larvi 2013
ghent university, belgium, 2-5 september 2013
6th fish & shellfish larviculture symposium
Epigenetic regulation of muscledevelopment and growthin Senegalese sole larvae
Catarina Campos
a CIMAR/CIIMAR & ICBASCentro Interdisciplinar de InvestigaçãoMarinha e Ambiental
Univ. Porto,Porto, Portugal
b CIMAR/CCMARCentro de Ciências do Mar do Algarve,
Univ. Algarve,Faro, Portugal
c UiNFaculty of Biosciences and Aquaculture
University of Nordland,Bodø, Norway
d SPAROS Lda,CRIA - Universidade do Algarve, Campus de
Gambelas, 8005-139 Faro,Portugal.
EPIGENETIC REGULATION OF MUSCLE DEVELOPMENTAND GROWTH IN SENEGALESE SOLE LARVAE
Catarina Campos a,b,c, Luísa M.P. Valente a, Luís E.C. Conceição b,d,Sofia Engrola b and Jorge M.O. Fernandes c
LARVI 2013 – 6th fish & shellfish larviculture symposium
LARVI 2013 – 6th fish & shellfish larviculture symposium
Senegalese sole (Solea senegalensis) is a marine flatfish with highpotential to the aquaculture industry.
However, great fluctuations of temperature during production arestill found, which can contribute to variations on sole’s growth and
muscle cellularity.
Such thermal plasticity must arise through changes in a multitude ofphysiological and molecular pathways, in which epigenetic gene
regulation plays an essential role → epigenetics is a bridgebetween genotype and phenotype.
DNA methylation
LARVI 2013 – 6th fish & shellfish larviculture symposium
DNA methylation usuallyoccurs on CpG
dinucleotides and is oftenassociated with a
repressed chromatin stateand inhibition of
transcription.
The demethylation of regulatory regions in myogenic genes at thebeginning of the differentiation program is essential to the commitment of
cells towards the muscle lineage.
miRNAs
LARVI 2013 – 6th fish & shellfish larviculture symposium
18–24 nt endogenous non-coding RNAs, that are repressive post-transcriptional regulators of gene expression
Some miRNAs are strongly expressed in muscle and known to interactwith the transcriptional networks involved in myogenesis
Exportin 5Exportin 5Exportin 5
Cytoplasm
Nucleus
Target mRNAPasha
Drosha Pre-miRNA
DicerDicerDicer
RISCMature miRNA
Argonaute
Transcript degradation(requires perfect complementarity)
Translational repression(requires partial complementarity)
Passenger strandPri-miRNA
LARVI 2013 – 6th fish & shellfish larviculture symposium
Experimental approach
Two experiments
Incubation experiment Larval rearing experiment
Three embryonic temperatures:15, 18 or 21 ºC
One rearing temperature:21 ºC until 30 dph
Three rearing temperatures duringpelagic phase: 15, 18 or 21 ºC
Transfer to common temperature:20 ºC until 121 dph
Muscle morphometry, gene expression, DNA methylation levels andprotein metabolism were analysed.
Blastula Epiboly 20 som Finalsom
Hatching Mouthopening
Met 30 dph
15 ºC
21 ºC
18 ºC
9
9.5
10
12
14
22
20
24
36
24
28
40
34
42
62
Hours post-fertilisation Days post-hatch
21 ºC
Pre-Met
2
2
3
8
8
9
14
14
15
30
30
30
Post-Met
22
22
23
LARVI 2013 – 6th fish & shellfish larviculture symposium
Incubation experiment
By 30 dph:• Weight of 18 and 21 ºC larvae wassuperior to the 15 ºC ones.
• The 18 ºC group had the highest numberof fast fibres, but no differences werefound between the 15 and 21 ºCtreatments, which instigates the idea that18 ºC can be an optimal temperature toincubate sole embryos.
• Total muscle area was similar across alltreatments, probably due to rearing alllarvae at a common temperature.
A (m
m2 )
0.0
0.1
0.2
0.3
0.4
0.5
a
bb
ab b
a b b
ab b
aa,b b
aa,b
b
N
0
500
1000
1500
2000
Hatch MOPreM Met
PostM30 dph
Fibr
e di
amet
er (µ
m)
0
5
10
15
20
15ºC18ºC21ºC
ac
b
ab c
ab c
a b b a b b
ab b
LARVI 2013 – 6th fish & shellfish larviculture symposium
Transient gene expression during development
Genes up-regulated in early development (embryo-hatching)
Genes up-regulated later in development (larvae)
LARVI 2013 – 6th fish & shellfish larviculture symposium
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
15°C
18°C
21°C
BL 75EP FS20S 30 dphMO PreM MetHatch
myf5, fst, myod2,mylc2, myHC and
mrf4 were highest at18 and/or 21 ºC at
specific points,which might havecontributed to the
general morepronounced growth
of these groups
0,0E+00
2,0E+05
4,0E+05
6,0E+05
8,0E+05
1,0E+06
1,2E+06
1,4E+06
# re
ads
Length (nt)
75Ep 15 ºC75Ep 21 ºC20S 15 ºC20S 21 ºCHatch 15 ºCHatch 21 ºCMet 15 ºCMet 21 ºC
SOLiD sequencing of small RNAs
Sample # Total reads# Trimmed
(% Total)
Average
length (nt)
# Annotated
miRBase (% Total)
# Conserved
miRNAs75Ep 15 ºC 6 642 089 2 802 986 (42.2) 23.5 200 224 (3.0) 232
75Ep 21 ºC 8 842 550 2 169 069 (24.5) 21.1 174 027 (2.0) 231
20S 15 ºC 6 566 636 3 320 266 (50.6) 22.4 993 981 (15.1) 265
20S 21 ºC 6 101 835 2 564 327 (42.0) 22.7 689 954 (11.3) 265
Hatch 15 ºC 7 268 950 4 312 425 (59.3) 23.0 1 467 039 (20.2) 285
Hatch 21 ºC 6 200 277 3 256 433 (52.5) 22.4 1 162 239 (18.7) 281
Met 15 ºC 7 105 697 4 965 849 (69.9) 23.0 2 391 674 (33.7) 288
Met 21 ºC 6 325 932 4 247 722 (67.2) 25.2 804 587 (12.7) 279
Increasingnumber and
diversity fromEpiboly to
Metamorphosis• 320 conserved miRNAs amongst 149 families
LARVI 2013 – 6th fish & shellfish larviculture symposium
• The very high expression of several miRNAs ata pre-metamorphic stage can be associated with
a high growth rate and preparation for themetamorphic process.
• qPCR validation of miR-26a, miR-181a-5p andmiR-206 revealed higher expression at 21 ºCthan at 15 ºC during embryogenesis and/or athatching, pointing to a higher activation of the
myogenic process at a higher temperature.
LARVI 2013 – 6th fish & shellfish larviculture symposium
miR-130c
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
20
40
60
80
100
*
*
*
miR-26a
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
1
2
3
4
5
6
**
miR-181a-5p
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
10
20
30
40
50
60
miR-181a-3p
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
5
10
15
20
25
30
35
*
*
miR-17a-5p
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
20
40
60
80
100
120
140 15 ºC21 ºC
miR-199a-5p
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
20
40
60
80
100
120
140
160
180
*
miR-206-3p
75Ep 20S Hatch PreMet Met 30dph
Rel
ativ
e ex
pres
sion
0
5
10
15
20
25
30
35
*
*
A B
C D
E F
G
*
*
*
miR-17a miR-26a
miR-130c miR-181a-5p
miR-181a-3p miR-199
miR-206
Larval rearing experiment
21 ºC
18 ºC
15 ºC
20 ºC
Experimental setupMet
16 d
ph
22 d
ph
35 d
ph
8 dph 35 dph
39 dph
51 dph
20 ºC
Hatching
12 dph
9 dph
12 dph
15 dph
23 dph
83 dph
83 dph
83 dph
100 dph
100 dph
100 dph
Pre-Met Post-Met 100 dph83 dph 121 dph
121 dph
121 dph
121 dph
LARVI 2013 – 6th fish & shellfish larviculture symposium
0
50
100
150
200
250
300
DW
(mg)
15 ºC18 ºC21 ºC
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
MO Pre-met Met Post-met
a
cb
a
cb
a
ba
a
cb
a
bb
a
b
a
During pelagic phase:
• High mortality andlower growth of the
15 ºC group.
By 121 dph:• Weight of juveniles
from 15 ºC was similarto 21 ºC, and both were
larger than 18 ºC.
Pre-met Met Post-met 83 dph 100 dph
Fibr
e di
amet
er (µ
m)
0
5
10
15
20
25
abc
abc
a
bc
ab
c
ab c
Metamorphosis
myod1 myod2 myf5 mrf4 myog pax7 mylc2 myHC mstn1 fst fgf6 igf-I igf1r igf-II hsp70
Rel
ativ
e ex
pres
sion
0.0
0.4
0.8
1.2
1.6
2.0
b
a
a,b
b
aa,b
b
aa,b
c
a
bb
a
a,bb
a
a,b
b
a
a,bba,b
a
LARVI 2013 – 6th fish & shellfish larviculture symposium
During pelagic phase:
• Fibre diameter was larger at 18and 21 ºC relatively to 15 ºC.
• During pelagic phase, themajority of MRFs as well as
myosins, fgf6 and igf-I were up-regulated at 18 ºC and/or 21 ºC
relatively to 15 ºC.
Methylation analysis
LARVI 2013 – 6th fish & shellfish larviculture symposium
• Higher methylation levels of myog at 15 ºC
Total number of methylated cytosines Methylation at CpG sites
Senegalese sole myogenin promoter:• Highly conserved amongst teleost species.
• Can DNA methylation of myog promoter insole muscle be affected by rearing
temperature?
Muscle - 100 dph
myod1 myod2 myf5 mrf4 myog pax7 mylc2 myHC mstn1 fst fgf6 igf-I igf1r igf-II hsp70
Rel
ativ
e ex
pres
sion
0.0
0.4
0.8
1.2
1.6
2.0
a
a,b
b
aa
b
a
a,b
b
Pre-met Met Post-met 83 dph 100 dph
Fibr
e di
amet
er (µ
m)
0
5
10
15
20
25
abc
abc
a
bc
ab
c
ab c
LARVI 2013 – 6th fish & shellfish larviculture symposium
By 100 dph:• Increased hypertrophy of muscle fibres inthe18 ºC group, which might induce a lower
growth capacity.
15 ºC 18 ºC 21 ºC
< 10
µm
(%) 1
00 d
ph
0
3
6
9
12
15
b
a a
LARVI 2013 – 6th fish & shellfish larviculture symposium
Artemia radiolabelling feeding trials:metamorphosis and benthic post-larvae.
LARVI 2013 – 6th fish & shellfish larviculture symposium
• Protein digestibility and retention is affected by rearingtemperature during metamorphosis – lowest at 15 ºC.
0%
20%
40%
60%
80%
100%
15 ºC 18 ºC 21 ºC
Ret
entio
n an
d ca
tabo
lism
(% A
bsor
ptio
n)
CatabolismRetention
0%
20%
40%
60%
80%
100%
15 ºC 18 ºC 21 ºC
Dig
estib
ility
(% o
f tot
al fe
d)
EvacuationAbsorption
ab c
a bc
a b a,b
a,bba
LARVI 2013 – 6th fish & shellfish larviculture symposium
• After transfer to a common temperature, post-larvae from 15 ºCshowed the highest protein digestibility.
0%
20%
40%
60%
80%
100%
15°C 18°C 21°C
Dig
estib
ility
(% to
tal f
ed)
24 HAF Evacuation
Digestibility
0%
20%
40%
60%
80%
100%
15°C 18°C 21°C
Ret
entio
n an
d C
atab
olis
m(%
Abs
orpt
ion)
24 HAF Catabolism
Retention
a
a
b
b
b
b
Conclusions
• Senegalese sole pelagic larvae → more susceptible to 15 ºC than embryos.
• Incubation at 18 ºC + larval rearing at 21 ºC → promoted larval musclehyperplastic growth, which can have positive implications on muscle growthpotential.
• Thermal plasticity of myogenic genes and miRNAs may be responsible forthe observed growth effects during the incubation experiment.
• Rearing pelagic larvae at 15 ºC → greatly decreased growth and survival,decreased protein absorption and retention and increased DNA methylationlevels of myog promoter in skeletal muscle → however, a mechanism ofcompensatory growth was later observed in the 15 ºC juveniles.
• Rearing pelagic larvae at 21 ºC → promoted a good growth and survival andtherefore is appropriate for Senegalese sole pelagic larvae.
LARVI 2013 – 6th fish & shellfish larviculture symposium
Take home message
• Water temperature during early ontogeny has major effectson Senegalese sole growth potential, both in short and long-
term, with implications on muscle cellularity, gene expression,gene regulation and protein utilisation.
LARVI 2013 – 6th fish & shellfish larviculture symposium
SFRH / BD / 43633 / 2008
João SendãoMaria Filipa CastanheiraPaulo GavaiaHelena TeixeiraFilipa RochaVera RodriguesElsa CabritaAndré Santos
Alessia GiannettoArvind SundaramTeshome Bizuayehu
COST Action FA0801
Acknowledgements
LARVI 2013 – 6th fish & shellfish larviculture symposium