Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes.

The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes, atp4a, atp4b, pgc, and pga2 have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, were co-deleted or pseudogenized in most agastric fishes of the four major groups. kcne2 and vsig1 were also deleted or pseudogenized in the agastric monotreme echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of cldn18 was retained in some agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the four major groups of agastric fish, as well as a single gene loss in the echidna and platypus.

Metabolic Water As a Route for Water Acquisition in Vertebrates Inhabiting Dehydrating Environments.

Vertebrates have expanded their habitats during evolution, which accompanies diversified routes for water acquisition. Water is acquired by oral intake and subsequent absorption by the intestine in terrestrial and marine animals which are subjected to constant dehydration, whereas most water is gained osmotically across body surfaces in freshwater animals. In addition, a significant amount of water, called metabolic water, is produced within the body by the oxidation of hydrogen in organic substrates. The importance of metabolic water production as a strategy for water acquisition has been well documented in desert animals, but its role has attracted little attention in marine animals which also live in a dehydrating environment. In this article, the author has attempted to reevaluate the role of metabolic water production in body fluid regulation in animals inhabiting desiccating environments. Because of the exceptional ability of their kidney, marine mammals are thought to typically gain water by drinking environmental seawater and excreting excess NaCl in the urine. On the other hand, it is established that marine teleosts drink seawater to enable intestinal water and ion absorption, and the excess NaCl is excreted by branchial ionocytes. In addition to the oral route, we suggest through experiments using eels that water production by lipid metabolism is an additional route for water acquisition when they encounter seawater. It seems that metabolic water production contributes to counteract dehydration before mechanisms for water regulation are reversed from excretion in freshwater to acquisition in seawater.

Circulating isotocin, not angiotensin II, is the major dipsogenic hormone in eels.

Angiotensin II (AngII) is generally known as the most important dipsogenic hormone throughout vertebrates, while two other neurohypophysial hormones, vasopressin and oxytocin, are not dipsogenic in mammals. In this study, we found that systemic isotocin, but not vasotocin, is the potent dipsogenic hormone in eels. When injected intra-arterially into conscious eels, isotocin, vasotocin and AngII equally increased ventral aortic pressure dose dependently at 0.03-1.0 nmol kg-1, but only isotocin induced copious drinking. The dipsogenic effect was dose dependent and occurred significantly at as low as 0.1 nmol kg-1. By contrast, a sustained inhibition of drinking occurred after AngII injection, probably due to baroreflexogenic inhibition. No such inhibition was observed after isotocin injection despite similar concurrent hypertension. The baroreceptor may exist distal to the gill circulation because the vasopressor effect occurred at both ventral and dorsal aorta after AngII but only at ventral aorta after isotocin. By contrast, intra-cerebroventricular (i.c.v.) injection of isotocin had no effect on drinking or blood pressure, but AngII increased drinking and aortic pressure dose dependently at 0.03-0.3 nmol per eel. Lesioning of the area postrema (AP), a sensory circumventricular organ, abolished drinking induced by peripheral isotocin, but not i.c.v. AngII. Collectively, isotocin seems to be a major circulating hormone that induces swallowing through its action on the AP, while AngII may be an intrinsic brain peptide that induces drinking through its action on a different circumventricular site, possibly a recently identified blood-brain barrier-deficient structure in the antero-ventral third ventricle of eels, as shown in birds and mammals.

Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

Molecular mechanisms underlying guanylin-induced transcellular Cl- secretion into the intestinal lumen of seawater-acclimated eels.

Guanylin (GN) stimulates Cl- secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl- transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl- secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3- from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl- and Na+, which was blocked by serosal DNDS. These results suggest that Cl- is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+-HCO3- cotransporter (NBC) but not by Na+-K+-2Cl- cotransporter 1 (NKCC1), and Cl- is secreted by unknown DPC-sensitive Cl- channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42- transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl- secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl- secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Evolution of the membrane/particulate guanylyl cyclase: From physicochemical sensors to hormone receptors.

Guanylyl cyclase (GC) is an enzyme that produces 3',5'-cyclic guanosine monophosphate (cGMP), one of the two canonical cyclic nucleotides used as a second messenger for intracellular signal transduction. The GCs are classified into two groups, particulate/membrane GCs (pGC) and soluble/cytosolic GCs (sGC). In relation to the endocrine system, pGCs include hormone receptors for natriuretic peptides (GC-A and GC-B) and guanylin peptides (GC-C), while sGC is a receptor for nitric oxide and carbon monoxide. Comparing the functions of pGCs in eukaryotes, it is apparent that pGCs perceive various environmental factors such as light, temperature, and various external chemical signals in addition to endocrine hormones, and transmit the information into the cell using the intracellular signaling cascade initiated by cGMP, e.g., cGMP-dependent protein kinases, cGMP-sensitive cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases. Among vertebrate pGCs, GC-E and GC-F are localized on retinal epithelia and are involved in modifying signal transduction from the photoreceptor, rhodopsin. GC-D and GC-G are localized in olfactory epithelia and serve as sensors at the extracellular domain for external chemical signals such as odorants and pheromones. GC-G also responds to guanylin peptides in the urine, which alters sensitivity to other chemicals. In addition, guanylin peptides that are secreted into the intestinal lumen, a pseudo-external environment, act on the GC-C on the apical membrane for regulation of epithelial transport. In this context, GC-C and GC-G appear to be in transition from exocrine pheromone receptor to endocrine hormone receptor. The pGCs also exist in various deuterostome and protostome invertebrates, and act as receptors for environmental, exocrine and endocrine factors including hormones. Tracing the evolutionary history of pGCs, it appears that pGCs first appeared as a sensor for physicochemical signals in the environment, and then evolved to function as hormone receptors. In this review, the author proposes an evolutionary history of pGCs that highlights the emerging role of the GC/cGMP system for signal transduction in hormone action.

The digestive tract as an essential organ for water acquisition in marine teleosts: lessons from euryhaline eels.

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/CaCO3) precipitates promoted by HCO3- 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.

Spreading of River Water Guides Migratory Behavior of Homing Chum Salmon Oncorhynchus keta in Otsuchi Bay, a Narrow Inlet with Multiple River Flows.

The Sanriku-ria coast of Japan, a homing area for chum salmon, Oncorhynchus keta, is characterized by a large number of small closed bays into which one or multiple short rivers flow. The present behavioral investigation of chum salmon in this region was designed to gain deeper insight into the migration of chum salmon to their natal rivers. Eighty-three fish caught at the middle part of Otsuchi Bay were tracked using an acoustic transmitter in the narrow inlet into which flow three rivers: the Otsuchi, Koduchi, and Unosumai. The majority of 18 fish that entered the Unosumai River, which flows into the southwest side of the bay, directly approached the river along the southern coast. More than half of fish that entered the Otsuchi and Koduchi Rivers, which flow into the northwest side, also migrated into the inner bay via the southerly route, and then entered these rivers frequently after passing the mouth of the Unosumai River. In the inner bay, the salinity of sea surface water suggested that water from the three rivers circulates in a counterclockwise direction at a depth of less than 1.0 m, flowing eastwardly along the southern coast. The observed migratory paths of homing salmon in Otsuchi Bay thus correspond well with the counterflow of surface river water in the bay. The present results suggest that homing migration of salmon in the Sanriku narrow inlet is guided by natal river flows.

Hormonal regulation of thirst in the amphibious ray-finned fish suggests the requirement for terrestrialization during evolution.

Thirst has evolved for vertebrate terrestrial adaptation. We previously showed that buccal drying induced a series of drinking behaviours (migration to water-taking water into the mouth-swallowing) in the amphibious mudskipper goby, thereby discovering thirst in ray-finned fish. However, roles of dipsogenic/antidipsogenic hormones, which act on the thirst center in terrestrial tetrapods, have remained unclear in the mudskipper thirst. Here we examined the hormonal effects on the mudskipper drinking behaviours, particularly the antagonistic interaction between angiotensin II (AngII) and atrial natriuretic peptide (ANP) which is important for thirst regulation in mammalian 'forebrain'. Expectedly, intracerebroventricular injection of ANP in mudskippers reduced AngII-increased drinking rate. ANP also suppressed the neural activity at the 'hindbrain' region for the swallowing reflex, and the maintenance of buccopharyngeal water due to the swallowing inhibition may attenuate the motivation to move to water. Thus, the hormonal molecules involved in drinking regulation, as well as the influence of buccopharyngeal water, appear to be conserved in distantly related species to solve osmoregulatory problems, whereas hormonal control of thirst at the forebrain might have been acquired only in tetrapod lineage during evolution.

Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels.

The intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, the hormonal regulation of HCO3- secretion is largely unknown. Here, mucosally added guanylin (GN) increased HCO3- secretion, measured by pH-stat, across isolated seawater-acclimated eel intestine bathed in saline at pH 7.4 (5% CO2). The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. As mucosal DNDS failed to inhibit the GN effect, apical DNDS-sensitive AE may not be involved. To identify molecular species of transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, AE genes (draa, b, and pat1a, c) on the apical membrane, and NBC genes (nbce1a, n1, n2a) and an AE gene (sat-1) on the basolateral membrane were candidates involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAs and basolateral DNDS-sensitive NBCs to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

Preparatory Mechanisms for Salinity Tolerance in Two Congeneric Anuran Species Inhabiting Distinct Osmotic Habitats.

Anurans occupy a wide variety of habitats of diverse salinities, and their osmoregulatory ability is strongly regulated by hormones. In this study, we compared the adaptability and hormonal responses to osmotic stress between two kajika frogs, Buergeria japonica (B.j.) and B. buergeri, (B.b.), which inhabit coastal brackish waters (BW) in the Ryukyu Islands and freshwater (FW) in the Honshu, respectively. Both hematocrit and plasma Na+ concentration were significantly higher in B.j. than in B.b. when both were kept in FW. After transfer to one-third seawater (simulating the natural BW environment), which is slightly hypertonic to their body fluids, their body mass decreased and plasma Na concentration increased significantly in both species. After transfer, plasma Na+ concentration increased significantly in both species. We examined the gene expression of two major osmoregulatory hormones, arginine vasotocin (AVT) and atrial natriuretic peptide (ANP), after partial cloning of their cDNAs. ANP mRNA levels were more than 10-fold higher in B.j. than in B.b. in FW, but no significant difference was observed for AVT mRNA levels due to high variability, although the mean value of B.j. was twice that of B.b. Both AVT and ANP mRNA levels increased significantly after transfer to BW in B.b. but not in B.j., probably because of the high levels in FW. These results suggest that B.j. maintains high plasma Na+ concentration and anp gene expression to prepare for the future encounter of the high salinity. The unique preparatory mechanism may allow B.j. wide distribution in oceanic islands.

The Amphibious Mudskipper: A Unique Model Bridging the Gap of Central Actions of Osmoregulatory Hormones Between Terrestrial and Aquatic Vertebrates.

Body fluid regulation, or osmoregulation, continues to be a major topic in comparative physiology, and teleost fishes have been the subject of intensive research. Great progress has been made in understanding the osmoregulatory mechanisms including drinking behavior in teleosts and mammals. Mudskipper gobies can bridge the gap from aquatic to terrestrial habitats by their amphibious behavior, but the studies are yet emerging. In this review, we introduce this unique teleost as a model to study osmoregulatory behaviors, particularly amphibious behaviors regulated by the central action of hormones. Regarding drinking behavior of mammals, a thirst sensation is aroused by angiotensin II (Ang II) through direct actions on the forebrain circumventricular structures, which predominantly motivates them to search for water and take it into the mouth for drinking. By contrast, aquatic teleosts can drink water that is constantly present in their mouth only by reflex swallowing, and Ang II induces swallowing by acting on the hindbrain circumventricular organ without inducing thirst. In mudskippers, however, through the loss of buccal water by swallowing, which appears to induce buccal drying on land, Ang II motivates these fishes to move to water for drinking. Thus, mudskippers revealed a unique thirst regulation by sensory detection in the buccal cavity. In addition, the neurohypophysial hormones, isotocin (IT) and vasotocin (VT), promote migration to water via IT receptors in mudskippers. VT is also dipsogenic and the neurons in the forebrain may mediate their thirst. VT regulates social behaviors as well as osmoregulation. The VT-induced migration appears to be a submissive response of subordinate mudskippers to escape from competitive and dehydrating land. Together with implications of VT in aggression, mudskippers may bridge the multiple functions of neurohypophysial hormones. Interestingly, cortisol, an important hormone for seawater adaptation and stress response in teleosts, also stimulates the migration toward water, mediated possibly via the mineralocorticoid receptor. The corticosteroid system that is responsive to external stressors can accelerate emergence of migration to alternative habitats. In this review, we suggest this unique teleost as an important model to deepen insights into the behavioral roles of these hormones in relation to osmoregulation.

Distribution and co-localization of diversified natriuretic peptides in the eel heart.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8 M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.

Drinking by amphibious fish: convergent evolution of thirst mechanisms during vertebrate terrestrialization.

Thirst aroused in the forebrain by angiotensin II (AngII) or buccal drying motivates terrestrial vertebrates to search for water, whereas aquatic fish can drink surrounding water only by reflex swallowing generated in the hindbrain. Indeed, AngII induces drinking through the hindbrain even after removal of the whole forebrain in aquatic fish. Here we show that AngII induces thirst also in the amphibious mudskipper goby without direct action on the forebrain, but through buccal drying. Intracerebroventricular injection of AngII motivated mudskippers to move into water and drink as with tetrapods. However, AngII primarily increased immunoreactive c-Fos at the hindbrain swallowing center where AngII receptors were expressed, as in other ray-finned fish, and such direct action on the forebrain was not found. Behavioural analyses showed that loss of buccal water on land by AngII-induced swallowing, by piercing holes in the opercula, or by water-absorptive gel placed in the cavity motivated mudskippers to move to water for refilling. Since sensory detection of water at the bucco-pharyngeal cavity like 'dry mouth' has recently been noted to regulate thirst in mammals, similar mechanisms seem to have evolved in distantly related species in order to solve osmoregulatory problems during terrestrialization.

Renoguanylin stimulates apical CFTR translocation and decreases HCO3- secretion through PKA activity in the Gulf toadfish (Opsanus beta).

The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO3- secretion, likely by limiting apical HCO3-/Cl- exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3- secretion.

Molecular and evolutionary perspectives of the renin-angiotensin system from lamprey.

The recent advance and revision on the renin-angiotensin system in lamprey were summarized and we emphasized that presence of two types of angiotensins (Angs) in lamprey. Due to the parasitic nature on fish blood, teleost-type Angs were produced in their buccal gland and secreted into the lamphredin to evade the host immunorejection. A native lamprey angiotensinogen (AGT) was identified in genome and it retains serine-protease inhibitor activity for thrombin that regulates the blood coagulation pathway. The native lamprey angiotensin II (Lp-Ang II) is hypotensive instead of hypertensive, suggesting a functional divergence on cardiovascular regulation from the main vertebrate groups. The renin gene was absent from the lamprey genome so far, and the mutation on the renin-recognition site on lamprey AGT suggested that other proteases may have replaced the role of renin. Lp-Ang II was shown to bind to AT1 receptor and internalized, but the downstream signaling was still unknown. Molecular and phylogenetic evidence on invertebrate ACE-like proteins indicated that they were not homologous to those in vertebrates and could be acting on other native peptides. Although it was generally believed that the RAS was a well-conserved hormone system in vertebrates and invertebrates, revision by molecular data indicated that invertebrates lack homologous RAS components while lamprey possess an almost complete RAS. This suggests that the hormone cascade system was first evolved around cyclostome emergence and invertebrates could have taken up the RAS components from vertebrates through horizontal gene transfer.

A sodium binding system alleviates acute salt stress during seawater acclimation in eels.

BACKGROUND: Teleosts transiting from freshwater (FW) to seawater (SW) environments face an immediate osmotic stress from ion influxes and water loss, but some euryhaline species such as eels can maintain a stable plasma osmolality during early SW exposure. The time course changes in the gene expression, protein abundance, and localization of key ion transporters suggested that the reversal of the ion transport systems was gradual, and we investigate how eels utilize a Na-binding strategy to slow down the ion invasion and complement the transporter-mediated osmoregulation. RESULTS: Using an electron probe micro-analyzer, we localized bound Na in various eel tissues in response to SW transfer, suggesting that the Na-binding molecules were produced to sequester excess ionic Na+ to negate its osmotic potential, thus preventing acute cellular dehydration. Mucus cells were acutely activated in digestive tract, gill, and skin after SW transfer, producing Na-binding molecule-containing mucus layers that fence off high osmolality of SW. Using gel filtration HPLC, some molecules at 18 kDa were found to bind Na in the luminal secretion of esophagus and intestine, and higher binding was associated with SW transfer. Transcriptome and protein interaction results indicated that downregulation of Notch and ß-catenin pathways, and dynamic changes in TGFß pathways in intestine were involved during early SW transition, supporting the observed histological changes on epithelial desquamation and increased mucus production. CONCLUSIONS: The timing for the activation of the Na-binding mechanism to alleviate the adverse osmotic gradient was temporally complementary to the subsequent remodeling of branchial ionocytes and transporting epithelia of the digestive tract. The strategy to manipulate the osmotic potential of Na+ by specific binding molecules is similar to the osmotically inactive Na described in human skin and muscle. The Na-binding molecules provide a buffer to tolerate the salinity changes, which is advantageous to the estuary and migrating fishes. Our data pave the way to identify this unknown class of molecules and open a new area of vertebrate osmoregulation research.

Changes in Plasma and Tissue Long-Chain Polyunsaturated Fatty Acid (LC-PUFA) Content in the Eel Anguilla japonica After External and Internal Osmotic Stress.

We investigated the effect of external and internal osmotic stress on the profile of long-chain polyunsaturated fatty acids (LC-PUFA) in euryhaline eels Anguilla japonica. Freshwater (FW) fish were transferred to seawater (SW) for external osmotic stress or subjected to internal stress through injection with hypertonic saline. FW eels injected with isotonic saline served as controls. Plasma osmolality, Na+ concentration, and gill Na+/K+ -ATPase activity increased, but hematocrit decreased compared with controls in eels exposed to external or internal osmotic stress. The expression of two major transporter genes for SW adaptation, the Na+ -K+ -2Cl - co-transporter 1a (NKCC1a) in the gill and NKCC2b in the intestine, was up-regulated only in SW-transferred eels, suggesting a direct impact of SW on the gill and intestine via SW ingestion. Total LC-PUFA contents and DHA (22:6 n-3) increased in the gill and liver of SW-transferred eels and in the intestine of hypertonic saline-injected eels. However, total LC-PUFA content in plasma decreased after both external and internal osmotic stimuli. In contrast, the gene expression of two key enzymes involved in the LC-PUFA biosynthesis, Δ6 fatty acid desaturase and elongase, did not change in the gill, intestine and liver of osmotically stressed eels. These results indicate that LC-PUFA is possibly involved in osmoregulation and the increased LC-PUFA contents of osmoregulatory organs might be a result of LC-PUFA transport via circulation, rather than through de novo biosynthesis.

Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater.

Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl- from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl- transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4'-diisothiocyano-2,2'-disulfonic acid-sensitive Cl-/[Formula: see text] exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+-K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl- channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase 2a (CA2a) supplies H+ and [Formula: see text] for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl- cotransporters and basolateral Na+-[Formula: see text] cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes (aqp1a and aqp3) and upregulated claudin gene (cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to provide a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.