Stress inhibition of melatonin synthesis in the pineal organ of rainbow trout (Oncorhynchus mykiss) is mediated by cortisol.

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1 First posted online on 16 Jnury 2014 s /je J Exp Biol Advnce Access Online the most Articles. recent version First t posted online on 16 Jnury 2014 s doi: /je Access the most recent version t Stress inhiition of meltonin synthesis in the pinel orgn of rinow trout (Oncorhynchus mykiss) is medited y cortisol Mrcos A. López-Ptiño, Mnuel Gesto, Mrt Conde-Sieir, José L. Soengs, nd Jesús M. Míguez. 8 The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Lortorio de Fisioloxí Animl. Deprtmento de Bioloxí Funcionl e Ciencis d Súde, Fcultde de Bioloxí, Universidde de Vigo Vigo. Spin. Running Title: Cortisol nd meltonin in trout pinel Keywords: meltonin, pinel orgn, AANAT2, cortisol, rinow trout, stress. Author to whom correspondence should e ddressed: Dr. Mrcos A. López-Ptiño Deprtmento de Bioloxí Funcionl e Ciencis d Súde Fcultde de Bioloxí Universidde de Vigo, Vigo. Spin Tel Fx e-mil: mlopezpt@uvigo.es Pulished y The Compny of Biologists Ltd

2 26 SUMMARY The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Cortisol hs een suggested to medite the effect of stress on pinel meltonin synthesis in fish. Therefore, we imed to determine how pinel meltonin synthesis is ffected y exposing rinow trout to different stressors, such s hypoxi, chsing nd high stocking density. In ddition, to test the hypothesis of cortisol s meditor of such stress-induced effects, set of nimls were IP implnted with coconut oil lone or contining cortisol (50 mg.kg -1 w) nd smpled 5 h or 48 h post injection t mid-dy nd mid-night. The specificity of such effect ws lso ssessed in cultured pinel orgns exposed to cortisol lone or with the generl glucocorticoid receptor ntgonist, mifepristone (RU486). The ptterns of plsm nd pinel orgn meltonin content displying highest vlues t night were ffected y stressors (in prticulr chsing nd high stocking density), resulting in decresed plsm nd pinel orgn meltonin content in oth time periods, ut with the most roust effect eing found t night. The decrese in nocturnl meltonin levels in the pinel orgn of stressed fish ws ccompnied y incresed serotonin content nd decresed AANAT2 enzymtic ctivity nd mrna undnce. Similr effects on pinel meltonin synthesis to those elicited y stress were oserved in trout implnted with cortisol for either 5 h or 48 h. These dt indicte tht stress influences negtively the synthesis of meltonin in the pinel orgn, thus ttenuting the dy-night vritions of circulting meltonin. The effect might e e medited y incresed cortisol levels which ind to trout pinel orgn specific glucocorticoid receptors to modulte meltonin rhythms. Our results in cultured pinel orgns re on its support. Considering the relevnt role of meltonin conveying photoperiodicl informtion to the synchroniztion of dily nd nnul rhythms, the results suggest tht stress-induced ltertions in meltonin synthesis could ffect the vilility of fish to integrte rhythmic environmentl informtion. 2

3 60 INTRODUCTION The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT In teleost fish the pinel orgn perceives nd trnsduces the light-drk signl (Bromge et l., 2001) into neurl nd humorl signls from which the hormone meltonin is well recognized. Meltonin is rhythmiclly synthesized minly from the pinel orgn nd relesed into the lood, showing highest plsm levels t night nd sl meltonin vlues occurring t dy-time. The penultimte step of meltonin synthesis in the pinel orgn is crried out y the enzyme ryllkylmine N-cetyltrnsferse (AANAT), which is considered s the rte-limiting enzyme sed on its dily vritions of ctivity tht prllel those of meltonin (Klein, 2007). Once relesed into lood, meltonin rhythmic profile conveys photic informtion to the orgnism (see rev. Flcón et l., 2010) nd cts s synchronizer of vriety of processes including lrvl development, locomotor ctivity, sedtion, skin pigmenttion, oxygen consumption, thermoregultion nd food intke ehvior (Ekstrom nd Meissl, 1997; Rees, 2002; Flcón et l., 2010; Zhdnov nd Rees, 2006). In ddition, nnul rhythms of reproduction, growth, immune response nd migrtion, re lso timed y meltonin in different fish species (Bromge et l., 2001; Oliveir nd Sánchez-Vázquez, 2010). The dily meltonin profile persists even fter exposing fish to constnt drkness s descried for most teleost species (Chill, 2002; Migud et l., 2007). This is due to the fct tht pinel orgn hosts true circdin light sensitive pcemker which drives meltonin rhythms. Only slmonids, including rinow trout, represent n exception to this rule. In ll slmonid species investigted to dte it hs een demonstrted tht pinel meltonin synthesis does not involve n endogenous clock, so tht lck of meltonin oscilltion hs een descried under constnt conditions (Thiult et l., 1993; Gern nd Greenhouse, 1988; Mizusw et l., 2000; Migud et l., 2007). However even under constnt drkness severl core circdin genes continue to cycle in other trout neurl regions (retin nd hypothlmus) (López-Ptiño et l., 2011), which re involved in the regultion of dily rhythms of severl prmeters such s feeding ehvior nd locomotor ctivity (Cuenc nd De l Higuer, 1994; Sánchez-Vázquez nd Tt, 1998) In ddition to externl fctors nd the circdin influence, severl internl fctors modulte meltonin synthesis in fish (Ekström nd Meissl, 1997). Among those some studies suggested role for prolctin (De Vlming nd Olcese, 1981), estrogens (Bégy et l., 1994; Forlno et l., 2005), glucocorticoids (Flcón, 1999; Benyssi et l., 3

4 The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT ) nd ctecholmines (Mrtin nd Meissl, 1992; Smejim et l., 1994; Ekström nd Meissl, 1997). Cortisol is glucocorticoid synthesized in the interrenl tissue of fish which plys n importnt role in severl spects of fish physiology, including energy metolism, ionic nd osmotic regultion, growth, immune function, nd stress response (Henderson nd Grlnd, 1980; McCormick, 1995; Wendelr Bong, 1997; Mommsen et l., 1999). Plsm cortisol levels disply circdin rhythm in teleost species like common dentex, Dentex dentex (Pvlidis et l., 1999), ut such dily pttern ppers to depend on the fish species (Grci nd Meier, 1973; Pickering nd Pottinger, 1983; Pvlidis et l., 1999; Sito et l., 2004; Eesson et l., 2008). Mny studies hve reported for rinow trout incresed plsm cortisol t night-time, peking efore the light onset, then flling down nd remining low during the dy (Rnce et l., 1982; Boujrd nd Letherlnd, 1992). Such dily profile is lso influenced y feeding time (Boujrd nd Letherlnd, 1992), in support of rhythmic cortisol secretion eing synchronized y oth photoperiod nd feeding ctivity, with differences mong sesons. Bsed on findings descriing tht i) cortisol levels pper to show dily vritions, ii) the interction within severl neurohumorl signls nd meltonin production, iii) cortisol circulting levels increse right fter fish stress exposure (Wendelr Bong, 1997; Mommsen et l., 1999), nd iv) the inhiitory effect exerted y glucocorticoids on AANAT ctivity of cultured pinel orgns in trout (Benyssi et l., 2001; Ynthn nd Gupt, 2007), we hypothesized tht, in the sme wy thn tht previously descried for other teleost species such s tipli, Oreochromis mossmicus (Nikido et l., 2010), nd tht previously suggested for trout (Lrson et l., 2004), stress negtively ffect meltonin synthesis in pinel orgn of rinow trout with cortisol mediting such inhiitory effect The im of the present study ws therefore to evlute the impct of stress on meltonin synthesis in rinow trout pinel orgn, nd to evlute the role of cortisol on such effect. Thus, we evluted dy-night vritions of plsm cortisol nd meltonin levels, pinel content of meltonin, serotonin (immedite meltonin precursor) nd its min oxidtive metolite, 5-hydroxyindolecetic cid, s well s AANAT2 enzyme ctivity nd mrna undnce in fish kept under norml housing conditions, or exposed to different stressors, or receiving cortisol implnts. An in vitro ssy of pinel orgns ws lso performed in order to corroorte the specificity of the effect. 4

5 RESULTS Stress ffects plsm cortisol nd meltonin levels The effect of exposing trout to different stressors on plsm cortisol nd meltonin content is shown in Figure 1. Plsm cortisol displyed significnt (P=0.004) dy-night vrition in control fish (higher levels oserved t night) nd those under high stocking density (lower levels t night). Exposing nimls to different stress condition significntly incresed cortisol levels t oth mid-dy nd mid-night, reltive to the respective control non-stressed group. The increse of cortisol levels ws more notorious in nimls exposed to cute stress, i.e., hypoxi nd chsing. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Meltonin levels in control group showed dy-night vrition with higher levels eing oserved during the night (P< 0.001). The sme trend ws oserved for ll the stressed groups. However, significnt decrese of plsm meltonin levels ws noticed fter stressing nimls t mid-dy-time (P=0.047; P=0.006 nd P=0.012 for hypoxi, chsing nd high stocking, compred to controls), wheres t mid-night-time it hppened under chsing nd high stock conditions (P=0.041 nd P=0.008 respectively). Effect of stress on meltonin content, AANAT2 ctivity nd mrna undnce in trout pinel orgn Meltonin content, AANAT2 enzyme ctivity nd mrna undnce in pinel orgn of trout exposed to different stressors re shown in Figure 2. Similrly to tht found for plsm meltonin, dy-night vrition of meltonin content in pinel orgn ws oserved, with higher vlues occurring t night (P<0.001 reltive to dy-time). No effect of stress ws noticed t mid-dy, wheres meltonin levels significntly decresed in fish exposed to high stocking density t mid-night (P=0.042 reltive to control) AANAT2 enzyme ctivity in trout pinel orgn showed cler dy-night vrition in control, hypoxi nd high stocking groups, with higher ctivity noticed t mid-night. Stress did not ffect AANAT2 ctivity t dy-time, ut significntly decresed it t night (P=0.002; P<0.001; P<0.001 reltive to control t night for hypoxi, chsing nd high stocking). This effect ws more roust in fish exposed to chsing, in which the dy-night vrition disppered. A significnt dy-night vrition of nt2 mrna undnce ws oserved in pinel orgn of control nd high stocking fish, with higher vlues t mid-night (P=

6 nd P=0.029 respectively). Decresed nocturnl nt2 mrna undnce, reltive to control group, ws found in fish exposed to cute stress (hypoxi nd chsing), leding the dy-night vrition of nt2 expression to dispper in oth groups. 5-HT nd 5-HIAA contents in pinel orgn of stressed trout The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Dy-night vritions of serotonin nd its min metolite, 5-HIAA, nd the rtios 5-HIAA/5-HT nd meltonin/5-ht re shown in Figure 3. Serotonin content in pinel orgn ws significntly lower t mid-night in ll the experimentl groups. Stress did not significntly ffect 5-HT levels t dy-time, wheres significnt increse ws oserved t mid-night only in fish exposed to high stocking (P=0.001; P=0.002; nd P=0.008, reltive to the other groups). Similrly to tht descried for serotonin, 5-HIAA dy-night chnges were found in ll the experimentl groups, with significntly higher levels t mid-dy. During the night, significnt decrese of 5-HIAA content ws found in trout exposed to hypoxi, reltive to tht of control (P=0.034) nd high stocking groups t this time period. The 5-HIAA/5-HT rtio did only show dy-night vritions in the hypoxi-exposed group, which ws higher t night (P=0.040). In ddition, exposing fish to high stocking density tended to decrese the 5-HIAA/5-HT rtio t night, ut this effect did not rech significnce when compred to control t night. A cler dy-night vrition of the rtio Meltonin/5-HT ws found in ll the experimentl groups, with higher vlues occurring t night. During the dy, hypoxi nd high stocking density significntly decresed the Meltonin/5-HT rtio, compred to control nd chsing groups. In contrst, only the high stocking density significntly decresed the rtio t mid-night, reltive to control, hypoxi nd chsing groups. Plsm cortisol nd meltonin levels fter cortisol IP dministrtion Plsm cortisol nd meltonin levels fter coconut oil dministrtion lone or with cortisol re shown in Figure 4. Dt otined from the control non-implnted group re not shown ut remined quite similr to those of the control implnted fish. Though no significnt dy-night vrition of plsm cortisol levels ws found in control group, tendency to higher nocturnl hormone levels persisted (P=0.082) in the sme wy thn tht oserved in the previous experiment (see Figure 1). As expected, 6

7 significnt increse of cortisol levels ws found in trout smpled 5-h or 48-h fter the IP dministrtion, compred to control group t oth time periods. Only significnt dy-night vrition ws found in trout IP injected with cortisol t 5-h post injection, with higher levels t night, reltive to the sme group t dy-time. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Plsm meltonin levels in control group showed the sme dy-night vrition s ove descried, with higher levels t night (P<0.001). The dministrtion of cortisol enhnced levels of meltonin fter 48-h t dy nd decresed fter 48-h t night, in oth cses reltive to control group (P=0.016 nd P=0.038 respectively), then mking the mplitude of the dy-night vrition to decrese in those 48-h cortisol implnted trout. Pinel content of meltonin nd AANAT2 ctivity nd mrna undnce in cortisol implnted trout Figure 5 shows meltonin content, AANAT2 enzyme ctivity, nd mrna undnce in pinel orgn of implnted trout. Dt in non-implnted fish (not shown) were consistent with those oserved in control-implnted trout for the three prmeters ssessed. A significnt dy-night vrition ws found for meltonin content in ll the experimentl groups with higher levels occurring t mid-night. The IP cortisol dministrtion significntly reduced nocturnl meltonin levels reltive to tht found in controls t night (P=0.011 nd P=0.027 for 5-h nd 48-h respectively). No effects of cortisol dministrtion were found during the dy. AANAT2 ctivity displyed significnt dy-night vrition in control trout, with higher levels t mid-night. In contrst, cortisol significntly inhiited the enzyme ctivity only t night (P=0.018 nd P=0.022 for 5-h nd 48-h respectively), leding the typicl dy-night vrition of the enzyme ctivity to dispper t oth 5-h nd 48-h post injection. This inhiitory effect of cortisol ws not oserved t mid-dy The nlysis of nt2 mrna undnce in pinel orgn of rinow trout reveled significntly higher expression t night-time in control trout, reltive to tht mesured during mid-dy. Cortisol dministrtion showed time-dependent effect. Thus, nt2 expression ws significntly enhnced during dy-time t 5-h post-cortisol injection (P=0.041), ut decresed fter 5-h nd 48-h t night-time (P=0.042 nd P=0.006 for 5-h nd 48-h respectively), with the effect eing more effective fter 48-h. 7

8 This inhiitory effect of cortisol on mrna expression did led the dy-night vrition of nt2 expression to dispper in oth cortisol-implnted groups. 5-HT nd 5-HIAA contents in pinel orgn of implnted nimls The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Figure 6 shows the dily vrition of serotonin nd 5-HIAA levels, nd the rtios 5-HIAA/5-HT nd meltonin/5-ht. group displyed significnt dy-night vrition for 5-HT content in the pinel orgn, with higher levels t mid-dy. Wheres cortisol dministrtion for 5-hours hd no effect on 5-HT content (reltive to control group) the 48-h dministrtion significntly decresed the diurnl 5-HT (P=0.024 nd P=0.038 reltive to control nd 5-h t dy-time) nd incresed the nocturnl 5-HT content (P=0.005 nd P=0.011 reltive to control nd 5-h t dy-time). Thus, the dy-night vrition of pinel 5-HT content displyed higher levels t night nd lower levels during the dy, which ws the opposite profile thn tht oserved in control. No dily vritions of 5-HIAA pinel content were oserved in control group nd tht IP dministered with cortisol for 5 hours. However, nocturnl significnt increse of the metolite ws oserved in nimls dministered with cortisol for 48 hours (P=0.049 reltive to control). Then 5-HIAA content in trout pinel orgn displyed significnt dy-night vrition only in nimls IP implnted with cortisol for 48 hours, with higher 5-HIAA levels occurring t night (P=0.004 reltive to dy-time). The rtio 5-HIAA/5-HT did not significntly chnge mong groups t oth dynd night-time. Dy-night significnt vritions of the rtio were found only in trout IP dministered with cortisol for 5-h, with higher vlues t night (P=0.007 reltive to dy). All the experimentl groups displyed significnt dy-night vrition in the rtio Meltonin/5-HT, with higher vlues t mid-night. Cortisol dministrtion significntly decresed the rtio only t night compred to control group (P<0.001 nd P=0.007 for 5-h nd 48-h reltive to control), with the more importnt effect eing oserved fter 48-h. No such effect ws found in nimls IP injected during the dy t oth 5-h nd 48-h post injection. Effect of cortisol tretment on meltonin production in vitro Meltonin production in cultured pinel orgn in the presence/sence of light ws compred mong experimentl groups (Figure 7). Meltonin ws detected in the culture medium of smples collected under ech lighting condition. There were no 8

9 group-specific differences in sl meltonin relese t oth light nd drk. However, the ddition of medium contining cortisol resulted in significnt decrese of meltonin production in drkness, reltive to control (P=0.037) t the sme lighting condition nd to tht oserved efore cortisol ddition within the sme group. This inhiitory effect of cortisol ws prevented y RU486 (P=0.032, reltive to cortisol) when oth chemicls were dded together. DISCUSSION The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT In the present study, different stressors were evluted, i.e., hypoxi, chsing nd high stocking density, mimicking those potentilly stressing situtions to which fish cn e exposed when rered. The response to stress in fish involves the ctivtion of the hypothlmus-sympthetic nervous system-chromffin tissue, nd the hypothlmuspituitry-interrenl tissue xes, followed y fst increse of ctecholmine nd cortisol levels in plsm, which in fct induce metolic nd functionl ltertions (Iwm et l., 2006), nd ffect fish physiology (Brton et l., 2002). Little is known regrding the effects of those hormones t the trout pinel orgn, ut previous studies descrie tht ctecholmines pper not to hve ny effect t this tissue loction, in contrst to tht in other teleost for which regultory role hs een proposed (Flcón et l., 1991). Regrding cortisol, meltonin synthesis in trout pinel orgn ws reported to e influenced y glucocorticoid hormones (Benyssi et l., 2001), with cortisol eing serious cndidte s meditor of such effect. Dy-night vritions of cortisol nd meltonin levels in plsm, the pinel orgn content of 5-HT, 5-HIAA nd meltonin, nd the AANAT2 ctivity nd mrna undnce t the pinel level were evluted in non-stressed, stressed nd cortisol-implnted trout. Those fish rered under norml housing conditions showed significnt (Experiment 1) or tendency (Experiment 2) to dy-night vritions of plsm cortisol with night vlues eing higher thn those mesured during the dy, in concordnce with previous studies in the sme species (Rnce et l., 1982; Boujrd nd Letherlnd, 1992) nd others such s the rown trout (Pickering nd Pottinger, 1983) nd tilpi (Mrtínez-Chvez et l., 2008; Nikido et l., 2010). Our results show tht cortisol levels were higher in those cutely stressed groups (hypoxi nd chsing) wheres such increse ws lower in high-stocked fish in prticulr during the night. Also, the increse in cortisol ws higher in those fish stressed t dy-time compred 9

10 with the sme groups t night, independently of the stress condition. This result suggests tht the integrted response to stress could e influenced y the time of the dy in which the stressor is present. Also the fct tht the higher cortisol increse ws coincident with the time of dy in which trout re more ctive suggest tht reltionship etween ehviourl components nd the response to stress might exist. Further reserch should e crried out. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Plsm meltonin levels were negtively ffected y stress in wy tht the highest reduction of dy-night vrition of hormone levels ws oserved in fish exposed to long-term stress (high stocking), rther thn those cutely stressed (chsing, hypoxi), which in fct lso showed decresed plsm meltonin levels, with those of the pinel orgn remining unltered. This indictes tht time periods longer thn 1-5 minutes (hypoxi nd chsing, respectively) might e required for significnt inhiition of meltonin content to e oserved in pinel orgn. In contrst, chnges ffecting AANAT2 enzyme ctivity nd mrna undnce immeditely occur s our results indicte. However, one might hypothesize the presence of correltion etween stress durtion nd the mgnitude of the inhiition of the night-time meltonin production in the pinel orgn, which is supported y the existence of similr effects in oth pinel orgn meltonin content nd plsm vlues. On the contrry, dy-time chnges in pinel meltonin fter ny stressor were minor, reflecting tht reduced levels of the hormone in lood of stressed trout during dy-time might involve ltertions in meltonin clernce rtes or, lterntively, tht hormone synthesis in other tissues ws lso defective, i.e., retin nd the gstrointestinl trct tht were suggested to contriute to lood meltonin levels during the dy (Lepge et l,. 2005; Muñoz et l., 2009). Further reserch is needed to discrd ny explntion The effect of stress on the dy-night meltonin secretion pttern hs een studied in severl fish species nd shows contrdictory results. Then, Lrson et l (2004) reported higher night-time meltonin nd cortisol levels in socilly suordinted rinow trout reltive to the dominnt fish. Similr results were oserved in our lortory in trout exposed to incresed slinity (López-Ptiño et l., 2011), ut tht ws in contrst to tht reported for Europen se ss (López-Olmed et l., 2009). Incresed circulting meltonin levels were lso found in gilthed se rem sujected to high stocking density, with such effect eing prevented y fsting (Mncer et l., 2008). Similr results hve een lso descried for rinow trout in which fsting 10

11 The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT decreses pinel orgn meltonin synthesis t night (Ceinos et l., 2008), nd disturnce stress negtively ffect severl prmeters including meltonin (Kulczykowsk, 2001), which is in consistency with our dt herein reported for trout exposed to different stressors. Since different species pper to specificlly respond to ny stressor, we might speculte with the ide of species-specific stress effect on meltonin synthesis t the pinel orgn. Tking in mind tht i) pinel meltonin synthesis is differentilly regulted mong teleost in reltion with the environmentl signls, i.e., light/drk cycle (trout meltonin system generting rhythm seems to lck functionl clock in contrst to tht of most non-slmonids), nd ii) the different socil ehviours nd physiologicl dpttions to the qutic environments in which fish inhit, it might not e surprising different species-specific response to stress. In ddition, the nture nd the durtion of the stressor pper to lso influence the response of the pinel orgn, s reveled y our study. In spite of tht ove mentioned for meltonin, stress lso diminished the AANAT2 enzymtic ctivity nd mrna undnce in the pinel orgn t night. It is generlly ccepted tht the nocturnl increse in AANAT2 enzyme ctivity is the min responsile of the dily rhythm of meltonin synthesis. In trout, light y directly cting on pinel photoreceptors, exerts n inhiitory influence on oth AANAT2 gene expression (López-Ptiño et l., 2011) nd enzyme ctivity (Flcón, 1999; Ceinos et l., 2005), with immedite consequences on pinel meltonin content (Ceinos et l., 2005). This is in contrst to tht previously reported for AANAT2 ctivity nd gene expression in trout pinel orgn, in which the nt2 expression dily profile ws not oserved (Bégy et l., 1998; Coon et l., 1998; Flcón et l., 2001). The resons for these discrepncies re not known, ut methodologicl differences or different trout strins might e the most plusile explntions s we previously reported (López-Ptiño et l., 2011). Further reserch needs to e crried out in order to understnd the nture of. Thus ccording to our previous dt nd tht descriing light effect, our present results indicte tht meltonin synthesis in trout pinel orgn is inhiited y stress y specificlly ffecting AANAT2 ctivity, which is proly consequence of the inhiition oserved in nt2 mrna expression. In ddition, our dt showing incresed nocturnl levels of 5-HT, ut not its min oxidtive metolite 5-HIAA, in pinel orgns of stressed trout lso support n inhiitory role for stress on meltonin synthesis. Therefore, it is likely tht, when stressed, the N-cetyltion 11

12 pthwy of 5-HT is inhiited due to decresed of AANAT2 enzyme ctivity nd expression, leding to decresed meltonin levels nd intrcellulr ccumultion of 5- HT, which is oxidized to 5-HIAA. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Previous in vitro studies on the hormonl regultion of fish pinel meltonin synthesis reveled the presence of glucocorticoid receptors in trout pinel orgn, nd when in vitro ssyed with the glucocorticoid nlogue dexmethsone decresed AANAT2 ctivity ws oserved (Benyssi et l., 2001), which is in consistency with our results from the pinel orgn in vitro ssy showing decresed meltonin relese in pinel orgns exposed to cortisol in drkness. Also Ynthn nd Gupt (2007) found decresed AANAT ctivity in cultured pinel orgn of the North Africn ctfish (Clris griepinus) fter milimolr corticoid tretment, n effect similr to tht reported recently in cultured pinel orgns of tilpi with nnomolr cortisol concentrtions (Nikido et l., 2010). Therefore glucocorticoid regultion of pinel meltonin production is possile under physiologicl conditions, nd this might occur when cortisol levels re elevted due to stressful condition. On the sis of these studies, n in vivo experiment ws conducted in trout to evlute whether or not cortisol tretment might result in chnges in plsm nd pinel prmeters similrly to those elicited y stress. Our results show tht cortisol implnts for 48 hours increse plsm meltonin levels t dy-time, wheres oth plsm (t 48-h) nd pinel meltonin (5-h nd 48-h) levels decresed during the night in cortisol-implnted trout. This differentil effect of cortisol on meltonin secretion seems to e independent of the cortisol increse (i.e., cortisol levels in implnted fish were higher thn those of controls ny time). Thus, we might speculte with the ide tht incresed cortisol could differentilly ffect the photoreceptor cell properties t dy nd night (i.e., y ltering memrne permeility or light trnsduction pthwys) with meltonin synthesis eing ffected. In greement with this hypothesis, we previously reported for rinow trout tht exposure to chemicl stressor (i.e., polycyclic romtic hydrocrons; Gesto et l., 2009) elicited similr effect thn tht herein reported for pinel orgn meltonin synthesis. In ddition, cortisol implnts lso decrese AANAT2 enzyme ctivity nd mrna undnce in the pinel orgn t night. These results clerly demonstrte the negtive effect exerted y cortisol on AANAT2 undnce nd ctivity. In ddition, the specificity of the negtive effect of cortisol on pinel orgn meltonin synthesis hs een proved, s our results from the in vitro ssy with cortisol nd its ntgonist (RU486) clerly demonstrte. In 12

13 The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT support of tht, we lso oserved tht trout receiving cortisol implnts for 48-h displyed incresed 5-HT levels t night. This effect ws similr to tht oserved for 5- HIAA, which suggests tht the inhiitory effect of cortisol on meltonin synthesis leds to incresed 5-HT content, which is then oxidized. A similr effect of cortisol ws not seen t mid-dy, which my suggest tht those vritions oserved during the night might e minly due to decresed 5-HT utiliztion through the N-cetyltion pthwy to synthesize meltonin t this time period. Thus we my discrd ny possile specific effect of 5-HT synthesis in trout pinel orgn. Tken together, we conclude tht cortisol, through the ctivtion of specific glucocorticoid receptors, might e the min responsile of the influence of stress on teleost pinel orgn physiology, especilly in those species in which ctecholmines might ply minor role (Flcón et l., 1991). Therefore, the effects oserved in oth stressed, cortisol-implnted trout, nd cultured pinel orgns tended to decrese the dy-night vrition of oth pinel content nd plsm meltonin levels. It is likely tht cortisol effect on pinel meltonin is meditted y either specific glucocorticoid receptors s indicted previously nd for other teleost species (Benyssi et l., 2001; Nikido et l., 2010), which might ctivte glucocorticoid-responsive elements t the AANAT gene promoter (Benyssi et l., 2001), nd/or other non-genomic ctions t the cell-surfce (Mommsen et l., 1999). Our results from cultured pinel orgns re in support of this hypothesis. In fish, plsm cortisol levels re known to cycle diurnlly (Hollowy et l. 1994; Reddy nd Letherlnd 1995) nd to chnge through the sesons (McLeese et l. 1994), lthough cortisol profile exhiits species-specific dily nd sesonl ptterns ffected y severl fctors like photoperiod (Pickering nd Pottinger, 1983) or feeding time (Boujrd nd Letherlnd, 1992), mong others. In some species, such s common dentex, dily fluctutions of plsm cortisol levels show endogenous rhythmic chrcteristics (Pvlidis et l., 1999), nd this hormone is tking incresed relevnce s n importnt output of the circdin clock system in vertertes (Lilley et l., 2012). However, temporl reltionship etween physiologicl clocks nd plsm levels of cortisol nd meltonin hs not een estlished in fish. Tking into ccount tht trout ppers to lck pinel circdin signlling tht controls meltonin synthesis, one might not discrd the possiility for dily chnges in cortisol to ply modultory role in the light-drk regultion of trout meltonin rhythm. In fct, cortisol vlues were shown to e especilly high t the end of the night nd in the erly morning, time in which pinel 13

14 meltonin synthesis hs decresed (Ceinos et l., 2005). On the contrry, high meltonin levels hve een reported to reduce cortisol secretion in goldfish (Azpelet et l., 2010) nd to counterct the stress-induced cortisol increse in Seneglese sole (López-Ptiño et l., 2013). This hormonl correltion is in support of our dt showing in trout n inverse reltionship etween high cortisol levels nd meltonin levels in stressed, cortisol-implnted fish nd in cultured pinel orgns. Then, digging into cortisol rhythms nd its interction with meltonin is promising topic to work with in order to understnd the regultion of pinel rhythmic physiology nd the circdin orgniztion which is elieved to exist in trout (Sánchez-Vázquez nd Tt, 1998). The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT In summry, our results provide evidence for the inhiitory effect exerted y cortisol on meltonin synthesis y the pinel orgn of rinow trout. This steroid likely medites the effects of different stressful situtions on the pinel orgn y ctivting specific glucocorticoid receptors. Such ctivtion seems to directly influence AANAT2 enzyme ctivity nd expression which re normlly incresed t night, llowing the meltonin secretory pek. Our results lso indicte tht cortisol, either in non-stressful or stressful conditions, might hve modultory role of the pinel rhythms, in prticulr those relted to meltonin synthesis. Considering the pivotl role of meltonin in synchronizing rhythmic physiologicl events to the cyclic environmentl chnges (minly light-drk cycle), the effect of stress on meltonin synthesis might e trnslted into process tht cn jeoprdize the vilility of the niml to respond to such fluctutions nd in consequence, to compromise its physiologicl integrtion. MATERIALS AND METHODS Animls Immture rinow trout (7.0±0.5 months old; 100±5 g ody mss) were otined from fish frm (Soutorredondo, Noi, Spin) nd trnsferred to our fcilities t the Vigo University. Animls were cclimted for 15 dys in 120 L tnks under our lortory conditions: 12:12 light (L):drk (D) photoperiod (lights on t 08:00 h, 400 lux intensity t the wter surfce), 14±1ºC wter temperture, nd continuously renovted nd erted wter. During cclimtion fish were fed dily to stiety (10:00 h) with commercil dry pellets (Diq-diproteg, Segovi, Spin). All the experimentl procedures nd niml mnipultion were designed ccording to the Europen Union Council (2010/63/EU), nd the Spnish Government (RD 53/2013) legl requirements. 14

15 Smple collection The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Animls were deeply nesthetized y fst immersion in MS-222 (50 mg/l) uffered to ph 7.4 with sodium icronte nd lood ws collected from the cudl vein, using 1 ml heprinized syringes. Then, fish were scrificed y decpittion nd pinel orgns were removed with sterilized mteril nd plced into RNse-free 1.5 ml Eppendorf tues. Smples were immeditely frozen in liquid nitrogen, nd stored t -80 C until ssyed. Plsm ws otined y centrifuging lood t 9,000 rpm for 10 min t 4 C. Aliquots were immeditely frozen nd stored t -80 C until nlyzed. Experimentl designs Three experiments were designed to evlute the influence of stress on meltonin production in rinow trout pinel orgn. In first experiment, fish were rndomly distriuted in four groups: control, or stressed y hypoxi, chsing, nd high stocking density (n=20 nimls ech). Following cclimtion, quntity of wter ws removed from those tnks hosting the high stocking density group until reching stressful high density (70 kg fish mss m -3 ), where fish remined for 4 dys efore scrificed. Stocking density conditions remined unltered for the other groups (10 kg fish mss m -3 ). On the dy of scrifice, nimls from the other stressed groups were exposed to different mnipultions nd then scrificed t mid-dy nd mid-night s follows. Fish from the hypoxi group were normlly hndled nd netted ut remined in the net for 60 seconds efore eing deeply nesthetized in MS-222 solution, lood smpled nd scrificed. Chsing group ws sujected to stndrdized hndling disturnce consisting on 5 min of repeted chsing followed y 15 min of recovery. Then trout were netted nd nesthetized efore lood collection nd fish scrifice for smple collection. The high stocking nd the control groups were exposed to norml hndling procedures, then netted nd rpidly trnsferred into new tnks where they were deeply nesthetized. Once nesthetized, lood ws individully collected nd fish were immeditely scrificed. When scrificed t mid-night, ll the mnipultions nd smple collection were done under low intensity (< 0.4 lux) dim red light. In ech group (n=20), lood ws collected from 8 nimls for plsm cortisol nd meltonin quntifictions, nd their pinel orgns were ssyed for indole content nd AANAT2 15

16 ctivity y HPLC. Addition pools (n=4/group) of pinel orgns (n=3 orgns/pool) were processed for mrna quntifiction, with the remining 12 fish from ech group. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT In second experiment the effect of cortisol on pinel orgn metolism ws evluted t dy nd night. Four groups of trout were divided into two 120 L tnks ech (n=20 nimls/tnk). Following cclimtion, fish were nesthetized, weighed nd intrperitonelly dministered with slow-relese coconut oil implnts (control group included) following procedures previously descried (Soengs et l., 1992). Another extr control group ws not implnted controls ut lso injected. Implnts consisted on coconut oil lone (10 g.g -1 ody weight) in controls, or contining dose of cortisol (50 mg.kg -1. w.) ccording to previous studies in this species (Vijyn et l., 2003). After implnted, fish were plced ck to their respective tnk. Fish from ech tnk were scrificed nd smpled t 5-h or 48-h post-implnt dministrtion, t mid-dy (the first tnk of ech group) or mid-night (the second tnk). No mortlity ws oserved during the experiment. Animls scrificed t night were mnipulted s ove. Smples were processed s indicted (experiment 1). A third experiment ws performed to corroorte tht pinel orgn meltonin synthesis is inhiited y specific ction exerted y cortisol. Thus, individul pinel orgns were tken out from fish nd immeditely plced in 96-well culture pltes ech contining 250 μl of modified Hnks medium ccording to Yñez nd Meissl (1995), ut supplemented with 0.1 mmol L -1 tryptophn. Assys were crried out under controlled temperture (16ºC) nd in the presence or sence of light. After 3-h incution the culture medium ws removed nd stored t -80ºC, nd replced with 250 μl modified Hnks medium lone (control group) or contining cortisol (100 ng ml -1 ), the generl glucocorticoid receptors ntgonist, mifepristone (RU486; 1.0 μg ml -1 ), or RU486+cortisol (n=8 orgns/group). After 3 hours, medium ws removed nd stored t -80ºC until ssyed. Meltonin content ws ssessed on ech medium frction. Hormones nd metolites quntifiction Plsm cortisol levels were mesured using commercilly ville Enzyme Immunossy Kit (Cymn, Ann Hror, MI, USA), ccording to mnufcturer s indictions. Plsm meltonin levels were ssyed y HPLC with fluorimetricl detection s descried (Muñoz et l, 2009). The resultnt residue fter the extrction procedure ws dissolved in 100 l moile phse nd filtered (0.5 m filter). An liquot 16

17 (50 l) of the filtrte ws injected into the HPLC system. Dt from the nlysis re expressed s pg ml -1 of plsm. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Meltonin content in culture medium ws ssyed y directly injecting 20 l from ech collected frction (n=8 per group) into the HPLC system. For pinel meltonin quntifiction, ech orgn (n=8 per group) ws homogenized y ultrsonic disruption in 100 l of 0.2 mol L -1 phosphte uffer (ph 6.7) nd centrifuged t 16,000 g for 10 min. A 60 l liquot ws ssyed for meltonin nd pinel indole contents. The resulting 40 l liquot ws immeditely ssyed for AANAT2 enzyme ctivity. From the 60 l liquot, pinel meltonin content ws mesured y direct injection of 20 l volume into the HPLC system, similrly to tht descried for plsm meltonin ssessment. Other conditions were s previously descried (Ceinos et l., 2008). Pinel 5-HT nd its cidic metolite, 5-hydroxindolecetic cid (5-HIAA) were ssyed y HPLC with electrochemicl detection from 10 µl liquot of the pinel homogentes s descried y Ceinos et l. (2005). AANAT2 enzyme ctivity ws ssyed y in vitro incution of 40 l of the ove mentioned smple homogentes with the sustrte, 40 l of 27 mmol L -1 tryptmine nd 40 l of 1.0 mmol L -1 cetyl-coa (finl concentrtions in ssy: 9 mmol L -1 tryptmine nd 0.5 mmol L -1 cetyl-coa). Assy conditions were s previously descried nd performed (Ceinos et l., 2008) with modifiction consisting of 60 min incution period t 16 C (temperture within the optiml rnge for trout). 20 l of the finl solution were directly injected into the HPLC system in order to quntify the rection product (N-cetyl tryptmine; NAT) formed. The system ws HPLC pump (Gilson M101) with n Ultrsphere Beckmn column (3 m prticles, 75 mm nd 4.6 mm i.d.) nd Jsco FP-1520 fluorescence detector set t 285/360 nm excittion/emission wvelengths. All nlyses were performed t room temperture nd 1 ml min -1 flow rte. Smple pek res were quntified reltive to tht of suitle stndrds. Anlysis of pinel orgn nt2 mrna undnce After dissection, pinel orgns from fish receiving the sme tretment were pooled (n=3 orgns/pool). Totl mrna ws extrcted from ech pool (n=4 pools/group) using the TRIzol method (Gico BRL, Githersurg, MD, USA) ccording 17

18 to mnufcturer s instructions. The isolted RNA qulity nd quntity were spectrophotometriclly determined. From ech smple, 2 g RNA were converted into cdna s descried (López-Ptiño et l., 2011). A negtive control of ech smple ws ssessed without reverse trnscriptse in order to discrd ny genomic contmintion. The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Rel-time quntittive RT-PCR (qpcr) ws performed using MximTM SYBR Green qpcr Mster Mix (K0252, 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 AF ) forwrd 5 -CATTCGTCTCTGTGTCTGGT-3, reverse 5 -TTTCTGGGA TATGCTGGGT-3 ; nd -ctin (AJ438158) forwrd 5 -GATGGGCCAGAAAGACA GCTA-3, reverse 5 -TCGTCCCAGTTGGTGACGAT-3. Gene expression ws normlized to tht of -ctin for ech smple. Reltive mrna expression ws clculted ccording to the comprtive Ct method. For ech gene, smples collected t the sme time point were processed in prllel nd the expression ws mesured in triplicte. Sttisticl nlysis Differences were evluted y Two-Wy ANOVA nlyses of vrince, with tretment 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. Significnce level ws set t P < Acknowledgements This work ws supported y the Spnish Ministerio de Cienci e Innovción nd Europen Fund for Regionl Development (AGL C03-03) to JL Soengs nd Xunt de Glici (Consolidción e estructurción de uniddes de investigción competitivs no sistem universitrio de Glici, CN 2012/004) to J.L. Soengs. M.A. López-Ptiño is n Isidro Prg Pondl Resercher (P.P S 14008). Specil thnks to L. Mnrique for her vlule corrections on the mnuscript, nd to S. González-Silv, A. Benedetti nd S. Usndizg for excellent technicl ssistnce. 18

19 REFERENCES Azpelet, C., Mrtínez-Alvrez, R.M., Delgdo, M.J., Isorn, E. nd De Pedro, N. (2010). Meltonin reduces locomotor ctivity nd circulting cortisol in goldfish. Horm. Behv. 57(3): Brton, B.A., Morgn, J.D. nd Vijiyn M.M. (2002). Physiologicl nd conditionrelted indictors of environmentl stress in fish. In: S. Mrshll Adms (ed.) Biologicl Indictors of Aqutic Ecosystem Stress, Americn Fisheries Society, Mrylnd, pp The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Bégy, V., Flcón, J., Chill, G.M., Klein, D.C. nd Coon, S.L. (1998). Trnscripts encoding two meltonin synthesis enzymes in the teleost pinel orgn: circdin regultion in pike nd zerfish, ut not in trout. Endocrinology 139: Bégy, V., Vlotire, Y., Rvult, J.P., Collin, J.P. nd Flcón J. (1994). Detection of estrogen receptor mrna in trout pinel nd retin: estrdiol-17 et modultes meltonin production y cultured pinel photoreceptor cells. Gen. Comp. Endocrinol. 93: Benyssi, A., Schwrtz, C., Ducouret, B. nd Flcón J. (2001). Glucocorticoid receptors nd serotonin N-cetyltrnsferse ctivity in the fish pinel orgn. Neuroreport. 12(5): Boujrd, T. nd Letherlnd, J.F. (1992). Circdin pttern of heptosomtic index, liver glycogen nd lipid content, plsm non-esterified ftty cid, glucose, T3, T4, growth hormone nd cortisol concentrtions in Oncorhynchus mykiss held under different photoperiod regimes nd fed using demnd-feeders. Fish Physiol. Biochem. 10: Bromge, N., Porter, M. nd Rndll, C. (2001). The environmentl regultion of mturtion in frmed finfish with specil reference to the role of photoperiod nd meltonin. Aquculture. 197: Chill, G.M. (2002). Clock mechnisms in zerfish. Cell. Tissue Res. 309:

20 Ceinos, R.M., Polkof, S., Illmol, A.R., Soengs, J.L. nd Míguez J.M. (2008). Food deprivtion nd refeeding effects on pinel indoles metolism nd meltonin synthesis in the rinow trout Oncorhynchus mykiss. Gen. Comp. Endocrinol. 156: Ceinos, R.M., Ráde, S., Soengs, J.L. nd Míguez J.M. (2005). Indolemines nd 5-methoxyindoles in trout pinel orgn in vivo: dily chnges nd influence of photoperiod. Gen. Comp. Endocrinol. 144(1): The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Coon, S., Bégy, V., Flcón, J. nd Klein, D.C. (1998). Expression of meltonin synthesis genes is controlled y circdin clock in the pike pinel orgn ut not in the trout. Biol. Cell 90: Cuenc, E.M. nd de l Higuer, M. (1994). A microcomputer-controlled demnd feeder for the study of feeding ehvior in fish. Physiol. Behv. 55(6): De Vlming, V. nd Olcese, J. (1981). The pinel nd reproduction in fish, mphiins nd reptiles. In: Reiter R.J., ed. The pinel Glnd, Vol. II. Reproductive effects. Boc Rton, Florid: CRC Press; pp Eesson, L.O.E., Björnsson, B.T., Ekström, P. nd Stefnsson, S.O. (2008). Dily endocrine profiles in prr nd smolt Atlntic slmon. Comp. Biochem. Physiol. A. 151: Ekström, P. nd Meissl, H. (1997). The pinel orgn of fishes. Rev. Fish Biol. Fisheries. 7: Flcón, J. (1999). Cellulr circdin clocks in the pinel. Prog. Neuroiol. 8: Flcón, J., Migud, H., Muñoz-Cueto, J.A. nd Crrillo, M. (2010). Current knowledge on the meltonin system in teleost fish. Gen. Comp. Endocrinol. 165: Flcón, J., Thiult, C., Mrtin, C., Brun-Mrmillon, J., Clustrt, B. nd Collin-, J.P. (1991). Regultion of meltonin production y ctecholmines nd denosine in photoreceptive pinel orgn. An in vitro study in the pike nd the trout. J. Pinel Res. 11(3-4):

21 Forlno, P.M., Deitcher, D.L. nd Bss, A.H. (2005). Distriution of estrogen receptor lph mrna in the rin nd inner er of vocl fish with comprisons to sites of romtse expression. J. Comp. Neurol. 483: Grci, L.E. nd Meier, A.H. (1973). Dily rhythms in concentrtion of plsm cortisol in mle nd femle gulf killifish, Fundulus grndis. Biol. Bull. 144: The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Gern, W.A. nd Greenhouse, S.S. (1988). Exmintion of in vitro meltonin secretion from superfused trout (slmo girdneri) pinel orgns mintined under diel illumintion or continous drkness. Gen. Comp. Endocrinol. 71: Gesto, M., Tintos, A., Rodríguez-Illmol, A., Soengs, J.L. nd Míguez, J.M. (2009). Effects of nphthlene, et-nphthoflvone nd enzo()pyrene on the diurnl nd nocturnl indolemine metolism nd meltonin content in the pinel orgn of rinow trout, Oncorhynchus mykiss. Aqut. Toxicol. 92(1):1-8. Henderson, I.W. nd Grlnd, H.O. (1980). The interrenl glnd in piscis. Prt 2. Physiology. In: Chester Jones I, Henderson IW, editors. Generl, Comprtive nd Clinicl Endocrinology of the Adrenl Cortex. New York: Acdemic Press. Vol. 3. p Hollowy, A.C., Reddy, P.K., Sheridn, M.A. nd Letherlnd, J.F. (1994). Diurnl rhythms of plsm growth hormone, somtosttin, thyroid hormones, cortisol nd glucose concentrtions in rinow trout, Oncorhynchus mykiss, during progressive food deprivtion. Biol. Rhythm Res. 25: Iwm, G.K., Afonso, L.O.B. nd Vijyn, M.M. (2006). Stress in fishes. Eds. D.H. Evns nd J.B. Cliorne. The Physiology of Fishes, CRC Press, Boc Rton, Florid, pp Klein, D.C. (2007). Aryllkylmine N-cetyltrnsferse: the Timezyme. J. Biol. Chem. 282:

22 Kulczykowsk, E. (2001). Responses of circulting rginine vsotocin, isotocin, nd meltonin to osmotic nd disturnce stress in rinow trout (O. mykiss). Fish Physiol. Biochem. 24: Lrson, E.T., Winerg, S., Myer, I., Lepge, O., Summers, C.H. nd Øverli, Ø. (2004). Socil stress ffects circulting meltonin levels in rinow trout. Gen. Comp. Endocrinol. 136: The Journl of Experimentl Biology ACCEPTED AUTHOR MANUSCRIPT Lepge, O., Lrson, E.T., Myer, I. nd Winerg, S. (2005). Tryptophn ffects oth gstrointestinl meltonin production nd interrenl ctivity in stressed nd nonstressed rinow trout. J. Pinel Res. 38(4): Lilley, T.R., Wotus, C., Tylor, D., Lee, J.M. nd De l Iglesi, H.O. (2012). Circdin regultion of cortisol relese in ehviorlly split golden hmsters. Endocrinology. 153(2): López-Olmed, J.F., Oliveir, C., Klmrz, H., Kulczykowsk, E., Delgdo, M.J. nd Sánchez-Vázquez, F.J. (2009). Effects of wter slinity on meltonin levels in plsm nd peripherl tissues nd on meltonin inding sites in Europen se ss (Dicentrrchus lrx). Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 152(4): López-Ptiño, M.A., Conde-Sieir, M., Gesto, M., Lirán-Pérez, M., Soengs, J.L. nd Míguez, J.M. (2013). Meltonin prtilly minimizes the dverse stress effects in Seneglese sole (Sole seneglensis). Aquculture. 388: López-Ptiño, M.A., Rodríguez-Illmol, A., Conde-Sieir, M., Soengs, J.L. nd Míguez J.M. (2011). Dily rhythmic expression ptterns of clock1, Bml1, nd Per1 genes in retin nd hypothlmus of the rinow trout, Oncorhynchus mykiss. Chronoiol. Int. 28(5): López-Ptiño, M.A., Rodríguez-Illmol, A., Gesto, M., Soengs, J.L. nd Míguez, J.M. (2011). Chnges in plsm meltonin levels nd pinel orgn meltonin synthesis following cclimtion of rinow trout (Oncorhynchus mykiss) to different wter slinities. J. Exp. Biol. 214: