished the decrease in muscle water content after SW transfer. In SW-adapted medaka, prolactin (PRL; 10 μg/g body weight) injection reduced muscle water content. However, when cortisol (50 μg/g) was injected simultaneously with PRL, cortisol abolished the decrease in muscle water. We concluded that cortisol plays an important role in the SW adaptation of the medaka, whereas PRL may be involved in ion uptake. Medaka seems to be a good model fish useful for the study of osmoregulatory mechanisms in general.
Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved because it is difficult to separate transcellular from TJ-controlled paracellular water flux. Using an alternative approach, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation channel-forming TJ proteins, which were not endogenously expressed in that cell line. Claudin-2 is typical for leaky, water-transporting epithelia like proximal tubule, while claudin-10b is present in numerous epithelia, including water-impermeable segments of Henle's loop. Both transfections did not alter the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na(+) gradient, or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared to vector controls, indicating claudin-2-mediated paracellular water permeability. Na(+)-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. In contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and by this mediates the paracellular part of water transport in leaky epithelia.
A primary culture system of rainbow trout gill pavement cells grown on permeable support (single-seeded insert, SSI) was used to examine histological and physiological changes induced by the addition of the corticosteroid hormone cortisol. Pavement cell epithelia were cultured under symmetrical conditions (L15 apical/L15 basolateral) and developed a high transepithelial resistance (TER, 6.84 ± 1.99 kΩ cm(2), mean ± SEM) with a low phenol red diffusion rate (PRD, 0.15 ± 0.03 μmol l(-1)/day). Addition of cortisol to the basolateral compartment increased TER twofold and reduced PRD threefold over a 5-day period. A similar increase in TER could be seen after 24 h apical freshwater (FW) in control cultures. In cortisol-treated cultures FW exposure did not change TER, but PRD increased significantly. Histochemical staining of the cytoskeleton of cells in SSI culture revealed a morphological partitioning into a single mucosal layer of polarized, polygonal cells featuring cortical F-actin rings which were comparable to F-actin rings of epithelial cells on the lamellar and filamental surface, and several unorganized serosal layers of cells with F-actin stress fibers. Addition of cortisol increased cell density by 18% and in the mucosal layer it led to smaller, less polygonal cells with increased height and increased cell contact area. In transmission electron microscopic images two pairs of cytoplasmatic electron-dense structures confining the zonula occludens apically and basally toward the zonula adhaerens were found. Addition of cortisol increased the distance between those paired structures, hence led to deeper tight junctions. The cortisol-induced increase in barrier properties, therefore, involves a structural fortification of the tight junctions which was not generally modified by a short 24-h apical freshwater stress. These results identify cortisol as a regulator of tight junction morphology between pavement cells of euryhaline fish such as the trout.
In seawater fishes, osmotic homeostasis requires uptake of ions and water in the intestine and these processes are governed by the combined trans- and paracellular pathways. The current study examined mRNA expression of two tight junction proteins (claudin-15 and -25 b) predominantly expressed in the intestine of Atlantic salmon. We examined the response in pyloric caecae, middle and posterior intestine to seawater challenge, during smoltification and after injection with osmoregulatory hormones. Seawater (SW) transfer elevated levels of claudin-15 and -25 b while no change was induced throughout the smolt stage. In freshwater, cortisol and growth hormone inhibited claudin-15 expression in the two anterior segments. Claudin-25 b was elevated in all intestinal segments by growth hormone, while cortisol had an inhibitory effect. In seawater, prolactin and cortisol inhibited claudin expression. The data suggest that claudin expression is involved in the reorganisation of intestinal epithelium and possibly change paracellular permeability during SW acclimation. The lack of preparatory change during smoltification suggests that this process is not completed during smolt development. The effects of the tested hormones cannot explain the sum of changes induced by salinity, which, like the smoltification data, suggests the importance of additional factors and possibly contact with the imbibed SW per se.
A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability.
We recently showed that a series of tight junction proteins of the claudin family are regulated in the gill of salmon during salinity acclimation. The aim of the present study was to investigate the role of cortisol, growth hormone (GH) and prolactin (PRL) on regulation of expression of these isoforms. Experiments on primary cultures of gill tissue showed that cortisol stimulates claudin 10e, 27a and 30 mRNA levels while no significant effects were observed on claudin 28a and 28b. The associated receptor signalling pathway was examined using glucocorticoid and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The observed in vitro responses were blocked by RU486, suggesting the involvement of a glucocorticoid type receptor. Injections of FW salmon with cortisol increased the expression of claudin 10e, 27a, and 30 but did not affect claudin 28a and 28b significantly. While GH had no effect on its own, the combination of GH and cortisol reduced claudin 28b levels. Injection of SW salmon with PRL selectively increased the expression of claudin 28a but had no effect on the other examined isoforms. The data shows that FW- (27a and 30) and SW-induced (10e) claudins are all stimulated by cortisol while the major osmoregulatory hormones GH and PRL had no effect on these salinity sensitive isoforms. This suggests that other hormones and/or osmotic conditions interact with cortisol to determine claudin composition in the gill.
Genes encoding for claudin-8 and -27 tight junction proteins in the euryhaline puffer fish (Tetraodon nigroviridis) were identified using its recently sequenced genome. Phylogenetic analysis indicated that multiple genes encoding for claudin-8 proteins (designated Tncldn8a, Tncldn8b, Tncldn8c and Tncldn8d) arose by tandem gene duplication. In contrast, both tandem and whole genome duplication events appear to have generated genes encoding for claudin-27 proteins (designated Tncldn27a, Tncldn27b, Tncldn27c and Tncldn27d). Tncldn8 and Tncldn27 mRNA were widely distributed in Tetraodon, suggesting involvement in various physiological processes. All Tncldn8 and Tncldn27 genes were expressed in gill and skin tissue (i.e., epithelia exposed directly to the external environment). A potential role for claudin-8 and -27 proteins in the regulation of hydromineral balance in Tetraodon was investigated by examining alterations in mRNA abundance in select ionoregulatory tissue of fish acclimated to freshwater (FW) and seawater (SW). In FW or SW, Tetraodon exhibited alterations in Na(+)-K(+)-ATPase activity (a correlate of transcellular transport) typical of a euryhaline teleost fish. Simultaneously, tissue and gene specific alterations in Tncldn8 and Tncldn27 transcript abundance occurred. These data provide some insight into the duplication history of cldn8 and cldn27 genes in fishes and suggest a possible role for claudin-8 and -27 proteins in the osmoregulatory strategies of euryhaline teleosts.
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.
A population of proximal tubules when isolated from the glomerular kidneys of seawater-adapted (SW) and freshwater-adapted (FW) killifish (Fundulus heteroclitus) spontaneously secrete fluid. Regardless of SW or FW adaptation, Na and Cl are the dominant electrolytes in secreted fluid. Mg concentrations in fluid secreted by both tubules are significantly greater than those in the peritubular bath, and Mg concentrations are inversely related to Na concentrations. Proximal tubules from either SW or FW fish exhibit low transepithelial voltage (-1 to -2 mV) and low transepithelial resistances (20-30 omega.cm2) typical of other vertebrate proximal tubules. Transepithelial diffusion potentials for Na, Cl, Mg, and SO4 suggest that the paracellular pathway is Na selective and impermeable to divalent ions. Consideration of transepithelial electrochemical potential differences for Na, Cl, Mg, and SO4 suggests active transport of Mg, SO4, and Cl in proximal tubules isolated from SW- and FW-adapted fish. The similarities in the functional properties of secretory proximal tubules isolated from SW- and FW-adapted killifish are striking and raise questions about the in vivo role of these tubules in the renal adaptations to seawater and freshwater.
Gill cell suspensions from freshwater (FW)- and seawater (SW)-adapted teleosts were obtained by density gradient centrifugation. The proportion of chloride cells (CCs) in the mixed cell suspensions was estimated using the fluorescent mitochondrial stain, DASPMEI, and ranged from less than 1 % (FW-adapted tilapia) to approximately 13% (SW-adapted toadfish). The gill cells displayed relatively high viability based on Trypan Blue exclusion (>75%), lactate dehydrogenase leakage (<6·5% h−1), oxygen consumption rates (5–15μmolg−1 cell wet mass h−1) and ATP levels (1–3μmol g−1 cell wet mass). There were no obvious differences between the viability of CCs and the other cell types present.
An initial comparison of gill oxidative metabolism in SW-adapted tilapia (Oreochromis mossambicus) and toadfish (Opsanus beta) demonstrated that both species oxidized glucose and lactate at substantially greater rates than alanine or oleate. Metabolic rates were significantly higher in toadfish cell suspensions. Kinetic experiments revealed that toadfish gill cells displayed lower values of Km and higher values of Vm for both lactate and glucose, in comparison to tilapia. The elevated metabolism in toadfish gill cells was correlated with increased activities of the oxidative enzyme citrate synthase and Na+/K+-ATPase. The toadfish cell suspensions had a greater proportion of CCs and it is likely that the difference in CC numbers between the two species is the basis for the observed differences in enzyme activities and rates of oxidative metabolism. This idea is supported by the highly significant correlation between Na+/K+-ATPase activity (or CC numbers) and rates of lactate oxidation in gill cell suspensions from FW-and SW-adapted tilapia and toadfish, as well as SW-adapted tilapia chronically treated with cortisol to elevate CC numbers.
Although it has been assumed widely that the high metabolic rate of gill tissue reflects, in part, the oxidative demands of the chloride cell, the results of this study provide the first experimental, albeit indirect, evidence for differential rates of metabolism in the various cell types that comprise the gill.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way
to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform,
provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification
of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters
into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to
a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated
only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model
needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA
integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler
PCR using the established mathematical model.
Tight junctions regulate paracellular conductance and ionic selectivity. These properties vary among epithelia but the molecular basis of this variation remains unknown. To test whether members of the claudin family of tight junction proteins influence paracellular ionic selectivity, we expressed human claudin-4 in cultured MDCK cells using an inducible promoter. Overexpression increased the complexity of tight junction strands visible by freeze-fracture microscopy without affecting the levels of claudin-1, -2, or -3, occludin, or ZO-1. A decrease in conductance correlated directly with the kinetics of claudin-4 induction. Dilution potentials revealed that the decrease in paracellular conductance resulted from a selective decrease in Na(+) permeability without a significant effect on Cl(-) permeability. Flux for an uncharged solute, mannitol, and the rank order of permeabilities for the alkali metal cations were unchanged. A paracellular site for these effects was supported by the lack of apical/basal directionality of the dilution potentials, the linearity of current-voltage relationships, and the lack of influence of inhibitors of major transcellular transporters. These results provide, to our knowledge, the first direct demonstration of the ability of a claudin to influence paracellular ion selectivity and support a role for the claudins in creating selective channels through the tight-junction barrier.
Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl-. These results support a model where claudins create charge-selective channels in the paracellular space.
In this short review of fish gill morphology we cover some basic gross anatomy as well as in some more detail the microscopic anatomy of the branchial epithelia from representatives of the major extant groups of fishes (Agnathans, Elasmobranchs, and Teleosts). The agnathan hagfishes have primitive gill pouches, while the lampreys have arch-like gills similar to the higher fishes. In the lampreys and elasmobranchs, the gill filaments are supported by a complete interbranchial septum and water exits via external branchial slits or pores. In contrast, the teleost interbranchial septum is much reduced, leaving the ends of the filaments unattached, and the multiple gill openings are replaced by the single caudal opening of the operculum. The basic functional unit of the gill is the filament, which supports rows of plate-like lamellae. The lamellae are designed for gas exchange with a large surface area and a thin epithelium surrounding a well-vascularized core of pillar cell capillaries. The lamellae are positioned for the blood flow to be counter-current to the water flow over the gills. Despite marked differences in the gross anatomy of the gill among the various groups, the cellular constituents of the epithelium are remarkably similar. The lamellar gas-exchange surface is covered by squamous pavement cells, while large, mitochondria-rich, ionocytes and mucocytes are found in greatest frequency in the filament epithelium. Demands for ionoregulation can often upset this balance. There has been much study of the structure and function of the branchial mitochondria-rich cells. These cells are generally characterized by a high mitochondrial density and an amplification of the basolateral membrane through folding or the presence of an intracellular tubular system. Morphological subtypes of MRCs as well as some methods of MRC detection are discussed.