Daily endocrine profiles in parr and smolt Atlantic salmon

Comparative Biochemistry and Physiology, Part A 151 (2008) 698–704

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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p a

Daily endocrine profiles in parr and smolt Atlantic salmon Lars O.E. Ebbesson a,c,⁎, Björn Th. Björnsson b, Peter Ekström c, Sigurd O. Stefansson a a b c

Department Biology, University of Bergen, High Technology Centre, N-5020 Bergen, Norway Fish Endocrinology Laboratory, Department of Zoology/Zoophysiology, University of Gothenburg, Box 463, SE-405 30 Göteborg, Sweden Department of Cell and Organism Biology, Lund University, Helgonavägen 3, SE-22362 Lund, Sweden

a r t i c l e

i n f o

Article history: Received 7 May 2008 Received in revised form 13 August 2008 Accepted 18 August 2008 Available online 24 August 2008 Keywords: Development Diurnal Fish Hormone Photoperiod Rhythm Salmo salar Smoltification

a b s t r a c t To elucidate possible mechanisms behind the endocrine control of parr–smolt transformation, the daily plasma profiles in thyroid hormones (TH; free thyroxine (FT4), total thyroxine (TT4), and total 3,5,3′triiodothyronine (TT3)), growth hormone (GH) and cortisol were studied in Atlantic salmon parr and smolts under simulated-natural winter (8 L:16D) and spring (16.5 L:7.5D) photoperiods, respectively. Overall, TT4, TT3 and GH levels were higher in smolts than in parr, whereas FT4 levels fluctuated within the same range in parr and smolts. Significant diurnal changes in plasma TH were present in parr. Both FT4 and TT4 levels increased during the photophase and decreased during the scotophase, while TT3 levels followed an inverse pattern. Growth hormone showed no significant changes in parr. Changes in FT4, TT4, GH, and cortisol, but not TT3, levels, were observed in smolts with peak levels during both the photophase and scotophase for FT4, TT4 and GH. Plasma cortisol was not assayed in parr but in smolts the peaks were associated with dusk and dawn. In addition to the general increases in TH, GH and cortisol, the distinct endocrine differences in nighttime levels between parr in the winter and smolts in the spring suggest different interactions between TH, GH, cortisol and melatonin at these different time points. These spring scotophase endocrine profiles may represent synergistic hormone interactions that promote smolt development, similar to the synergistic endocrine interactions shown to accelerate anuran metamorphosis. The variations in these diurnal rhythms between parr and smolts may represent part of the endocrine mechanism for the translation of seasonal information during salmon smoltification. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Many vertebrates use the annual cycle of changes in daylength to time their seasonal physiological and behavioral functions. For anadromous salmonids, the change in photoperiod in spring and/or autumn is well accepted as the major driving force behind seasonally triggered events, such as parr–smolt transformation (smoltification) and sexual maturation (Hoar, 1988; Randall and Bromage, 1998; Bromage et al., 2001; Björnsson et al., 2002, Stefansson et al., 2008). Smoltification is a developmental process which changes the resident freshwater parr into seaward migrating smolts, prepared for seawater existence and imprinted on their natal stream enabling them to return as adults to reproduce (Hoar, 1988; McCormick et al., 1998). This transitional period involves specific sequences of events involving neural, endocrine, physiological, structural and behavioural changes (Hoar, 1988; Boeuf, 1993; McCormick et al., 1998; Ebbesson et al., 2003; Stefansson et al., 2008). Thyroid hormones (TH), growth hormone (GH) and cortisol are known to have important roles in these processes and in recent years considerable progress has been made in resolving ⁎ Corresponding author. Department of Biology, University of Bergen, PO Box 7800, N-5020 Bergen, Norway. Tel.: +47 730598600; fax: +47 46 2224425. E-mail address: [email protected] (L.O.E. Ebbesson). 1095-6433/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.08.017

various aspects of their biology in salmonid fishes, including their role in smoltification and the influence of photoperiod (Leatherland, 1994; Specker et al., 2000; Power et al., 2001; Björnsson et al., 2002; Reddy and Leatherland, 2003; Blanton and Specker, 2007; Ebbesson et al., 2007, Stefansson et al., 2007, 2008). However, it is still largely unknown if seasonal changes in diurnal endocrine rhythms are associated with parr–smolt transformation in salmon. Fluctuations in circulating TH, GH and cortisol levels, in relation to photoperiod and feeding schedules, have been shown to display at least as many profiles as species studied (Rydevik et al., 1984: Laidley and Leatherland, 1988; Bates et al., 1989; Boujard and Leatherland, 1992; Holloway et al., 1994; Reddy and Leatherland, 1994; Gomez et al., 1996; Gomez et al., 1997; Leiner et al., 2000; Boujard, 2001; Leiner and MacKenzie, 2001; Reddy and Leatherland, 2003; Loter et al., 2007). Generally, T4 levels increase postprandially during the photophase and decrease during the scotophase, yet this varies among species, feeding time and season (Laidley and Leatherland, 1988; Bates et al., 1989; Boujard and Leatherland, 1992; Holloway et al., 1994; Reddy and Leatherland, 1994; Leiner et al., 2000; Boujard, 2001; Leiner and MacKenzie, 2001; Reddy and Leatherland, 2003; Loter et al., 2007), with additional increases occurring during the scotophase or extending from the photophase into the scotophase (Laidley and Leatherland, 1988; Leiner and MacKenzie 2001). In trout, GH levels display a variety of

L.O.E. Ebbesson et al. / Comparative Biochemistry and Physiology, Part A 151 (2008) 698–704 Table 1 Overall mean values of measured parameters of parr in December (n = 84) and smolts in April (n = 90)

FT4 (ng 100 mL− 1) TT4 (ng mL− 1) TT3 (ng mL− 1) GH (ng mL− 1) Cortisol (ng mL− 1) Mass (g) Fork Length (cm) Condition Factor NKA activity

Parr

Smolt

P-value

0.60 ± 0.04 3.6 ± 0.08 2.0 ± 0.06 0.81 ± 0.05 – 59.6 ± 1.0 17.4 ± 0.12 1.10 ± 0.008 3.0 ± 0.4

0.94 ± 0.06 9.4 ± 0.33 2.9 ± 0.10 6.15 ± 0.37 84.1 ± 6.2 78.8 ± 1.6 20.0 ± 0.14 0.96 ± 0.006 12.7 ± 0.8

b0.0001 b0.0001 b0.0001 b0.0001 – b0.0001 b0.0001 b0.0001 b0.0001

The data are presented as mean ± SEM. Significant differences between parr and smolts were determined using the Mann–Whitney U test.

profiles with increases in the scotophase and/or the photophase (Gelineau et al., 1996; Reddy and Leatherland 2003) and in salmon nocturnal GH increases have been reported in parr (Bates et al., 1989). Circulating cortisol levels vary with season in trout with increases during the scotophase at specific times of the year (Pickering and Pottinger, 1983; Laidley and Leatherland, 1988) and in salmon plasma cortisol levels show no diel pattern in parr under a winter photoperiod, but are elevated during the scotophase in smolts under a spring photoperiod (Thorpe et al., 1987). With the exception of cortisol, less is known about the influence of photoperiod on daily hormone profiles at different developmental states. There is a general agreement that increases in circulating hormone levels regulate smolt development, similar to their roles during metamorphosis in flatfish (Power et al., 2001, Blanton and Specker, 2007). In salmon, increases in daylength stimulate the development (Stefansson et al., 2008), yet continuous Exposure to 24 h light disrupts it (Björnsson et al., 2000; Stefansson et al., 2007; Ebbesson et al., 2007). This suggests that seasonal changes in the cycling between day and night promote this development, possibly through their influence on diel hormone cycles. It is noteworthy that in salmon melatonin levels increase rapidly in the scotophase and remain elevated until the onset of the photophase (Randall et al., 1995; Bromage et al., 2001), and in amphibian metamorphosis, increases in corticosteroids, thyroid hormones and melatonin concomitantly accelerate development and play specific roles in the transformation (Wright, 2002, Wright et al., 2003, Wright and Bruni, 2002). For these reasons, the present study examines diurnal patterns between parr in a winter photoperiod and developing smolts in a spring photoperiod to determine if interactions occur in the spring that do not occur in the winter. 2. Materials and methods 2.1. Fish Four hundred juvenile Atlantic salmon (Salmo salar) parr were transferred in November to indoor flow-through tanks with 400 L rearing volume maintained at 7 ± 1 °C freshwater and exposed to a simulated natural photoperiod (SNP, 60°N) including twilight at the High Technology Centre, Bergen, Norway. Each tank was illuminated during the photophase by one 36 W fluorescent daylight tube. Photon irradiance at the tank bottom was ca. 5 μmol m− 2 s− 1. Fish were fed a commercial dry diet (Nutra Svev, T. Skretting A/S, Stavanger, Norway) in excess from automatic feeders at approximately hourly intervals during the light phase. Feeding was halted the evening prior to each experiment.

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10 mM EDTA, 50 mM imidazole, pH 7.3) for determination of branchial Na+,K+-ATPase (NKA) activity as a criterion of smolt status (McCormick 1993). Condition factor (CF) was calculated as CF = (W × (L)− 3) × 100. Blood samples were obtained as described below. 2.2.1. Parr under winter photoperiod The four hundred fish were divided equally among six identical tanks two weeks prior to sampling and maintained on SNP, corresponding to 8 L:16D in early December. During the experiment, samples were taken every four hours for 48 h, starting at 10:00 h on December 8. Following the 10:00 h sampling on December 9, sampling was shifted two hours to 12:00 h, so that subsequent sample times would fall between times from the previous 24 h. Six fish were removed from different tanks at each sampling within each 24 h period, allowing 24 h between sampling from the same tank. After the sampling, the remaining 300 fish were combined into two tanks and maintained on SNP until two weeks prior to the spring experiment. 2.2.2. Smolts under spring photoperiod In mid-April, three hundred fish were divided equally among 12 identical tanks two weeks prior to sampling. Photoperiod in mid-April corresponded to 16.5 L:7.5D. Six fish were removed from different tanks 12 times at two-h intervals, over a 24-h period, starting at 12:00 h on April 21. 2.3. Na+,K+-ATPase activity Branchial NKA activity to assess smolt status was determined in 12 parr and 12 smolts according to the method of McCormick (1993). 2.4. Plasma collection Six fish were quickly dip netted out of the tank and anaesthetised directly in MS222 (Finquel, USA) and blood collected from the caudal vessels using heparinized syringes. The fish sampled during the dark periods were collected and anaesthetised under darkness and covered with black plastic during the blood collection. Blood was placed on ice until centrifuged within 15 min of first sample and plasma collected and stored at −80 °C. 2.5. Hormone analyses All assays in this study were performed in duplicate and averaged. 2.5.1. Thyroid hormones (TH) Commercially available radioimmunoassay (RIA) kits were used to measure TT4 and TT3 (RIA-gnost T4 and T3 coated tube kits, Cis-Bio, France), and FT4 (Free-T4 Equilibrium Dialysis RIA Kit, Nichols Institute, USA) according to Ebbesson et al. (1998). Note that FT4 levels are expressed in ng/100 mL and not ng/mL as are the other hormones, i.e. FT4 levels represent approximately 0.1% of the plasma TT4.

Table 2 Correlations between TH, GH and cortisol levels in Atlantic salmon parr and smolts FT4 FT4 TT4 TT3 GH

1 − .09 (.50) − .48 (− .20) .06 (− .12) (− .13)

TT4

TT3

GH

Cortisol

1 .36 (.31) .10 (− .21) (−.33)

1 − .12 (.14) (− .10)

1

2.2. Sampling

Cortisol

The weight (W) and fork length (L) of each fish were measured, and the 2nd gill arch taken and quickly frozen SEI buffer (250 mM sucrose,

The data represents data from the first 24 h period for parr in December (10.00–10.00, n = 48) and smolts (in parentheses) in April (12.00–12.00, n = 78). Values in bold indicate a significant difference using Fisher's r to z test.

(.05)

1

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2.5.2. Growth hormone (GH) Plasma GH levels were quantified by RIA for sGH according to Björnsson et al. (1994).

2.5.3. Cortisol Plasma cortisol levels were quantified by a direct immunoassay (EIA) as outlined by Carey and McCormick (1998). Briefly, 96-well microtiter

Fig. 1. Daily hormone profiles of plasma free thyroxine, total thyroxine, total triiodothyronine, growth hormone in Atlantic salmon parr (left) and smolts (right). Shaded areas indicate scotophase. Each data point represents the mean ± SEM. (n = 6) and points are significantly different if they are labelled with different letters.

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plates were coated with polyclonal rabbit anti-cortisol antibody (Cat # F3314, Endocrine Science Products; diluted 1:16000) and incubation performed using either 100 µL cortisol-horseradish peroxidase conjugate (obtained from Coralee Munro, University of California, Davis, CA, USA; diluted 1:120 000)+2.5 µL sample or cortisol standard (Cat # T0440, Sigma) in each well. Color development using 3,3′,5,5′-tetramethylbenzidine (Cat # T0440, Sigma) was terminated with 0.5 M HCl and absorbance read at 405 nm by a temperature-controlled plate reader. Maximum binding (B0; 2.5 μL of EIA buffer+100 µL cortisol-horseradish peroxidase) and non-specific binding (NSB; 102.5 μL EIA buffer) were determined on each plate. 2.6. Statistics Statistical differences were defined using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference test for multiple pair-wise comparisons. When necessary, data were logtransformed to remove heterogeneity in variance between groups. The data in Table 1 were evaluated by the Mann–Whitney test. The relationships between plasma FT4, TT4, TT3, GH and cortisol levels in parr and in smolts were evaluated through correlations between these hormones during the first 24 h period only, to minimize starvation effects (Table 2). Statistically significant differences were accepted at p b 0.05. All data are given as means ± standard error of the mean (SEM).

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3.3.2. Smolts under spring photoperiod Plasma TT3 levels were on average higher in smolts than parr during the first 24-hour period (Table 1). In smolts, no significant daily changes in plasma TT3 levels were found (Fig. 1). 3.4. Total thyroxine (TT4) 3.4.1. Parr under winter photoperiod In parr, plasma TT4 levels changed over the sampling period (p b 0.002, Fig. 1). TT4 levels increased during the photophase, with lower levels were observed during the late scotophase. 3.4.2. Smolts under spring photoperiod Plasma TT4 levels were on average higher in smolts than parr during the first 24-hour period (Table 1). In smolts, plasma TT4 levels showed a daily change (p b 0.01, Fig. 1), with TT4 levels peaking during the day to 11.6 ng mL− 1, decreasing to 7.6 ng mL− 1 in early scotophase, followed by an increase to 10.6 ng mL− 1 at mid scotophase and then a decline to the lowest levels of 5.1 ng mL− 1 at the end of the scotophase, at which time TT4 levels rapidly increased to similar high levels observed in the previous photophase. 3.5. Growth hormone (GH) 3.5.1. Parr under winter photoperiod In parr, plasma GH levels showed no significant daily change (Fig. 1).

3. Results 3.1. Characteristics of parr and smolts In December, the parr displayed characteristic parr marks and had a mean weight of 59.6 ± 1.0 g and fork length of 17.4 ± 0.12 cm (Table 1). In April, the smolts were silvery and had a mean weight of 78.8 ± 1.6 g and fork length 20.0 ± 0.14 cm. The condition factor declined from parr to smolts, 1.10 ± 0.008 and 0.96 ± 0.006, respectively. Gill NKA activity increased from parr to smolts, from 3.0 ± 0.4 to 12.7 ± 0.8 μmol ADP × mg prot.− 1 × h− 1, respectively (Table 1).

3.5.2. Smolts under spring photoperiod Plasma GH levels were on average higher in smolts than parr during the first 24-hour period (Table 1). In smolts, significant changes in plasma GH levels were observed (p b 0.0001, Fig. 1) with levels increasing significantly during the late photophase from 4.7 to 8.4 ng mL− 1 followed by a rapid decrease to 3.5 ng mL− 1 at the beginning of the scotophase. GH levels increased to peak levels between 7.1 and 9.5 ng mL− 1 for the duration of the scotophase and beginning of the photophase, followed by a rapid decline to basal levels (3.0–4.2 ng mL− 1, Fig. 1). 3.6. Cortisol

3.2. Free thyroxine (FT4) 3.2.1. Parr under winter photoperiod In parr, plasma FT4 levels showed significant daily changes (pb 0.0001, Fig. 1) where levels increased from 0.4 to 1.4 ng dl− 1 during the photophase followed by a rapid decline to basal levels of 0.4 ng dl− 1 during the scotophase. There were no differences between peak FT4 levels during the first and second photophases, although FT4 levels were higher during the second scotophase (0.9 ng dl− 1) compared with the first (0.4 ng dl− 1).

3.6.1. Parr under winter photoperiod Due to limited plasma volume, cortisol levels could not be measured in the parr samples.

3.2.2. Smolts under spring photoperiod Plasma FT4 levels were on average higher in smolts than parr during the first 24-h period (Table 1). In smolts, FT4 levels showed significant daily changes (p b 0.0001, Fig. 1) with day levels increasing to 1.1 ng 100 mL− 1, decreasing again before the scotophase to 0.5 ng 100 mL− 1 and increasing during the scotophase to 1.1 ng 100 mL− 1. FT4 levels increased during the early photophase and by noon the levels ended on levels higher than 12.00 h the previous day. 3.3. Total triiodothyronine (TT3) 3.3.1. Parr under winter photoperiod In parr, plasma TT3 levels showed a significant daily change (p b 0.003, Fig. 1). Plasma TT3 levels decreased during the first photophase from 2.1 to 1.6 ng mL− 1 followed by a gradual increase during the first scotophase to 2.3 ng mL− 1. During the second 24 h period, TT3 levels followed the same general pattern as for the first cycle.

Fig. 2. Daily plasma cortisol levels in Atlantic salmon smolts. Shaded areas indicate scotophase. Each data point represents the mean ± SEM. (n = 6) and points are significantly different if they are labelled with different letters.

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3.6.2. Smolts under spring photoperiod In smolts, cortisol levels showed significant daily changes (p b 0.0001, Fig. 2) with levels increasing during the early scotophase from 65 to 137 ng mL− 1, followed by a rapid decrease to 53 ng mL− 1. The levels remained low during the middle of the scotophase followed by an increase to peak levels at the end of the scotophase where they remained high during the early photophase, dropping again to low levels. 3.7. Relationships between plasma TH and GH levels over a 24 h period The relationships between plasma FT4, TT4, TT3 and GH levels in parr and smolts were evaluated through correlations between these hormones in parr and smolts during the first 24 h period (Table 2). In parr, FT4 was only correlated with TT3, while in smolts FT4 was positively correlated with TT4. Plasma TT4 levels were positively correlated with TT3 in parr and smolts, while it was negatively correlated with cortisol (Table 2). 4. Discussion Smoltification-related increases in hormone levels are well documented in salmon (Boeuf, 1993; Stefansson et al., 2008), yet only cortisol diel profiles have been demonstrated during smoltification (Thorpe et al., 1987). This is the first study in salmonids to investigate daily profiles in plasma FT4 levels and the first study in salmon to compare daily profiles of plasma THs and GH levels between parr and smolt stages under their respective photoperiods. The present comparison between parr and smolt Atlantic salmon under winter and spring photoperiods, respectively, demonstrates that plasma THs and GH levels change in their daily profiles and/or their overall levels. Interestingly, thyroxine availability (FT4 levels) is inverse to T3 levels in parr, with increasing FT4 levels during the day and decreasing during the night. In smolts however, FT4 levels increase during the day, decease at dusk and increase again in the middle of the night. The elevated and daily change in GH and cortisol levels in smolts are among the factors that most likely influence changes in TH profiles as they interplay with both T4 production and conversion of T4 to T3. Thyroid hormones have been suggested to play a role in a number of processes during smoltification, including the control of body growth and silvering, metabolism, lipid mobilization, downstream migration, osmoregulation and olfactory imprinting (Hoar, 1988; Boeuf, 1993; Morin et al., 1997; Specker et al., 2000; McCormick, 2001; Blanton and Specker, 2007). The present study demonstrates that daily plasma FT4 profiles change significantly in parr and smolts with peak values attained in the photophase. In parr, a rapid decline in FT4 levels is observed in the early scotophase, whereas in smolts, FT4 levels decrease already in the late photophase. This decrease may result from negative feedback to the neuroendocrine system after FT4 levels reach a certain threshold level (see Leatherland, 1994; Blanton and Specker, 2007) and appears to be consistent with previous studies on salmon using this method of FT4 measurement (Ebbesson et al., 2000). The fact that FT4 levels decline during the photophase in smolts and not in parr suggests that either T4 production is higher in smolts, and/or with a longer photophase the postulated threshold level is reached prior to the scotophase (see below). It is also possible that the decrease in T4 levels in smolts is a consequence of increased cortisol and GH levels during the late photophase and late scotophase (Figs. 1 and 2) as cortisol regulates aspects of TH system (Redding et al., 1991; Blanton and Specker, 2007) and GH increases the conversion of T4 to T3 (Maclatchy and Eales, 1990). The combination of these increases in smolts, together with the roles of melatonin in TH regulation and development in vertebrates, could result in increased conversion of T4 to T3, increasing its distribution into target tissues and promoting development (Wright, 2002).

Circulating FT4 levels have two main functions in thyroid hormone regulation. Firstly, FT4 provides the substrate for 5′-monodeiodinase in the peripheral tissues, which converts L-thyroxine to T3. T3 is believed to be the more biologically active of the two hormones. Secondly FT4 is thought to provide a negative feedback on T4 production (Blanton and Specker, 2007). In the present study, the role of FT4 as a substrate for T3 is indicated in parr, which showed increased TT3 levels during the scotophase as FT4 levels decreased. Furthermore, the role of FT4 as a feedback messenger was indicated in smolts, where FT4 levels appeared to have a specific maximum threshold level, which, when reached, was followed by a rapid decline in FT4 accompanied by a more gradual decline in TT4. As reported previously by Ebbesson et al. (1998, 2000), the FT4 assay used in the present study has been designed to minimize stripping of T4 from low affinity plasma binding sites in order to determine trends, rather than absolute changes in FT4 levels, which would otherwise be masked by changes in plasma binding sites. The present study confirms that this method for measuring FT4 levels avoids stripping of T4 from plasma binding sites as demonstrated by no difference in the diel range of FT4 levels between parr and smolts, even though the TT4 levels increase 3-fold in smolts. The present findings further suggest that plasma thyroxine binding sites change during smoltification, increasing the TT4 pool, as FT4 levels stay within the same range in parr and smolts, despite the increase in TT4 levels. This is supported by recent data showing that a specific thyroxine distributor protein appears during smoltification (Richardson et al., 2005). This interpretation of the present data is contrary to previous results in Atlantic salmon which show that FT4 and TT4 levels mimic each other and the suggestion that no change in thyroxine binding proteins occur during smoltification (Boeuf et al., 1989). The present study demonstrates that the changes in FT4 levels observed by Ebbesson et al. (2000) result mainly from daily FT4 changes and not a seasonal increase in FT4 levels, as there is a similar increase in FT4 levels following lights-on in both parr and smolts and the levels stay within the same range during both periods. The parr TT4 levels in the present study are in general agreement with the only previous study reporting daily profiles in plasma TT4 and TT3 levels in salmon (Rydevik et al., 1984). In that study, plasma TT4 levels were significantly higher in May than in March, in agreement with the present data. Plasma TT3 levels on the other hand differ between studies. Rydevik et al., 1984 found no significant daily change in plasma TT3 levels in March, whereas in May, a significant daily change was observed. Further, overall TT3 levels were significantly lower in May compared with March parr, while in the present study the reverse pattern is observed (see Table 1). These differences in circulating thyroid hormone levels demonstrate that either developmental and/or seasonal changes occur in this system. At this point the specific roles and functions of the differences remain unknown although recent evidence suggests the role of TH on CRF neurogenesis during smoltification (L. Ebbesson et al. unpublished observations). It should be noted that the TT4 and TT3 in circulation are mainly coming from the thyroid follicles with additional TT3 resulting from the hepatic deiodinase conversion of TT4 to TT3. Further, conversion of TT4 to TT3 through specific deiodinases in peripheral tissues has been established as a key regulator of TH actions in specific tissues including their development (see Loter et al., 2007). Therefore until diurnal changes in deiodinases is studied in parr and smolts, a clear picture of how the TH system is activated during smoltification will remain unresolved. The present study is in line with the general findings that plasma GH levels increase during smoltification (Young et al., 1989; Stefansson et al., 1991; Björnsson et al., 1995; Ebbesson et al., 2000). In the present study, daily changes in plasma GH levels are absent in parr, whereas significant daily changes are seen in smolts (Fig. 1). In contrast with the present study, Bates et al. (1989) reported that plasma GH levels in coho salmon parr (9.5 L:14.5D at 3 °C) increase 8-fold from basal levels at 24:00 h. suggesting species and/or temperature differences in the control of plasma GH levels in parr. THs and GH may be synergistic

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towards many of the processes that occur during smoltification (Hoar, 1988; Boeuf, 1993; McCormick, 2001). Thus, GH stimulates the hepatic 5′-monodeiodinase conversion of T4 to T3 (Maclatchy and Eales, 1990) and T3 increases the synthesis of GH mRNA (Moav and Mckeown, 1992). Cortisol is another important hormone during smoltification that influences both the thyroidal status and the GH system. Synergy between GH and cortisol has been shown in their influence on gill NKA activity (McCormick, 2001). In agreement with Thorpe et al. (1987), circulating cortisol levels increase nocturnally during the spring in smolts (Fig. 2). Cortisol levels were not measured in parr in the present study, but previous studies have not reported nocturnal increases in parr in the winter. The function of these nocturnal increases during smoltification is as of yet unknown, although light– dark cycles are necessary for developmental progression (Björnsson et al., 2000; Ebbesson et al., 2007; Stefansson et al., 2007). The lengthening of photoperiod in the spring is a central factor regulating the endocrine changes which stimulate smoltificationrelated transformations (Hoar, 1988; Boeuf, 1993; McCormick, 2001; Björnsson et al., 2002, Stefansson et al., 2008). The neuroendocrine system undergoes developmental changes prior to the major hormonal surges (Ebbesson et al., 2003), and it has been hypothesized that these changes in the neuroendocrine system make the system more respondent to photoperiod information (Ebbesson et al., 2003, 2007). When comparing lower mode salmon parr (salmon not reaching the size threshold needed to undergo smolt development) with smolts under similar photoperiods, the development of the endocrine system is lacking in the lower mode fish. The present findings are in line with McCormick et al. (2007) and Nordgarden et al. (2007), suggesting that parr are unable to receive and/or translate the photic signals to endocrine responses to the same extent as smolts. Variations in brain development may be one explanation. For instance, fish raised under 24 L that do not display increases in hormone levels (Stefansson et al., 2007) do not develop characteristic increased retinal innervation to the preoptic area (Ebbesson et al., 2007). Further, landlocked salmon have a dampened endocrine and physiological smolt development compared to the anadromous salmon (Nilsen et al., 2003, 2007, 2008), as shown by decreased retinal innervation and dampened corticotropin-releasing factor neurogenesis (Ebbesson et al unpublished observations). In addition to development of the light-brain-pituitary axis, changes in plasma binding proteins during smoltification also contribute to changes in circulating TH levels. We have recently shown that a specific TH distributor protein (TTR-like) appears during smoltification (Richardson et al., 2005). This may also account for differences in TT4 and TT3 daily profiles during winter and spring photoperiods. Together, endocrine increases in the day as well as the night may be a critical factor in stimulating the smolt development. In conclusion, the present data are in general agreement with the literature showing increased resting daytime levels of thyroid hormones, growth hormone and cortisol from parr to smolt (Boeuf 1993; Stefansson et al., 2008). The data further show distinct endocrine differences in nighttime levels between parr in the winter and smolts in the spring. What is found in smolts, but not in parr, are increases in thyroxine in the middle of the scotophase concomitant with increased GH levels, which is straddled by increases in cortisol levels at dusk and dawn. In parr, thyroxine and GH levels do not increase during the scotophase, and overall cortisol levels are low (Nilsen et al., 2008) and have previously not shown daily changes in parr (Thorpe et al., 1987). The differences between winter and spring endocrine profiles in the present study may represent hormone interactions that promote smolt development, in particular TH, GH and cortisol interactions with melatonin during the scotophase. This is in line with what has been demonstrated in anuran metamorphosis where increases in corticosteroids, thyroid hormones and melatonin concomitantly accelerate development and play specific roles in the transformation (Wright, 2002, Wright et al., 2003, Wright and Bruni, 2002). The lack of endogenous

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melatonin rhythm in salmon differs from anurans where developmental changes in T4 and melatonin rhythms occur even on a constant light/ dark cycle (Wright et al., 2003). The present data do not separate developmental stage from photoperiod, thus additional experiments altering photoperiods associated with the different developmental stages may resolve whether the differences are due to development or photoperiod. Further research into the molecular and cellular processes associated with the dark in the spring may shed light on the mechanisms behind seasonal development in vertebrates. Acknowledgments The authors wish to thank Gunilla Eriksson, Carina Rasmussen, Bjørn Sveinsbø, Tom Nilsen and Anna Ebbesson for excellent assistance during the study. 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