Metamorphosis induces a light-dependent switch in Senegalese sole (Solea senegalensis) from diurnal to nocturnal behavior.

Abstract

Light plays a key role in the development of biological rhythms in fish. Recent research in Senegal sole has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions. However, the effect of light on the daily patterns of behavior remains unexplored. Therefore, the aim of this study was to investigate the impact of different photoperiod regimes and white, blue, and red light on the activity rhythms and foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To this end, eggs were collected immediately after spawning during the night and exposed to continuous white light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white (LD(W)), blue (LD(B), λ(peak) = 463 nm), or red light (LD(R), λ(peak) = 685 nm). A filming scenario was designed to video record activity rhythms during day and night times using infrared lights. The results revealed that activity rhythms in LD(B) and LD(W) changed from diurnal to nocturnal on days 9 to 10 DPH, coinciding with the onset of metamorphosis. In LD(R), sole larvae remained nocturnal throughout the experimental period, while under LL and DD, larvae failed to show any rhythm. In addition, larvae exposed to LD(B) and LD(W) had the highest prey capture success rate (LD(B) = 82.6% ± 2.0%; LD(W) = 75.1% ± 1.3%) and attack rate (LD(B) = 54.3% ± 1.9%; LD(W) = 46.9% ± 3.0%) during the light phase (ML) until 9 DPH. During metamorphosis, the attack and capture success rates in these light conditions were higher during the dark phase (MD), when they showed the same nocturnal behavioral pattern as under LD(R) conditions. These results revealed that the development of sole larvae is tightly controlled by light characteristics, underlining the importance of the natural underwater photoenvironment (LD cycles of blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae during early ontogenesis.

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Journal of Biological Rhythms
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DOI: 10.1177/0748730411435303
2012 27: 135J Biol Rhythms
Muñoz-Cueto and F.J. Sánchez-Vázquez
B. Blanco-Vives, M. Aliaga-Guerrero, J.P. Cañavate, G. García-Mateos, A.J. Martín-Robles, P. Herrera-Pérez, J.A.
to Nocturnal Behavior
) from DiurnalSolea senegalensisMetamorphosis Induces a Light-Dependent Switch in Senegalese Sole (


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135
1. To whom all correspondence should be addressed: B. Blanco-Vives, Department of Physiology, Faculty of Biology,
University of Murcia, 30100-Murcia, Spain; e-mail: borja@um.es.
2. These authors contributed equally to this work.
JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 27 No. 2, April 2012 135-144
DOI: 10.1177/0748730411435303
© 2012 The Author(s)
Metamorphosis Induces a Light-Dependent
Switch in Senegalese Sole (Solea senegalensis)
from Diurnal to Nocturnal Behavior
B. Blanco-Vives,*,1,2 M. Aliaga-Guerrero,†,‡,2 J.P. Cañavate,§ G. García-Mateos,|| A.J. Martín-Robles,†,‡
P. Herrera-Pérez,†,‡ J.A. Muñoz-Cueto,†,‡ and F.J. Sánchez-Vázquez*
*Department of Physiology, Faculty of Biology, University of Murcia, Espinardo Campus,
Murcia, Spain, †Department of Biology, Faculty of Marine and Environmental Sciences,
University of Cádiz, Cádiz, Spain, ‡CACYTMAR, Institutos de Investigación, Campus
Universitario de Puerto Real, Puerto Real, Spain, §IFAPA Centro El Toruno, Cádiz, Spain,
and ||Department of Informatics and Systems, Faculty of Computer Science,
University of Murcia, Espinardo Campus, Murcia, Spain
Abstract Light plays a key role in the development of biological rhythms in fish.
Recent research in Senegal sole has revealed that spawning and hatching rhythms,
larval development, and growth performance are strongly influenced by lighting
conditions. However, the effect of light on the daily patterns of behavior remains
unexplored. Therefore, the aim of this study was to investigate the impact of differ-
ent photoperiod regimes and white, blue, and red light on the activity rhythms and
foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To
this end, eggs were collected immediately after spawning during the night and
exposed to continuous white light (LL), continuous darkness (DD), or light-dark
(LD) 12L:12D cycles of white (LDW), blue (LDB, λpeak = 463 nm), or red light (LDR,
λpeak = 685 nm). A filming scenario was designed to video record activity rhythms
during day and night times using infrared lights. The results revealed that activity
rhythms in LDB and LDW changed from diurnal to nocturnal on days 9 to 10 DPH,
coinciding with the onset of metamorphosis. In LDR, sole larvae remained noctur-
nal throughout the experimental period, while under LL and DD, larvae failed to
show any rhythm. In addition, larvae exposed to LDB and LDW had the highest
prey capture success rate (LDB = 82.6% ± 2.0%; LDW = 75.1% ± 1.3%) and attack rate
(LDB = 54.3% ± 1.9%; LDW = 46.9% ± 3.0%) during the light phase (ML) until 9
DPH. During metamorphosis, the attack and capture success rates in these light
conditions were higher during the dark phase (MD), when they showed the same
nocturnal behavioral pattern as under LDR conditions. These results revealed that
the development of sole larvae is tightly controlled by light characteristics, under-
lining the importance of the natural underwater photoenvironment (LD cycles of
blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae
during early ontogenesis.
Key words activity rhythms, light, photoperiod, development, fish larvae, behavior
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136 JOURNAL OF BIOLOGICAL RHYTHMS / April 2012
In nature, the spectral characteristics of underwater
light are determined by a combination of the ambient
skylight and the optical properties of the water. Light
becomes increasingly monochromatic with depth
because the spectral profile is selectively attenuated
as light passes into deep water (Jerlov, 1968). Thus,
clear ocean waters transmit maximally at blue wave-
lengths (~470 nm), while coastal waters transmit bet-
ter at blue-green wavelengths (~500 nm) and estuarine
waters at green wavelengths (~580 nm) (Cohen and
Forward, 2002). Furthermore, fish have adapted their
photopigment sensitivity according to their surround-
ing environment (Kusmic and Gualtieri, 2000). The
effects of artificial lighting conditions on the perfor-
mance, development, and welfare of some fish larvae
of commercial interest have recently been reviewed by
Villamizar et al. (2011a), who pointed out the impor-
tance of light during early development to optimize
rearing protocols at hatcheries and to sustain juvenile
supply (one of the main production bottlenecks in
aquaculture).
Under an LD cycle, fish show daily patterns of
locomotor activity that can be classified into several
types: diurnal, nocturnal, crepuscular, and a combination
of them (Herrero et al., 2003; Schulz and Leuchtenberger,
2006; Vera et al., 2009). In adult sole, the activity is
maximal in the first part of the dark period, progressively
decreasing during the night (Bayarri et al., 2004;
Rubio et al., 2009). The daily locomotor rhythms of
the Senegal sole larvae are still unknown, despite the
fact that such knowledge is of prime importance for
optimizing larval rearing and production.
A successful feeding response is not solely dependent
on prey contact but rather on a continuum of events,
proceeding from the encounter through the attack and
capture of prey (O’Brien, 1979; Wanzenböck and
Schiemer, 1989; MacKenzie et al., 1994). For the majority
of teleost larvae, vision is the primary sense involved
in the execution of these feeding events (Blaxter, 1969).
Thus, a lack of correlation between prey density and
larval growth and survival may result from the
influence of light. In fish, the photosensory systems
and extraretinal photoreceptors are complex and far
from fully understood, as the response to light is often
species specific, depending on phylogenetic and
ecological factors (Marchesan et al., 2005).
The Senegal sole (Solea senegalensis) is a flatfish of
great commercial and scientific interest (Dinis et al.,
1999). Recent work performed in this species reported
that light spectra, intensities, and photoperiods have
a great influence in terms of larval growth, development,
malformation, survival (Blanco-Vives et al., 2010),
and hatching rhythms (Blanco-Vives et al., 2011).
Nevertheless, the behavioral responses of fish larvae
and their prey capture success rate under different
lighting conditions remain unexplored.
The purpose of this article was to investigate the
foraging behavior and daily activity rhythms in sole
larvae exposed to different light spectra and photoperiods.
To this end, we built a video recording/analysis system
that enabled us to quantify accurately, from 1 to 40
DPH, the distribution and behavioral patterns (modal
action patterns [MAPs]: swimming duration, orientation,
capture, miss, and pass frequency).
MATERIALS AND METHODS
Animals and Housing
The experiment was carried out at the facilities of
IFAPA Centro El Toruño (Puerto de Santa María,
Spain). Fertilized eggs were collected before dawn in
complete darkness from naturally spawning tanks to
ensure that eggs did not receive any light before the
experimental treatments. The brood stock was kept
in tanks exposed to natural lighting conditions in a
12L:12D cycle.
At 3 days posthatching (DPH), coinciding with the
onset of exogenous feeding, larvae from each
experimental condition were transferred to three 1-L
aquaria per treatment to continue the analysis of
morphometric parameters. To feed the larvae, Brachionus
plicatilis rotifers were cultured and enriched with
commercially available freeze-dried green algae
Nannochloropsis sp. (Easy Algae, Fitoplancton Marino,
Cádiz, Spain) in a proportion of 300,000 cells/mL/d
from 3 to 7 DPH. Enriched rotifers were added to tanks
twice a day, both during the light (ZT1, with ZT0
representing the beginning of light) and dark (ZT13)
phases, as an early live food at a density of 20
individuals/mL from 3 to 7 DPH. Artemia sp. nauplii
were added to tanks twice a day both during the light
(ZT1) and dark (ZT13) at a density of 2 to 3 nauplii
mL−1/d−1 that were provided from 8 to 30 DPH. Before
being fed to the larvae, the nauplii were enriched with
a mixture with optimal DHA/EPA ratio (INVE DC
DHA Selco Dendermonde, Belgium). Three to 5
metanauplii mL−1/d−1 were provided from 27 to 40
DPH. Before being fed to the larvae, the metanauplii
were also enriched for 24 hours with a mixture (ORI-GO,
ORI-PRO, Skretting AS, Burgos, Spain) of phytoproteins
and highly unsaturated fatty acids (HUFAs).
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Blanco-Vives et al. / BEHAVIORAL RHYTHMS IN SOLE LARVAE 137
Experimental Design
To study the effects of different light spectra and
photoperiods, the eggs were distributed into 5
experimental groups: 12L:12D cycle with red (LDR,
λpeak = 685 nm, half-peak bandwidth = 592-668 nm),
blue (LDB, λpeak = 463 nm, half-peak bandwidth = 435-
500 nm), or white (LDW) lights; constant white light
(24L:0D or LL); and constant dark (0L:24D or DD)
conditions. The white light had a broad spectrum,
with 95% irradiance within the range of 367 to 757
nm. The spectral analysis of the lights was performed
using a spectroradiometer (FieldSpec HH, ASD Inc.,
Boulder, CO) (Table 1). To avoid the effects of any
background light on the experiments, the experimental
aquaria were covered with a black light-tight screen.
For the different spectral trials, lamps were constructed
using light-emitting diodes (LEDs) mounted on
fiberglass plaques (160 × 232 mm). Each red and blue
lamp was made of a clustered panel with 17 LEDs
homogeneously distributed. White lamps had 18
white and 4 red LEDs to produce a broader range
wavelength. Each lamp was encased in a waterproof
container placed approximately 50 cm above the water
surface. The lamps were powered by a 3-V DC supply
connected to a variable resistor (0-2 kΩ) that allowed
the light intensity to be adjusted to 0.42 W/m−2, which is
low but well above the light threshold (0.053 W/m−2)
required to modify melatonin contents in both the eye
and plasma in Senegal sole (Oliveira et al., 2007). Fish
were reared and manipulated following Spanish
legislation on Animal Welfare and Laboratory
Practices (EC Directive 86/609/EEC).
Experiment 1: Daily Activity Rhythm
To record the behavior of larvae during day and night
times, 5 video recording systems equipped with infrared
lights were designed and built (Fig. 1). A webcam
(Logitech QuickCam
E3500 USB Webcam
Fremont, USA) was
modified by replacing
the UV optical filter
with a negative film
that acted as a filter
letting infrared light
pass through. Two
infrared lights (they
consist of a cluster of
30 high efficiency
LEDs, Cebek C-2290,
Barcelona, Spain) were placed 0.4 m from the aquarium,
opposite the camera, which was placed 0.5 m from the
aquarium. For a better diffusion of light, white acrylic
plates were fixed to the back wall of the aquarium,
which greatly improved the contrast of larvae. The
webcam was connected to a computer, and the
movement of larvae was recorded until 40 DPH.
Previous observations have shown that sole are capable
of feeding in the dark from the very early hatching stage
(Blaxter, 1969). The larvae were kept at a constant
temperature (20.9 °C ± 1.4 °C), and water was changed
every day using seawater hyperfiltered by means of a
biological filter (mod. 2227, EHEIM, Deizisau, Germany)
and a bio-balls filter system, with the filtering surface
ratio being approximately 1 L of bio-balls per 10 L of
water.
Due to the small size of larvae, most existing
methods for visual object tracking cannot be applied
to the images. Thus, in collaboration with the
Computer Vision Research Group of the University
of Murcia, in-house specialized software for video
analysis of fish larvae was developed to measure and
quantify the behavior of larvae in long-lasting video
sequences. The program provided an estimate of
overall locomotor activity value by using image
differences. The recorded video sequences consisted
of a set of images, I0, I1, I2 . . . In–1, where n is the total
length of the sequence; the videos were recorded at 1
frame per second. For each time instant, t, from 1 to
n–1, the difference image, Dt, was obtained with
Dt(x,y) = abs(It(x,y) – It–1(x,y)) for all pixels (x,y) in the
images. Then, the average of each difference image
was computed as motion(t) = 1/HW???∑ for all pixels (x,y)
(Dt(x,y)), where H is the height of the images, and W
is the width (in pixels).
A low value of motion(t) indicates that the overall
locomotor activity of larvae is very reduced at time t.
In the extreme case, a value of 0 means that images It
and It–1 are exactly the same, so there is no movement
Table 1. Experimental conditions and total length (mm) of Senegal sole larvae under different light
spectra and photoperiods.
LDW
LDB
LDR
LLDD
Photoperiod, h
Half-peak bandwidth (λ), nm
Temperature, °C
Length (3 DPH), mm
Length (10 DPH), mm
Length (20 DPH), mm
Length (30 DPH), mm
Length (40 DPH), mm
12L:12D
367-757
12L:12D
435-500
12L:12D
592-668
22.9 ± 1.4
1.12 ± 0.08
1.95 ± 0.08a
2.37 ± 0.08c
3.97 ± 0.08c
5.41 ± 0.07c
24L24D
—367-757
1.09 ± 0.08
2.07 ± 0.09a
4.21 ± 0.08a
6.01 ± 0.09a
8.52 ± 0.08a
1.11 ± 0.08
2.32 ± 0.10b
4.38 ± 0.12a
6.38 ± 0.11a
9.57 ± 0.09a
1.11 ± 0.09
2.14 ± 0.09a
5.12 ± 0.11a
7.12 ± 0.09a
9.51 ± 0.09a
1.10 ± 0.09
1.45 ± 0.09c
—
—
—
DPH = days posthatching. Data are expressed as mean ± SEM. Different letters indicate statistical
differences between groups (p < 0.05).
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138 JOURNAL OF BIOLOGICAL RHYTHMS / April 2012
at instant t. On the other hand, the higher the
motion(t) values, the greater the movement of larvae
at second t. The values given by this parameter
motion(t) were analyzed in relative terms (i.e.,
considering the rest of the sequence) because the
range of the measure depends on many factors:
the number of larvae, the contrast in the images, and
the lighting conditions.
Experiment 2: MAPs and Gut Content
To check the proportion of feeding and prey
capture rate per larva, 15 larvae per treatment were
collected from 0 to 10 DPH every 2 days, and total
length was measured from 3 to 40 DPH. Each
sampled larva was immediately observed under a
microscope. Larval gut contents were determined in
the day time (ZT6) and night time (ZT19) under
pressure of a cover slip, and the number of rotifers
consumed was counted. Although teleost larvae
digestion rates can be rapid (Fossum, 1983; Tilseth
and Ellertsen, 1984), in the present study, whole
rotifers were clearly visible within the larval gut, and
partially digested rotifers were easily recognized
and counted using the rotifer’s undigested mastax
apparatus (often enclosed within the remnants of the
lorica).
The focal animal technique (Altman, 1974) was
applied to collect information from randomly chosen
larvae during 2 minutes (5 larvae per sampling point
and treatment). Larval behavioral observations were
performed every 2 days in the middle of the light
(ZT6, ML) and dark (ZT18, MD) cycles in 1-L glass
aquaria illuminated using the LED lamps (red, blue,
or white). The occurrence of 5 MAPs including
swimming duration, orientation, capture, miss, and
pass frequency (Puvanendran and Brown, 1998)
(Table 2) was registered. The resulting data for each
MAP, larva, and replicate were averaged to obtain a
mean value of a given MAP from each treatment. The
frequencies of miss and capture were pooled to
generate the variable “attack”, which was then used to
calculate the capture success and attack rates (Drost,
1987) using the following relationship: Capture
success = (frequency of capture / frequency of attack)
× 100, Attack rate = (frequency of attack / frequency of
orientation) × 100.
Data Analysis
The analysis was carried out using Excel 2003
(Microsoft, Redmond, WA), SPSS (Chicago, IL), and
chronobiology software designed by Professor Díez
Noguera (El Temps, University of Barcelona, Barcelona,
Spain). The locomotor activity was recorded for each
treatment box separately. To establish statistical
differences in larval development (total length)
between treatments, a 1-way ANOVA was performed.
Regarding the gut contents (feeding proportion and
prey per larva) and MAPs (swimming duration,
capture success rate, and attack rate), 2-way ANOVA
tests were carried out with group and time as factors,
and a Tukey post hoc test was performed. All percentage
data were normalized and arcsin transformed before
statistical analysis. All statistical analyses were carried
out with SPSS 15.0 for Windows. Data are expressed
as mean ± SEM values. p < 0.05 was taken as the
statistically significant threshold.
RESULTS
Larval Development and Gut Content
Larvae exposed to different light regimes differed
in size as the experiment progressed. At 10 DPH,
larvae reared under LDB were longer than those
maintained under LDW, LDR, and LL conditions, while
the larvae under DD were the smallest (Table 1). The
longest larvae at 40 DPH were those maintained
under LDB (9.6 ± 0.09 mm), LDW (8.5 ± 0.08 mm), and
LL (7.1 ± 0.09 mm), which were longer than those
reared under LDR (5.4 ± 0.07 mm) (ANOVA, Duncan
test, F = 4.55, p = 0.035).
Figure 1. Detailed scheme of the system used to analyze
locomotor and feeding behaviors in developing sole: aquarium
with infrared illumination, webcam connected to a computer,
and lamps provided with light-emitting diodes of different light
spectra.
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Blanco-Vives et al. / BEHAVIORAL RHYTHMS IN SOLE LARVAE 139
We also observed differences in foraging behavior:
during the light phase (ZT6), larvae under LDR
showed a lower proportion of feeding than those
under the other treatments (ANOVA, Tukey test, F =
5.11, p = 0.034). At night (ZT19), larvae under LDR
and LL showed a higher proportion of feeding than
LDW and LDB treatments (ANOVA, Tukey test, F =
4.68, p = 0.037). Larvae under LL and DD showed no
difference in the proportion of feeding and prey per
larva at different sampling times, but at ZT6, larvae
under LL, LDB, and LDW exhibited more prey per
larva than those reared under the LDR and DD
condition (ANOVA, Tukey test, F = 5.32, p = 0.033).
The LL and LDR groups showed higher proportions
of prey per larva than LDB, LDW, and DD at ZT19
(ANOVA, Tukey test, F = 4.36, p = 0.028) (Fig. 2).
Locomotor Activity Rhythm
Sole larvae quickly develop activity rhythms, so that
they showed a clearly marked daily pattern of activity
from 3 DPH onwards under LD cycles. The larvae
reared under LDB and LDW showed a strictly diurnal
activity pattern before the onset of metamorphosis at 10
DPH (the criterion for defining the beginning of
metamorphosis was the eye migration), when they
inverted their activity pattern and became nocturnal
(Fig. 3A and 3B). Moreover, the LDB group underwent
the diurnal-nocturnal activity switch 1 day earlier (9
DPH) with regard to larvae maintained under LDW,
which became nocturnal from day 10 DPH (Fig. 3A and
3B). However, larvae under LDR showed a clear pattern
of nocturnal activity throughout the experimental
period, independently from the beginning of the
metamorphosis at 13 DPH, and there was no change in
their locomotor activity, as occurred under the LDB and
LDW conditions (Fig. 3C).
The fish reared under
LL and DD did not show
any marked rhythmic
activity pattern. The
larvae under LL began
metamorphosis at 10
DPH, like LDB and LDW.
The activity recorded
under DD was low, and
it was impossible to
continue the study in the
DD condition from 10
DPH onwards due to the
high mortality rate of
larvae (Fig. 3D and 3E).
MAPs
From the swimming duration of the larvae under
different conditions (Fig. 4), we see that the larvae
maintained under LDW changed their behavior from
7 DPH onwards, swimming more in MD than in ML
(ANOVA, Tukey test, F = 5.31, p = 0.027), while those
under LDB and LL showed a gradual increase in the
duration of swimming from 7 DPH in both ML and MD
(ANOVA, Tukey test, F = 4.96, p = 0.034). Also, from 9
DPH, the LDR and DD treatments showed shorter
swimming times than the other treatments in both ML
and MD (ANOVA, Tukey test, F = 4.75, p = 0.031).
There were also significant differences between
capture success and attack rates (Fig. 5). The larvae
under LDW showed a higher rate of capture and
attack during ML than during MD until 9 DPH
(ANOVA, Tukey test, F = 5.57, p = 0.038), whereas the
inverse pattern was observed from 11 to 15 DPH (Fig.
5A and 5B). Larvae under LDB showed a similar
pattern to those in LDW (ANOVA, Tukey test, F =
3.38, p = 0.066) (Fig. 5C and 5D), while those reared
under LDR, unlike other larvae, showed in all cases
higher percentages of capture and attack in MD
(Fig. 5E and 5F). In larvae maintained under LL,
captures and attacks increased over time (Fig. 5G
and 5H), unlike in the DD condition, in which larvae
capture and attack rates were lower and almost
invariable during development (Fig. 5I and 5J). In LL
and DD, there was no clear daily pattern of behavior
(Fig. 5G and 5J), as in the LDB, LDW, and LDR treatments.
DISCUSSION
Our observations revealed that lighting conditions
modified the onset of daily activity rhythms and
brought out different behavioral responses in sole
Table 2. Operational definitions of feeding modal action patterns (MAPs) for sole larvae.
MAP
Swim
Definition
Forward movement of larva through water column accomplished by movements of
caudal area of body.
Larva motionless (similar to “nonswimming” of Munk [1995]).
Larva motionless and fixates (determined by larva’s head and/or eye movement) on a
prey item (similar to “approach and attack position” of Munk [1995]).
Larva bites and ingests prey. Larva moves towards prey by a posterior drive of the tail
(similar to “attack” of Munk [1995]).
Larva fails to capture prey after a bite.
Larva orients and fixates on a prey item and moves toward the prey but does not bite.
Larva then swims in another direction.
Attacks = Misses + Captures.
Pause
Orient
Capture
Miss
Pass
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140 JOURNAL OF BIOLOGICAL RHYTHMS / April 2012
larvae. LDB and LDW cycles provided the best results
in terms of swimming duration, capture success rate,
and attack rate during the light phase, and most
interestingly, these treatments triggered behavioral
changes. Indeed, both LDW and LDB cycles produced
well-defined daily locomotor activity rhythms, which
shifted from diurnal to nocturnal following meta
morphosis. Contrasting constant light conditions
(LL and DD) prevented the development of daily
rhythms, leading to arrythmicity, while LDR induced
nocturnal activity from 3 DPH.
Previous research indicates that depending on the
fish species studied, contrasting outcomes can be
found, suggesting that the responses to light depend
on phylogenetic and ecological factors (Downing,
2002; Marchesan et al., 2005). If a given lighting
regime enhances the growth and development of fish
larvae (Vallone et al., 2007), it will in turn improve
their capacity to swim and to detect and capture
food. In our study, larvae reared under LDB and LDW
conditions showed higher percentages of prey attacks
and captures than the other treatments. This may be
related to the fact that the larvae showed advanced
development under LDB and LDW lighting regimes,
the biometric parameters responsible for carrying out
the MAPs (opening of the mouth, appearance of fins,
and pigmentation of the eyes) being developed
earlier under these conditions than under LDR and
DD (Blanco-Vives et al., 2010, 2011). In addition to
development, our present results revealed that the
behavior of larvae was also affected by light. Indeed,
larvae under LDB and LDW were the most active
(longest swimming duration) compared with larvae
reared in DD or LDR conditions. These experimental
conditions (LD cycles and blue wavelengths) closely
mirror the underwater environmental conditions that
this benthonic fish finds in the wild because the
water column acts as a chromatic filter, quickly
absorbing long wavelengths, so that blue becomes
predominant as depth increases (Jerlov, 1968).
It seems that light is a key factor not only for fish
larvae but also for the hatching rate and timing of
embryos (Blanco-Vives et al., 2011). Light and/or
melatonin effects on hatching have also been reported
in fish eggs (Helvik and Bernt, 1993; Danilova et al.,
2004). In sole, light information is transduced through
the pineal into melatonin rhythms (Oliveira et al.,
2007). The ontogenetic expressions of the pineal
(Aanat2) and retinal (Aanat1a and Aanat1b) melatonin-
synthesizing enzymes have been characterized in
sole, and cone opsin and rodopsin photoreceptor
cells have been identified in the developing and
adult pineal organ of this species (Confente et al.,
2008; Isorna et al., 2009a, 2011). Most curiously, the
expression of Aanat2 was detected as early as 12
hours postfertilization (0 DPF), and this expression
increases between 0 and 2 DPF, when hatching
occurs (Isorna et al., 2009a). At 2 DPF, pineal
photoreceptors (but not retinal photoreceptors)
already contain opsin photopigments, suggesting
that the pineal organ is mediating the effects that
light has on sole hatching and early development.
Basic questions concerning the biological clock are
when the system starts to cycle and how light affects
its development. In rainbow trout, Davie et al. (2011)
found recently that clock systems are present and
functional during embryonic development. In this
context, our results point to differences in the
development of the circadian system of sole according
to the environmental conditions in which they
develop. The Period gene family has been proposed
as key clock genes for the control of activity rhythms
in fish (Pando et al., 2001; Kaneko et al., 2006). As
recently reported in adult Senegal sole, Per3 exhibits
a significant daily oscillation in visually related
neural tissues such as the retina and optic tectum
(Martín-Robles et al., 2011). Ongoing research aims to
characterize the ontogeny of clock gene expression in
sole larvae, which appears to be functional very early
(A. Martín-Robles and J.A. Muñoz-Cueto, personal
communication), and it is most likely influenced by
light and temperature cycles during larvae
development. This information, together with the
0
0.5
1
1.5
2
2.5
LLLD R
LD B
LDW
DD
Treatment
Prey per larva
b
b
b
B
a
B
A
A
a
A
0
Proportion feeding
b
b
b
a
b
B
B
A
A
AB
100
80
60
40
20
ZT6
ZT19
Figure 2. The proportion of sampled sole larvae with rotifers in
their guts (A) and the mean number of rotifers found in larval
guts (B) for larvae from 3 to 7 days posthatching exposed to dif-
ferent light regimes and spectra. Different letters indicate means
within treatments significantly different from each other (lower-
case letters refer to ZT6, and capital letters refer to ZT19)
(ANOVA, Tukey test, p < 0.05).
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Blanco-Vives et al. / BEHAVIORAL RHYTHMS IN SOLE LARVAE 141
present behavioral findings, will
contribute to a better understanding
of the molecular clockwork sustaining
circadian rhythms and the switch
from diurnal to nocturnal behavior
during metamorphosis, which might
provide useful information of practical
interest for sole aquaculture.
Our results are consistent with
those obtained by Bayarri et al. (2004),
who observed that adult sole are
strictly nocturnal. In our experiment,
sole larvae during metamorphosis in
the LDW, LDB, and LDR treatments
also showed strict nocturnal activity.
However, no studies have previously
taken into account the activity rhythm
of flatfish
metamorphosis, and so our data
revealed for the first time that the
larvae of this species show a diurnal
pattern before 9 DPH, shifting to
nocturnalism during metamorphosis.
A similar phase
phenomenon occurs in juvenile
Atlantic salmon (Salmo salar), in
which a temperature-dependent
switch from diurnal to nocturnal
activity has been described (Fraser et
al., 1993), in sea bass that undergo
seasonal inversions (Sánchez-Vázquez et al., 1995,
1998), and even in mole rats (Spalax ehrenbergi) (Oster et
al., 2002). In contrast, this developmental pattern was
disrupted in sole larvae under LDR, which were
nocturnal throughout the experiment, and under LL or
DD, which became arrhythmic. Taken together, these
findings indicate that light and photoperiod are key
factors in setting the pace of rhythmic activity in this
species. Recent studies in sole showed that under LD
conditions, the expressions of different melatonin-
generating enzymes (Aanat1a, 1b, and 2) were much
higher in early ontogenetic stages and declined
dramatically before the onset of metamorphosis,
while thyroid hormone levels and Aanat1b expression
showed the inverse temporal profile, increasing
during metamorphosis (Isorna et al., 2009a, 2009b,
2011). The possible relation between these changes in
the melato ninergic system and the shift from diurnal
to nocturnal activity rhythms requires further
investigation.
larvae before
inversion
Figure 3. Representative actograms of activity of sole larvae (Solea senegalensis)
reared with a 12L:12D (LD) photoperiod of (A) blue light (LDB), (B) white light (LDW),
(C) red light (LDR), (D) constant light (LL), and (E) constant dark (DD) conditions. The
actograms are double plotted (time scale, 48 hours) for better visualization. The white
and black bars at the top indicate the light and dark phases of the LD cycle, respec-
tively.
0
10
20
30
LL
LD B
LD W
LD R
DD
0
10
20
30
35791113151719
ML
MD
Swimming duration (s)
Age larval (DPH)
b
b
a
a
c
c
a
a
b
b
a
a
a
a
a
a
C
B
B
b
b
b
b
b
b
c
c
a
b
c
c
c
a
b
B
B
C
a
b
B
B
C
b
b
b
b
a
b
b
b
b
a
b
b
b
b
b
b
b
b
b
b
b
b
C
C
C
b
a
a
a
a
B
B
B
a
a
b
Figure 4. Swimming duration of sole larvae exposed to differ-
ent light regimes and spectra. Data are expressed as mean ±
standard deviation. Different letters indicate statistical differ-
ences between groups and developmental time (ANOVA, Tukey
test, p < 0.05).
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142 JOURNAL OF BIOLOGICAL RHYTHMS / April 2012
In nature, the most common foraging mode used by
carnivorous larvae is visual detection, and so the
encounter rate between fish and prey seems to depend
on factors such as the contrast of the prey against the
background, the visual capabilities of the predator, and
the ambient light (Aksnes and Utne, 1997; Utne-Palm,
1999; Huse and Fiksen, 2010). In general, zooplankton
become more visible to predators when the background
illumination contains this particular spectrum profile
(blue wavelengths, 470 nm), as occurs in
nature (Novales
Browman, 2001). In the present study, the
poor foraging behavior displayed by
larvae under red light suggested that the
larvae needed better illumination (color)
in order to find and successfully capture
prey. Many studies (Utne-Palm and
Bowmaker, 2006; Downing and Litvak,
2001; Max and Menaker, 1992; Bayarri et
al., 2002) on larvae foraging behavior
considered only 1 or 2 critical factors that
influence prey detection and ingestion
(Huse and Fiksen, 2010). Despite the clear
evidence of the impact of light, no model
to date has incorporated the impact of
light quality on larval rhythmic foraging
behavior and their switch from diurnal to
nocturnal. In our findings in sole, feeding
was consistently better under blue and
white light, in accordance with Villamizar
et al. (2011b), who observed that sea bass
larvae under blue light could detect and
capture more prey than larvae under red
and white light regimes. A visual system
refined for the rapid identification and
capture of prey in the natural environment
allows larvae to exploit a patchy prey
distribution both temporally and spatially
(Villamizar et al., 2011a). Poor feeding
performance under red light was probably
due to a lack of photons of the appropriate
wavelength to stimulate the visual
pigments. In the dark, sole larvae were
also found to consume prey, albeit
infrequently and in lower numbers. This
may have unlikely been a consequence
of accidental ingestion
osmoregulatory drinking (Huse, 1994),
but a consequence of high food abundance,
which may have increased the probability
of chance encounters between larvae and
prey (Connaughton et al., 1994). The
ability of sole larvae to feed with varying degrees of
success under all light conditions tested is not surprising
given that climactic conditions may expose larvae to a
wide range of spectral environments in nature. The
results of the present study suggest that spectral light is
a critical factor for feeding success and, by inference, the
visual range of sole larvae.
In summary, our findings reveal for the first time the
existence of daily rhythmic behavioral patterns in
Flamarique and
during
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
35
7
9
11
13 15
35
7
9
11
13 15
Capture success (%)
Attack rate (%)
AB
C
D
E
F
G
H
b
IJ
b
b
b
B
B
A
A
A
a
a
b
A
B
B
B
a
a
a
b
b
b
b
B
B
B
A
A
A
A
a
a
a
A
A A
A
a
a
a
b
b
b
b
B
B
B
B
B
B
B
B
BB
a
a
a
a
a
a
a
a
a
a
a
B
B
B
B
B
a
B
a
A
A
b
B
a
b
A
b
A
B
a
Larval age (DPH)
Larval age (DPH)
MLMDML
MD
Figure 5. Capture success rate (# min -1) of sole larvae exposed to different light
spectra: (A) white light (LDW), (C) blue light (LDB), (E) red light (LDR), (G) constant
light (LL), and (I) constant dark (DD) conditions. Attack rate (# min -1) of sole larvae
exposed to different light spectra: (B) white light (LDW), (D) blue light (LDB), (F) red
light (LDR), (H) constant light (LL), and (J) constant dark (DD) conditions. Data are
expressed as mean ± standard deviation. Different letters indicate statistical differ-
ences between groups (lowercase letters refer to ML, and capital letters refer to MD)
(ANOVA, Tukey test, p < 0.05).
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Blanco-Vives et al. / BEHAVIORAL RHYTHMS IN SOLE LARVAE 143
developing sole larvae reared under different lighting
conditions and confirm that the early development of
Senegal sole eggs and larvae is strongly affected by
light characteristics (both photoperiod and spectrum).
Because “unnatural” or inappropriate environmental
conditions seriously compromise the welfare of fish
larvae and negatively affect their survival and
performance (Villamizar et al., 2009), light/dark cycles
of blue wavelengths appear to be the optimal conditions
and a prerequisite for the proper development of the
biological clock and behavior of sole larvae.
ACKNOWLEDGMENTS
This research was funded by the Spanish Ministry of Science
and Innovation (MICINN) by projects “CRONOSOLEA”
AGL2010-22139-C03 to F.J. Sánchez-Vázquez and J.A. Muñoz-
Cueto, AQUAGENOMICS (Consolider-Ingenio Program)
and SENECA (Ref. 08743/PI/08) to F.J. Sánchez-Vázquez,
and the Regional Government of Andalusia (P06-
AGR-019399) to J.A. Muñoz-Cueto. Thanks are also due to
COST ACTION 867 (Fish Welfare) and FA0801 (Larvanet), in
which part of these results were presented and discussed. B.
Blanco-Vives was supported by an FPI scholarship from the
MICINN. The authors thank Abdeslam El M’Rabet for his
help in sampling animals.
CONFLICT OF INTEREST STATEMENT
The author(s) have no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
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