Chronobiology International, 2013; 30(5): 699–710
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ISSN: 0742-0528 print / 1525-6073 online
DOI: 10.3109/07420528.2013.782313
Differential effects of transient constant light-dark conditions on daily
rhythms of Period and Clock transcripts during Senegalese sole
metamorphosis
Águeda J. Martı́n-Robles1,2,3, David Whitmore4, Carlos Pendón2, and José A. Muñoz-Cueto1,3
Departamento de Biologı́a, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Campus de Excelencia
Internacional del Mar (CEIMAR), Puerto Real, Spain, 2Departamento de Biomedicina, Biotecnologı́a y Salud Pública,
Facultad de Ciencias, Universidad de Cádiz, Puerto Real, Spain, 3CACYTMAR, Institutos de Investigación, Campus
Universitario de Puerto Real, Puerto Real, Spain, and 4Department of Cell and Developmental Biology, Centre for Cell and
Molecular Dynamics, University College London, London, United Kingdom
Studies on the developmental onset of the teleost circadian clock have been carried out in zebrafish and, recently,
in rainbow trout and Senegalese sole, where rhythms of clock gene expression entrained by light-dark (LD) cycles
have been reported from the first days post fertilization. However, investigations of molecular clock rhythms during
crucial developmental phases such as metamorphosis are absent in vertebrates. In this study, we documented the
daily expression profile of Per1, Per2, Per3, and Clock during Senegalese sole pre-, early-, middle-, and postmetamorphic stages under LD 14:10 cycles (LD group), as well as under transient exposure to constant light (LL-LD
group) or constant dark (DD-LD group) conditions. Our results revealed that robust rhythms of clock genes were
maintained along the metamorphic process, although with declining amplitudes and expression levels. All daily
profiles were affected by transient constant conditions, in particular Per1, Per3, and Clock amplitudes and Per2
acrophase. Rhythm parameters were progressively restored upon reversion to LD cycles but even after 9 d under
cycling conditions, a prolonged effect on clock function was observed, especially in the LL-LD group. These results
reflect the differential sensitivity of clock machinery of sole to transitory light cues, being Per1 and Per3 predominantly
clock regulated and supporting the role of Per2 as part of the light input pathway. Interestingly, there is no reversal
in the phase of clock gene rhythms between pre- and post-metamorphic animals that would be coincident with
the switch from diurnal to nocturnal locomotor activity, which occurs in this species just before the beginning of this
process. Whether specialized central pacemakers dictate the phase of locomotor activity or this control is exerted
outside of the core clock mechanism remains to be elucidated. Our results emphasize the importance of maintaining
cycling light-dark conditions in aquaculture practices during ontogeny of Senegalese sole. (Author correspondence:
munoz.cueto@uca.es or carlos.pendon@uca.es)
Keywords: Circadian system, clock genes, development, fish, photoperiod
INTRODUCTION
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prenatal to the postnatal period. Synchronized oscillations with high amplitude appear earlier in the suprachiasmatic nucleus of the hypothalamus than in the
peripheral clocks such as liver and heart (Kovacikova
et al., 2006; Sakamoto et al., 2002; Sladek et al., 2007).
Maternal signals such as feeding time and melatonin
production play a key role in setting the phase of these
early clocks, whereas photic entrainment appears
later on development (Seron-Ferre et al., 2007; Sumova
et al., 2006).
The timing of biochemical, physiological, and behavioral daily functions is controlled by the circadian
system in most living organisms. One of the central
elements of this system is the endogenous oscillator or
molecular clock, which is maintained by complex
transcriptional-translational feedback loops of clock
genes and their protein products (Pegoraro & Tauber,
2011). In mammals, the emergence of clock gene
rhythms during ontogenesis arises gradually from the
Submitted November 23, 2012, Returned for revision February 20, 2013, Accepted March 1, 2013
Correspondence: José A. Muñoz-Cueto, Departamento de Biologı́a, Facultad de Ciencias del Mar y Ambientales, Universidad de
Cádiz, Campus Rio San Pedro, E-11510, Puerto Real, Spain. Tel.: +34 956016023. Fax: +34 956016019. E-mail: munoz.cueto@uca.es;
or to Carlos Pendón, Departamento de Bioquı́mica, Facultad de Ciencias, Universidad de Cádiz, Campus Rio San Pedro, E-11510,
Puerto Real, Spain. Tel.: +34 956016391. Fax: +34 956016288; E-mail: carlos.pendon@uca.es
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Á. J. Martı́n-Robles et al.
Fish have ultimately proven to be valuable complementary models for studying various aspects of clock
biology (Idda et al., 2012). Early development in this
vertebrate group is strongly influenced by photoperiod
conditions (Villamizar et al., 2011). Importantly in
zebrafish, embryos, cell lines, as well as most tissues
and organs appear directly light responsive (Carr et al.,
2006). Circadian rhythms of behavior are first detected
soon after hatching, and are dependent on exposure
of larvae to light-dark cycles (Hurd & Cahill, 2002).
Rhythmic clock gene expression begins much earlier,
and a functional circadian clock has been shown
to autonomously oscillate within the first 12 h of development, prior to the complete differentiation of specialized light-receptive structures (Davie et al., 2011;
Dekens & Whitmore, 2008; Delaunay et al., 2003).
These embryonic molecular clocks require exposure
to environmental stimuli for the entrainment and
generation of synchronized oscillations in the embryo
(Dekens & Whitmore, 2008; Lahiri et al., 2005).
The Senegalese sole (Solea senegalensis) shows a
rapid larval development, which is also tightly influenced by lighting conditions (Blanco-Vives et al., 2010;
Cañavate et al., 2006; Parra & Yúfera, 2001; Yúfera
et al., 1999). Rhythms in behavior appear early at 3 d
post hatching (dph), roughly coinciding with the
complete organization of molecular clock rhythms, at
least for Per1, Per2, Per3, and Clock genes (Martı́nRobles et al., 2012a). In the course of their development, flatfish species as Senegalese sole undergo a real
metamorphic process that represents a dramatic transition from symmetric pelagic larva to an asymmetric
benthic juvenile and involves tissue differentiation,
biochemical, molecular, and physiological changes
(Fernández-Dı́az et al., 2001; Isorna et al., 2009b;
Parra & Yúfera, 2001; Power et al., 2001). Regarding
biological rhythms, it has been reported that this
rearrangement of the body plan encompasses a lightdependent switch from diurnal to nocturnal locomotor
activity rhythms and feeding behavior, which occur
just before the onset of this process (Blanco-Vives
et al., 2012; Cañavate et al., 2006). In previous studies,
we showed that constant light (LL) or dark (DD)
photoperiods markedly influenced clock gene expression rhythms during the first days of development and
prolonged DD conditions led to a high mortality in sole
larvae, which do not accomplish the metamorphic
process (Blanco-Vives et al., 2012; Martı́n-Robles et al.,
2012a). Nevertheless, the existence of clock molecular
rhythms and the effect of lighting conditions during
this remarkable phase of larval ontogeny are still
unknown. The aim of this work was to determine, for
the first time, clock gene expression profiles during
Senegalese sole pre- and post-metamorphic stages
and investigate the effects of transitory constant light
or dark conditions in Per1, Per2, Per3, and Clock daily
rhythms.
MATERIALS AND METHODS
Animals and rearing system
Senegalese sole fertilized eggs were supplied by IFAPA
El Toruño (Puerto de Santa Marı́a, Cádiz, Spain) from
naturally spawning tanks during the main reproductive
season (spring). They were collected early in the morning (0 d post fertilization [dpf]) and transferred to the
indoor fish facilities of the ‘‘Laboratorio de Cultivos
Marinos’’ (Faculty of Marine and Environmental
Sciences, University of Cádiz, Puerto Real, Spain). Eggs
were maintained in 1-m3 tanks equipped with a cover
connected to an individual automatic photoperiod
control system. Tanks were filled up with 200 L of
seawater at a temperature and salinity of 19 1 C and
39 ppt, respectively, in an open circuit system with
gentle aeration and a central draining pipe with 132-mm
mesh. Larvae were maintained from the day of fertilization (0 dpf) to 24 dpf when metamorphosis had been
completed in all groups. They were fed with Brachionus
plicatilis rotifers and Artemia sp. nauplii. Rotifers were
supplied from 2 to 14 d post hatching (dph) and its
density was gradually increased from 3 to 15 rotifers
mL1 for 9-d-old larvae onwards. Live brine shrimp
Artemia sp. nauplii were supplied from 10 to 24 dph,
its density being daily adjusted and also gradually
increased from 0.1 to 8 nauplii mL1. The unicellular
marine microalgaes Isochrysis galbana (T-ISO strain)
and Nannochloropsis gaditana were used for enrichment of live preys and were also added to larval tanks
during rotifer use (green waters technique). The experimental protocols were approved by the Institutional
Animal Care and Use Committee at the University of
Cádiz and were conducted in accordance with international ethical standards (Portaluppi et al., 2010).
Experimental design and sampling
Senegalese Sole eggs were split and initially stocked at a
density of 100 individuals per liter. In order to analyze
the diel expression of clock genes during sole metamorphosis as well as the effect of transient constant lighting
conditions, three different experimental groups were
made. In the first experimental group, larvae were
maintained under LD (14:10) from 0 to 24 dpf (LD
group). The two remaining groups were reared under
LD from 0 to 9 dpf, followed by transient constant light
(LL-LD group) or dark (DD-LD group) conditions
from 10 to 14 dpf and then they were returned to LD
conditions from 15 to 24 dpf. Light was switched on at
zeitgeber time (ZT) 0 (08:00 h local time) and off at ZT 14
(22:00 h local time). The artificial photophase under
LD conditions was adjusted to 14 h, since this is the
local average duration of day light following the main
natural spawning period of Solea senegalensis (Anguis &
Cañavate, 2005). Animals were sampled at four developmental stages during the metamorphic process,
coinciding with pre- (11 dpf), early (14 dpf), middle
(18), and post- (24 dpf) metamorphosis. We have
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Clock Genes During Senegalese Sole Metamorphosis
tested the expression of clock genes by using different
sampling points along a daily cycle. Cosinor analysis
does not markedly differ by using four (ZT 0, ZT 7, ZT 12,
ZT 19) or six (ZT 0, ZT 4, ZT 8, ZT 12, ZT 16, ZT 20)
sampling points (data not shown). Therefore, we have
considered that four sampling points/24-h cycle were
enough to perform a comprehensive developmental
study. Pools of whole animals containing 5–10 specimens were then collected at ZT 0, ZT 7, ZT 12, and ZT 19
(n ¼ 5). In constant light and dark conditions, these
hours represent circadian time (CT). Animals were
starved during the sampling days and collected at the
same developmental stage, measured according to
their position in the water column. During darkness,
sampling was performed under a dim red light.
All samples were rapidly frozen in liquid nitrogen and
stored at 80 C until used.
Gene expression analysis
Real-time quantitative PCR (RT-qPCR) was used to
analyze Per1, Per2, Per3, and Clock mRNA relative
expression. Total RNA from sole larvae was extracted
using TRIsure Reagent (Bioline, London, UK) according
to the manufacturer’s instructions. Samples were
homogenized in a mixer mill MM400 (Retsch, Haan,
Germany) with 4–6 stainless steel beads (2 mm diameter). After extraction, RNA was resuspended in 20–30 mL
diethylpyrocarbonate (DEPC)-treated water (Bioline).
Total RNA yield and purity were determined by the
260/280 nm absorbance ratio in an Eppendorf biophotometer (Eppendorf, Hamburg, Germany). Aliquots of
100 ng were reverse transcribed into cDNA (20 mL reaction volume) using random hexamers and the Quantitec
reverse transcription kit (Qiagen, Hilden, Germany),
which includes a previous genomic DNA removal step.
RT-qPCR reactions were developed in a Chromo 4 FourColor Real-Time System using the iTaqTM SYBR Green
Supermix with ROX (Bio-Rad, Alcobendas, Spain) as
previously described (Martı́n-Robles et al., 2011, 2012a,
2012b). Samples were run in duplicate. Nontemplate
control and non-retrotranscribed total RNA sample were
used as negative controls. Relative mRNA expression
was determined by means of the DDCt method (Livak &
Schmittgen, 2001), using Senegalese sole -actin
(GenBank accession number DQ485686) as a reference
gene, which was revealed as an adequate housekeeping
gene for developmental studies in this species (Infante
et al., 2008). Moreover, we used a second housekeeping
gene for normalisation, rps4 (GenBank accession
number AB291557), which provided similar developmental profiles of clock gene expression.
Data analysis
Statistical tests were performed using the Statgraphics
Plus 5.1 software (Statpoint Technologies, Warrenton,
VA, USA). Data of clock gene relative expression within a
given developmental stage (11, 14, 18, or 24 dpf) were
subjected to a two-way analysis of variance (ANOVA)
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701
followed by a Tukey’s post hoc comparisons test to
determine significant differences between photoperiods
and daily time points. When a significant correlation
between both factors was found, a one-way ANOVA was
used followed by the Tukey test. In all cases, significance
was accepted at p50.05. Data are presented as
mean SEM (standard error of the mean). Rhythmicity
in daily gene expression values was evaluated by cosinor
analysis using the chronobiological software El Temps
(version 1.228; www.el-temps.com) developed by Prof.
A. Dı́ez Noguera (University of Barcelona). Expression
profiles were considered to display a significant daily
rhythm when p50.05.
RESULTS
Period genes expression during senegalese sole
metamorphosis
The three Period genes currently studied (Per1, Per2,
and Per3) were rhythmically expressed in sole from premetamorphic larvae to post-metamorphic juveniles, as
revealed by ANOVA and cosinor analysis (Figures 1–3
and Table 1). Regarding Per1 expression, a significant
interaction between photoperiod and time of day was
found at 11, 14, 18, and 24 dpf (two-way ANOVA,
p50.05; Figure 1). The interaction also yielded significant differences in Per2 and Per3 at 11, 14, and 18 dpf
(two-way ANOVA, p50.05; Figures 2, 3). In these cases,
Period genes expression significantly varied with time in
all photoperiods (Figures 1–3). At 24 dpf, statistically
significant differences were found across time points
and also between photoperiod conditions in the case of
Per3, and only across time points in Per2 mRNA levels
(Figures 2, 3).
In larvae exposed to light-dark conditions (LD group),
an overall reduction of amplitudes and transcript levels
was observed from early metamorphosis (14 dpf) to
middle- and post-metamorphic stages (18 and 24 dpf,
respectively; Figures 1–3, Table 1). Acrophases of Per1
and Per3 were observed at dawn in all cases, being Per1
peaks slightly phase advanced in relation to Per3. They
were placed just prior to lights on for Per1 (between ZT
23.52 and ZT 0.61; Table 1) and immediately subsequent
to lights on for Per3 (between ZT 0.49 and ZT 1.62;
Table 1). Per2 phase was also maintained during pre and
metamorphic stages, its peak expression occurring later
during daytime between ZT 4.42 and ZT 7.19 (Table 1).
Under transient conditions of constant light (LL-LD
group), amplitudes and transcript levels were also
reduced, although in this case it was evident from preto early metamorphosis (11–14 dpf), when the continuous light treatment was applied. Rhythmicity in Period
genes was conserved, but their amplitudes and acrophases were differentially affected. During the second
day under LL (11 dpf), amplitudes were similar to those
observed in LD conditions, and expression peaks at CT
20.69 and 22.71 for Per1 and Per3, respectively (Table 1,
Figures 1, 3). Per2 phase was markedly influenced by
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Á. J. Martı́n-Robles et al.
Per1
Relave expreession
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ZT0
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LL-LD
cd
a
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abc
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abc
ZT7 ZT12 ZT19
FIGURE 1. Relative Per1 mRNA expression during sole metamorphosis. The LD group was maintained under a 14 h light:10 h dark regime,
and transient constant conditions of light and dark were applied from 10 to 14 dpf in the LL-LD and DD-LD groups, respectively. Larvae
were collected during pre- (11 dpf; PreM), early- (14 dpf; EarlyM), middle- (18 dpf; MidM), and post- (24 dpf; PostM) metamorphic stages at
ZT/CT 0, 7, 12, and 19. The bars above each graph indicate the photoperiod conditions within a given 24-h period, white bars representing
light phases and black bars representing phases of darkness. Relative expression was quantified by RT-qPCR. Each value represents the
mean SEM (n ¼ 5). Daily expression data from different photoperiods were analyzed at each metamorphic stage by two-way ANOVA
followed by a Tukey’s post hoc test. When a significant correlation between hours and photoperiods was found, a one-way ANOVA was
used followed by the Tukey test. Different letters indicate significant differences between time points from different photoperiod regimes
within a given developmental stage.
constant lighting conditions and its peak expression was
displaced from daytime at ZT 7.19 under LD conditions
to the subjective night at CT 21.17 under LL (Table 1,
Figure 2). By the fifth consecutive day under this regime
(14 dpf), amplitudes of Per1 and Per3 were significantly
reduced in relation to LD conditions (Table 1).
Acrophases of Per1 and Per3 resembled those observed
under LD (Table 1, Figures 1, 3), but Per2 peak occurred
again during the subjective night at CT 20.29 (Table 1,
Figure 2). From 15 dpf onwards, animals were reverted
to LD conditions and by the next two sampling days
(18 and 24 dpf) lower amplitude was still detected in
Chronobiology International
Clock Genes During Senegalese Sole Metamorphosis
Per2
Relave expreession
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p
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ZT7
Relave expression
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l
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ZT0
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FIGURE 2. Relative Per2 mRNA expression during sole metamorphosis. For further details, see the legend of Figure 1.
Per1 compared to LD conditions (Table 1). Three days
after the restoration of LD regime (18 dpf), the phases of
the three Period genes remained similar in the LL-LD
group and the LD group (Table 1, Figures 1–3).
In animals kept under transient conditions of continuous dark during 10–14 dpf (DD-LD group), Period
genes expression was revealed as rhythmic by ANOVA
and cosinor analysis (Figures 1–3, Table 1). Amplitudes
were also decreased compared to LD conditions but
after 5 d of constant darkness, the effect was not as
noticeable as in LL conditions (Table 1). Acrophases of
Per1 and Per3 at 11 and 14 dpf were comparable to their
peaks in LD conditions (Table 1, Figures 1, 3). Per2
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phase was the most affected by the DD conditions,
showing its peak of expression during the subjective day
but 4–6 h phase advanced in relation to LD conditions,
at CT 0.89 and 1.64 during 11 and 14 dpf, respectively
(Table 1, Figure 2). At 18 dpf, 3 d after reversion to LD
cycles, amplitudes of Period genes were even higher to
those observed in LD conditions (Table 1). At 24 dpf,
Per2 and Per3 amplitudes were similar to the LD group
but Per1 amplitude was lower than that observed in LD
conditions (Table 1). The phase of Per2 was resynchronized to the new LD cycle, showing its acrophase at ZT
3.73 and ZT 4.84 during 18 and 24 dpf, respectively
(Table 1, Figure 2).
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Á. J. Martı́n-Robles et al.
Per3
LD
LL-LD
11 dpf
p
PreM
Relave expreession
120
100
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d
ab
20
Relave exp
pression
40
de
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Relavee expression
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f
20
120
24 dpf
PostM
100
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e
ZT0
18 dpf
MidM
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0
14 dpf
E l M
EarlyM
DD-LD
bcd
abc
20
0
ZT0
ZT7 ZT12 ZT19
ZT0
ZT7 ZT12 ZT19
FIGURE 3. Relative Per3 mRNA expression during sole metamorphosis. For further details, see the legend of Figure 1.
Clock expression during senegalese sole
metamorphosis
The expression profile of Clock gene was also clearly
rhythmic during sole metamorphosis (Figure 4, Table 1).
A significant interaction between time points and
photoperiod conditions was observed at 11, 14, and
18 dpf (two-way ANOVA, p50.05; Figure 4). At 11 and
18 dpf, Clock mRNA levels significantly varied with time
in all photoperiods, but at 14 dpf only the LD group
shows significant daily variations (Figure 4). At 24 dpf,
both time of day and photoperiod condition yielded
significant differences (Figure 4).
As occurred with Period genes, Clock expression
levels, mesor, and amplitudes showed a general decline
in the LD group from pre- (11 dpf) and early metamorphosis (14 dpf) to middle- (18 dpf) and post-metamorphic (24 dpf) stages (Figure 4, Table 1). The phase
of the rhythm was maintained over the four stages
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Clock Genes During Senegalese Sole Metamorphosis
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TABLE 1. Cosinor analysis of Per1, Per2, Per3, and Clock daily expression during Senegalese sole metamorphosis under the different
lighting conditions tested.
11 dpf; PreM
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Gene
Per1
Mesor (r.e.)
Amplitude (r.e.)
Acrophase
Variance (%)
Significance
Per2
Mesor (r.e.)
Amplitude (r.e.)
Acrophase
Variance (%)
Significance
Per3
Mesor (r.e.)
Amplitude (r.e.)
Acrophase
Variance (%)
Significance
Clock
Mesor (r.e.)
Amplitude (r.e.)
Acrophase
Variance (%)
Significance
14 dpf; EarlyM
18 dpf; MidM
24 dpf; PostM
LD
LL
DD
LD
LL
DD
LD
LL
DD
LD
LL
DD
65.55
63.04
23.94
91.77
58.99
53.04
20.69
88.61
42.67
43.37
22.95
95.96
61.87
71.36
23.52
93.68
37.38
9.25
20.91
94.85
41.29
26.36
.36
96.21
27.41
30.48
23.61
95.22
19.60
17.45
23.05
97.73
25.45
36.24
.37
89.78
32.91
37.85
.61
92.53
17.66
19.64
.57
91.72
18.71
21.67
1.32
85.83
9.67
6.32
7.19
97.68
19.25
7.40
21.17
94.02
5.91
5.65
.89
87.23
6.93
4.71
5.29
96.45
14.37
2.26
20.29
97.30
6.30
3.49
1.64
94.83
4.51
2.38
5.88
89.84
4.99
2.61
7.37
94.84
7.95
5.19
3.73
88.76
3.67
2.39
4.42
94.02
4.23
2.14
4.49
92.30
5.29
3.82
4.84
76.00
35.65
44.80
1.62
90.90
44.59
39.11
22.71
95.14
28.05
36.77
.43
88.86
34.60
43.97
.49
89.85
33.38
14.10
.77
96.53
25.16
23.28
23.98
95.44
19.95
20.21
1.25
96.07
22.11
18.45
2.03
98.17
34.81
44.26
1.33
90.46
24.51
28.68
1.24
91.95
19.26
21.16
1.11
93.04
27.47
30.62
1.40
92.41
28.19
31.27
10.63
94.70
15.48
8.90
10.43
90.06
6.81
5.56
9.72
86.57
34.45
33.29
9.27
96.44
13.77
2.59
13.15
92.11
N.S.
9.85
6.02
9.44
89.37
21.87
16.38
9.37
98.42
16.09
9.22
9.47
97.66
26.54
13.82
10.40
93.61
18.00
8.41
10.73
96.98
12.77
7.29
11.12
95.53
24.62
10.98
11.85
92.24
Light-Dark (LD) group was maintained under a 14L:10D photoperiod regime all over the metamorphic process. Constant light or dark
conditions were applied from day 10 to day 14 of development in the LL-LD group (LL) and DD-LD group (DD), respectively. Samples
were collected during pre- (11 dpf; PreM), early- (14 dpf; EarlyM), middle- (18 dpf; MidM), and post- (24 dpf; PostM) metamorphic stages.
Rhythm parameters (mesor, amplitude, acrophase, variance, and significance) are indicated. Mesor and amplitude are given as relative
expression values (r.e.), acrophase as zeitgeber/circadian time (ZT/CT), and hours and variance as percentage of experimental data
explained by the cosine function calculated by the cosinor method. Significance: p50.01; p50.001; p50.0001; p50.00001;
p50.00000001.
analyzed, with the peak expression occurring at the
second half of the light period (ZT 9.27 to ZT 10.73;
Table 1).
In the LL-LD and DD-LD groups, where LL or DD
conditions were applied from 10 to 14 dpf, Clock transcript levels did not change over the metamorphic
process or even increased in the DD-LD animals after
reversion to LD cycles (Figure 4). Rhythm amplitudes
and mesor were remarkably diminished at 11 and 14 dpf
in relation to the LD group (Table 1, Figure 4). The effect
of the transient constant conditions was particularly
important in the LL-LD group, where Clock expression
did not show significant daily variations and did not fit a
cosinor analysis at 14 dpf (Figure 4, Table 1, cosinor,
p40.2). After reversion to LD conditions, Clock expression was again significantly rhythmic at 18 dpf, with its
peak expression at ZT 9.47, similar to LD conditions.
However, its amplitude was still lower than that of the
LD group (Table 1). At 24 dpf, all groups showed similar
amplitudes and acrophases (Table 1, Figure 4).
DISCUSSION
In the present study, we have analyzed the diel expression of Per1, Per2, Per3, and Clock genes in Senegalese
!
Informa Healthcare USA, Inc.
sole during the metamorphic process. Previously, we
have revealed that the locomotor activity rhythms in this
species exhibited a switch from diurnal to nocturnal
before the onset of metamorphosis (Blanco-Vives et al.,
2012). In this paper, we have shown that the mesor
and amplitudes of Periods and Clock rhythms decline
notably along this process, but their phases do not
markedly change through metamorphosis or in relation
to early developmental stages (Martı́n-Robles et al.,
2012a). In addition, we have evidenced that transient
constant light and dark conditions have differential
effects on the phases and amplitudes of some of the core
clock gene rhythms, which in some cases remain
affected even after the restoration of cycling conditions.
Metamorphosis drastically modifies the anatomy
of Pleuronectiform species (Padrós et al., 2011).
Importantly, this process implies eye migration towards
the upper-pigmented side of the body (Amaoka, 1971;
Fernández-Dı́az et al., 2001; Policansky, 1982) and
craniofacial remodeling, which determines its characteristic juvenile and adult asymmetry (Brewster, 1987;
Okada et al., 2001; Rodrı́guez-Gómez et al., 2000a,
2000b; Schreiber, 2006; Wagemans et al., 1998). This
transformation is accompanied by changes in their
habitats, where they come across markedly different
706
Á. J. Martı́n-Robles et al.
Clock
LD
LL-LD
11 dpf
p
PreM
Relave expreession
80
f
70
60
ef
50
20
bc
bc
Relave exp
pression
10
40
de
de
30
cd
bc
ZT12 ZT19
ZT7
ZT12 ZT19
ZT0
80
70
70
60
60
60
50
50
50
40
40
40
30
30
20
b
a
a
20
a
a
a
a
10
0
ZT7
ZT12 ZT19
30
20
10
ZT0
ZT7
ZT12 ZT19
80
80
70
70
60
60
60
50
50
50
40
40
d
30
20
bc
30
a
a
20
10
10
0
0
ZT0
ZT7
ZT12 ZT19
bc
a
30
a
ZT7
ZT12 ZT19
80
70
60
60
60
50
50
50
40
40
40
20
abc
30
20
10
10
0
0
ZT0
ZT7
ZT12 ZT19
ZT7
ZT12 ZT19
d
cd
ab
ZT0
70
abcd
a
ab
0
ZT0
80
def
a
10
70
de
a
ZT12 ZT19
20
80
30
ZT7
a
ZT0
70
cd
ab
0
ZT0
80
40
cd
a
0
ZT0
80
b
70
cd
20
10
0
ZT7
0
e
Relave expression
50
20
10
Relavve expression
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60
50
30
80
24 dpf
PostM
70
60
30
ZT0
18 dpf
MidM
80
70
40
0
14 dpf
EEarlyM
l M
80
40
10
DD-LD
abcde
cde
30
ab
a
ZT7
ZT12 ZT19
f
ef
bcde
abcde
20
10
0
ZT0
ZT7
ZT12 ZT19
ZT0
ZT7
ZT12 ZT19
FIGURE 4. Relative Clock mRNA expression during sole metamorphosis. For further details, see the legend of Figure 1.
environmental light conditions as pre-metamorphic
pelagic larvae swimming in the water column develop
into benthic juveniles living on the bottom of the sea.
Moreover, adaptive mechanisms intended to enhance
light sensitivity have been described in Senegalese sole
photoreceptive structures such as the pineal gland
(Confente et al., 2008). Of particular interest in this
species is the light-dependent switch from diurnal to
nocturnal locomotor activity and feeding behavior that
takes place just before the onset of this process, at 9–10
dph (Blanco-Vives et al., 2012). Studies on animals that
show the ability to switch their chronotypes, including
mammalian (Blanchong et al., 1999; Kas & Edgar, 1999;
Oster et al., 2002; Vivanco et al., 2010) and fish species
such as the European sea bass, goldfish, and Senegalese
sole (Blanco-Vives et al., 2012; Reebs, 2002; SánchezVázquez et al., 1995, 1996), are of special interest in
deciphering the mechanisms responsible for temporal
preference. Once the molecular oscillations of Per1,
Per2, Per3, and Clock are fully organized at 4 dpf in
Senegalese sole (Martı́n-Robles et al., 2012a), all of them
were actively expressed throughout the metamorphic
process. Daily expression levels were also found to be
rhythmic, showing the usual profiles described for fish
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Clock Genes During Senegalese Sole Metamorphosis
central tissues, where acrophases of Per1 and Per3
were placed at dawn, Per2 during the first half of the
day, and Clock at the second half of the light phase
(Park et al., 2007; Patiño et al., 2011; Sánchez et al., 2010;
Velarde et al., 2009; Whitmore et al., 1998; Zhdanova
et al., 2008). These rhythms could sustain the day-night
variations in the expression of Aanats and melatonin
receptors as well as the daily pattern of locomotor
activity observed in sole during the metamorphic process (Blanco-Vives et al., 2012; Isorna et al., 2009a,
2011a, 2011b). Our results, together with previous works,
revealed that the molecular clock oscillations are established very early during development and are preserved
during the whole ontogeny of sole. All phases of the
rhythms were maintained during pre-, early-, middle-,
and post-metamorphic stages and were clearly similar
to those found earlier at 4 dpf and in adult neural tissues
(Martı́n-Robles et al., 2011, 2012a, 2012b). This fact is
consistent with a growing body of evidence suggesting
that the key for diurnality/nocturnality does not lie upon
changes on the molecular clock, at least in the genes
currently studied. However, specialized central pacemakers could dictate the phase of locomotor activity,
which cannot be visualized in a whole animal RNA
extract. Otherwise, it could arise from mechanisms
operating upstream, downstream from the neural oscillators, or both, i.e., in the light input pathway or in the
neural/humoral output connecting to the physiological
and behavioral rhythmic processes (Mrosovsky &
Hattar, 2005).
In the course of metamorphosis, an important
decrease of clock gene amplitudes was observed. This
decline could be associated with changes in tissuedependent zeitgebers and reflect a reorganization of the
circadian clock in the different tissues, which appear
masked when whole animals are used. As mentioned
before, locomotor activity and feeding behavior of
Senegalese sole changes from diurnal to nocturnal
preference. Peripheral tissues can be entrained by
food-related cues in fish (Feliciano et al., 2011; LópezOlmeda et al., 2010), affecting the phases of clock genes
in these organs. Indeed, in sole adult (nocturnal)
specimens, clock gene rhythms in central tissues displayed and inverted phase in relation to peripheral
tissues such as liver (Martı́n-Robles et al., 2012b). The
phases of rhythms in clock gene expression also vary
throughout mammalian development in liver and
heart (Sakamoto et al., 2002; Sladek et al., 2007).
Changes in pups’ feeding behavior appear to account
for these phase differences (Weinert, 2005). However,
the possibility that the metamorphic process itself may
have an effect on the expression levels of clock genes
should not be ruled out, since the mesor of the rhythms
also decrease as metamorphosis proceed (see Table 1)
and the most important fall in rhythm amplitudes do
not coincide in time with the diurnal-nocturnal shift in
locomotor activity and feeding behavior (Blanco-Vives
et al., 2012). Further investigation at tissue or organ level
!
Informa Healthcare USA, Inc.
707
is needed in sole metamorphic larvae as well as studies
in other metamorphic species.
Embryonic development and larval growth of marine
fish are greatly affected by biotic and abiotic conditions
(Blanco-Vives et al., 2010; Villamizar et al., 2009). Fish
appear to be highly light responsive from eggs and
embryos to larvae and juveniles (Tamai et al., 2004;
Whitmore et al., 2000). Moreover, multitude of rhythms
and physiological parameters such as hatching, locomotor activity, feeding time, and cortisol or melatonin
secretion are influenced by lighting conditions (BlancoVives et al., 2011, 2012; López-Olmeda et al., 2009;
Oliveira et al., 2007). In previous studies, it has also
been demonstrated that constant light or darkness have
important effects on developing sole larvae, affecting
total length, yolk sac absorption, mouth opening, jaw
malformations, development of pectoral fins, and eye
pigmentation (Blanco-Vives et al., 2010, 2011, 2012).
Some of these effects could be mediated, at least in part,
by changes in clock gene expression rhythms, which are
entrained by the LD cycle. In fact, a differential response
in clock gene rhythms was observed in sole during
exposure to transient constant light and dark conditions,
which affected particularly Per1, Per3, and Clock amplitudes and Per2 acrophase. Upon release into DD and
after 5 d in these conditions (DD-LD group), rhythmic
gene expression consistent with the previous LD cycles
persisted in Per1, Per3, and Clock, demonstrating its
circadian nature (Pando et al., 2001; Whitmore et al.,
2000). Amplitudes were reduced but acrophases did
not vary considerably in relation to the LD group.
In contrast, Per2 expression was rhythmic by the cosinor
analysis but was immediately 6-h phase advanced in
relation to the LD group, peaking during the subjective
dawn. Therefore, its daily expression pattern was significantly different from the LD conditions. Under
transient LL conditions (LL-LD group), clock gene
expression rhythms were much more affected, supporting that sustained light treatment markedly influences
clock function (Tamai et al., 2007). Amplitudes of Per1
and Per3 were significantly reduced after 5 d of continuous light, but their phases were almost unaltered.
In contrast, Per2 phase was markedly disrupted, with
the acrophase located during the subjective night. Per2
was also up-regulated under these conditions (compare
expression values between groups and significance,
Figure 2). It should be noted that Clock rhythm was
lost at 14 dpf, after 5 d in LL conditions. Whether this
absence of rhythmicity is related to the decrease in the
amplitude of Per oscillation remains to be elucidated.
Altogether, our results in sole are consistent with
previous works supporting that the three Per genes
have different regulatory mechanisms. Whereas Per1
and Per3 are predominantly clock regulated, alteration
in the light regime clearly influences Per2 expression
(Besharse et al., 2004; Okabayashi et al., 2003; Pando
et al., 2001; Shearman et al., 1997; Zhuang et al., 2000;
Ziv et al., 2005; Zylka et al., 1998). As an element of the
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708
Á. J. Martı́n-Robles et al.
light input pathway, and together with Cry1a, Per2 has
been proposed in fish to play key roles in circadian
entrainment (Tamai et al., 2004, 2007; Vatine et al.,
2009). An intriguing observation of this study is that
although Per2 is markedly affected by lighting conditions, a rhythmic expression was maintained either
under DD and LL transient regimes. This evidence could
suggest a clock-regulated control of Senegalese sole
Per2 in the absence of cycling conditions. In contrast,
in zebrafish cell lines and transgenic embryos, Per2
rhythmic expression dampens immediately and remains
constant following transfer to DD (Pando et al., 2001;
Vatine et al., 2009). However, under LL conditions,
oscillations in Per2 are maintained in the transgenic
embryos, suggesting also both light- and clock-regulated
transcriptional control of Per2 in this species (Vatine
et al., 2009). Whether these differences in Per2 rhythmic
expression under constant conditions represent real
species-specific differences or a consequence of the
different experimental approaches and sensitivity of the
techniques used remain to be elucidated. Additionally,
other environmental cues could be sustaining Per2
rhythms under constant conditions in sole.
When larvae were reverted to LD cycles at 15 dpf, all
phases were recovered at 18 dpf and robust Clock
expression rhythm was restored. Nevertheless, 3 d after
the reestablishment of the LD conditions, amplitudes
for some clock genes such as Per1 and Clock were not as
robust as those observed for the LD group, and even
after 9 d under LD cycles (24 dpf) Per1 amplitude was
still lower than in the LD group. Moreover, significant
differences were found at 24 dpf between different
photoperiods in both Per1 and Per3. These data suggest
that constant photoperiod regimes, especially continuous illumination, even applied for a short period of time
have a prolonged effect on the molecular clock of sole.
This evidence could have a practical interest because
transient constant light conditions are commonly used
in sole aquaculture to accelerate growth and development. Consequently, outputs directly regulated by the
clock might be altered and this fact should be taken
into account to improve aquaculture practices and
welfare in this species. Our present results, together
with recent studies performed in this species (BlancoVives et al., 2010, 2012), emphasize the high sensitivity
of the sole circadian system to light cues and also
underline the important role of maintaining LD cycles
to the proper development of sole larvae.
In summary, our results confirm that clock gene
rhythms are maintained throughout Senegalese sole
metamorphosis with similar phase, providing evidence
that molecular rhythms are established very early during
development and preserved during the diurnal-nocturnal switching period. However, a general decline of
amplitudes and expression levels was observed during
this hallmark process that could be related to changes in
tissue-specific clock gene expression patterns. In addition, our results reveal the differential sensitivity of sole
clock components to transitory light cues and reinforce
the role of Per2 in light entrainment. The prolonged
effect that transient light changes applied during this
process has in the molecular clock machinery should
be considered when rearing Senegalese sole larvae in
aquaculture facilities.
ACKNOWLEDGEMENTS
We would like to thank Esther Isorna and Rosa Ma
Martı́nez-Álvarez for their help during sampling, and
all staff from the ‘‘Planta de Cultivos Marinos’’
(University of Cádiz) for the maintaining of animals
used in these studies. This is the CEIMAR Journal
Publication no. 26.
DECLARATION OF INTEREST
This work was supported by grants from the Spanish
MICINN (AGL2007-66507-C02-01 and AGL201022139-C03-03) and Junta de Andalucı́a (P06-AGR01939) to José A. Muñoz-Cueto, and a predoctoral
fellow of the Spanish MICINN (BES-2005-8629) to
Águeda J. Martı́n-Robles.
The authors report no conflicts of interest. The
authors alone are responsible for the content and
writing of the paper.
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