Marcos A. López-Patiño, Arnau Rodríguez-Illamola, Manuel Gesto, José L. Soengas and Jesús M. Míguez*

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1 928 The Journl of Experimentl Biology 214, Pulished y The Compny of Biologists Ltd doi:1.1242/je RESEARCH ARTICLE Chnges in plsm meltonin levels nd pinel orgn meltonin synthesis following cclimtion of rinow trout (Oncorhynchus mykiss) to different wter slinities Mrcos A. López-Ptiño, Arnu Rodríguez-Illmol, Mnuel Gesto, José L. Soengs nd Jesús M. Míguez* Deprtmento de Bioloxí Funcionl e Ciencis d Súde, Fcultde de Bioloxí, Universidde de Vigo, 3631 Vigo, Spin *Author for correspondence (jmmiguez@uvigo.es) Accepted 16 Novemer 21 SUMMARY Meltonin hs een suggested to ply role in fish osmoregultion, nd in slmonids hs een relted to the timing of dptive mechnisms during smolting. It hs een descried tht cclimtion to different environmentl slinities lters levels of circulting meltonin in numer of fish species, including rinow trout. However, nothing is known regrding slinity effects on meltonin synthesis in the pinel orgn, which is the min source of rhythmiclly produced nd secreted meltonin in lood. In the present study we hve evluted, in rinow trout, the effects of cclimtion to different slinities on dy nd night plsm meltonin vlues nd pinel orgn meltonin synthesis. Groups of freshwter (FW)-dpted rinow trout were plced in tnks with four different levels of wter slinity (FW, 6, 12, 18 p.p.t.; prts per thousnd) nd mintined for 6 h or 5 dys. Meltonin content in plsm nd pinel orgns, s well s the pinel content of serotonin (5-HT) nd its min oxidtive metolite (5- hydroxyindole-3-cetic cid; 5-HIAA) were mesured y high performnce liquid chromtogrphy. In ddition, dy night chnges in pinel orgn ryllkylmine N-cetyltrnsferse (AANAT2) ctivity nd nt2 gene expression were studied. Plsm osmollities were found to e higher in rinow trout exposed to ll slinity levels compred with the control FW groups. A slinity-dependent increse in meltonin content ws found in oth plsm nd pinel orgns. This effect ws oserved during the night, nd ws relted to n increse in nt2 mrna undnce nd AANAT2 enzyme ctivity, oth of which lso occurred during the dy. Also, the levels of indoles (5-HT, 5-HIAA) in the pinel orgn were negtively ffected y incresing wter slinity, which seems to e relted to the higher recruitment of 5-HT s sustrte for the incresed meltonin synthesis. A stimultory effect of slinity on pinel nt2 mrna expression ws lso identified. These results indicte tht incresed externl slinity promotes meltonin synthesis in the pinel orgn of rinow trout y enhncing synthesis of AANAT protein independently of its regultion y light. The possiility tht pinel meltonin is trget for hormones involved in the response of fish to osmotic chllenge is discussed, s well s the potentil role of meltonin in the timing of osmoregultory processes. Key words: meltonin, slinity, pinel orgn, serotonin, osmoregultion, rinow trout. INTRODUCTION The pinel orgn of teleost fish is photo-neuro-endocrine structure locted on the roof of the rin nd immeditely elow the skull. Pinel photoreceptor cells respond to environmentl photoperiod y relesing neurl or endocrine signls, the ltter eing involved in the synthesis of meltonin (Ekström nd Meissl, 1997). This hormone is not stored nd its plsm concentrtion reflects the synthesizing cpcity of the glnd, which is higher during drkness (Flcón, 1999). Dily rhythms of circulting meltonin re elieved to entrin the temporl co-ordintion of numer of physiologicl processes ssocited with dily nd sesonl rhythms, including locomotor nd feeding ehviours, reproduction nd smoltifiction, mong others (Porter et l., 1998; Bromge et l., 21; López- Olmed et l., 26; Flcón et l., 27; Flcón et l., 21). In ll clsses of fish, rhythms of meltonin in the lood re primrily dependent on its formtion in the pinel cells. The rhythm results from the dily vritions in the ctivity of the second lst enzyme of meltonin iosynthesis, ryllkylmine N- cetyltrnsferse (AANAT; EC ), which controls the rte t which serotonin (5-HT) is converted into N-cetylserotonin nd is thought to e the limiting step in the pthwy (Klein et l., 1997). In most fish species there is complete meltonin-rhythm-generting system in ech individul photoreceptor cell in the pinel glnd, so lrge chnges in meltonin re typiclly endogenous nd persist on circdin sis when fish re exposed to constnt drkness or when pinel glnds re plced in culture (Bolliet et l., 1994; Flcón, 1999). However, there re exceptions in slmonids, which pprently lck functionl intrpinel circdin clock (Gern nd Greenhouse, 1988; Iigo et l., 27; Migud et l., 27), nd lrge chnges in meltonin production re ssocited with rpid non-clock-dependent light suppression of AANAT ctivity (Flcón et l., 27). Two AANAT genes, nmed nt1 nd nt2, hve een descried in teleosts, with nt2 eing the dominnt form expressed in the pinel orgn (Flcón et l., 27). Regultion of AANAT2 protein vries mong fish species, ut in most cses chnges in nt mrna undnce determines AANAT ctivity (Bégy et l., 1998; Benyssi et l., 2). The importnce of this mechnism is poorly understood nd might vry mong fish species. Thus, in rinow trout the nt2 gene hs een reported to e expressed constitutively, nd meltonin synthesis is strongly ffected y dily AANAT proteosoml proteolysis (Flcón et l., 21). Even though light is the principl regultor of meltonin production in the fish pinel orgn, other fluctuting environmentl fctors, such s temperture, cn lso modulte meltonin secretion

2 Wter slinity nd meltonin in trout 929 in the pinel orgn through the regultion of AANAT2 ctivity (Zchmnn et l., 1992; Flcón et l., 1994). Recently, it hs een reported tht chnges in environment slinity could lso influence circulting meltonin levels in numer of euryhline fish, with the results vrying depending on the fish species studied or the wter cclimtion periods. Thus, Kleszcynsk et l. hve shown tht cclimtion of gilthed se rem (Sprus urt) to hypersline wter or sewter (SW) for 2 weeks results in decresed plsm meltonin levels compred with the levels in low slinity conditions (Kleszcynsk et l., 26). Similrly, López-Olmed et l. reported decresed plsm meltonin in Europen se ss (Dicentrrchus lrx) cclimted to SW, with respect to fish cclimted to freshwter (FW) or rckish wter (BW) (López-Olmed et l., 29). In contrst, in rinow trout, plsm meltonin levels hve een shown to increse fter 2 weeks exposure to BW (Kulczykowsk, 1999), nd to e unltered fter n osmotic stress (Kulczykowsk, 21). Euryhline fish re le of living in environments tht re sujected to vritions in slinity ecuse they hve developed physiologicl strtegies to dpt to such chnges. The osmoregultory function involves different endocrine responses tht directly or indirectly promote chnges in trget tissues involved in osmotic lnce nd moility of metolic resources (Sngio- Alvrellos et l., 23; McCormick nd Brdshw, 26). Recently, meltonin hs een proposed to ply role in osmoregultion (Kleszczynsk et l., 26; Sngio-Alvrellos et l., 27), s it hs lso een implicted in the timing of slmonid smolting (Porter et l., 1998; Iigo et l., 25). Indeed, meltonin-inding sites hve een found in fish osmoregultory tissues such s the gills, smll intestine nd kidney (López-Ptiño, 24; Kulzczykowsk et l., 26), suggesting possile role of meltonin in wter ion lnce in fish. Therefore, there my e n increse in circulting meltonin levels once the fish encounter new sline environments, thus fvouring environmentl dpttion. Although the osmotic influence on lood meltonin levels is likely to derive from chnges in the pinel meltonin production, no specific studies hve een crried out t this level. However, López-Olmed et l. hve reported tht, in the Europen se ss, in ddition to plsm meltonin chnges, wter slinity lso ffected meltonin content in intestine nd gills, suggesting tht extrpinel tissues could lso contriute to n ltertion in lood meltonin levels in response to osmotic chnges (López-Olmed et l., 29). We therefore focused the present study on the evlution of chnges in meltonin synthesis in the pinel orgn of the rinow trout exposed to different slinity levels, nd how it ws reflected in plsm meltonin levels. It is known tht exposing fish to new sline environments induces differentil shortnd long-term chnges in their physiology (Lery et l., 1981; Sngio-Alvrellos et l., 23). Consequently, two cclimtion periods (6 h nd 5 dys) were tested in order to ssess the timedependent meltonin response to chnges in the osmotic environment. MATERIALS AND METHODS Animls All experiments were designed ccording to the guidelines of the Europen Union Council (86/69/EU), nd the Spnish Government (RD 121/25) legl requirements. Rinow trout (Oncorhynchus mykiss Wlum 1792) with ody mss of 11±22 g were provided y commercil htchery (Soutorredondo, A Coruñ, Spin). Animls were kept in experimentl 1-litre tnks with filtered nd continuously replenished fresh wter, under constnt photoperiod (12 h:12 h light:drk) nd temperture (14±1 C) for 15 dys efore ny experimentl procedure. Feeding time ws scheduled t zeitgeer time (ZT) 3 (ZT=lights on), nd consisted of commercil (Diq diproteg, Segovi, Spin) dry pellet diet (1% ody mss). Experiments nd smpling Two sets of experiments were designed in order to test the timedependent effect of slinity on rinow trout meltonin production. In the first experiment, fish were cutely exposed for 6 h to different wter slinities 6 p.p.t. (12 mosm l 1 ), 12 p.p.t. (25 mosm l 1 ) nd 18 p.p.t. (38 mosm l 1 ) wheres the control group ws kept in FW. To chieve the required slinity, one hlf of the wter ws slowly removed from every tnk nd replced with FW to which different concentrtions of mrine slts were dded to chieve the finl slinities, given ove. Two 1-l tnks were used for ech wter condition. In the first tnk, nimls (N=8 per group) were exposed to FW or one of the different slinities for up to 6 h, strting t ZT2; fish were scrificed nd smples collected t ZT8. In the second tnk the sme protocol ws performed ut tretments were scheduled to strt 1 h efore the end of the light phse (ZT11); 6 h lter, i.e. ner to the middle of the scotophse (ZT17), nimls (N=8 per tretment) were scrificed nd smples collected under dim red light. A second experiment, with similr experimentl protocol involving two tnks of fish for ech of the slinity condition tested (FW, 6, 12 nd 18 p.p.t.) ws performed, ut in this cse trout were llowed to cclimte for 5 dys efore smpling. After this time fish (N=8 per group) were scrificed nd smpled t either light phse (ZT8) or ner to drk phse (ZT17), depending on the experimentl groups. At the smpling time fish were deeply nesthetized with MS- 222 (5 mg l 1 ) uffered to ph 7.4 with sodium icronte, nd killed t either the dy or night points ssyed. Blood ws collected seprtely from the cudl vein of ech fish using 1 ml heprinized syringes. After tht, ech fish ws immeditely killed y decpittion nd the pinel orgn ws removed with the id of sterilized mteril nd plced into RNse-free 1.5 ml Eppendorf tues. Then, smples were immeditely frozen in liquid nitrogen, nd stored t 8 C until ssyed. Plsm ws otined y centrifuging lood t 12,9 g for 1 min t 4 C. Aliquots were then frozen on dry ice nd stored t 8 C until nlysis. Plsm N + nd osmollity ssys Plsm N + content, nd plsm osmollity were mesured in order to determine the vritions in oth prmeters fter exposing the nimls to different wter slinities. N + content ws mesured using n tomic sorption sprectrophotometer (Vrin SpectrAA-25 Plus, Vrin Medicl Systems, Zug, Switzerlnd) nd osmollity ws mesured with Roeling (Berlin, Germny) 13DR Autocl osmometer. Plsm nd pinel glnd meltonin quntifiction Plsm meltonin ws ssyed ccording to Muñoz et l. (Muñoz et l., 29), with modifictions. Briefly, 2 l liquot of plsm ws mixed (1:1 v/v) with.1 mol l 1 cetic cette uffer (ph 4.6) nd 2 ml chloroform dded. The mixture ws mixed for 1 min, centrifuged (38 g, 1 min) nd the queous phse spirted. The orgnic lyer ws seprted nd 5 l.1 mol l 1 NOH were dded. After stirring nd centrifugtion, the queous phse ws spirted nd the orgnic lyer ws dried out under n ir strem t room temperture. The residue ws dissolved in 1 l of moile phse nd filtered through.5 m filter. An liquot (5 l) of the filtrte

3 93 M. A López-Ptiño nd others ws injected into the HPLC system. Dt from the nlysis re expressed s pg ml 1 of plsm. The chromtogrphic system consisted of Gilson (Middleton, WI, USA) 321 solvent delivery pump equipped with 5 l Rheodyne (IDEX-HSS, Ok Hror, WA, USA) injection vlve, nd Jsco Anlitic (Mdrid, Spin) FP-152 fluorescence detector set t 28/345 nm excittion/emission wvelengths. Meltonin ws seprted on Beckmnn Ultrsphere ODS column (3 m prticles, mm i.d.; Fullerton, CA, USA). The moile phse consisted of solution of 85 mmol l 1 cetic cette,.1 mmol l 1 N 2 -EDTA nd cetonitrile (14% of finl volume), with the ph djusted to 4.7. All nlyses were performed t room temperture t flow rte of 1. ml min 1. For pinel meltonin quntifiction, ech orgn (N=8 per group) ws homogenized y soniction in 1 l of.2 mol l 1 phosphte uffer (ph 6.7) nd centrifuged t 16 g for 1 min. The superntnt ws divided into two liquots. A 6 l liquot ws ssyed for meltonin nd pinel indole (see elow). The second, 4 l liquot, ws immeditely ssyed for AANAT2 ctivity (see elow). From the 6 l liquot, pinel meltonin content ws mesured following direct injection of 2 l volume into the HPLC system, which ws similr to tht descried for plsm meltonin ssys. Other conditions were s descried y Ceinos et l. (Ceinos et l., 28). Acquisition nd integrtion of chromtogrms were performed using the BiocromXP softwre (Micron Anlític, Mdrid, Spin). Smple pek res were compred with those of pproprite stndrds to estimte the quntities of compounds present. Mesurement of indole content in pinel orgns Pinel 5-HT nd its cidic metolite, 5-hydroxindole cetic cid (5-HIAA) were mesured y HPLC ccording to Ceinos et l. (Ceinos et l., 25). Briefly, 2 l of 1:4 dilution from the 6 l originl liquot from ech pinel smple were injected into the HPLC system, which consisted of Jsco PU28 pump nd n ESA Coulochem detector (Bedford, MA, USA). The moile phse ws solution of 85 mmol l 1 NH 2 PO 4,.72 mmol l 1 octnosulfonic cid, 18% methnol, which ws djusted to ph 3.. All seprtions were performed t room temperture t flow rte of.8 ml min 1. The detection system consisted of doule nlyticl M511 ESA cell with the electrode potentils set t +2 nd +3 mv. Acquisition nd integrtion of chromtogrms were performed using the BiocromXP softwre. Pinel AANAT ctivity ssys Pinel AANAT2 ctivity ws ssyed y in vitro incution of smple homogentes with the sustrte (tryptmine), nd cetyl- CoA s cofctor, ccording to Ceinos et l. (Ceinos et l., 28) with some modifictions. Pinel orgns were individully sonicted in 1 l of.2 mol l 1 phosphte uffer (ph 6.2) on ice, nd the homogente ws centrifuged t 16, g for 1 min. Then, 4 l of the superntnt were mixed with 4 l of 27 mmol l 1 tryptmine nd 4 l of 1. mmol l 1 cetyl-coa (finl concentrtions in ssy: 9 mmol l 1 tryptmine nd.5 mmol l 1 cetyl-coa) nd incuted for 6 min t 16 C. The rection ws stopped y dding 1 ml of 4 C chloroform, nd the smples were then vortexed (1 min) nd centrifuged t 12,9 g for 1 min. The resulting queous superntnt ws discrded nd the orgnic lyer ws evported to dryness under n ir strem, t room temperture. Then, the extrcts were dissolved in 1 l of moile phse (65 mmol l 1 NH 2 PO 4,.123 mmol l 1 N 2 -EDTA nd 15% cetonitrile; ph 4.7). 2 l of this solution were directly injected into the fluorescence HPLC system in order to quntify the rection product (N-cetyl tryptmine; NAT) formed. The system consisted of HPLC pump (Gilson M11) with Ultrsphere Beckmn column (3 m prticles, 75 mm nd 4.6 mm i.d.) nd Jsco FP-152 fluorimetric detector set t 285/36 nm excittion/emission wvelengths. All nlyses were performed t room temperture t flow rte of 1 ml min 1. Smple pek res were quntified with pproprite stndrds using the Biochrom XP softwre. Anlysis of pinel orgn nt2 mrna Immeditely fter dissection pinel orgns from fish receiving the sme tretment were pooled (N=2). Totl mrna ws extrcted from these pooled pinel orgns using the TRIzol method (Gico BRL, Githersurg, MD, USA) ccording to mnufcturer s instructions. The isolted RNA qulity nd quntity ws spectrophotometriclly determined. From ech smple, 2 g RNA were trnsferred to new vil nd sterile nuclese-free H 2 O ws dded to mke it up to 1 l. Then 1 l rndom primers (C1181, Promeg, Mdison, WI, USA) ws dded, nd vils were then incuted for up to 5 min t 7 C. After tht, 9 l of mixture contining.2 l RNAgurd TM RNse inhiitor ( , Promeg), 1 l of 1 mmol l 1 dntp Mix (U1511, Promeg), 1 l of M-MLV reverse trnscriptse (M171, Promeg), 4 l M-MLV RT 5 uffer (M531A, Promeg) nd 2.8 l sterile nuclese-free H 2 O, were dded to ech vil. Vils were then incuted t 37 C for 1 h nd 65 C for 5 min. A negtive control for ech smple ws ssessed without reverse trnscriptse in order to confirm the sence of ny genomic contmintion. Rel-time quntittive RT-PCR (qpcr) ws performed using Mxim TM SYBR Green qpcr Mster Mix (K221, Ferments, Burlington, ON, Cnd) nd Bio-Rd (Hercules, CA, USA) MyIQ rel-time PCR system. The primers nd proes were sed on previously reported sequences of rinow trout genes nd otined from Sigm-Genosys (St Louis, MO, USA), including: nt2 (ccession numer AF166.1) forwrd 5 -CATTCG TCTCT - GTGTCTGGT-3, reverse 5 -TTTCTGGGATA TGCTGGGT-3 ; nd -ctin gene (AJ438158) forwrd 5 -GATGGGCCAGAAA- GACAGCTA-3, reverse 5 -TCGT CCC AGT TGGTGACGAT-3. Gene expression for ech smple ws normlized to tht of the - ctin gene. Reltive mrna expression ws clculted y using the stndrd comprtive C t method. For ech gene, smples collected t the sme time point were processed in prllel nd the expression ws mesured in triplicte. Sttisticl nlysis Comprisons etween groups were performed t ech time (6 h nd 5 dys) using two-wy ANOVA with slinity degree nd time of dy s min fctors. When significnt effect ws identified within fctor, post hoc comprisons were crried out within tht fctor using Student Newmn Keuls test, nd differences were considered sttisticlly significnt t P<.5. RESULTS Plsm N + content nd osmollity Plsm osmotic prmeters re shown in Tle 1. There ws n pprent increse in plsm osmollity nd N + concentrtion with incresing wter slinity fter oth 6 h nd 5 dys exposure. Significnt differences in plsm N + content were oserved in the 12 p.p.t. slinity group (5 dys) nd 18 p.p.t. slinity group (6 h nd 5 dys) compred with their respective control (FW) groups. For plsm osmollity, there were significnt increses in the 12 nd

4 Wter slinity nd meltonin in trout 931 Tle 1. Plsm N + content (meq l 1 ) nd osmollity (mosm l 1 ) under the different experimentl conditions Tretment Plsm N + content Plsm osmollity 6 h Control 123±8 298±4 6 p.p.t. 132±8 311±4 12 p.p.t. 136±8 323±4* 18 p.p.t. 149±4* 359±3* 5 dys Control 128±8 286±4 6 p.p.t. 14±8 293±2 12 p.p.t. 17±8* 322±2* 18 p.p.t. 191±8* 365±7* Vlues re mens ± s.e.m. (N=8 per group). *P<.5 vs the control group. 18 p.p.t. slinity groups (6-h nd 5-dy experiments) compred with their respective controls. Plsm nd pinel orgn meltonin contents There were significnt dy night vritions in plsm meltonin levels in ll groups of fish fter oth 6 h nd 5 dys of exposure (Fig. 1A,B). In ddition, nimls cclimted to different slinities (6, 12 nd 18 p.p.t.) did not show ny significnt chnge in dytime plsm meltonin concentrtions fter 6 h or 5 dys, reltive to their respective FW control group. In contrst, nocturnl circulting meltonin levels t oth 6 h nd 5 dys were significntly incresed y slinity. This effect ws not dependent on the slinity concentrtion in the 6 h tretment ut it ws clerly slinity dependent fter 5 dys exposure ecuse it ws oserved only in the smples from fish exposed to 12 nd 18 p.p.t. slinity. Similr to plsm, pinel orgn meltonin content lso showed dy night vritions with the mximl levels oserved during the drk phse in ll the experimentl groups (Fig. 1C,D). No significnt differences were detected in the dytime pinel meltonin content for ech slinity tretment t oth 6 h nd 5 dys. However, nighttime pinel meltonin levels did show slinity-dependent effect, with fish exposed to 18 p.p.t. for 6 h showing significntly increse in meltonin content (P<.5 vs control, 6 nd 12 p.p.t.; Fig. 1C), nd fish cclimted for 5 dys to 12 nd 18 p.p.t. lso showed significnt nocturnl meltonin increse reltive to control nd 6 p.p.t. slinity (Fig. 1D). 5-HT nd 5-HIAA levels in rinow trout pinel orgn The effects of slinity on the dy night vrition of indoles in the pinel glnd re shown in Fig. 2. After oth 6 h nd 5 dys, significnt dy night vritions were oserved in the 5-HT content of fish exposed to FW nd 6 nd 12 p.p.t. wter slinity (Fig. 2A,B). In ll of them, levels of 5-HT were higher during the dy thn t night. However, fish cclimted for 6 h to 18 p.p.t. slinity showed lower pinel 5-HT content in the dytime reltive to the other groups (P<.5 vs control, 6 nd 12 p.p.t.), nd no significnt dy night fluctution ws found in fish in this group (Fig. 2A). In ddition, trout exposed for 6 h to 12 nd 18 p.p.t. slinity showed significntly lower nocturnl levels of 5-HT in the pinel orgn compred with the nocturnl vlues of the FW control group. After 5 dys cclimtion, the dy night vrition in 5-HT in the pinel orgn ws mintined in ll groups exposed to the different tretments (Fig. 2B). A low level of 5-HT in the 18 p.p.t. slinity group in the dytime, which might hve een expected from the 6-h smples, ws not found; indeed the levels were higher in oth the 12 nd 18 p.p.t. groups thn in the control group, lthough this ws not significnt. At night, there ws significnt decline in pinel glnd 5-HT content in ll groups of fish cclimted to the different slinities (6, 12 nd 18 p.p.t.) reltive to the control FW group (P<.1 in ll cses). The levels of the min 5-HT cid metolite, 5-HIAA, hd similr dy night vritions to those of 5-HT (Fig. 2C,D). This fluctution occurred in ll experimentl slinity groups. The min effect of wter slinity on pinel 5-HIAA levels ws significnt decrese in dytime 5-HIAA levels fter exposing nimls to 18 p.p.t. for 6 h (P<.5 vs FW, 6 nd 12 p.p.t. slinity). This effect ws lso oserved fter 5 dys of cclimtion to the different slinity conditions (P=.5 vs FW, 6 nd 12 p.p.t. slinity). Meltonin (pg ml 1 ) Control 6 p.p.t. 12 p.p.t. 18 p.p.t. Plsm 5 dys * *, *, *,, *,c A B Meltonin (pg pinel orgn 1 ) , 5 dys *, *, Dy Night Dy Night,, Pinel orgn *, *, C D Fig. 1. Dy night chnges of plsm (A,B) nd pinel orgn (C,D) meltonin levels in rinow trout cclimted for 6 h (A,C) or 5 dys (B,D) to freshwter (control) or severl slinities (6, 12 nd 18 p.p.t.). Dt re presented s mens ± s.e.m. (N=8 per group). *Significntly different from their respective tretment group during the dytime (P<.5). Different letters ove the rs indicte significnt difference (P<.5) etween tretment groups t the sme time point.

5 932 M. A López-Ptiño nd others 5-HT (pg pinel orgn 1 ) Control 6 p.p.t. 12 p.p.t. 18 p.p.t. 5-HT 5 dys *, *,,, A B 5-HIAA (pg pinel orgn 1 ) dys 15 *, 1, 5 Dy Night Dy Night 5-HIAA *, *, *, C D Fig. 2. Chnges in the levels of serotonin (5-HT; A,B), nd 5-hydroxindole cetic cid (5-HIAA; C,D) in the pinel orgn of trout following 6 h (A,C) nd 5 dys (B,D) cclimtion to freshwter (control) or incresed wter slinity (6, 12 nd 18 p.p.t.). Dt re presented s mens ± s.e.m. (N=8 per group). *Significntly different from their respective tretment group during the dytime (P<.5). Different letters ove the rs indicte significnt difference (P<.5) etween tretment groups t the sme time point. Pinel orgn AANAT2 ctivity nd nt2 mrna expression Dy night vritions in rinow trout pinel orgn AANAT2 ctivity re shown in Fig. 3. In ll the experimentl groups, nocturnl AANAT2 ctivities were higher thn the diurnl ones in oth the 6-h nd 5-dy experiments. In ddition, oth diurnl nd nocturnl pinel AANAT2 ctivities were progressively incresed with incresing slinity in the 6 h experiment (Fig. 3A), in such wy tht the enzyme ctivity of fish in ll slinities ws significntly higher tht the respective dy nd night FW control groups. Similr results were found fter 5 dys of tretment in oth the dy nd night mesurements, with pinel AANAT2 ctivity eing significntly higher in trout exposed to the different slinities (P<.5 vs respective control groups; Fig. 3B). In contrst, no meningful differences in AANAT2 ctivity ws found etween slinities in oth the dy nd the night smples. mrna levels of the nt2 gene were exmined in the control nd 12 p.p.t. slinity groups fter 6 h nd 5 dys cclimtion. Significnt dy night vrition in nt2 mrna expression ws found in oth groups, with the highest ctivity eing mesured during the night phse (Fig. 4). Consistent with the AANAT2 ctivity, there ws significnt increse in nocturnl nt2 mrna expression fter cute (6 h) exposure to 12 p.p.t. slinity, compred with its respective FW control group (P<.1; Fig. 4A). In ddition, trout cclimted for 5 dys to 12 p.p.t. slinity showed significntly incresed nt2 mrna expression oth during the dy nd t night (P<.5 vs respective FW groups; Fig. 4B). DISCUSSION Rinow trout re euryhline teleosts so they cn survive in reltively wide rnge of slinities, which llows them to mte nd spwn in freshwter nd then the juveniles cn migrte to the open se for growth (Boeuf, 1993). Acclimtion of rinow trout to SW occurs in two distinct phses: n initil djustment phse tht is chrcterized y incresing plsm osmotic concentrtion, nd lter regultive phse during which control is estlished over the osmotic concentrtion. The djustment phse is crucil for cclimtion to sewter when fish re ruptly exposed to n elevted wter osmollity (42 45 mosm l 1 ), which could result in generl physiologicl filure. However, for slinities lower thn 3% SW slight osmotic shock occurs tht permits the fish to lnce etween ion flux nd efflux for 1 or 2 dys fter trnsfer (Jckson, 1981). Our results show moderte increse in plsm osmollity in fish exposed for 6 h to low nd moderte slinity (rnging from 13 to 38 mosm l 1 ), which persisted for 5 dys (see Tle 1). These dt re in greement with those previously reported for oth short- nd long-term exposure to different wter slinities AANAT2 ctivity (pmol pinel 1 h 1 ) Control 6 p.p.t. 12 p.p.t. 18 p.p.t., Dy,c 5 dys *, *, *,c *,c Night Fig. 3. AANAT2 ctivity during the dy nd night in pinel orgns of rinow trout cclimted for 6 h (A) or 5 dys (B) to freshwter (control) or different environmentl slinities. Dt re presented s mens ± s.e.m. (N=8 per group). *Significntly different from their respective tretment group during the dytime (P<.5). Different letters ove the rs indicte significnt difference (P<.5) etween tretment groups t the sme time point. A B

6 Wter slinity nd meltonin in trout 933 nt2 mrna (reltive fold chnge) Control 12 p.p.t. Dy (Jckson, 1981; Lery et l., 1981; Kulczykowsk, 1997; Kulczykowsk, 1999). It is interesting lso tht in the present study the two lowest slinities used (6 nd 12 p.p.t.) were hypo-osmotic compred with plsm. Therefore, chnges in osmollity of the plsm were proly due to incresed ionic concentrtions (i.e. N + levels) rther thn to n osmotic dehydrtion y reduction of plsm volume. Circulting meltonin levels were ffected y incresed wter slinity. Nocturnl meltonin levels incresed fter oth 6 h (6, 12 nd 18 p.p.t. slinities) nd 5 dys (12 nd 18 p.p.t. slinities) of tretment, in greement with previous studies in rinow trout exposed for 2 weeks to BW (Kulczykowsk, 1999). Dt from other slmonid species re scrce, nd re not in greement with tht herein reported. For exmple, higher plsm meltonin concentrtion in prllel with high N + nd Cl vlues ws demonstrted in coho slmon during SW dpttion (Folmr nd Dickhoff, 1981), nd in yerling coho slmon during 1-dy exposure to SW (Gern et l., 1984). Sito et l. lso reported incresed dytime plsm meltonin in SW-cclimted chum slmon compred with FW-cptured fish (Sito et l., 24). However, some mjor controversies exist in mrine species. Thus, plsm meltonin levels were found to increse in the dytime ( night-time effect ws not studied) in gilthed sem rem (Sprus urt) during cclimtion to low environmentl slinity (Kleszczynsk et l., 26), wheres meltonin levels incresed during the night (no chnges occurred during the dy) in the Europen se ss (Dicentrrchus lrx) (López-Olmed et l., 29). All these dt suggest tht meltonin 5 dys *, *, Night Fig. 4. Dy night vritions in pinel orgn nt2 mrna expression in rinow trout exposed for 6 h (A) nd 5 dys (B) to freshwter (control) or sline (12 p.p.t.) wter. Reltive fold chnge ws estimted y tking s reference the dytime vlues of the control group fter 6 h (gene expression ws considered s 1). Dt re presented s mens ± s.e.m. (N=4 per group). *Significntly different from their respective tretment group during the dytime (P<.5). Different letters ove the rs indicte significnt difference (P<.5) etween tretment groups t the sme time point. A B is only prt of the process tht enles fish to cope with osmotic chllenge, nd tht responses of meltonin might vry in euryhline teleosts depending on species-specific tolernces. Furthermore, our results show tht slinity-induced chnges in plsm meltonin levels occurred predominntly during drkness, nd no effect ws oserved during dytime in ny of the slinity conditions ssyed. Similrly, pinel orgn meltonin content ws ffected y slinity only t night, with incresed meltonin levels fter 6 h (18 p.p.t. slinity) nd 5 dys (12 nd 18 p.p.t. slinities). However, chnges in AANAT2 ctivity, which were lso enhnced fter incresing wter slinity, were found in fish smpled during the dy nd the night. An effect of slinity on AANAT2 enzyme ctivity ws oserved in fish exposed for 6 h nd 5 dys, ut the effect of the ltter exposure ws not dependent on slinity concentrtion. Furthermore, significnt dy night difference in AANAT2 ctivity ws still found in ll groups of fish. Considering the smll mount of meltonin produced in the pinel orgn during the dy (in comprison with tht synthesized t night) it is plusile tht chnges in meltonin during cclimtion to sline wter were not so evident in the pinel orgn during the dytime, in contrst to the chnges oserved in AANAT enzymtic ctivity. Also, meltonin is relesed from the pinel orgn into lrge volume of plsm. This would explin the cler effect of slinity on plsm meltonin levels t night (when pinel meltonin secretion is high), nd its sence during dytime (when meltonin secretion from the pinel orgn is low). Moreover, ltertions in the plsmtic clernce of the hormone could contriute to the pprently different dy night effect of slinity on plsm nd pinel meltonin content, compred with pinel AANAT ctivity. In mny fish species light inhiits meltonin synthesis in the pinel orgn, thus modulting the dily pttern of severl meltonin-relted indoles (Iigo et l., 1991; Migud et l., 27). In teleosts, AANAT2 limits the rte t which 5-HT is converted into N-cetylserotonin, which proly limits the pinel orgn meltonin synthesis, s long s dily rhythmic pttern for AANAT2 ctivity prllels tht of meltonin content (Flcón, 1999). Thus, AANAT2 ctivity is incresed during the scotophse (Flcón et l., 1992; Ceinos et l., 25), leding to nocturnl increse in meltonin synthesis nd its secretion into the lood. Our dt clerly showed dy night vritions in AANAT2 ctivity in trout kept in FW, nd lso in most groups of trout exposed for either 6 h or 5 dys to ny of the slinity conditions studied. These dt indicte tht incresed slinity enhnces meltonin synthesis y incresing AANAT ctivity, n effect tht ws independent of the environmentl light phse. Moreover, decresed levels of 5-HT (the AANAT2 sustrte) nd its min oxidtive metolite (5-HIAA) were found under incresed sline concentrtions, oth during the dy (18 p.p.t. slinity) nd t night (ll slinities ssyed). In FW control fish, cler dy night rhythm ws oserved for 5-HT content, with lowest vlues t night due to its use s sustrte for the elevted meltonin synthesis (Ceinos et l., 25). Accordingly, the oserved decrese in 5-HT content in fish cclimted to the different slinity conditions re likely to derive from n ctivted meltonin synthesis in response to the slineinduced increse in AANAT ctivity. Also for 5-HIAA, decreses due to slinity exposure seem to e consequence of the reduced vilility of 5-HT ecuse of its higher utiliztion to form meltonin. All these dt llow us to clerly conclude tht pinel orgn function in trout is ctivted during exposure to sline environments, resulting in incresed meltonin synthesis nd relese into the lood. The slinity effect seems to e medited y enhncing dily AANAT2 ctivity nd pprently did not distur processes interpreting the lighting messge in the pinel photoreceptor cells.

7 934 M. A López-Ptiño nd others Teleost fish hve two different genes encoding for AANAT: nt1 nd nt2 (Bégy et l., 1998). However, t lest in pike nd rinow trout only the nt2 gene is expressed in the pinel orgn, with no orthologue in other verterte clsses (Flcón et l., 27). The nt2 expression hs een shown to follow dily vritions, with the higher vlues occurring during the scotophse (Bégy et l., 1998), nd presumly it is involved in pinel orgn meltonin synthesis. However, the rinow trout studies filed to demonstrte chnges in the nt2 expression pttern in the pinel orgn (Bégy et l., 1998; Coon et l., 1998; Flcón et l., 21). In contrst, in the present study we oserved significnt increse in nocturnl nt2 gene expression compred with tht in the dytime, in correspondence with dily chnges in AANAT enzyme ctivity. The resons for these discrepncies re not known. In the rinow trout studies, nt2 trnscripts were quntified y conventionl northern lot nlysis (Bégy et l., 1998; Coon et l., 1998), which usully requires elevted totl RNA mounts. Owing to the smll size of pinel orgn, severl orgns were pooled in order to otin tissue enough for ssys. Moreover, we used the more sensitive qpcr technique with RNA extrcted from two pinel orgns. Therefore, it is plusile tht dily nt2 chnges were not oserved in other studies, in contrst to the cler dy night fluctution oserved in this study, ecuse of the nlyticl techniques used. However, it is lso possile tht rinow trout used in the different studies were of different strins or differentilly ffected y other environmentl fctors. Independently of tht, our dt clerly show tht light is le to suppress nt2 gene expression nd enzymtic ctivity, n effect tht involves photoreceptor cell hyperpolristion, leding to decresed intrcellulr clcium nd camp levels, with the susequent dephosphoryltion of AANAT2 nd its degrdtion through proteosoml proteolysis (Flcón et l., 21; Gnguly et l., 21). During the scotophse, the pinel photoreceptor cells re depolrized nd then intrcellulr clcium levels re incresed. This leds to the increse in the expression of severl genes, including nt2, with the susequent increse in the enzyme synthesis nd ctivity (Kroeer et l., 2; Flcón et l., 27). According to our dt, AANAT expression nd ctivity chnged when the fish were exposed to 12 p.p.t. slinity during oth dy nd night for 6 h, with the effect eing lrger fter 5 dys. In ddition, AANAT ctivity incresed during the dy fter exposure to 18 p.p.t., reching levels even higher thn tht oserved in the FW control group during the night. This lso suggests tht meltonin synthesis might e influenced y other non-identified limiting fctors (i.e. hydroxindole- O-methyltrnsferse ctivity). Whether these time-dependent chnges in gene expression fter slinity chllenge re relted to the two distinct phses ffecting osmoregultory cpcity in trout deserves further investigtion. Osmoregultory mechnisms re ctivted when fish re exposed to ltertions in their sline environment, with the susequent ctivtion of numer of hormones nd metolic processes (Gern et l., 1984; McCormick nd Brdshw, 26). It hs een reported tht exposure of rinow trout to hypersline environments induces rpid nd reltively persistent increse in ctecholmines nd cortisol secretion (Wendelr-Bong, 1997; Lieert nd Schreck, 26). This is known s the osmotic stress response, nd helps fish to cope with the demnds of the osmoregultory response during the sline-dpttion process (Sngio-Alvrellos et l., 23; Sngio-Alvrellos et l., 27). A physiologicl role for ctecholmines in the trout pinel orgn hs een suggested, ut they hve not een shown to control meltonin production, s they hve in the pike pinel orgn (Flcón et l., 1991). Cortisol, however, is serious cndidte for involvement in meltonin formtion in fish. In fct, glucocorticoid receptors hve een reported in the rinow trout pinel orgn, nd dexmethsone, glucocorticoid nlogue, ws oserved to negtively influence meltonin synthesis in cultured pinel orgns (Benyssi et l., 21). Also cortisol ws shown to inhiit in vitro nocturnl meltonin production in the tilpi pinel glnd (Nikido et l., 21). This does not gree with the increses found in our study following sline cclimtion of trout. Therefore, role of elevted cortisol levels in the sline-induced increse in meltonin synthesis in trout is difficult to predict. Moreover, the results of studies on the effect of stress on fish meltonin levels vry. Thus, incresed levels of plsm meltonin were found in gilthed se rem sujected to high stocking density (Mncer et l., 28), wheres disturnce stress reduced plsm meltonin levels in trout (Kulczykowsk, 21). Besides ctecholmines nd cortisol, other hormones could e mediting chnges in meltonin synthesis during osmotic cclimtion in trout. Thus, the neurohypophysel hormone vsotocin, which is thought to ply role in dpttion of teleost fish to externl slinity chnges (Wrne et l., 25), hs lso een shown to interct with meltonin (Kulczykowsk, 21; Kulczykowsk, 22) to control the physiologicl dpttion of fish to dily nd sesonl environmentl chnges. However, no specific studies evluting the influence of vsotocin nd other stress-relted hormones in fish meltonin synthesis in the pinel orgn hve een done to dte. The physiologicl significnce of osmoticlly triggered chnges in meltonin synthesis is not known, ut role of meltonin in osmoregultion hs een strongly suggested. Thus, meltonin tretments were shown to influence osmoregultory processes in trout, including gill nd kidney N + /K + -ATPse ctivity nd plsm ion lnce (Sngio-Alvrellos et l., 27). Meltonin tretments lso enhnced survivl in SW tolernce tests in Atlntic slmon (Slmo slr) (Porter et l., 1998) nd were suggested to ffect timing of smoltifiction in msu slmon (Oncorhynchus msou) (Iigo et l., 25). The existence of meltonin-inding sites in osmoregultory tissues of severl teleost species, i.e. flounder, trout nd se rem (Kulczykowsk et l., 26) lso pointed to these orgns s potentil trget sites for meltonin ction in ion wter ion lnce. Our present results, showing tht circulting meltonin levels in rinow trout re ltered fter externl osmotic chnges, lso reinforce the ide tht meltonin is one more of the endocrine sustnce involved in cclimtion nd homeostsis in different slinity conditions. Although meltonin might modulte processes in fish osmoregultory orgns, the hormone is frequently linked to timing of physiologicl functions (Flcón et l., 21). Therefore, it would e of interest to evlute how other rhythmic functions of fish re influenced y environmentl osmotic chnges, s well s the potentil of meltonin to temporlly djust metolic, hormonl nd ehviourl processes during fish osmotic cclimtion. To summrize, we demonstrte tht slinity increses trout meltonin synthesis nd secretion. The pinel orgn is mjor trget for the effects of slinity since osmoticlly relted increses in pinel orgn meltonin synthesis were found nd they prlleled those oserved in plsm fter short- (6 h) nd long-term (5 dys) exposure of rinow trout to sline wter. We lso show tht the effects of slinity on pinel meltonin synthesis re medited y higher nt2 mrna expression nd AANAT2 enzyme ctivity. Thus, the enzymtic ction tht typiclly controls light-induced dily chnges in meltonin synthesis lso ppers to integrte informtion out the sline environment. Moreover, incresed pinel orgn meltonin synthesis in response to elevted externl slinity ws found during oth dy nd night, suggesting tht there is no interference with

8 Wter slinity nd meltonin in trout 935 cellulr mechnisms mediting the cyclicl light drk, inhiition ctivtion of meltonin formtion in pinel cells. More reserch is needed to clrify the role of the pinel glnd in osmoregultory nd/or stress hormones tht re moilized during fish cclimtion to chnges in environmentl slinity. 5-HIAA 5-HT AANAT BW FW HPLC SW LIST OF ABBREVIATIONS 5-hydroxyindole-3-cetic cid 5-hydroxytryptmine. Serotonin ryllkylmine N-cetyltrnsferse rckish wter freshwter high performnce liquid chromtogrphy sewter ACKNOWLEDGEMENTS This work ws supported y the Spnish Ministerio de Educción y Cienci (MEC) project AGL C3-1/ACU to J.M.M. M.A.L.-P. nd M.G. re Reserchers from the Isidro Prg Pondl (P.P. 3S 148) nd A. Alvriño progrms (Xunt de Glici), respectively. The uthors re grteful to Centro de Apoyo Cientifico y Technológico l Investigción (CACTI) for mesuring plsm nd wter ionic content nd osmollities. Specil thnks to Lucí Grcí Ledo nd José L. 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