Desarrollo Del Sistema Circadiano en Peces

MASTER EN ACUICULTURA Y PESCA: RECURSOS
MARINOS Y SOSTENIBILIDAD
CURSO: BASES FISIOLÓGICAS DE

LA ACUICULTURA
Desarrollo del sistema circadiano en peces
José A. Muñoz-Cueto
Departamento de Biología. Facultad de Ciencias del Mar y Ambientales.
Universidad de Cádiz
José A. Muñoz-‐Cueto
Why are we interested in the study

of the circadian system in
developing fish, in general, and in
sole, in par9cular?
Melatonin and /or light effects on:

! HATCHING (Forsell et al., 1997; Gothilf et al., 1999;
Falcón et al., 2003; Danilova et al., 2004)
! PIGMENTATION (Castrucci et al., 1997)
! FEEDING (Pinillos et al., 2001; Rubio et al., 2004; De Pedro
et al., 2008)
! LOCOMOTOR ACTIVITY (Bayarri et al., 2004; Herrero et al., 2007)
! GROWTH (De Vlaming, 1980; Porter et al., 1998; Boeuf and
Falcón, 2001; De Pedro et al., 2008)
! CELLULAR PROLIFERATION (Danilova et al., 2004)
! METAMORPHOSIS (Parmen9ern et al., 2004;
Cañavate et al., 2006; Delgado et al., 1987; Wright et
al., 2003)
Solea senegalensis: an interes9ng species for developmental studies

! It exhibits a real metamorphic process during development

! It changes from pelagic to benthic habits of life during metamorphosis
Pre-‐met Early-‐met Milddle-‐met Final-‐met
0 dpf 4 dpf 9 dpf 12 dpf 15 dpf 21 dpf

Solea senegalensis: an interes9ng species for Chronobiology

ØRHYTHMIC SPECIES:
• Daily locomotor ac9vity and feeding
profiles (nocturnal)
Boluda Navarro et al., 2009

Bayarri et al., 2004
• Daily and annual melatonin rhythms.

Vera et al., 2007

ØRHYTHMIC SPECIES:
• Daily and annual rhythms in sexual steroids and spawning.
Oliveira et al., 2009

• Larval development is strongly influenced by ligh9ng condi9ons

• Growth
• Yolk sac absor9on
• Comple9on of metamorphosis
• Jaw malforma9ons
Blanco-‐Vives et al., 2010

ORGANIZATION OF THE CIRCADIAN SYSTEM

Our objec9ves:
! The input:
! Ontogenic analysis of photoreceptor cells in the pineal
organ and re9na of sole
! Effects of environmental factors (photoperiod and
light spectrum) on early development and
metamorphosis of sole
! The oscillator:
! Developmental study of the molecular clock
machinery of sole
! The output:
! Developmental analysis of melatoninergic systems
(melatonin-‐synthesizing enzymes, melatonin
receptors)
! Behavioral rhythms during ontogenesis
Development of the pineal organ:
Coronal sectiions
2 dpf Sagital sections
! The pineal organ is evident at 1-‐2 days post-‐

fer9liza9on (dpf)
A. El M’Rabet and J.A. Muñoz-‐Cueto, unpublished
Development of the pineal organ:

pin
tel
L R
Sagital sections
21 dpf Coronal section
! In post-‐metamorphic animals, the pineal organ adopts

an asymmetric disposi9on being directed to the right
side of the head where both eyes are also placed
Development of photorecep9on in the pineal organ:
! The first photorecep9ve cells appear in the 2 dpf

pineal organ at 1-‐2 days post-‐fer9liza9on (dpf),
before than in re9na
Development of photorecep9on in the re9na:
3 dpf
! These re9nal photoreceptors become evident at

3-‐4 days post-‐fer9liza9on (dpf)
Effects of photoperiod and light spectrum on sole development

Experimental design
Light inputs (LEDs) Photoperiod Sampling daysSampling

post-fertilization (dpf)
Aquaculture facilities
Early developmen
Light-Dark 12L:12D cycles of white light (LDW)
0dpf 1dpf 2dpf 4dpf
Light-Dark 12L:12D cycles of blue light (LDB)

Metamorphosis
White light: ! range: 367-757nm
Blue Wavelengths: ! peak: 463nm Light-Dark 12L:12D cycles of red light (LDR) 0-‐2
9dpfdpf:
15dpfhatching
25dpf study
Red Wavelengths: ! peak: 685nm
Tanks were covered with Lights intensity: 0.42 W/m2 0-‐7 dph: morphomethic study
Constant light (LL)
a black light-tight screen
Constant dark (DD)
• Hatching rate
• Hatching rhythms
• Eye pigmenta?on
• Mouth opening
• Pectoral fins
! Light spectra and photoperiods affect hatching rate
Blanco-‐Vives et al., Chronobiology Interna4onal,

28(4): 300–306, (2011)
! Eggs exposed to LDB had significantly highest hatching rate


! Light spectra and photoperiod affect acrophase of hatching rhythms
! Hatching peak was advanced
under LDB light
LDW
Hatching (%)
50
5’00h 40 LDB
30 LDR
20
10
50
Hatching (%)
40
LL
5’00h 30 DD
20
10 28(4): 300–306, (2011)
0 2 4 6 8 10 12 14 16 18 20 22

Zeitgeber ?me
! Light spectra and photoperiod affect acrophase of hatching rhythms

28(4): 300–306, (2011)
! From 4 dph onwards, larvae under LDB showed best growth and quickest
development (advanced eye pigmenta9on, mouth opening and pectoral fins)
Our objec9ves:
! The oscillator:
! Developmental study of the molecular clock machinery of sole
Most of the works on fish embryonic clocks have been mainly focused on the
zebrafish

Zebrafish Atlan?c salmon

(Danio rerio) (Salmo salar)
RabbiYish
(Siganus guVatus) European Sea bass
(Dicentrarchus labrax)
Goldfish
(Carassius auratus) Cavefish
(PhreaXchthys andruzzii)
Rainbow trout
(Oncorhynchus mykyss)
The molecular clock during early development of Senegalese sole: effect
of photoperiod condi9ons
Experimental design
1 2 3 4
05:00 h
ZT0 (08:00 h)

6-‐10 hpf LD 14:10
Spawn 19 ± 1oC LL
collec?on
DD
before dawn Sampling every 4h
Real Time qPCR ΔΔCt: Per1, Per2, Per3, Clock

βacXn, Rps4 as housekeeping genes
Gene expression analysis
Cosinor analysis
1400
Per1 LD Rhythm from 0-‐1 dpf
Gradual entrainment under LD cycles Per1 expression
Per1 LL
1200 Increase in mesor and amplitude
Per1 DD Rhythm from 0-‐1 dpf
1000
Maintained with low amplitude tending to arrhythmia
Phase considerably affected
Rela?ve expression
Rhythm lost
800
High mortality
ZT 23.52
600
CT 15.27
-‐-‐-‐-‐
400
ZT 5.10 ZT 1.93 ZT 23.52
CT 5.43 CT 4.88 CT 13.16
200
Mar[n-‐Robles et al.,
Chronobiology Interna4onal, 29
(9): 1195–1205, (2012)
0
0 4 8 12 16 20 0 4 8 12 16 20 0 4 8 12 16 20 0 4 8 12 16 20 0 4
0-‐1 dpf 1-‐2 dpf 2-‐3 dpf 3-‐4 dpf 4 dpf

300
Per2 LD Rhythm from 0-‐1 dpf
Acrophases d uring t he l ight p hase Per2 expression
Per2 LL
250
Increase in mesor and amplitude, 0-‐1 to 1-‐2 and 2-‐3 to 3-‐4 dpf
Per2 DD Photorecep?on in re?na
Rhythm maintained with low amplitude,
tending to arrhythmia
Mar[n-‐Robles et al., ZT 6.18
Rela?ve expression
Chronobiology Interna4onal,
200
29(9): 1195–1205, (2012)
Non significant rhythms CT 2.96
High mortality -‐-‐-‐-‐
150
ZT 5.33 ZT 7.86
CT 13.03
CT 4.03 in pineal gland
Photorecep?on
N.S.
100 ZT 5.64 N.S.
N.S.
CT 2.93
50
0
0 4 8 12 16 20 0 4 8 12 16 20 0 4 8 12 16 20 0 4 8 12 16 20 0 4
0-‐1 dpf 1-‐2 dpf 2-‐3 dpf 3-‐4 dpf 4 dpf

The molecular clock during metamorphosis: effect of transient constant condi9ons
Experimental design
dpf
0 Sampling points:
11dpf 14dpf 18dpf 24dpf ZT
0 , Z T 7 , ZT 12, ZT19
Pre-‐met Early-‐met Milddle-‐met Final-‐met
19 ± 1oC
0-‐9 dpf 10-‐14 dpf 15 dpf onwards

LD group
LL-‐LD group
DD-‐LD group
Real Time qPCR

Cosinor analysis
ΔΔCt: βacXn, Rps4

LD 11 dpf PreM 14 dpf EarlyM 18 dpf MidM 24 dpf PostM
LL-‐LD
DD-‐LD
160
140 Per1: ZT 23.52-‐0.61
120 Per3: ZT 0.49-‐1.62
100
Phases were 80
maintained during 60
Per1 LD
metamorphosis 40
20 Per3 LD
0
160 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19
140
Per2: ZT 4.42-‐7.19
120
Clock: ZT 9.27-‐10.73
100
80
Decrease in 60 Per2 LD
amplitudes and 40
Clock LD
mesors along 20
metamorphosis 0
ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19
Mar[n-‐Robles et al., Chronobiology Interna4onal, 2013; 30(5): 699–710
LL-‐LD
DD-‐LD
180
Decrease in 160 Per1

140
amplitudes and 120
mesors 100
80
Even aler 10 d under 60

cycling condi9ons, a 40 20
prolonged effect on 0
clock func9on was 35 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19
Per2
observed. 30
25
Mar[n-‐Robles et al.,
20 Chronobiology Interna4onal,
Phase markedly 15
2013; 30(5): 699–710
affected
10
5
0
LL-‐LD
DD-‐LD
180
160 Per1
Decrease in 140
amplitudes, with 120
phases comparable to 100
LD 80
60
40
10 days aler the cycling 20
condi9ons are restored, 0
Per1 amplitude is s9ll 35 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19 ZT0 ZT7 ZT12 ZT19
lower. 30
Per2
25
20
Phases markedly 15
affected 10
5
0
Mar[n-‐Robles et al., Chronobiology Interna4onal, 2013; 30(5): 699–710
Exposure to different light spectra affects the organiza9on of the biological
clock during early development of Senegalese sole
Experimental design
Aquaculture facilities Light inputs (LEDs) Photoperiod Sampling days post-fertilization (dpf)
Early development
Light-Dark 12L:12D cycles of white light (LDW)
0dpf 1dpf 2dpf 4dpf
Light-Dark 12L:12D cycles of blue light (LDB)

White light: ! range: 367-757nm Metamorphosis
Blue Wavelengths: ! peak: 463nm
Light-Dark 12L:12D cycles of red light (LDR) 9dpf 15dpf 25dpf
Red Wavelengths: ! peak: 685nm
Tanks were covered with Lights intensity: 0.42 W/m2
a black light-tight screen
Sampling every 4h
Real Time qPCR ΔΔCt: Per1, Per2, Per3, Clock

βacXn, Rps4 as housekeeping genes
Gene expression analysis
Cosinor analysis
Exposure to different light spectra affects the organiza9on of the biological
clock during early development of Senegalese sole
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(! (! (!
-
M. )!Aliaga and, B. Blanco-‐Vives, J.P.
- Cañavate, F.J. Sánchez-‐Vázquez
. and J.A. Muñoz-‐Cueto,
. in
!"#preparaXon
!$% &'(
4@A%B ,
, )! )!
%! ! Molecular clock
. %! mature early u, nder LDB light
%!)*+ //-,/ /,-1, 22222
Our objec9ves:
! The output:
! Developmental analysis of melatoninergic systems:
! melatonin-‐synthesizing enzymes
! melatonin receptors
! Behavioral rhythms during ontogenesis
Melatonin biosynthesis:
RETINA PINEAL
AA-‐NAT1 AA-‐NAT2
(plasma)
AA-‐NAT1a AA-‐NAT1b
?
Cloning of melatoning-‐synthesizing enzymes in sole:
! We have cloned three

different AA-‐NAT
enzymes in sole, AA-‐
NAT1a, AA-‐NAT1b and
AA-‐NAT2
Isorna et al., Gen Comp Endocrinol 161 (2009) 97–102

Isorna et al., J. Pineal Res. 2011; 51:434–444
Developmental expression of AA-‐NAT2 in sole:

! Real 9me
quan9ta9ve
PCR study
Isorna et al., Gen Comp Endocrinol 161 (2009) 97–102
! The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf,
with increased expression at night
! A significant 60-‐fold reduc9on in Aanat2 expression was seen just
before metamorphosis
Developmental expression of AA-‐NAT2 in sole:

A 2 dpf an9sense 21 dpf
pin
pin
E F
an9sense
pin 4 dpf
G
an9sense
B C D H I
! In situ hybridiza9on provides highly consistent results, showing

highest Aanat2 expression at 2 and 4 dpf, in rela9on to 21 dpf
! Aanat2 expression was pineal-‐specific, being detected in
photoreceptor cells. Isorna et al., Gen Comp Endocrinol 161 (2009) 97–102
Developmental expression of AA-‐NAT1a in sole:

! Real 9me
Adults
quan9ta9ve
PCR and in situ
ML MD
120 1000 hybridiza9on
g
studies
ssNAT1a (rela9ve expression)
rela9ve expression
14:30 (ML)
100
2:30 (MD)
80 100
60
f
40 f f 10
de ef
20 d d
d
d d
a c c bc bc bc ab
0 1
RETINA BRAIN
0 2 4 6 9 12 15 19 21
Developmental stage (days posmer9liza9on) Isorna et al., J. Pineal Res. 2011; 51:434–444
! The expression of Aanat1a increased between 0 and 4 dpf, decreasing therealer
! Aanat1a expression was rhythmic at early developmental stages but rhythmic
expression disappears during metamorphosis and in adult brain and
re9na
Developmental expression of AA-‐NAT1b in sole:

! Real 9me
quan9ta9ve
PCR and in situ
hybridiza9on
studies
ML MD

45 1000
ssNAT1b (rela9ve expression)
i

40 14:30 (ML) Adults

35 hi ghi
rela9ve expression
2:30 (MD)

30 100

25 fghi

20 efg fghi

15 efgh ef

10 de cd bc
10

5 a ab ab abc ab bc bc Isorna et al., J. Pineal Res.

0 2011; 51:434–444
0 2 4 6 9 12 15 19 21 dpf 1
RETINA BRAIN
! The expression of Aanat1b remains low during early stages of development
and increased significantly during metamorphosis
! Aanat1b expression was non-‐rhythmic at early developmental stages ,
acquiring its rhythmicity at the end of metamorphosis and in adults
Cloning of melatonin receptors in sole:

zebrafish
sole
seabass
pike
chicken
zebrafinch
! We have cloned three
xenopus
different MelR in sole,
human
chicken
MT2
MT1, MT2 and Mel1c
Mel1c
seabass
sole
rabbitfish mouse
MT1 chicken
zebrafish
zebrafish
trout Confente et al., Gen Comp Endocrinol 167 (2010) 202–214
seabass
mouse sole
rat human cow rabbitfish
sheep
0.1
Developmental expression of melatonin receptors:
O. Lan-‐Chow-‐Wing et al., IJMS. 2014
! The expression of melatonin receptors increased between 2 and 6 dpf
! A significant reduc9on in melatonin receptor expression was seen

along metamorphosis
hatching feeding metamorphosis
MT1 O. Lan-‐Chow-‐Wing et al., IJMS. 2014
MT2
Locomotor ac9vity rhythms during sole development:

LDB LDR
! Ac9vity rhythms in LDB and
changed from diurnal to
nocturnal on days 9 to 10 DPH,
coinciding with the onset of
metamorphosis
! In LDR, sole larvae remained

DD
nocturnal throughout the
experimental period

! Under and DD, larvae failed
to show any rhythm
Blanco-‐Vives et al. J. Biol. Rhythms, 27 (2), 2012 135-‐144)
Locomotor ac9vity rhythms during sole development:

! The key for diurnality/nocturnality does not lie
upon changes in the molecular clock (Per1,
Diurnal to nocturnal preference Per2, Per3, Clock)
!!"#$%"&'()" !*"#$%"+,'-.)" !/"#$%")0#)" 1*"#$%"&234)"
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Feeding behavior during sole development:

! Larvae exposed to LDB and
had the highest
LDB capture success and anack
rate during ML un9l 9
DPH.

! Aler the metamorphosis,
the anack and capture
success was higher at MD
in LDB and condi9ons
LDR
! Under LDR they showed
the same nocturnal
panern of behavior
Feeding behavior during sole development:
! Under and DD, there was no

clear daily panern of feeding
behavior in developing sole.
DD
In conclusion:
! The pineal organ develops at early ontogenic stages and photorecep9on in the
pineal organ of sole precedes the re9nal photorecep9on (2 dpf vs 3 dpf)
! Photoperiod and light spectrum have important effects on hatching rate,
hatching rhythms, eye pigmenta9on, mouth opening and development of
pectoral fins.

! Rhythms of Clock genes have been revealed in sole from the first hours post
fer9liza9on. During the following days, these rhythms are gradually entrained
under LD cycles, and at 4 days post fer9liza9on high amplitude rhythms
resembling adult central profiles were evident
! Light-‐dark condi9ons are required for the proper organiza9on of the
endogenous clock in sole, because rhythmicity in clock gene expression was
progressively anenuated or lost with 9me under constant light (LL) and dark
(DD) condi9on.
In conclusion:
! Exposure to different light spectra during early development of Senegalese
sole also affects the organiza9on of the biological clock, which matures early
under LDB light.

! Transitory constant light (LL) and dark (DD) condi9ons at the onset of
metamorphosis condi9ons have prolonged effects on the molecular clock and
should be considered when rearing sole larvae

! The onset of photoreceptor ability in the pineal organ, and the rhythmic
expression of AA-‐NAT2, AA-‐NAT1a and melatonin receptors between 2 dpf
and 4 dpf suggest an important role of pineal and melatonin in early
development of sole (e.g. hatching, feeding rhythms, pigmenta9on, etc)
! AA-‐NAT2, AA-‐NAT1a and melatonin receptors (MT1 and MT2) down-‐
regula9on before the onset of sole metamorphosis suggest that, similar to
anurans, flamish melatoninergic and thyroid hormone systems have
antagonizing effects on this process.
In conclusion:
! Locomotor ac9vity rhythms in LDB and LDW changed from diurnal to
nocturnal coinciding with the onset of metamorphosis, whereas animals
reared under LDR remain nocturnal throughout the development and those
maintained in LL and DD condi9ons were arrhythmic.

! Feeding behavior in sole is consistent with the locomotor ac9vity panern. Sole
larva reared in LDB and LDW are diurnal feeders before the onset of
methamorphic process and becomes nocturnal feeders aler the
metamorphosis. Larva maintained under LDR condi9ons exhibit nocturnal
feeding habits all over the development.

Desarrollo Del Sistema Circadiano en Peces

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Desarrollo Del Sistema Circadiano en Peces

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MASTER EN ACUICULTURA Y PESCA: RECURSOS

CURSO: BASES FISIOLÓGICAS DE

Desarrollo del sistema circadiano en peces

Why are we interested in the study

Melatonin and /or light eﬀects on:

Solea senegalensis: an interes9ng species for developmental studies

Pre-­‐met Early-­‐met Milddle-­‐met Final-­‐met

0 dpf 4 dpf 9 dpf 12 dpf 15 dpf 21 dpf

Solea senegalensis: an interes9ng species for Chronobiology

Boluda Navarro et al., 2009

• Daily and annual melatonin rhythms.

Solea senegalensis: an interes9ng species for Chronobiology

Oliveira et al., 2009

Solea senegalensis: an interes9ng species for Chronobiology

• Larval development is strongly inﬂuenced by ligh9ng condi9ons

Blanco-­‐Vives et al., 2010

ORGANIZATION OF THE CIRCADIAN SYSTEM

Development of the pineal organ:

! The pineal organ is evident at 1-­‐2 days post-­‐

Development of the pineal organ:

! In post-­‐metamorphic animals, the pineal organ adopts

Development of photorecep9on in the pineal organ:

! The ﬁrst photorecep9ve cells appear in the 2 dpf

Development of photorecep9on in the re9na:

! These re9nal photoreceptors become evident at

Eﬀects of photoperiod and light spectrum on sole development

Light inputs (LEDs) Photoperiod Sampling daysSampling

Light-Dark 12L:12D cycles of blue light (LDB)

Eﬀects of photoperiod and light spectrum on sole development

! Light spectra and photoperiods aﬀect hatching rate

Blanco-­‐Vives et al., Chronobiology Interna4onal,

! Eggs exposed to LDB had signiﬁcantly highest hatching rate

Eﬀects of photoperiod and light spectrum on sole development

0 2 4 6 8 10 12 14 16 18 20 22

! Light spectra and photoperiod aﬀect acrophase of hatching rhythms

Blanco-­‐Vives et al., Chronobiology Interna4onal,

Zebraﬁsh Atlan?c salmon

ZT0 (08:00 h)

Real Time qPCR ΔΔCt: Per1, Per2, Per3, Clock

0-­‐1 dpf 1-­‐2 dpf 2-­‐3 dpf 3-­‐4 dpf 4 dpf

0-­‐1 dpf 1-­‐2 dpf 2-­‐3 dpf 3-­‐4 dpf 4 dpf

0-­‐9 dpf 10-­‐14 dpf 15 dpf onwards

Real Time qPCR

LD 11 dpf PreM 14 dpf EarlyM 18 dpf MidM 24 dpf PostM

LD 11 dpf PreM 14 dpf EarlyM 18 dpf MidM 24 dpf PostM

Decrease in 160 Per1

Even aler 10 d under 60

LD 11 dpf PreM 14 dpf EarlyM 18 dpf MidM 24 dpf PostM

Light-Dark 12L:12D cycles of blue light (LDB)

Real Time qPCR ΔΔCt: Per1, Per2, Per3, Clock

Cloning of melatoning-­‐synthesizing enzymes in sole:

! We have cloned three

Isorna et al., Gen Comp Endocrinol 161 (2009) 97–102

Developmental expression of AA-­‐NAT2 in sole:

Isorna et al., Gen Comp Endocrinol 161 (2009) 97–102

Developmental expression of AA-­‐NAT2 in sole:

! In situ hybridiza9on provides highly consistent results, showing

Developmental expression of AA-­‐NAT1a in sole:

Developmental expression of AA-­‐NAT1b in sole:

Cloning of melatonin receptors in sole:

Developmental expression of melatonin receptors:

O. Lan-­‐Chow-­‐Wing et al., IJMS. 2014

Pre-‐met Early-‐met Milddle-‐met Final-‐met

Blanco-‐Vives et al., 2010

! The pineal organ is evident at 1-‐2 days post-‐

! In post-‐metamorphic animals, the pineal organ adopts

Blanco-‐Vives et al., Chronobiology Interna4onal,

Blanco-‐Vives et al., Chronobiology Interna4onal,

0-‐1 dpf 1-‐2 dpf 2-‐3 dpf 3-‐4 dpf 4 dpf

0-‐1 dpf 1-‐2 dpf 2-‐3 dpf 3-‐4 dpf 4 dpf

0-‐9 dpf 10-‐14 dpf 15 dpf onwards

Cloning of melatoning-‐synthesizing enzymes in sole:

Developmental expression of AA-‐NAT2 in sole:

Developmental expression of AA-‐NAT2 in sole:

Developmental expression of AA-‐NAT1a in sole:

Developmental expression of AA-‐NAT1b in sole:

O. Lan-‐Chow-‐Wing et al., IJMS. 2014

MT1 O. Lan-‐Chow-‐Wing et al., IJMS. 2014