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CNRS INEEFRE 3484 BioMEA,
«Biologire des Mollusques Marins
et des Ecosystèmes Associés»
Factors involved in sex determination
(early sex differentiation)
in the pacific oyster Crassostrea gigas
… Knowledge for other mollusc species
and applications in aquaculture…
Anne-Sophie MARTINEZ
What is sex determination?
Sex determination
♂
Spz
Molecular cascade
Fertilization
♀
Testis
Gonadic differentiation
Ovary
Ooc
GSD
ZZ/ZW
(chicken)
XY/XX
(mouse)
XY/XX/XO
TSD
Turtle Lepidochelys sp.
and its eggs
GSD +TSD
Pleurodele
Pleurodeles waltl
Initial questions about sex determination …
What are the
molecular actors
(+ regulations)?
When does it happen
during the development and/or
in adults?
Sex determination?
Is sex determination
under environmental
influence ?
Which information is available to study mollusc sex determination?
1. Genomes
C. gigas
In each cell
and tissue
1st draft genome recently published
Other molluscs …
Bivalves
Genomic information tends to increase
Gastropods
Giant owl limpet, Lottia gigantea (2007)
California sea hare, Aplysia californica (2008)
Biomphalaria glabrata (2013)
2. Transcriptomes (reproduction)
C. gigas
In specific cells
and tissues
Gonad transcripts differentially
expressed between sex and
gametogenetic stage (Dheilly et al., 2012)
Gonad transcripts
differentially expressed
between R and S oysters
(Fleury et al., 2010)
Other molluscs …
> Pecten maximus and Ruditapes decussatus: Reproseed project
> Mytilus galloprovincialis : array-based gene expression of mantle tissue from animals at
different gonad maturation stages (Banni et al., 2011)
> Haliotis asinina : cDNA microarray on eggs to adult tissues (2008)
Increasing transcriptomic (reproduction) information
3. Reproductive traits/features
C. gigas
10 pairs of
chromosomes;
no sexual
chromosomes
identified
‘Protandrous ‘ hermaphrodite
then
Other farmed molluscs (main famillies grown in the world) …
Bivalves
Gastropods
Famillies
Karyotype (2n)
« Way » of reproduction
Mytilidae
Veneridae
26, 28, 30, 32, 38
Essentially gonochoric
Pectinidae
26, 32, 38
Gonochoric (Patinopecten yessoensis -Japon)
Simultaneous hermaphrodites (Pecten maximus)
Ostreidae
20
Essentially successive/alternative hermaphrodites
(Crassostrea virginica, C. japonica, Ostrea edulis)
Pteriidae
28/26
Successive hermaphrodites (Pinctada margaritifera)
Haliotididae
28, 32, 36
Gonochoric (Haliotis tuberculata)
Devauchelle, Cochard, 1980
Different karyotypes and reproduction amongst molluscs (between/within families)
4. Gonad (Anatomy, structure) and gametogenetic cycle
C. gigas
Tr
Tr
Ov
T
Stade 0
Winter
Stade I
Fall
Opv
Stade II
Spring
T
Stade III
Summer
Sexual rest
Tr
Spawning
T
Sexual maturity
Tr
Gonia proliferations
Stage 0
T
Sptz
Stage III male
Differenciation of
gametes
T
Sptd
Sptz
Om
Go
Tr
Stage I
Spg + Spc
Tr
Stage III female
Stage II male
Tr
Ov
Opv
T
Stage II female
C. gigas: Anatomy/structure of gonad and gametogenetic cycle well known
Other european farmed molluscs …
Anatomy/structure of gonad and gametogenetic cycle also well known
♀
> Mytilus edulis
♂
> Ruditapes decussatus
♂
♀
> Ruditapes philippinarum (Lee et al., 2012)
> Ostrea edulis
> Pecten maximus
> Mytilus galloprovincialis (Ruiz ret al., 2011)
> Haliotis tuberculata
5. Sex-ratio (field and experiments)
C. gigas and molluscs …
Hypothesis: environmental influence on sex determination (some points reviewed in Soyez
et al., 2011)
Abiotic factors (temperature, photoperiod…) or biotic (food, …) (Korpelainen, 1990; Yusa, 2007:
Dutertre et al., 2009)
> C. gigas: more ♂ [environment less favorable for reproduction; low food
levels; constant 8°C during a one-year experiment (Steele & Mulcahy 1999; Lango
Reynoso, 1999; Fabioux et al. 2005)]
> Mussel Mytella charruana: more ♂ (1 month low food level) (Stenyakina et al.,
2010)
> Tropical oyster Cortez corteziensis: ♂/18°C - ♀/9°C
Rodriguez-Jaramillo et al., 2008)
(Chavez-Villalba et al., 2008;
Which information is available on mollusc sex determination?
C. gigas
1. Genetic models
50%
Major gene
Dominant M allele
Year 1
Protandrous F allele
2 genotypes
(Guo et al., 1998)
Major gene
M and F alleles
3 genotypes
Year x
50%
♂
♀
♂
♂
♀
♀ ♂
f
1-f
Year 1
♂
♀
♂
♀
Year x
♂
♀
♀ ♂
♀
(Hedrick et
Hedgecock, 2010)
Environment ?
Genes ?
2. Our contribution …before Reproseed…
What are the
molecular actors
(+ regulations)?
Hypothetical models of molecular
cascades of sex determining actors (+
some
regulations)
(adults
and
development)
C. gigas
When does it happen
during the development
and in adults?
Kinetics:
> Sex determination (adult and development)
> Gonadic differentiation in spat
Cellular cartography of transcripts (adults)
Sex determination?
Is sex determination under
environmental influence ?
Other molluscs …
Few information; Hypothesis: Different sex determination systems amongst molluscs
(experimental crosses and triploids)
Sex determination?
Class/family
XY
Majority of gonochoric
molluscs (gastropods,
some bivalves)
> Dwarf surf clam
Mulinia lateralis
Allen et al., 1986
Guo and Allen,
1994
XO
(XX♀ ; XO ♂)
Widespread amongst
gastropods (Neritidae,
Littorinidae)
> Water snail
Theodoxus sp
> common winkle Littorina sp
Nakamura, 1995
Vitturi et al/and
Catalano,
1986,1988, 1995
> Soft-shell clam
Mya arenaria
Allen et al., 1986
X/A
Dosage-dependent
Bivalves
ZW/Z0
+ father and mother
mt genome (Breton
et al., 2007, 2011)
Bivalves
Oligogenic /
Polygenic
Bivalves
Example
Refs
> Mytilus galloprovincialis
> M. edulis
Kiyomoto et al.,
1996
Danton, 1996
Brake et al., 2004
> Crassostrea
virginica
Haley, 1977 and
1979
C. gigas
3. Reproseed contribution…
What are the
molecular actors
(+ regulations)?
Hypothetical models of molecular
cascades of sex determining actors (+
some
regulations)
(adults
and
development)
When does it happen
during the development
and in adults?
Kinetics:
> Sex determination (adult and development)
> Gonadic differentiation in spat
Cellular cartography of transcripts (adults)
Sex determination?
Is sex determination under
environmental influence ?
Food:
Optimal and constant concentration
(1500 μm3.μL− 1)
Conditioning system (spring 2011)
50 Isochrisis affinis
galbana (T)/ 50
Chaetoceros calcitrans
forma pumilum (Cg)
1-monthold spat
C. gigas
500 ml cylindricoconical tubes in a
flow-through system
18°C
22°C
25°C
28°C
(Rico-Villa et al., 2006)
> Mesure of the effect of temperature on :
- spat sex-ratio
- first gonadic differentiation
- expression of actors of the sex-determining cascade (Cg-DMl,
Cg-Foxl2/Cg-Foxl2os, Cg-SoxE, Cg-Betacatenin) + Oyvlg
Techniques:
Histology, qPCR
Growth (size and weight)
18°C
22°C
25°C
28°C
3.69 mm
6.23 mm
8.14 mm
9.26 mm
Size
(49 dpf)
(Growth
rate µg.d-1)
Robert et al. unpublished results
Increasing growth rates
Gonadic differentiation
18°C
22°C
28°C
25°C
Number of
gonadal tubules
(49 dpf)
spg
Inside the tubules:
Number and
‘maturity’
of germ cells
(49 dpf)
spg
spc
spg
spc
sptd
sptz
Accelerated kinetics of gonadic differentiation and gametogenesis
Disturbed but complete gametogenesis
> Growth and these physiological kinetics not necessarily correlated
> Different effects of increasing T° amongst molluscs:
- Accelerate gonadic differentiation/gametogenesis in C. gigas adults (Fabioux et al., 2005)
- Reduce rates of gametogenesis in M. edulis, Pinctada imbricate (Urban, 2000; Fearman et al., 2010)
Sex-ratio
22°C
18°C
♀/♂
100.0
1.4
1.3
♀/♂
Disturbed gametogenesis
0.9
55.6
58.6
60.0
0.5
4.0
2.7
80.0
28°C
25°C
45.9
32.0
Herma.
♀
40.0
20.0
0.0
44.4
51.4
64.0
n=44
n=24
n=42
n=53
♂
Increasing number of hermaphrodites and males
% of ♂
pourcentage de mâles
%
41.4
70.0
65.0
60.0
55.0
50.0
45.0
40.0
35.0
30.0
F
M
« ♀ ♂ » pattern of
temperature-sensitivity in spat of C. gigas
15
20
25
Température en °C
Temperature (°C)
30
Expression of sex-determining actors – time-window of sex determinination
22°C
18°C
28°C
25°C
Disturbed gametogenesis
18°C
♀<♂
I
Oyvlg
Cg-SoxE
0.16
Cg-b-catenin
Cg-DMl
0.12
♀ actor
0.10
DG
0.06
♂ actors
0.08
0.08
QR Ct
QR Ct
Transcript
expressions
0.10
K
0.14
0.06
0.04
0.02
0.00
d 28
d 32
d 36
d 40
d 44
d 48
d 52
d 56
No visible
Gonad
(histology)
40-44
Oyvlg
Cg-SoxE
Cg-b-catenin
Cg-DMl
♂ actors
**
0.04
♀ actor
*
0.02
0.00
d 28 d 29 d 30 d 31 d 32 d 33 d 34 d 35 d 36 d 37 d 38 d 39
days post fecundation
Time-window
(dpf)
28°C
♀ >♂
days post fecundation
34-37
32-34
34-36
Inversion of general pattern of expression
How to use these data for field applications?
> Careful use of information:
- Results not fully confirmed/hypotheses
- Not everything can be extrapolated to adults (physiology very different)
- The effect of T° can’t be dissociated from the availability of food
- Sex determination and gonadic differentiation: A specific physiological phenotype
associated to specific «environmental / rearing conditions» and genetic origin
C. gigas
1.
28
30
Food ad libitum
40 44 48
60
2. FTS, constant food concentration
18°C
80 90 100
dpf
Start of gonadic
differentiation
28
30
40 44
48
60
80 90 100
Gonadic
differentiation
dpf
Some potential applications in aquaculture
> Kinetics of first gonad differentiation and gametogenesis and how to « control » them?
- accelerate/slow dow these kinetics in oyster’s spat >> Improve their physiological
status and their potential resistance to stress when transferred to the field?
- Studies in other farmed molluscs: tools in C. gigas and knowledge about gonad and
reproductive cycles in other molluscs
> Physiological mechanisms controlling sex change: How to sex animals early? How to
orientate sex-ratio?
Physiological time-window of sensitivity
- adult oysters : stage III (cycle n) / stage 0 (cycle n+1); spat: after metamorphosis
- Information on anatomy/structure of gonad and gametogenetic cycle >> asset to future
studies in other farmed molluscs
Molecular mechanisms
- most information available in C. gigas
- increasing genomic/transcriptomic information in C. gigas and in other molluscs >>
conserved and early expressed actors of gonadic differentiation >> sex markers for
different farmed molluscs
Influence of age and size on sex determination
- no information available for the moment
- potential answers on C. gigas from an ongoing project supported by the Normandy
region (size)
Orientate sex (adult or spat)
- knowledge from sex-ratio in C. gigas >> contol the stock of genitors
- Reproseed results
But: Careful transfer of information to other molluscs
≠ types of sex determination hypothesized
≠ « ways » of reproduction (hermaphrodites vs gonochoric)
≠ issues in aquaculture
Interactions between professionals and researchers needed
Need of :
- Pool of information from mollusc farmers, hatcheries and researchers
- Link between genetics / physiology / «environment »
Professionals
Researchers
> Sex-ratio in the field and in hatcheries for specific conditions (rearing
system, T° conditions, nature and type of delivery of food…)
> Genetic origin of animals
> Size, age…
> Sex determination/gonadic
differentiation studies
> Genetic studies
Many thanks for REPROSEED experiments …
> Financial support
Reproseed (7th Framework Programme)
> Experimental support
BioMEA: Christelle Santerre, Béatrice Adeline, Pascal
Sourdaine, Jeanne Mathieu…; PROTEOGEN Platform (SFR
ICORE)
Argenton hatchery and its members (René Robert,
Christian Mingant, Nicolas Marc, Isabelle Queau, Luc
Lebrun ...)
THANK YOU !!
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