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 !!