Exploring novel hormones essential for seawater adaptation in teleost fish

General and Comparative Endocrinology 157 (2008) 3–13

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General and Comparative Endocrinology

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Review

Exploring novel hormones essential for seawater adaptation in teleost fish

Yoshio Takei *

Laboratory of Physiology, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 February 2008Revised 11 March 2008Accepted 12 March 2008Available online 30 March 2008

Keywords:Natriuretic peptide familyGuanylin familyAdrenomedullin familyComparative genomicsEcological evolutionOsmoregulationCardiovascular regulation

0016-6480/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ygcen.2008.03.021

* Fax: +81 3 5351 6463.E-mail address: [email protected]

Marine fish are dehydrated in hyperosmotic seawater (SW), but maintain water balance by drinking sur-rounding SW if they are capable of excreting the excess ions, particularly Na+ and Cl�, absorbed withwater by the intestine. An integrative approach is essential for understanding the mechanisms for SWadaptation, in which hormones play pivotal roles. Comparative genomic analyses have shown that hor-mones that have Na+-extruding and vasodepressor properties are greatly diversified in teleost fish. Phys-iological studies at molecular to organismal levels have revealed that these diversified hormones aremuch more potent and efficacious in teleost fish than in mammals and are important for survival inSW and for maintenance of low arterial pressure in a gravity-free aquatic environment. This is typifiedby the natriuretic peptide (NP) family, which is diversified into seven members (ANP, BNP, VNPand CNP1, 2, 3 and 4) and exerts potent hyponatremic and vasodepressor actions in marine fish.Another example is the guanylin family, which consists of three paralogs (guanylin, uroguanylin andrenoguanylin), and stimulates Cl� secretion into the intestinal lumen and activates the absorptive-typeNa–K–2Cl cotransporter by local luminocrine actions. The most recent addition is the adrenomedullin(AM) family, which has five members (AM1, 2, 3, 4 and 5), with AM2 and AM5 showing the most potentor efficacious vasodepressor and osmoregulatory effects among known hormones in teleost fish. Accumu-lating evidence strongly indicates that members of these diversified hormone families play essential rolesin SW adaptation in teleost fish. In this short review, the author has attempted to propose a novelapproach for identification of new hormones that are important for SW adaptation using comparativegenomic and functional studies. The author has also suggested potential hormone families that are diver-sified in teleost fish and appear to be involved in SW adaptation through their ion-extruding actions.

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1. Introduction

It is generally accepted that vertebrates first appeared in thecoastal sea where the salinity is slightly lower than seawater(SW) (Carroll, 1988). The early vertebrates seem to have enteredinland fresh water (FW) once, then they expanded their habitatsto the sea and onto the land (Romer and Grove, 1935). This isthought to be one of the reasons why most extant vertebrateshave tonicity of extracellular fluids between FW and SW (ca.one-third of SW) irrespective of their habitats. It is thought thatthis intermediate tonicity of plasma may ensure a survivable lifein both FW and SW. During the course of this ecological evolu-tion to diverse habitats, vertebrates have developed many mech-anisms for body fluid regulation to maintain water and ionbalance and this preferred plasma composition. Hormones playpivotal roles in such homeostatic regulation. In terrestrialanimals where both water and ion retention are usually priori-ties for regulation, disruption of genes related to antidiuretichormone (vasopressin/vasotocin) or antinatriuretic hormone

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(aldosterone) profoundly influences body fluid balance andthreaten survival (White, 2004; Fujiwara and Bichet, 2005). InFW fish where water excretion and ion retention are the keychallenges, removal of prolactin, a FW-adapting hormone, fromthe circulation results in an inability to survive in FW (Pickfordand Phillips, 1959), although there do seem to be species differ-ences in the relative importance of this hormone (Manzon,2002). On the other hand, an essential or indispensable hormonefor SW adaptation does not seem to have been demonstrated,although growth hormone (insulin-like growth factor) and corti-sol are established SW-adapting hormones in teleost fish(McCormick, 2001). In fact, removal of these hormones fromplasma increased plasma Na+ concentration significantly aftertransfer of salmonid fish to SW. However, this disturbance wasnot severe enough to affect their survival in SW.

Although critical SW-adapting hormones have not yet beenidentified in teleost fish, maintenance of water and ion balancein SW is not an easy task because they lose water by osmosisand gain excess ions down a concentration gradient through thegills because of the much lower plasma ion concentrations andosmolality compared with SW (Marshall and Grosell, 2005). Thisis exemplified by the observation that when frogs and toads, which

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4 Y. Takei / General and Comparative Endocrinology 157 (2008) 3–13

have plasma ion concentrations similar to teleost fish, are im-mersed in SW, they lose body weight and increase plasma osmolal-ity at much faster rates than when they are exposed to dry air.Therefore, there must be multiple regulatory systems that ensureSW adaptation, in which hormones should play essential roles.Accordingly, it is reasonable to speculate that there is a criticalSW-adapting hormone that governs the survival of teleost fish inSW, as is the case in antidiuretic hormone for terrestrial animalsand in prolactin for FW fish. With this idea in mind, we startedan explorative study to identify new and essential SW-adaptinghormones using physiological, biochemical, and molecular biolog-ical techniques, and more recently a comparative genomic ap-proach. The recent generation of genome and expressed sequencetag (EST) databases along with the development of bioinformaticstools accelerated advancement of this study (Hsu and Hsueh,2000). In this review, I would like to summarize a brief history ofour research on identification of SW-adapting hormones in fishand suggest future prospects for this field.

2. Is life in the sea comfortable?

As a medium from which life originated, the sea appears to bean ideal habitat for living organisms. Among the factors that char-acterize the environment, water seems to be a primary factor.Water has the highest specific gravity among solvents, which nul-lifies the gravitational force that every creature on earth is exposedto. The high specific heat of water also contributes to the mainte-nance of a constant environmental temperature. Furthermore,water is an excellent solvent that dissolves a variety of solutes,both organic and inorganic. The dissolved organic salts are takenup by microorganisms, which ultimately nourish marine organ-isms higher in the food chain. Accordingly, sea water serves asthe body fluids of the earth to maintain the homeostasis of its envi-ronment. As mentioned above, however, water dissolves largeamounts of inorganic salts, which increases osmotic pressure ofSW. Accordingly, marine organisms have developed excellentmechanisms to cope with this osmotic problem and maintain bodyfluid homeostasis.

2.1. Aquatic versus terrestrial environment

As extant vertebrates contain 65–85% of their body weight aswater, water retention is an essential part in environmentaladaptation. The most prominent difference between the aquaticand terrestrial environments is the ease of access to water. Afterabandoning the life in water, terrestrial animals had to developmechanisms to retain water in the body. Water is lost by respi-ration, evaporation from the body surfaces and renal excretion.The former two routes are hardly controllable as they are indis-pensable for oxygen acquisition and thermoregulation, and thusfor life on the land. Accordingly, the major site of body waterregulation is the kidney, principally through re-absorption fromurine by the renal tubules. This explains why antidiuretichormone is essential for body fluid homeostasis in terrestrialanimals. By contrast, fish can obtain water by drinking thesurrounding media either in FW or SW, although water alsoenters the body passively across the gills when they are in FW.As discussed in detail in the following section, ion regulationis more important than water regulation for fish, as themaintenance of water balance is dominated by the extent towhich they take up ions from the environment in FW andextrude excess ions in SW. Therefore, the most importantdifference between fish and terrestrial tetrapods is that waterretention is essential for terrestrial life, whereas ion regulationis the primary challenge for aquatic life albeit in the oppositedirection in FW (retention) and SW (extrusion).

Another important difference between aquatic and terrestrialenvironments is gravity. Terrestrial animals circulate bloodthroughout the body against gravitational force and accordinglyhave a powerful heart pumping relatively large volumes of blood(ca. 10% of body mass) at high arterial pressure (ca. 100 mm Hg).By contrast, teleost fish circulate a small volume of blood (3–4%of body mass) at low arterial blood pressure (20–30 mm Hg) inthe almost gravity-free aquatic environment. Because of suchobvious differences in cardiac performance and arterial pressure,cardiotropic and hypertensive hormones, such as angiotensin,vasopressin and endothelin, dominate in terrestrial animals, whilehypotensive hormones such as natriuretic peptides and adrenome-dullins appear to play more important roles in teleost fish (Takeiet al., 2007).

2.2. Freshwater versus seawater environment

Among fish, body fluid regulation has reverse requirements forFW and SW species. In FW, fish are on the edge of overhydrationand hyponatremia, so they actively absorb NaCl from the environ-ment by the gills and from food in the intestine, while they excreteexcess water as dilute urine by the kidney (Marshall and Grosell,2005). By contrast, SW fish must cope with dehydration and hyper-natremia. They actively excrete excess monovalent ions by themitochondria-rich cells (MRCs) of the gills and excess divalent ionsby the kidney (Evans et al., 2005), while they drink copiousamounts of SW and absorb it by the intestine to rehydrate but inso doing add further to the saline load that must be excreted(Loretz, 1995). Therefore, although SW fish are in a dehydratingenvironment, they can maintain water balance by drinkingsurrounding SW and extruding excess ions from the body. Accord-ingly, the most important mechanism for body fluid regulation inSW is ion-extrusion not water retention. This differentiates SW fishfrom terrestrial animals which also live in a dehydrating environ-ment but where water retention is of primary importance.

3. Integrative physiology of seawater adaptation in fishes

While we are seeking for an essential hormone that is indis-pensable for SW adaptation, we are also aware that multiple hor-mones work in concert to ensure the success of SW adaptation infish. To understand the interlinked homeostatic processes involvedin SW adaptation, it is essential to look at the whole process froman integrative viewpoint. In this section, therefore, I would like tointroduce our integrative approach toward understanding of themechanism of SW adaptation.

3.1. Comparative study is an integrative approach

Fish have adopted diverse strategies for survival in the hyperos-motic SW environment. Hagfish, the most primitive extant speciesof marine cyclostomes, have plasma ionic concentrations almostidentical to SW (Rankin, 1997), which is a characteristic commonto marine invertebrates. According to the Goldman–Hodgkin–Katzequation, high extracellular Na+ and Cl� concentrations hyper-polarize the cell, which results in impaired excitability of neuronsand muscles. Therefore, the ion concentrations in cytoplasmshould be increased proportionally to maintain cell excitability.However, this may then impair cytoplasmic enzyme activity(Yancy et al., 1982; Hochachka and Somero, 2002). Cartilaginousfish, elasmobranchs and holocephalans, and a lobe-finned marinebony fish, the coelacanth, accumulate urea in plasma to increaseplasma osmolality to the SW level, while maintaining plasma ionconcentrations at a level lower than SW (Hazon et al., 1997). It isknown that unfavorable effects of these high urea concentrationson metabolic enzymes are ameliorated by the counter action of

Y. Takei / General and Comparative Endocrinology 157 (2008) 3–13 5

trimethylamine oxide (Yancy et al., 1982). Ray-finned fish, ofwhich teleosts are predominant in terms of biomass and numberof species, maintain plasma ionic concentration and osmolalityaround one-third of SW irrespective of the environmental salinity(Marshall and Grosell, 2005). Judging from the current success ofray-finned fish compared with cyclostomes, cartilaginous fish,and lobe-finned fish, maintenance of low plasma ion concentrationand osmolality appear to have some advantages for thriving in SW.Therefore, to trace the evolutionary process of SW adaptation fromion- and osmo-conforming hagfish, through ion-regulating andosmo-conforming cartilaginous fishes, to ion- and osmo-regulatingteleost fish should provide us with an important insight into theimportant mechanism of SW adaptation in fishes. Therefore, webelieve that such a comparative approach is one of the integrativeapproaches which allows us to understand the development ofmechanism across different evolutionary time scales.

3.2. Whole-body influx and efflux of water and ions

In order to maintain body fluid volume and composition, thebudget (gain and loss) of ion and water across body surfaces mustbe balanced within a narrow tolerance range. In teleost fish, gainand loss of ion and water occur across body surfaces, especiallyat the gills where the blood is separated from environmental waterby only the thin respiratory epithelia. The skin covering the body isa such smaller area than the gill surface and generally protectedfrom the external medium by scales and a mucous layer. In SW,water is lost and ions, particularly Na+ and Cl�, the major osmo-lytes in SW, are gained passively. However, the final balance ofNaCl at the gills is loss because of the active excretion by MRCsusing ATP-driven transport mechanisms (Evans et al., 2005). An-other major route for the gain of water and ions is oral intake ofwater and food with subsequent absorption by the digestive tracts(Hirano et al., 1976). Since the lumen of the digestive tract is effec-tively outside the body, water and ions have to be absorbed acrossthe intestinal epithelia to enter the body fluids. In SW teleost fish,drinking and subsequent intestinal absorption of water and ionsconstitute a major regulatory site for body fluid homeostasis. Thekidney primarily serves as a site for the removal of excess divalentions, Mg2+, Ca2+ and SO4

2�, in SW teleost fish (Beyenbach, 1995). Ashomeostasis of body fluids is achieved by the balance of the com-

drinking

gills

k

intestineesophagus380

1294

921(157 222)

379+

A

k

intestine

drinking

ANGIIAM2, AM5 ANP

ANPGLN

B

Fig. 1. (A) Gain and loss of Na+ ions at each osmoregulatory sites and total budget betweeThe gills and intestine (after drinking seawater) are major sites for Na+ handling in seawasites.

bined gain and loss of water and ions at these osmoregulatorysites, we must examine the integral effects of candidate hormonesfor SW adaptation at all these sites (Fig. 1A). This is the mostimportant integrative approach to evaluate the roles of specifichormones in SW adaptation.

3.3. From molecular to organismal level

Investigations at levels ranging from molecules (genes) to thewhole body are also regarded as an integrative approach. Whenwe focus on a candidate hormone, we first examine how its geneexpression or plasma level changes in response to osmoregulatorychallenges such as after transfer of a euryhaline fish from FW toSW. We also assess how exogenous administration of the hormoneaffects body fluid balance. If these effects are positive, we nextexamine which osmoregulatory site (tissue level) or which celltype in the osmoregulatory tissue (cell level) is responsible forthe changes seen at the whole-body level. Then, transporters/chan-nels and other molecules involved in the cellular response areidentified at the molecular or gene level. For instance, it was shownthat growth hormone is important for the maintenance of lowplasma Na+ concentration in SW (Bolton et al., 1987), which waslater shown to be accounted for by the development of SW-typeMRCs in the gills (Madsen, 1990). In turn in the MRC, expressionof the Na+,K+-ATPase and other transporter genes are increased inthe apical and basolateral membrane (Evans et al., 2005). Finally,we need to investigate how the expression of such osmoregulatorygenes is regulated by the hormone, and how the expression of thehormone gene, its secretion and perhaps tissue receptors are en-hanced after transfer of fish to higher salinity.

More recently, investigations driven from the opposite directionhave been performed. Novel genes whose expression is increasedafter SW challenges are first identified by the differential display,subtraction cloning, etc. Then, the role of identified genes in SWadaptation is evaluated at each osmoregulatory site and finally atthe whole-body level. As many genes are up-regulated after salin-ity challenges including stress-related genes, it is not easy to differ-entiate and extract the genes that respond solely to salinitychanges. Nonetheless, transcriptional factors that are up-regulatedafter SW exposure have been identified in the gills of tilapia (Fioland K}ultz, 2005). It is essential that the mechanisms for SW adap-

idney

6

idney

ANPAM2

n the body and the environment in seawater-adapted eel. Numbers are in lmol/kg/h.ter fish. (B) Biological actions of osmoregulatory hormones at major osmoregulatory


tation be investigated at different levels to understand how thewhole system functions integratively.

3.4. Value of genetically modified fish

In order to evaluate the role of specific candidate genes in thewhole process of SW adaptation at the organismal level, gain-of-function and loss-of-function experiments on osmoregulatorygenes using genetically modified fish can be used as a tool. Thesetechniques appear to offer ultimate examples of how to give thefruits of molecular studies back to the whole body (McGonnelland Fowkes, 2006). However, the introduction of such gene modi-fication techniques has been delayed in osmoregulatory studies,because there is a belief that homeostatic regulation involvingmany factors cannot be elucidated by modification of a single gene.It is also possible that a single gene is involved in many processes,so that the disruption of the gene affects too many processes topinpoint its function to osmoregulation. However, the gene modi-fication technique has advantages over other old techniques suchas immuno-neutralization of a circulating hormone, as the tech-nique is applicable to hormones that act in a paracrine/autocrinefashion and those that act in a luminocrine fashion as observedwith guanylin (see below). Therefore, this technique should bemore routinely used in fish models in the near future. Thus far,transgenic fishes have been produced with increased growth hor-mone and melanin concentration hormone, resulting in acceler-ated growth and albinic phenotype, respectively (Devlin et al.,1994; Kinoshita et al., 2001). Further, natural mutants of thesomatolactin gene in medaka revealed its important role in lipidmetabolism (Fukamachi et al., 2004). However, currently informa-tion is lacking with regard to altered osmoregulatory geneexpression.

There are several kin species of teleost fish which exhibit dis-tinct and differing pattern of adaptability to altered and variableenvironmental salinities. For instance, the Mozambique tilapia,Oreochromis mossambicus, is readily adaptable to both FW andSW, while Nile tilapia, Oreochromis niloticus, can only survive inhyperosmotic media less than 50% SW. There should be somegenes that are responsible for such profound differences in adapt-ability, but no attempts seem to have been made to identify andthen modify such genes. Another interesting species is medaka,genus Oryzias, which are distributed widely in Asia. We haveshown that Oryzias javanicus and Oryzias dancena are euryhalinespecies that normally live in brackish waters and can adapt to bothFW and SW. However, Oryzias marmoratus is a stenohaline FW spe-cies that is endemic to an inland lake of Sulawesi Island in Indone-

Fig. 2. Schematic drawing of gain and loss-of-function experiment using genetically moprofound differences in salinity tolerance.

sia and cannot adapt to the salinity greater than half strength SW(Inoue and Takei, 2002). It is important to note that adaptability istrue not only for adults but also for fertilized eggs in terms of sur-vival and hatching rates in different salinities. For instance, O. java-nicus can hatch and grow in SW but O. marmoratus show 65%mortality before hatching in 50% SW. Therefore, we can evaluatethe importance of a potential gene essential for SW adaptationby producing transgenic O. marmoratus to enable them to survivein SW (Fig. 2). We can also assess the role of the gene in SW adap-tation by knocking-down the gene in O. javanicus, by injecting anti-sense oligonucleotide into the egg and then assessing whethertheir SW-adaptability is impaired or not (Fig. 2). As an embryonicstem cell line appears to have been established in medaka (Honget al., 2004), and as homologous recombination at the cell levelhas been attempted in the zebrafish (Fan et al., 2006), the produc-tion of gene knockout O. javanicus may be realized in the near fu-ture, allowing assessment of change in SW-adaptability of adultfish. The nuclear transfer technique has been established in meda-ka fish eggs with cultured somatic cell line, which may help deci-phering gene function when homologous recombination is realizedin culture cells (Bubenshchikova et al., 2007). Recently, a methodfor gene knockout, named TILLING (Targeting Induced Local LesionIN Genomes), has been established to identify gene function, inwhich random mutagenesis of genes using N-ethyl-N-nitrosourea(ENU) is followed by sequencing for mutation screening of a targetgene (Wienholds et al., 2003). This method is now widely used inthe zebrafish (Sood et al., 2006) and medaka (Taniguchi et al.,2006).

4. Hormones that promote seawater adaptation in fish

There is a long history of research pursuing importanthormones for SW adaptation in fishes (Henderson et al., 1987;McCormick, 2001; Evans, 2002; Takei and Loretz, 2005). Theestablished SW-adapting hormones include growth hormone,cortisol, IGF-I, urotensins, angiotensin II, atrial natriuretic peptide(ANP), etc.

4.1. Fast-acting versus slow-acting hormones

Among the SW-adapting hormones, growth hormone, cortisoland IGF-I are so-called long-acting hormones, which induceexpression of new genes to re-organize the osmoregulatory organsto a SW-type for long-term adaptation to the hyperosmotic envi-ronment. They are also called slow-acting hormones because plas-ma levels increase slowly after transfer of fish from FW to SW

dified medaka fish. Oryzias marmoratus and O. javanicus are kin species but exhibit


(Balment et al., 1987; Sakamoto et al., 1993). It has been shownthat administration of these hormones to salmonid fish acceleratestranslocation of MRCs from secondary to primary lamellae of thegills and also transforms MRCs from absorptive FW type intoexcretory SW-type (Madsen, 1990). In addition to the gills, cortisolwas shown to induce changes in the intestine and urinary bladderto a SW-type in a number of teleost species (Johnson et al., 1972;Nagahama et al., 1975; Yamamoto and Hirano, 1978). Aldosterone,the major mineralocorticoid in tetrapods, is scarcely produced inteleost plasma as measured by specific radioimmunoassay(Reinking, 1983), and the aldosterone synthase gene (CYP11B2)could not be detected in the pufferfish genome and EST databases(Nelson, 2003). Therefore, cortisol seems to act as both mineralo-corticoid and glucocorticoid in teleost species (Balment et al.,1987). However, a mineralocorticoid receptor has been identifiedrecently in teleost fish, which has high affinity to a mineralocorti-coid-type steroid, 11-deoxycorticosterone (Prunet et al., 2006).Growth hormone may act directly on osmoregulatory tissues orthrough augmentation of IGF-I in plasma and at peripheral tissues(Sakamoto et al., 1993; Mancera and McCormick, 1998). IGF-I mayalso be involved in the re-organization of osmoregulatory tissuesby initiating differentiation of new cell types.

In contrast to the long-acting protein and steroid hormones, oli-gopeptide hormones such as angiotensin II, urotensins, vasoactiveintestinal polypeptide (VIP), guanylins, and natriuretic peptides arefast- and short-acting hormones, which may be involved in theinitial phase of SW adaptation. These hormones are secretedimmediately after changes in environmental salinity, and act onosmoregulatory organs to change the activities of existingtransporters, channels and pumps and probably the intercellularmatrices that regulate the movement of water and ionsthrough the intercellular space. It is of interest to note that suchshort-acting hormones often stimulate the secretion of long- andslow-acting growth hormone and cortisol before disappearingfrom the plasma. Therefore, the fast-acting hormones seem togovern the whole process of SW adaptation, affording integrationof fast and slow components of hormone regulation.

4.2. Diversification of Na+-extruding hormones in fish

The recent establishment of genome databases for teleost fishincluding pufferfish (Takifugu rubripes and Tetraodon nigroviridis),zebrafish (Danio rerio), medaka (Oryzias latipes), stickleback(Gasterosteus aculeatus), etc. allows us to trace the evolutionaryhistory of hormone genes within the vertebrate series. In particu-lar, comparison of the hormone genes between genome databasesfor teleosts and mammals reveals profound differences in the geneevolution, reflecting the difference in habitat (Hsu and Hsueh,2000; Sherwood et al., 2000; Inoue et al., 2003). One outstandingfeature of such evolution is that vasodepressor hormone genesshow greater diversification in teleost fish than in mammals, prob-ably reflecting the low blood pressure of fish (Takei et al., 2007).The second interesting feature is that Na+-extruding hormonesare more significantly diversified in teleost fish than in mammals.This may be related to the fact that ray-finned bony fishexperienced explosive speciation or species diversification afterthey expanded their occupation of the sea from FW and coastalwaters. This occurred in the late Jurassic to early Cretaceous periodof the Mesozoic era by virtue of developing an ability to extrudeexcess Na+ from the body via MRCs of the gills under appropriatehormonal regulations. This was much later than the emergenceof marine cartilaginous fish and lobe-finned body fish, whichre-entered the sea in the late Devonian to early Carboniferousperiod of the Paleozoic era (MacFarlane, 1923; Smith, 1932).Typical examples of diversified Na+-extruding hormones are theNP family, the adrenomedullin (AM) family and the guanylin

family. It is intriguing to note that the Na+-extruding hormonesare mostly vasodepressor as well, although a decrease in systemicpressure generally decreases glomerular filtration rate (GFR) andthus decreases Na+ excretion by the kidney.

4.2.1. The natriuretic peptide (NP) familyANP is a potent natriuretic and hypotensive hormone, which

was first isolated from the cardiac atrium of mammals (Kangawaand Matsuo, 1984). While the NP family consists of ANP, B-typeNP (BNP) and C-type NP (CNP) in mammals, it has seven members,ANP, BNP, ventricular NP (VNP) and four CNPs (CNP1, 2, 3 and 4) inteleost fish (Inoue et al., 2003). Since seven NPs already exist inchondrostean bony fish (sturgeon and bichir), the NPs werediversified before the third-round whole genome duplication (3R)that occurred early in the teleost lineage (Kawakoshi et al.,2004). Cartilaginous fishes (holocephalans and elasmobranchs)have only CNP3 and only CNP4 could be identified in cyclostomes(hagfishes and lampreys). Therefore, CNP4 seems to be an ancestralmolecule of the NP family and CNP3 produced at the second-roundwhole genome duplication (2R) that occurred at the transitionfrom agnathans (cyclostomes) to gnathostomes, and CNP4 mayhave subsequently disappeared in cartilaginous fishes over thesubsequent long evolutionary period. Cardiac hormones, ANP,BNP and VNP, were generated by tandem duplication from CNP3on the same chromosome. CNP1 and CNP2 were generated byblock duplication onto different chromosomes from the CNP3 gene,as the three CNPs are, respectively, linked with three enolase genes(Inoue et al., 2005). It is obvious that seven NP genes had existedwhen the lobe-finned bony fish, from which tetrapods are derived,diverged from the ray-finned bony fish more than 400 million yearago. In tetrapods, however, only three (ANP, BNP, and CNP4) wereretained in mammals, four (BNP, VNP, CNP1, and CNP3) in chicken(Trajanovska et al., 2007), and four (ANP, BNP, CNP3, and CNP4) inamphibians (Kojima et al., 1994). It is intriguing to consider why allseven NP genes still exist in the ray-finned fish, although anotherset of the NP genes, duplicated at the 3R, seem to have beensilenced during evolution in the teleost lineage.

In addition to the ligand, NP receptors are also diversified in tel-eost fish. Most biological actions of the NP family are mediated bythe second messenger, cGMP, which is produced by the cytoplas-mic guanylyl cyclase domain of the receptor. There are two typesof such receptors, A-type NP receptor (NPR-A or GC-A) for ANPand BNP, and B-type NP receptor (NPR-B or GC-B) for CNP in mam-mals (Hirose et al., 2001). An additional receptor that has only ashort cytoplasmic domain and binds all ligands with high affinities,named C-type NP receptor (NPR-C), has also been identified. Itsfunction is proposed to regulate the local concentration of ligandsat target tissues, but other biological functions are also suggested(Anand-Srivastava, 2005). In medaka, two types of NPR-A geneswere identified in addition to NPR-B and NPR-C (Yamagami andSuzuki, 2005). In the eel, a new type of guanylyl cyclase deficientreceptor, named NPR-D, has been identified in addition to NPR-A,NPR-B, and NPR-C (Kashiwagi et al., 1995). Since multiple candi-dates for NPRs can be detected in the genome and EST databasesof teleosts, the NP receptors may have been diversified in parallelwith the ligands in the teleost lineage.

4.2.2. The adrenomedullin (AM) familyAM is a multifunctional hormone with a potent hypotensive

and modest natriuretic activity, which was discovered from cancercells of adrenal medulla origin (Kitamura et al., 1993). AM was pre-viously known as a member of the calcitonin gene-related peptide(CGRP) family, which consists of CGRP, AM and amylin in mam-mals (López and Martínez, 2001). However, it is now evident thatfive AM peptides (AM1, 2, 3, 4 and 5) form an independent subfam-ily in teleost fish, of which mammalian AM is the apparent ortho-


log of teleost AM1 by synteny analysis (Ogoshi et al., 2003). Com-parative genomic analysis further showed that AM1/AM4 andAM2/AM3 were duplicated at the 3R in the teleost lineage, butthe counterpart of AM5 disappeared after the 3R (Ogoshi et al.,2006). Based on these findings, we subsequently discovered AM2and AM5 in mammals (Takei et al., 2004, 2008). Therefore, it isnow known that the AM family consists of three types of peptides,AM1/AM4, AM2/AM3 and AM5. The sequence identity of the dupli-cate teleost AMs differs greatly among three types; AM1 and AM4have 30–40% identity, AM2 and AM3 more than 80%, while thecounterpart of AM5 has changed into a pseudogene. Such differ-ences in mutation rate may imply differences in their relativeimportance for physiological functions.

The receptors for AM are suggested to be a complex of calcito-nin receptor-like receptor (CLR) and receptor activity-modifyingprotein (RAMP), as is the case for CGRP (Poyner et al., 2002). Inmammals, AM(1) binds to the complex of CLR and RAMP2 orRAMP3 with high affinity, and CGRP binds to the complex of CRLand RAMP1. In parallel with the diversification of the AM familyof ligands, CLR and RAMP are also diversified in teleost fish; 3 CLRs(CLR1–3) and 5 RAMPs (RAMP1–5) have been identified in the puf-ferfish, Takifugu obscurus (Nag et al., 2006), in contrast to the singleCLR and 3 RAMPs in mammals. It has been shown that, while AM1binds with high affinity to the complex of CLR1 + RAMP2/3/5 andCLR2 + RAMP2, AM2 and AM5 bind only to the complex ofCLR1 + RAMP3. In spite of the low affinity of AM2 and AM5 forthe known AM receptors, AM2 and AM5 are 100-fold more potentthan AM1 for the vasodepressor activity in the eel (Nobata et al.,2008). Therefore, additional receptors for AM2 and AM5 are likelyto be present in teleost fish.

4.2.3. The guanylin familyGuanylin potently inhibits Na+ and Cl� absorption by the intes-

tine, from which it was first isolated (Currie et al., 1992). Theguanylin family consists of guanylin and uroguanylin, the latterbeing also synthesized in the kidney where it exerts potent natri-uretic effect in mammals (Forte et al., 2000). Guanylin peptidesdo not seem to have vasoactive properties. In addition to guanylinand uroguanylin, a third guanylin, named renoguanylin because ofits robust expression in the kidney, has been identified in the eel(Yuge et al., 2003). The existence of multiple guanylins has alsobeen reported in other teleost species (Takei and Yuge, 2007).

The receptor for the guanylin family of peptides shares struc-tural similarity to the NP receptors in that it has a guanylyl cyclasedomain within its sequence (Forte et al., 2000). Therefore, theguanylin receptor was named GC-C and uses cGMP as an intracel-lular messenger. In the eel, two types of GC-Cs have been identi-fied, which exhibit differences in ligand selectivity to the threeligands (Yuge et al., 2006). The two types of GC-C were also iden-tified in the genome database of pufferfish and medaka (Takeiand Yuge, 2007).

Considering all the current information about the molecularspecies of NP, AM and guanylin families, it is apparent thatNa+-extruding hormones and their receptors are greatly diversifiedin teleost fish, probably because of their relative importance forfacilitating SW adaptation in these aquatic species.

4.3. Effects of Na+-extruding hormones on SW adaptation

The diversified Na+-extruding hormones of teleost fish havebeen shown to promote survival in SW. Examples of their actionsare summarized below.

4.3.1. Atrial natriuretic peptide (ANP)It is generally accepted that ANP is a volume-regulating hormone

secreted from the cardiac atrium in response to an increase in blood

volume (atrial stretch). It acts on the brain, intestine, adrenal glandand kidney to decrease both water and Na+ from the body, resultingin a restoration of normal blood volume (Takei, 2000; Toop andDonald, 2004). In contrast to these established actions in mammals,the major effect of ANP appears restricted to the reduction of Na+ inteleost fish, which facilitates SW adaptation (Fig. 1B). In the eel, ANPsecretion is augmented by an increase in plasma osmolality (Kaiyaand Takei, 1996b), and acts on various osmoregulatory organs todecrease body Na+ but not water content (Takei and Hirose, 2002).In fact, ANP decreased urine flow rate and increased urine Na+

concentration when infused at physiological doses into SW-adaptedeels (Takei and Kaiya, 1998). In terms of the antidipsogenic effectand the inhibition of Na+ absorption by the intestine, ANP is a feworders of magnitude more potent in the eel than in mammals(Tsuchida and Takei, 1998; Ando et al., 1992; Loretz and Takei,1997). ANP seems to act directly on MRCs of the gills to enhanceNaCl excretion in flatfish (Arnold-Reed et al., 1991), though theeffect was not detectable in the eel (Tsukada and Takei, 2006).

ANP decreases plasma Na+ concentration at physiological, non-depressor doses in SW eels (Takei and Kaiya, 1998). The decreasewas brought about principally by inhibition of drinking and subse-quent intestinal absorption of NaCl, as infusion of SW into thestomach at the normal drinking rate nullified the hyponatremic ef-fect of ANP (Tsukada et al., 2005). Further, removal of circulatingANP in plasma, by a specific antiserum administration, enhanceddrinking rate and plasma Na+ concentration in SW eels (Tsukadaand Takei, 2006). Therefore, it is apparent that ANP regulatesdrinking in SW to limit an excessive increase in plasma Na+ con-centration, thereby promoting SW adaptation. Consistently, plas-ma ANP concentration increases transiently after transfer of FWeels to SW (Kaiya and Takei, 1996a), which coincides well withthe transient reduction of the copious drinking that occurs afterSW transfer in response to Cl� ions in SW (Hirano, 1974). There-fore, the transiently increased plasma ANP appears to amelioratesudden and excess increases in plasma NaCl concentration thatmay have occurred after the SW challenge.

In addition to the direct actions, ANP stimulates the secretion oflong-acting, SW-adapting hormones and promotes long-termadaptation to the SW environment. It has been shown that in afew teleost species, ANP increases cortisol secretion, which thenacts on MRCs in the gills to newly synthesize Na+,K+-ATPase andfacilitates NaCl excretion (Arnold-Reed and Balment, 1994; Li andTakei, 2003). In Mozambique tilapia, ANP also stimulates growthhormone secretion in dispersed pituitary cells (Fox et al., 2007).Therefore, ANP seems to be involved in the whole process of SWadaptation through interaction with long-acting hormones (Takeiand Hirose, 2002).

4.3.2. AdrenomedullinsIn mammals, AM, equivalent to teleost AM1, is synthesized in

various peripheral tissues and exerts a variety of biological actionsincluding cardiovascular, osmoregulatory, respiratory andantimicrobial action (López and Martínez, 2001). These actionsare probably exerted in a paracrine/autocrine fashion, thoughimmunoreactive AM is also circulating in the blood at significantconcentrations in mammals (Kitamura et al., 1994). AM2 is synthe-sized in the brain and pituitary and exerts potent central effectssuch as regulation of drinking and pituitary hormone secretion,but further studies are required to delineate its physiological role(Takei et al., 2007). AM2 exhibits peripheral cardiovascular andrenal actions, but its potency is somewhat lower than AM, reflect-ing its lower affinity to the CLR and RAMP complex. AM5 has justbeen reported in mammals, and thus little is known of its biologi-cal actions except for cardiovascular effects (Takei et al., 2008).

In fish, on the other hand, studies on the biological actions ofthe various AM peptides have just started, revealing unexpectedly

ig. 3. Amino acid sequences of vasoactive intestinal peptide (VIP) and pituitarydenylate cyclase-activating peptide (PACAP) in fish. Human sequence is alsohown for reference. Accession number of DNA and protein or localization of theene is as follows. VIP: human, M36634; Tetraodon, chromosome 17; Takifugu,


potent cardiovascular and osmoregulatory actions of AM2 andAM5. In the eel, AM2 and AM5 are the most efficacious vasodepres-sor hormones that have ever been reported in teleost fish (Nobataet al., 2008). AM2 and AM5 induce drinking even more potentlythan angiotensin II, the most potent dipsogenic hormone thus farknown in the eel (M. Ogoshi, S. Nobata and Y. Takei, unpublisheddata) (Fig. 1B). Interestingly, AM1 has little cardiovascular orosmoregulatory effects in the eel compared with AM2 and AM5,which contrasts to the situation in mammals. As affinities ofAM2 and AM5 for the known AM receptors are lower than thatof AM1, other specific receptors for AM2 and AM5 are likely to ex-ist in teleost fish and wait to be described. Since AM2 was vaso-pressor but AM5 was vasodepressor when administered into theeel brain ventricle, AM2 and AM5 may have distinct receptors infish. The presence of a new AM2 receptor has also been suggestedin mammals from pharmacological experiments using AM receptorantagonists (Hashimoto et al., 2007). Judging from the distinct ac-tions of AM2 and AM5 in teleost fish, they appear to be an excellentmodel for identification of new receptors for AM2 and AM5 thatwill likely have relevance in mammals.

4.3.3. GuanylinsThe guanylin family of peptides exhibit distinct actions that

promote SW adaptation in the eel (Yuge and Takei, 2007). Expres-sion of guanylin and uroguanylin genes was profoundly aug-mented in the intestine and kidney after transfer of eels from FWto SW (Yuge et al., 2003). Since the expression of ANP and AMgenes was not altered in response to SW transfer, guanylins arethe first hormones that exhibit obvious upregulation in responseto the osmotic challenge. In addition, expression of their receptorgenes, GC-C1 and GC-C2, was also enhanced after transfer of eelsto SW (Yuge et al., 2006). In the isolated SW eel intestine, mucosalapplication of guanylin peptides strongly inhibited short-circuitcurrent (Yuge and Takei, 2007) (Fig. 1B). Since a specific blockerfor cystic fibrosis transmembrane regulator (CFTR)-type Cl� chan-nel impairs the effects, the guanylin-induced inhibition may be dueto the increased Cl� secretion into the intestinal lumen. Therefore,guanylins appears to exhibit similar actions in teleost fish as al-ready reported in mammals (Forte et al., 2000).

In mammals, Cl� secretion into the intestinal lumen accompa-nies parallel secretion of water, resulting in diarrhea. In teleost fish,however, the increased luminal Cl� mobilizes the absorptive-typeNa–K–2Cl cotransporter in the apical membrane of the epithelialcells, and thus total absorption of ions (Na, K, and 2Cl� absorptionin exchange of 2Cl� secretion) increases after guanylin activation.Therefore, intestinal water absorption in fish is enhanced byguanylin, although the direct action is Cl� secretion into the gut lu-men. It is also possible that not only Cl� but also HCO3

� is secretedinto the lumen through the CFTR in teleost fish, as shown in mam-mals (Wilson et al., 2002), although direct evidence is still lacking.Since Mg2+ and Ca2+ are highly concentrated in the lumen ofmarine fish intestine and white precipitates are usually found inthe lumen, it is possible that secreted HCO3

� supplies materialsfor forming CaCO3 and MgCO3 precipitates to decrease the osmoticpressure of luminal fluids and further enhance water absorption(Takei and Yuge, 2007). Therefore, guanylins increased waterabsorption by the intestine and promote SW adaptation. The renalaction of guanylins in fish has not been examined yet.

8DPE0; sea bream, DQ659329; flounder, EB038409; stickleback, DW648422;edaka, AM154517; cod, DQ09988; zebrafish-a, chromosome 11; zebrafish-b,

K397558; smelt, EL530269; rainbow trout, BX884408; bowfin, P84771; spottedogfish, P09685; spiny dogfish, none; elephantfish, AAVX01117807.PACAP: human,83513; Tetraodon-a, chromosome 6; Tetraodon-b, unassigned chromosome;akifugu-a, A8DPE1; Takigufu-b, A8DPE2; sea bream, DQ659328; stargazer, P810-9; mackerel, AB083647; medaka-a, chromosome 20; medaka-b, chromosome 17;od, E908648; rainbow trout, CX136329; sockey salmon, X73233; zebrafish-a,N019479; zebrafish-b, AF217251; grass carp, EF592488; catfish, X79078;turgeon, AB083648; elephantfish, AAVX01141077.

5. Perspectives

As noted above, Na+-extruding hormones and vasodepressorhormones are greatly diversified in teleost fish and they playimportant roles in the adaptation processes to hyperosmotic SWenvironments and in the maintenance of low arterial blood pres-

sure in the aquatic environment. In addition to NP, AM andguanylin, there are several other hormones that are eitherNa+-extruding or vasodepressor and candidates as importantSW-adapting hormones.

5.1. The VIP/PACAP family

Vasoactive intestinal polypeptide (VIP) has sequence similari-ties with pituitary adenylate cyclase-activating peptide (PACAP)(Fig. 3). VIP and PACAP are localized on the same precursor along

FasgAmCdST3cCs


with peptide histidine isoleucine (PHI)/peptide histidine methio-nine (PHM) and growth hormone releasing peptide (GHRH),respectively, which are also grouped in the VIP/PACAP family(Cardoso et al., 2007). VIP and PACAP have significant sequencesimilarities with the glucagon family of peptides that includeglucagon, secretin and other related peptides, so that they are alsogrouped as members of the glucagon/secretin superfamily (Sherwoodet al., 2000). VIP and PACAP exert biological actions throughaccumulation of cAMP after binding to Class II, G protein-coupledreceptors PAC1 and VPAC1 and VPAC2 (Laburthe et al., 2007).

VIP is one of the brain-gut peptides that is synthesized in thecentral and peripheral nervous system and exerts potent vasodila-tory and hypotensive actions (Sherwood et al., 2000).VIP actions onbody fluid regulation do not seem to be prominent in mammals; inthe intestine which is innervated by many VIPnergic fibers, VIPstimulates ion secretion into the lumen, and in the pituitary, VIPenhances prolactin and growth hormone secretion, which in fishare FW- and SW-adapting hormones, respectively. In non-mamma-lian species, however, VIP exhibits more prominent osmoregula-tory actions. In elasmobranchs, it has long been known that VIPstimulates Cl� secretion from the rectal gland (Stoff et al., 1979)and the mechanisms of VIP action including the receptor and re-lated transporters have been well characterized (Silva et al.,1996; Bewley et al., 2006). In teleost fish, VIP increased Na+ andCl� secretion from MRCs of the opercular epithelia of SW-adaptedMozambique tilapia (Foskett et al., 1982). In the intestine of winterflounder, VIP induced Cl� secretion through cAMP production(O’Grady and Wolters, 1990), which as described above is likelyto increase water absorption by way of activation of Na–K–2Clcotransporter localized on the apical membrane. All these actionsseem to promote survival of teleost fish in SW. However, thesestudies have largely used heterologous (mammalian) VIP in teleostfish preparations and future use of homologous VIP should providemore convincing data on its SW-adapting potential for teleosts.

Reflecting their potent vasodepressor and Na+-extruding ac-tions, VIP peptides and their receptors appear to have significantlydiversified in teleost fishes. It has been reported that databasesearches have identified four types of VIP receptors in the puffer-fish, Takifugu and Tetraodon (Cardoso et al., 2005). The two typesmay have duplicated at the 3R that occurred before radiation ofthe teleost lineage (Vandepoele et al., 2004). It is rather surprisingthat the duplicated genes are retained for more than 300 millionyears after the 3R. VIP has been identified in several teleost species,

Fig. 4. Amino acid sequences of relaxin 3 peptides in teleost fish in comparison withAF447451; Tetraodon-a, chromosome 3/15; Tetraodon-b, chromosome 18; Tetraodon-c, ccontig 4627; zebrafish-a, AY834223; zebrafish-b, chromosome 24; zebrafish-c; AY83422

and multiple VIP genes are detectable in the EST database of zebra-fish (Cardoso et al., 2007), which implies possible multiple ligandsfor the VIP receptors (Fig. 3). The PACAP is a potent secretagogue ofpituitary hormones and corticosteroids in tetrapods, but has onlyweak cardiovascular actions (Vaudry et al., 2000). However, twoPACAP genes (PACAPa and PACAPb) have been detected in all tele-ost species thus far examined (Cardoso et al., 2007) (Fig. 3), indicat-ing potentially important physiological functions in these aquaticvertebrates. Furthermore, amino acid sequences of both VIP andPACAP are highly conserved within fish species and between fishand mammals (Fig. 3). Therefore, the VIP/PACAP family of peptidesappear to have important functions generally in vertebrates. It willbe intriguing to examine osmoregulatory actions of VIP and PACAPin fish using homologous peptides.

5.2. The relaxin family

Relaxin was first identified more than 80 years ago as a femalereproductive hormone that helped delivery of fetuses at birth byrelaxing the pubic ligament (Hisaw, 1926). Subsequent studiesshowed that relaxin is a member of the insulin/relaxin superfamilythat includes insulin, insulin-like growth factor (IGF)-I and II, andmembers of the relaxin family of peptides (Sherwood, 2004). Themature peptides of the superfamily share common structural fea-tures that consist of two chains, termed A and B-chain, which arecovalently linked by two interchain disulfide bonds with an addi-tional intradisulfide bond in the A chain except for IGFs. The relax-in family consists of relaxin 1, 2 and 3, and insulin-like peptide(INSL) 3, 4, 5 and 6 (Wilkinson et al., 2005a). Only anthropoids (hu-man and chimpanzee) have relaxin 2, which is a major reproduc-tive hormone in human and an ortholog of relaxin 1 of othermammals. The amino acid motif, R-X-X-X-R-X-X-I/V, in the middlepart of the B-chain is required for biological activity of relaxins.

Accumulating evidence indicates that relaxin 1/2 is not only ahormone involved in reproductive function, but exerts also potentcardiovascular (vasorelaxant and hypotensive) and osmoregula-tory actions in mammals (Dachietzig and Stangl, 2003). Relaxin1/2 stimulates vasopressin secretion from the posterior pituitaryof rats (Dayanithi et al., 1987), induces thirst (Summerlee et al.,1998; Sunn et al., 2002), and promotes the secretion of anteriorpituitary hormones such as prolactin and growth hormone in therat and monkey (Bethea et al., 1989; Sortino et al., 1989). Relaxin3 is the newest member of the relaxin family and is produced

human sequence. Accession number and chromosomal localization are: human,hromosome 4; sticleback-a, contig 4416; stickleback-b, contig 3135; stickleback-c,4; Fathead minnow, DT343526; Atlantic salmon, CX357543.


almost exclusively in the brain, particularly in the brain nuclei thatare related to sensory function (Liu et al., 2005a). Functional studyof relaxin 3 is still in its infancy, but orexigenic action has beensuggested in the rat (McGowan et al., 2007).

Thanks to the recent development of the reverse pharmacologyapproach, a number of orphan G protein-coupled receptors(GPCRs) have been identified as functional receptors for the relaxinfamily of peptides (Hsu et al., 2002; Liu et al., 2003, 2005b). Insulinand IGFs are known to bind tyrosine kinase-coupled receptors, butrelaxins bind GPCRs (GPCR135/RXFP3 and GPCR142/RXFP4) orleucine-rich repeat-containing GPCR (LGR7/RXFP1 and LGR8/RXFP2)with high affinity (Bathgate et al., 2006). After binding to the recep-tors, relaxin generally transmits its information intracellularly bymodifying cAMP levels. It is now evident that LGR7 is a receptorfor relaxin 1/2, LGR8 for INSL3, GPCR134 for relaxin 3, andGPCR142 for INSL5 (Liu et al., 2005a; Bathgate et al., 2006).

Comparative studies have been performed with the relaxin pep-tides and their receptors (Hsu and Hsueh, 2000; Wilkinson et al.,2005a). These studies indicate that relaxin 3 and its receptor(CGRP135/RXFP1) show particular diversification in teleost fish(Wilkinson et al., 2005b) (Fig. 4). Relaxin 3 is suggested as anancestral molecule of the relaxin family and seems to be a neuro-peptide, principally functioning in the brain (Liu et al., 2005a).Since relaxin 1/2 plays a role in blood pressure and body fluid reg-ulation, it is possible that relaxin 3 also may have similar actions intetrapods and fishes. Having already recognized the important car-diovascular and osmoregulatory actions of the greatly diversifiedhormones in fish, it will be intriguing in the future to examine re-laxin 3 actions on SW adaptation in teleost fish.

Acknowledgments

I am deeply indebted to Dr. Hideshi Kobayashi, who was asupervisor during my graduate studies. I learned from him thejoy of research and value of comparative studies, particularly whendeveloping an original view of the evolution of structure and func-tion of hormones during phylogeny. I also thank Dr. Tetsuya Hir-ano, who was a senior disciple of Dr. Kobayashi and guided meto the field of marine physiology. Thanks are also due to the cur-rent and former staff of my laboratory, Susumu Hyodo, Koji Inoue,Makoto Kusakabe and Sanae Hasegawa for their continuous sup-port. I also thank many domestic and international collaboratorswho inspired me greatly with new ideas in research, and the grad-uate students and postdoctoral fellows who worked with me pa-tiently for pursuing higher levels of research. Finally, I thank Dr.Richard J. Balment, who is my lifelong friend and challenger, forthe critical reading of this manuscript.

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Exploring novel hormones essential for seawater adaptation in teleost fish