Department of Fish Physiology and Biotechnology, IATS/CSIC, Castellón, Spain. and

Transcription

1 CBP ms Revised prt A Impct of photoperiod mnipultion on dy/night chnges in meltonin, sex steroids nd vitellogenin plsm levels nd spwning rhythms in Senegl sole, Sole seneglensis Ctrin Oliveir, Evristo Mñnós b, Jesus Rmos b, Frncisco Jvier Sánchez-Vázquez c* CIMAR/CCMAR, Algrve University, Cmpus de Gmbels, Fro, Portugl. ccoliveir@ulg.pt b Deprtment of Fish Physiology nd Biotechnology, IATS/CSIC, Cstellón, Spin. evristo@its.csic.es nd rmos@its.csic.es c Deprtment of Physiology, Fculty of Biology, Murci University, Murci, Spin. Running title: Photoperiod effect on reproduction rhythms in sole * Corresponding Author: Dr. F. Jvier Sánchez Vázquez Deprtment of Physiology, Fculty of Biology University of Murci, Cmpus Espinrdo Murci, SPAIN jvisn@um.es Abstrct Photoperiod nd temperture re known s the min synchronizers of sesonl reproduction in fish. This pper studied the role of photoperiod on the synchroniztion of F1 Senegl sole s reproduction rhythms. Fish were mintined under constnt short-photoperiod (9L:15D) from the winter solstice onwrds (experimentl group) or under nturlly-chnging photoperiod (control group), nd wter temperture nturlly oscillted in both groups. Blood smples were collected during reproduction seson t pre-spwning (Mrch), spwning (April) nd 1

2 post-spwning (My) to determine the endocrine sttus. Spwning events nd egg qulity prmeters were lso monitored. The results reveled significnt increse in nocturnl meltonin concentrtion from Mrch to My in the control group, while in the experimentl group such sesonl chnge did not occur. As to plsm levels of vitellogenin, testosterone, estrdiol nd 11keto-testosterone, differences between groups were found mostly in Mrch, while in April nd My levels were often similr. Spwning ws observed in both groups, lthough the experimentl group strted slightly erlier nd lso finished erlier thn the control group, perhps s result of the increse in sex steroids nd VTG observed t prespwning. Briefly, reproduction rhythms persisted in the bsence of the nturl lengthening of photoperiod, lthough photoperiod mnipultion ltered the sesonl modultion of meltonin, incresed sex steroids nd vitellogenin t pre-spwning, nd slightly dvnced the timing of spwning. Keywords: meltonin, photoperiod, sex steroids, Sole seneglensis, spwning, vitellogenin 1. Introduction Reproduction in fish is rhythmic nd timed to gurntee the mximum survivl of the offspring. Environmentl cues, such s photoperiod nd temperture cycles, synchronize the internl timing system tht controls breeding (Bromge et l., 2001). The pinel orgn is possible cndidte to be the meditor between environmentl cycles nd the brin-pituitrygond (BPG) xis, controlling reproduction timing. This orgn trnsduces dily nd sesonl cycles into neurohormonl signl, producing meltonin (MEL) nd working both s clock nd clendr in order to provide the BPG xis with environmentl informtion, lthough the exct pthwy of its ction is yet unknown (Flcón et l., 2007). Sex steroids re produced in the gonds nd ct both on the brin nd nterior lobe of the pituitry to control the production of luteinizing hormone (LH) (Yron et l., 2003), while vitellogenin (VTG) is protein tht specificlly ppers in the blood of sexully mturing femle of oviprous nd ovoviviprous nimls during vitellogenesis in response to circulting estrogens (Specker et l., 1994). Thus, plsm concentrtions of both sex steroids nd VTG re good indictors of the sttus of the BPG xis nd hence the reproductive condition of the nimls. 2

3 The Senegl sole, Sole seneglensis, is species tht hs incited incresing interest in both quculture nd bsic reserch during the pst decdes. However, severl questions of sole quculture still need to be optimized, including the control of reproduction (Agulleiro et l., 2006; Dinis et l., 1999). The spwning seson of this species is observed minly in spring, lthough secondry seson my occur in utumn (Anguis et l., 2005). Sex steroids nd VTG exhibited nnul rhythms with high concentrtions during the pre-spwning period in spring, nd secondry, less pronounced increse round utumn (Grcí-López et l., 2006; Guzmán et l., 2008). The sesonl rhythms of meltonin hve lso been ssessed recently, with temperture modulting its nocturnl production (Ver et l., 2007). Temperture lso seemed to ply key role in reproduction timing, since Senegl sole deprived of its nturlly oscillting sesonl thermo cycle did not sustin reproduction rhythms (Grcí-López et l., 2006b; Oliveir et l., 2009), probbly due to the bsence of clock/clendr informtion provided by meltonin rhythms. However, to dte there re no studies focusing on the effect of the sesonl photoperiod cycle nd such phenomenon requires ttention in order to better understnd the synchroniztion of the sesonl reproduction rhythms in this species nd thus optimize breeding protocols. The im of this reserch ws to investigte the role of photoperiod in the synchroniztion of reproduction rhythms in Senegl sole subjected to two different photoperiodic conditions: nturlly fluctuting vs. constnt short-photoperiod from winter solstice onwrds. The levels of MEL, sex steroids (testosterone, T, 17β-estrdiol, E 2, nd 11keto-testosterone, 11KT) nd VTG were mesured during pre-spwning, spwning nd post-spwning periods, s well s spwning nd egg qulity prmeters. 2. Mteril nd Methods 2.1. Animls nd housing Twenty-five Senegl sole (Sole seneglensis Kup, 1858) from the reserch centre Instituto de Acuicultur de Torre l Sl (IATS) (40 6'N, 0 9'E, Cstellón, Spin) were used for this reserch. They were from n F1 genertion nd hd men body mss of ± 77.5 g nd men body length of 47.5 ± 0.6 cm. They were divided in n experimentl group, rered under constnt short photoperiod (L9:D15) from winter solstice onwrds (n=13, 7 femles nd 6 mles) nd control group, rered under nturl photoperiod conditions (n=12, 6 femles nd 6 mles). Both groups were rered indoors in three cylindricl fiberglss tnks 3

4 (4000 L, 1 m depth, 4.9 m -2 ) equipped with egg collectors, which were checked dily to verify the presence of eggs. The wter ws provided with constnt flow of running mrine wter from the Mediterrnen Se, with nturl nnul cycle of temperture (rnging between 13.5 nd 18 ºC during the smpling months) nd slinity of 37. Fish were fed d libitum in the morning five dys week, using dry pellet feed (ProAqu) nd nturl food, consisting of chopped fresh mussels nd frozen squid. All sole were individully lbeled with n internl pssive integrted trnsponder (PIT) tg system, enbling identifiction of ech fish in repeted smplings. Hndling of the fish ws in ccordnce with the Europen Union Directive (EEC, 1986) for the protection of nimls used for experimentl nd other scientific purposes Experimentl design To determine the influence of the photoperiod sesonl cycle on the regultion of Senegl sole reproduction, fish from the experimentl group were submitted to constnt short photoperiod from the 21 st December until the reproduction seson. This photoperiod ws obtined by covering the two experimentl group tnks with lightproof cover, nd illuminting the wter surfce with florescent bulb of 11 w nd 240 v (19E1000, RS Components, Ltd, Northnts, UK) with mximum light intensity of 500 lux. The tnk contining the control group ws just covered with rffi cloth, which provided nturl photoperiod, with mximum light intensity of 600 lx t the wter surfce during the dy. Smpling ws performed t three criticl moments of the reproductive cycle ccording to Guzmán et l., (2008): t pre-spwning (7/8 Mrch), t full spwning (23 April) nd t postspwning (27/28 My). At ech time intervl ech fish ws blood smpled t Mid-Drk (MD) nd t Mid-Light (ML), to detect dily fluctutions on hormone levels. During the drk phse, smpling ws crried out under dim red light nd fter covering the fish hed with luminum foil. Soles previously nesthetized with 2-phenoxyethnol (0.3 ml L -1 ), were individully weighed nd identified with pit-tg lector, nd then smpled by cudl puncture, with 1 ml heprinized syringe. Blood ws collected in heprinized eppendorf tubes on ice to lter be centrifuged (3 000 g, 4 ºC, 15 min), nd then plsm ws seprted into three liquots (for MEL, sex steroids nd VTG mesurement) nd frozen t -80 ºC. Spwning events were monitored dily during reproduction seson for both groups, nd on dys spwning took plce eggs were collected nd weighed. As mesure of egg qulity, buoynt eggs were seprted from non-buoynt eggs in grduted cylinders filled with se wter (slinity 37 ) nd buoyncy % clculted. Buoynt eggs from ech spwn 4

5 were incubted for 48 h to determine htching %. Totl number of eggs produced dily ws relted to the femle biomss of the group t tht time, to clculte the reltive dily fecundity (# of eggs kg -1 ). Totl fecundity for ech group ws obtined t the end of the spwning seson nd relted to the femle biomss of the group, to clculte the reltive totl fecundity (totl # of eggs kg -1 ) (Guzmán et l., 2008) Dt nlysis Meltonin nlysis MEL levels in plsm smples were mesured by Rdioimmunossy commercil Kit (Meltonin Direct RIA, Biosource, Belgium), with lower limit of quntifiction (LLOQ) of 2 pg/ml, s described by Oliveir et l. (2007). Vitellogenin nd Sex Steroid ELISA To ssess plsm VTG, T nd E 2 in femles, nd T nd 11KT in mles, smples were nlysed in the fcilities of the Institute of Aquculture of Torre l Sl (IATS) (Cstellón, Spin), by enzyme-linked immunosorbent ssy (ELISA) using protocols previously vlidted for Senegl sole plsm smples (Guzmán et l., 2008). Sttisticl nlysis Sttisticl tests were performed using SPSS. Dt re expressed s men ± stndrd error of the men (S.E.M.). Plsm concentrtions of MEL, VTG nd sex steroids, were subjected to one-wy ANOVA, followed by Duncn s post-hoc test (with degree of significnce of p<0.05) to determine the existence of significnt differences between smpling points. Finlly, t-student test ws performed (p<0.05) to detect the existence of differences in spwning qulity prmeters between the two groups. 3. Results In the short photoperiod group, plsm MEL concentrtions mesured t MD were sttisticlly higher thn the vlues registered t ML nd remined similr during the three months smpling took plce (oscillting slightly from ± 66.1 to ± 48.5 pg/ml). However in the control group plsm MEL vlues mesured in the MD-soles in My (274.0 ± 53.8 pg/ml) were significntly higher thn in Mrch (138.6 ± 34.1 pg/ml) (ANOVA, 5

6 Duncn s test, p<0.05), showing sesonl increse in nocturnl concentrtions (fig. 1). However, there were no significnt differences between groups t ny smpling point. Concerning the spwning rhythms, the experimentl group spwned eight times from 7 th to 29 th April, while the control group spwned lso eight times but lter, from 18 th April to 27 th My (fig. 2, tble 1). In wht refers to spwn weight per dy, egg buoyncy (%) nd dily men fecundity there were no significnt differences registered between groups (p<0.05 t- student test) lthough the vlues slightly differed (tble 1). In ll cses no fertilized eggs were obtined. Regrding VTG nd sex steroids in generl, the profiles differed between the two groups minly t pre-spwning, being very similr t full nd post-spwning. In femles, plsm VTG concentrtions in the experimentl group were significntly higher t MD in Mrch (pre-spwning) (6.2 ± 0.6 mg/ml) thn t ML in April (full spwning) (3.1 ± 0.6 mg/ml), while for the control group, MD vlues in Mrch (1.5 ± 0.4 mg/ml) were lower thn ll smplings in April nd My (men vlues 5.3 ± 0.6 nd 4.5 ± 0.5 mg/ml, respectively) (fig 3). Between groups, significntly different vlues were only observed t MD in Mrch (ANOVA, Duncn s test, p<0.05). For the sex steroids in femles the pttern ws very similr, with most differences between the two groups being found in Mrch. In the cse of T (fig. 4), the experimentl group vlues were significntly higher t ML in Mrch (1.5 ± 0.2 ng/ml) thn in ll the other smpling points, nd there were lso higher concentrtions observed t Mrch MD when compred to April MD (0.7 ± 0.2 nd 0.3 ± 0.1 ng/ml, respectively). Between groups, vlues just differed t ML in Mrch. With regrd to E 2, Mrch ML plsm concentrtion in the experimentl group were higher (7.4 ± 1.2 ng/ml) thn in the following months, while Mrch MD vlues (5.7 ± 0.6 ng/ml) just differed from April MD nd My ML. Moreover, in April nd in My MD nd ML vlues differed, thus showing dy/night differences on these months. In the control group, dy/night differences were lso observed in April, with higher vlues being detected t ML (4.6 ± 0.8 ng/ml ginst 1.5 ± 0.5 t MD ng/ml) (ANOVA, Duncn s test, p<0.05). Between groups, different plsm concentrtions of E 2 were observed in the smpling points ML in Mrch nd both t MD nd ML in My. In the cse of mles, sex steroid concentrtions (T nd 11KT) only showed different levels between groups in Mrch. With regrd to T rhythm, in both the experimentl nd control groups the plsm concentrtions registered in Mrch were significntly higher thn in the following months (3.0 ± 0.3 nd 2.4 ± 0.2 ng/ml, respectively) (fig. 5), just with the difference tht in the experimentl group dy/night differences were detected in Mrch. With 6

7 regrd to 11KT rhythms, only the experimentl group showed higher levels in Mrch, (18.3 ± 1.5 ng/ml), while control group vlues remined similr during the three smpling months (ANOVA, Duncn s test, p<0.05) (fig. 5b). 4. Discussion The group of Senegl sole subjected to constnt short photoperiod from the winter solstice onwrds filed to show the sesonl increse in nocturnl meltonin seen in the control group. However, these fish successfully spwned during the sme period (spring) s the control group but few dys erlier, perhps s result of the increse in sex steroids nd VTG observed t pre-spwning. These nimls were ble to time the spwning seson in the bsence of photoperiod informtion during three months prior to reproduction, which my suggest they used other environmentl cycles (sesonl chnges in wter temperture) or the presence of free-running reproduction rhythm. The sesonlity of reproduction in fish is known to be medited by the nturlly chnging pttern of photoperiod nd temperture during the yer, nd, depending on the species, either one or the other fctor (or both combined) is the min cue to synchronize these rhythms (Bromge et l., 2001; Flcón et l., 2010). As long-dy breeder, in sole the nturlly lengthening dy towrds spring should ply key role in triggering the onset of vitellogenesis, steroidogenesis nd spwning. According to recent investigtions (Ver et l., 2007) sole kept during the whole yer in conditions of controlled temperture suffered disruption of the MEL rhythms, lost their nnul environmentl informtion nd filed to spwn. Furthermore, under such constnt temperture conditions, the nimls lost their nnul sex steroid rhythms, preventing spwning (Grcí-López et l., 2006b; Oliveir et l., 2009). Bsed on ll the bove evidence, it seems tht the min cue Senegl sole uses is temperture, since the nimls successfully synchronized their reproduction seson without sesonl informtion on the photoperiod. In spite of the stted in Bromge et l., (2001) considering photoperiod s the principl determinnt of mturtion in fltfish, slmonids nd in most other teleosts, in Senegl sole we could be fcing different strtegy, since this fish hs evolved interesting wys to synchronize spwning. In the cse of the Eursin perch, species which lso spwns during spring, sesonl photoperiodic vritions were required to induce nd control the reproductive cycle, lthough temperture lso plyed n importnt role (Migud et l., 2006). For species such s se bss (Byrri et l., 2004; Prt et l., 1999; Znuy et l., 7

8 ) or cod (Dvie et l., 2003; Hnsen et l., 2001), studies hve demonstrted profound effect of photoperiod on gondl mturtion, fecundity, sex steroid levels nd spwning. In contrst, both cues combined re required by cyprinids for the ccurte synchroniztion of reproduction (Hontel et l., 1990). However, to the best of our knowledge, there is no evidence of other teleosts from temperte ltitudes using temperture s the min determinnt of the timing of reproduction. The MEL concentrtions reched during the night in the control group were similr to those seen in the sme species under nturl thermo nd photoperiod conditions for the sme months (Ver et l., 2007). However, in the bsence of the nturl lengthening of the photoperiod, the sesonl rise in nocturnl meltonin could not be detected within the time frme of the smplings performed, thus suggesting tht both photo nd thermo informtion re being trnsduced by the pinel orgn, modulting the meltonin rhythm. In the cse of the se bss, in different sesons of the yer with similr vlues of temperture, the rise in meltonin levels differed gretly ccording to the photoperiod (Grcí-Allegue et l., 2001), suggesting role for both these environmentl fctors in the modultion of nocturnl meltonin. The rhythm of VTG in femles ws similr in the two groups, except during prespwning, nd its concentrtions remined high in ll three months, generlly similr to those described before by Guzmán et l., (2008). According to this uthor, Senegl sole showed high circulting VTG levels during severl months, suggesting vitellogenesis in this species s long term process. In our study, femles from both groups lso presented high concentrtions of VTG during the three months, suggesting on-going vitellogenesis during this period. With regrd to sex steroids in femles, both E 2 nd T rhythms were similr to vlues observed for the VTG: differences between groups were observed t pre-spwning, but during spwning nd post-spwning vlues were similr in the cse of T nd lso differed in My for E 2. In mles the pttern ws lso similr, with higher concentrtions of sex steroid registered t pre-spwning in the experimentl group. The vlues recorded in our study were similr to those described in previous studies on sex steroids in Senegl sole (Grcí-López et l., 2006; Guzmán et l., 2008; Oliveir et l., 2009), lthough such comprisons should be treted crefully becuse the rhythms of steroids vry gretly between yers nd re relted with the nturl vritions in wter temperture. The fct tht the experimentl group presented significntly higher concentrtions of VTG nd sex steroid concentrtions during pre-spwning period could be responsible for the erlier spwning period observed in this group. 8

9 The ppernce of dy/night differences only in some smplings of ech steroid studied (T: in Mrch for both femles nd mles of the experimentl group; E 2 : in April nd My for the experimentl group nd April for the Control group) my be due to phse shift of the dily rhythms, chnge in its timing. However, the existence of dy/night differences in sex steroids strongly suggests tht these concentrtions do not remin sttic during the dy. Curiously enough, in ll points dy/night differences were observed, concentrtions were lwys higher during the dy, with the exception of E 2 in My, when the experimentl group seemed to invert this tendency, presenting higher vlues during the night, suggesting n ltertion of the dily rhythm, nd consequently of the dily pek. However, more time points would be necessry to confirm such result. The bsence of egg fertiliztion observed in the present study is common phenomenon in the F1 genertion of Senegl sole, s described previously in the studies of Guzmán et l., (2008) nd Agulleiro et l., (2006) nd is probbly due to lck of synchrony between femles nd mles t the time of spwning. Due to this fct, in the present pper it ws not possible to determine whether the photoperiodic mnipultion could be ffecting the finl spwn qulity. However, the other prmeters ccessed such s fecundity or egg buoyncy rte seemed similr between two groups, not showing ny tendency to n ltertion of qulity. 5. Conclusions In conclusion, Senegl sole deprived from photoperiod informtion during the lst three months prior to reproduction, sustined nocturnl MEL concentrtion from Mrch to My, bolishing the sesonl rise observed in nturl conditions. However, nimls mintined their VTG nd sex steroid rhythms, lthough t higher level t pre-spwning compred with the control group. As result, the spwning rhythm persisted, though it ws slightly dvnced under constnt short-photoperiod, stressing the possibility of mnipulting spwning seson in fish frms. These findings bring bout new insights on the understnding of reproduction rhythms of Senegl sole, which represents bottle neck for quculture production of this species. 6. Acknowledgments This work ws finnced by the MEC (project Reprosole AGL CO2-01/ACU grnted to FJSV nd project AGL C03-01 to EM) nd by SENECA (Project 05690/PI/07 to FJSV). CO received FPI Fellowship from the MEC. The uthors would lso 9

10 like to thnk Cridd Meseguer nd José Mri Gúzmn for their help during smple collection nd lbortory smple nlysis. References Agulleiro, M.J., Anguis, V., Cñvte, J.P., Mrtínez-Rodríguez, G., Mylons, C.C., Cerdá, J., Induction of spwning of cptive rered Senegl sole (Sole seneglensis) using different dministrtion methods for gondotropin-relesing hormone gonist. Aquculture 257, Anguis, V., Cñvte, J.P., Spwning of cptive Senegl sole (Sole seneglensis) under nturlly fluctuting temperture regime. Aquculture 243, Byrri, M.J., Rodríguez, L., Znuy, S., Mdrid, J.A., Sánchez-Vázquez, F.J., Kgw, H., Okuzw, K., Crrillo, M., Effect of photoperiod mnipultion on the dily rhythms of meltonin nd reproductive hormones in cged Europen se bss (Dicentrrchus lbrx). Gen. Comp. Endocrinol. 136, Bromge, N., Porter, M., Rndll, C., The environmentl regultion of mturtion in frmed finfish with specil reference to the role of photoperiod nd meltonin. Aquculture 197, Dvie, A., Porter, M.J.R., Bromge, N.R., Photoperiod mnipultion of mturtion nd growth of Atlntic cod (Gdus morhu). Fish Physiol. Biochem. 28, Dinis, M.T., Ribeiro, L., Sores, F., Srsquete, C., A review on the cultivtion potentil of Sole seneglensis in Spin nd in Portugl. Aquculture 176, EEC, Council Directive 86/609 EEC for the protection of nimls used for experimentl nd other scientific purposes. Officil Journl L358, Flcón, J., Besseu, L., Suzet, S., Boeuf, G., Meltonin effects on the hypothlmo-pituitry xis in fish. Trends Endocrinol. Metb. 18, Flcón, J., Migud, H., Muñoz-Cueto, J.A., Crrillo, M., Current knowledge on the meltonin system in teleost fish. Gen. Comp. Endocrinol. 165, Grcí-Allegue, R., Mdrid, J.A., Sánchez-Vázquez, F.J., Meltonin rhythms in europen se bss plsm nd eye: influence of sesonl photoperiod nd wter temperture. J. Pinel Res. 31, Grcí-López, A., Anguis, V., Couto, E., Cnrio, A.V.M., Cñvte, J.P., Srsquete, C., Mrtínez- Rodríguez, G., Non-invsive ssessment of reproductive sttus nd cycle of sex steorid levels in cptive wild broosdstock of seneglese sole Sole senglensis (Kup). Aquculture 254, Grcí-López, A., Pscul, E., Srsquete, C., Mrtínez-Rodríguez, G., 2006b. Disruption of gondl mturtion in cultured Seneglese sole Sole seneglensis Kup by continuous light nd/or constnt temperture regimes. Aquculture 261, Guzmán, J.M., Norberg, B., Rmos, J., Mylons, C.C., Mñnós, E., Vitellogenin, steroid plsm levels nd spwning performnce of cultured femle Seneglese sole (Sole seneglensis). Gen. Comp. Endocrinol. 156, Hnsen, T., Krlsen, Ï., Trnger, G.L., Hemre, G.I., Holm, J.C., Kjesbu, O.S., Growth, gondl development nd spwning time of Atlntic cod (Gdus morhu) rered under different photoperiods. Aquculture 203,

11 Hontel, A., Stcey, N.E., Cyprinide. In: Munro, A.D., Scott, A.P., Lm,T.J. (Eds.), Reproductive sesonlity in teleosts: environmentl influences, CRC Press, Boc Rtón, FL, pp Migud, H., Wng, N., Grdeur, J.-N., Fontine, P., Influence of photoperiod on reproductive performnces in eursin perch Perc fluvitilis. Aquculture 252, Oliveir, C., Ver, L.M., López-Olmed, J.F., Guzmán, J.M., Mñnós, E., Rmos, J., Snchez- Vzquez, F.J., Monthly dy/night chnges nd sesonl dily rhythms of sexul steroids in Senegl sole (Sole seneglensis) under nturl fluctuting or controlled environmentl conditions. Comp. Biochem. Physiol. A 152, Prt, F., Znuy, S., Bromge, N., Crrillo, M., Effects of constnt short nd long photoperiod regimes on the spwning performnce nd sex steroid levels of femle nd mle se bss. J. Fish Biol. 54, Specker, J.L., Sullivn, C.V., Vitellogenesis in fishes: sttus nd perspectives. In: Dvey, K.G., Peter, R.E., Tobe, S.S. (Eds.), Perspectives in Endocrinology, Ntionl Reserch Council, Ottw, pp Ver, L.M., Oliveir, C., López-Olmed, J.F., Rmos, J., Mñnós, E., Mdrid, J.A., Sánchez- Vázquez, F.J., Sesonl nd dily plsm meltonin rhythms nd reproduction in senegl sole kept under nturl photoperiod nd nturl or controlled wter temperture. J. Pinel Res. 43, Yron, Z., Gur, G., Melmed, P., Rosenfeld, H., Elizur, A., Levvi-Sivn, B., Regultion of fish gondotropins. Int. Rev. Cytol. 225, Znuy, S., Prt, F., Crrillo, M., Bromge, N.R., Effects of constnt photoperiod on spwning nd plsm 17ß-oestrdiol levels of se bss (Dicentrrchus lbrx). Aqut. Living Resour. 8, Figure Cptions Figure 1. Plsm concentrtions of meltonin during spwning seson (vlues expressed s men ± S.E.M.) in Senegl sole exposed to n experimentl short photoperiod (mid-drk: blck spotted brs; mid-light: white spotted brs) nd exposed to nturl oscillting photoperiod (mid-drk: blck brs; mid-light: white brs). Different letters (, b, c), indicte groups with significnt differences between smpling points (ANOVA, Duncn s test p<0.05). Figure 2. Spwning events observed during the spwning seson in group of Senegl sole exposed to n experimentl short photoperiod (blck brs) nd nother group exposed to nturl oscillting photoperiod (grey brs) (vlues expressed s g of eggs dy -1 ). Figure 3. Plsm concentrtions of vitellogenin during spwning seson (vlues expressed s men ± S.E.M.) in Senegl sole femles exposed to n experimentl short photoperiod (middrk: blck spotted brs; mid-light: white spotted brs) nd exposed to nturl oscillting 11

12 photoperiod (mid-drk: blck brs; mid-light: white brs). Different letters (, b, c) indicte groups with significnt differences between smpling points (ANOVA, Duncn s test p<0.05). Figure 4. Plsm concentrtions of sex steroids (testosterone, T nd estrdiol, E 2 ) during spwning seson (vlues expressed s men ± S.E.M.) in Senegl sole femles exposed to n experimentl short photoperiod (mid-drk: blck spotted brs; mid-light: white spotted brs) nd exposed to nturl oscillting photoperiod (mid-drk: blck brs; mid-light: white brs). Different letters (, b, c, d, e) indicte groups with significnt differences between smpling points (ANOVA, Duncn s test p<0.05). Figure 5. Plsm concentrtions of sex steroids (testosterone, T nd 11keto-testosterone, 11- KT) during spwning seson (vlues expressed s men ± S.E.M.) in Senegl sole mles exposed to n experimentl short photoperiod (mid-drk: blck spotted brs; mid-light: white spotted brs) nd exposed to nturl oscillting photoperiod (mid-drk: blck brs; midlight: white brs). Different letters (, b, c, d) indicte groups with significnt differences between smpling points (ANOVA, Duncn s test p<0.05). 12

13 Tble 1. Spwning chrcteristics for both control nd experimentl groups Control group Experimentl Group # femles 6 7 Sex rtio (f:m) 1:1 1:0.86 # spwns 8 8 1st spwning dy lst spwning dy Dily fecundity 12.50± ±1.89 Totl fecundity Egg buoyncy (%) 16.30± ±6.86 Htching rte (%)

14 Figure 1 c c b Mrch April My

15 Figure Eggs (g dy 1 ) Apr 13 Apr 20 Apr 27 Apr 4 My 11 My 18 My 25 My 2007

16 Figure 3 b b b b b b b b c Mrch April My

17 Figure 4 () d b d d d d d cd d d Mrch April My (b) b b de b d de cde e de Mrch April My

18 Figure 5 () b cd d d d d d d d Mrch April My (b) b b b b c Mrch April My