Nutrition and reproduction of the pacific oyster Crassostrea gigas : main results of the European project GIGANUGA (Gigas Nutrition and Gametogenesis)

JF. Samain1, P.Sorgeloos4, M.Caers4, C. Van Ryckeghem4, P.Soudant4, O.Garcia5, J.Espinosa5, Y.Marty2, E.Palacios1, M.Mathieu3, C.Berthelin3, C.Quere1, J.R.Le Coz1, C.Seguineau1, J.Moal1

1IFREMER Brest, 2UBO-CNRS Brest, 3University of Caen : France4ARC Gent University : Belgium5University of Santiago de Compostela : Spain

Project FAIR CT.96.1852 (France, Belgium, spain)

5 aspects were studied :

– Cellular reproductive and storage cycles– Biochemical level : compare natural reproductive

cycle and hatchery conditioning at two seasons– Define deficiencies and problems– Evaluate possibilities to supplement hatchery

conditioning standard diet or low cost diet using emulsions or other particules

– Evaluate possibility to solve the question of fall conditioning

GIGANUGA 1:

CELLULAR ASPECTS OF REPRODUCTIVE AND STORAGE TISSUE CYCLES OF CRASSOSTREA GIGAS.

C. Heude Berthelin (1), J. Espinosa (2), O. Garcia(2), G. Hernandez (2),

K. Kellner (1) and M. Mathieu (1)

1 IBBA ,Biologie et Biotechnologies Marines, Université de Caen, 14032 Caen, France2 Facultad de Farmacia, Universidad de Santiago de Compostela, Espana

Course of the oyster gametogenesis

M onth J F M A M J J A S O N DGonad I/II II II II IIIA II/IIIA IIIA IIIB IIID IIID/0 0/I I

Cellular aspects of storage tissue development and mobilisation

Sstage Age A

Stage B Stage C Stage D

Month J F M A M J J A S O N D

Storage B/C C C C D D D D A B B B

Gonad I/II II II II IIIA IIIA IIIA IIIB IIID IIID/0 0/I I

GIGANUGA 2:

Nutrient storage and transfer during the reproductive cycle in nature and in hatchery

of the Pacific Oyster Crassostrea gigas

Soudant P.(1), M. Caers(2), Y.Marty(3), E.Palacios, C.VanRyckeghem(2), C. Heude Berthelin(4), JR Le Coz(1), C.Quere(1),

C.Seguineau(1), J.Moal(1), JF.Samain(1)

1 IFREMER Brest, Physiologie des Invertébrés, BP 70, 29280 Plouzané, France;2 ARC, Laboratory of Aquaculture , University of Gent, Rozier 44, 9000 Gent, Belgium3UMR/CNRS 6521, Université de Bretagne Occidentale, BP 809, 29285 Brest, France

4 IBBA ,Biologie et Biotechnologies Marines, Université de Caen, 14032 Caen, France

1/ Natural cycle : lipidsTotal lipid percentage

Natural cycle

0

5

10

15

20

25

20-Jan 11-Mar 30-Apr 19-Jun 8-Aug 27-Sep 16-Nov 5-Jan 24-Feb

Sampling date

% o

f the

DW

Gonad+mantleDigestive glandMuscle

1/ Natural cycle : sterols 97-98STERYL ESTERS

0

50

100

150

200

250

janv févr mars avr mai juin juil août sept oct nov déc janv févr mars

µg o

f ste

ryl e

ster

s/10

0 m

g of

dry

wei

ght

Glycogen metabolism in vesicular cell during gametogenesis

0

5

10

15

20

25

30

35

40

J F M A M J J A S O N D

Glycogène (% du PS)

0,00

2,00

4,00

6,00

8,00

10,00

12,00

Glucose incorporé (nmol)

1/ Natural cycle : thiamine andriboflavin

RIBOFLAVIN

02468

101214161820

20/01 11/03 30/04 19/06 08/08 27/09 16/11 05/01

µg /

g dr

y w

eigh

t

stade 1 3 4 1stade 2

Gonade + mantledigestive gland

2/ Deficiencies : comparisonNature-Hatchery: Spring period

PUFAs Steryl Esters

COMPARISON NATURE / HATCHERY

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

2 0 - a v r 2 0 - m a i 1 9 - ju i n 1 9 - ju i l

% of

ste

ryl e

ster

s in

tota

l ste

rols

Comparison natural / hatchery

0

5

10

15

20

25

20-Apr 10-May 30-May 19-Jun 9-Jul

Sampling date or larvae stage

% o

f the

tota

l fat

ty a

cids

20:5(n-3) in NL20:5(n-3) in PL

•2/ Relation between diet qualityand gonad composition : PUFAs

Information from gonad neutral lipids (fatty acids)

Neutral lipids from female gonads

y = 0.8373x + 1.9856R 2 = 0.8162

05

1015202530

0 10 20 30% AG Algues

% A

G L

NG

onad

s

2/ Relation between diet quality and gonad composition : sterols

STERYL ESTERS

y = 0.27x + 0.57R2 = 0.76

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1 1.2Arc sin % sterols in diet

Arc

sin

% s

tero

ls in

Gon

ad

Brassicasterol24Methylene cholesterolCampesterol

Cholesterol

Giganuga 3: Advances in liposoluble and hydrosolublecompound supplementation for broodstockand larvae of the Pacific oyster Crassostreagigas

C.Seguineau(1), M.Cansell(2), P.Soudant(1), C.Langdon(3), B.PoncePinon(4), M.Val Sanles(4), J.R.Le Coz(1), C.Quere(1), J.Moal(1), M.Caers(5), P.Coutteau(6), P.Sorgeloos(5), J.F.Samain(1)

1 IFREMER Brest, Physiologie des Invertébrés, BP 70, 29280 Plouzané, France;2 ISTAB, Université Bordeaux I, avenue des Facultés, 33405 Talence, France3 Hatfield Marine Science Center, 2030S Marine Science Drive, Newport, OR 973654 Facultad de Farmacia, Universidad de Santiago de Compostela, Espana5 ARC, Laboratory of Aquavculture, University of Gent, Rozier44, 9000Gent, Belgium6 INVE Technologies NV Oeverstraat7-9200 Baasrode, Belgium

EMULSION AND ALGAL SIZE

0

5

10

15

20

25

30

35

40

0 10 20 30

diameter µm

% v

olum

e

microalgal mixture

emulsion

EFFECT OF EMULSION CONCENTRATION ON ALGAL FILTRATION

algae

algae + emulsion

No algal filtration pertubation for emulsion concentration < 1.2 mg/L

emulsion 1.2mg/L

y = -0,0051x + 1,5535

y = -0,004x + 1,5249

1,21,31,31,41,41,51,51,6

0 20 40 60minutes

log

DW

(mg)

EMULSION ASSIMILATION EXPERIMENT ON SPAT

Control

0,1

0719330

2

4

6

µg/in

divi

dual

Days

Stigmasterol

Stigmasterolassimilation wasdose and timedependent

Emulsion: 0%, 3%, 10%, 20%

2/ Emulsion supplementation : absorption efficiency was lowAbsorption rate of sterols

010

20

30

4050

6070

80

90

100

Control 3% 10% 20%Treatments

Inco

rpor

atio

n ra

te (%

)

CholesterolStigmasterol

010

20

30

4050

6070

80

90

100

Control 3% 10% 20%Treatments

Inco

rpor

atio

n ra

te (%

) BrassicasterolCampesterol

From microalgae From emulsion

SPRAY BEADS

Incorporated materialsdissolved in an aqueous phasedry particules

within a triglyceridebead composed of:60% tripalmitin40% fish oil

Riboflavin crystal

Lipid matrix

GIGANUGA 4: EFFECTS OF LIPID SUPPLEMENTATION DURING CONDITIONING OF Crassostrea gigas FED A STANDARD MIXED ALGAL DIET AND A LOW COST ALGAL DIET

M.Caers*1, P.Soudant, E.Palacios, K. Curé*1, P.Sorgeloos1, J.R.Le Coz2, C.Quere2, Y.Marty5, O. Garcia3, J.Espinoza3, C. Heude Berthelin4, E.Danton6, B.Diss6, J.F.Samain2, J.Moal2

3/ Spring : effect of emulsiondoses on biochemistry

– No effect at 2.5, 5.0, 10% emulsion FA/DW algae (Hatchery diet)

– Positive effect at 20 and 40% emulsion and Skeletonema (Low cost diet)

DHA/EPA in Polar Lipids

0

0,2

0,4

0,6

0,8

1

Natural Skeleto Skeleto + 40% Emulsion

Conditioning diet

DHA/

EPA

3/ Spring : biological effectNo biological effect at less than 10%Some compensatory effect at 20 and 40%, but

survival and fecundity were affected depending on rearing systems

Open circulating system

0

10

20

30

40

50

% larvae-D % abnormality

Ske +T-isoSkeSke + 40%

Enclosed recirculating system

0

10

20

30

40

50

60

% larvae-D % abnormality

Ske +T-isoSkeSke + 40%

gigas in fall, a combination of physical and nutritional factors?

◆ J.F. Samain, M. Mathieu, C.Heude Berthelin, C.Cure, J. Espinosa, P. Soudant, E.Palacios, J.R.Le Coz, C.Quéré, J. Moal

Perspectives for a better reproduction process in hatcheries

3/ Hatchery conditioning in spring/ fall :

In spring hatchery conditioning was normalIn fall, hatchery conditioning after spawning

was impossible– No effect of the increase in temperature and

food

Problems in fall conditioning:

Gonad dry weight 1997-98

0

0,5

1

1,5

2

2,5

01 02 03 04 05 06 07 08 09 10 11 12 01 02 03

months

g / o

rgan

NatureFall 97

Conditioning in fall did not allow storage restoration

Problems in fall conditioning:

Fall 97 conditioning

0%

20%

40%

60%

80%

100%

start (sept) end (jan)

mitosis

no gametogenesisfrequ

ency

Histology : no gametogenesis was observed (C stage).

Availability of oogonia ?Annual cycle

0,0

5,0

10,0

15,0

20,0

25,0 T°C

Photophase

Mitosis

J F M A M J J A S O N D

E1 E2 E2 E2 E3A1 E3A1 E3A2 E3A3 C C E0 E1

Hypothese : all oogonia weremobilized for the main spawning during summer.

Are temperature and low photoperiod necessary for reinitiation of the sexualcycle?

Protocol :Preconditioning : 4 weeks at 9°C,

decreasing photoperiod. Algal mixture.Then, conditioning at 19°C and high

photoperiod. Algal mixture +/- emulsion

0

5

1 0

1 5

2 0

2 5

2 4 /0 7 /9 8 1 2 /0 9 /9 8 0 1 /1 1 /9 8 2 1 /1 2 /9 8 0 9 /0 2 /9 9

tem

pera

ture

C o n t r o l

c o n t r o l w ith o u tp r e c o n d it io n in g

Preconditioning conditioning

1/ Preconditioning on gametogenesis0

5

10

15

20

25

24/07/98 12/09/98 01/11/98 21/12/98 09/02/99

tem

pera

ture

Control

control w ithoutpreconditioning

In fall, a decrease of temperature and photoperiod allowed the initiation of a new gametogenesis cycle

Gametogenesis

0

20

40

60

80

100

120

no preconditioning with preconditioning

mitosisno gametogenesis

1/ Preconditioning on storage

No storage tissue restorationNo gain in DW, lipid, and steryl esters.

0

5

10

15

20

25

24/07/98 12/09/98 01/11/98 21/12/98 09/02/99

tem

pera

ture

Control

control w ithoutpreconditioning

Storage tissue development

0,020,040,060,080,0

100,0120,0

no medium

nopreconditioning

w ithpreconditioning

Storage composition

02468

10121416

gonad DW % Lipid/DW % steryl ester

3/ Fall : Effect of supplementation on gonad biochemistry and weight

Emulsion increased vitellogenesis (biochemical criteria)However, weak gonad development

Gonad composition

05

10152025

lipid/DW % EPA inPL

% sterylester

Algae5% emulsion10% emulsion

Gonad development

00,5

11,5

22,5

gonad DW

Algae5% emulsion10% emulsion

spring reference

3/Fall : effect of supplementationon gametogenesis

0

20

40

60

80

100

indifferenciated differenciated

no precondAlgae5% EM3010% EM30

emulsion accelerated vitellogenesis rate

Temperature and photoperiod

FoodNo storage-?

High

No Mitosis-?

No gametogenesis

Temperature and photoperiod No storage Food

1/Low

Mitosis+?

Oogonia

Mitosis

Oogonia

1/Low 2/High

+?

+?

Temperature and photoperiod

2/FoodSmall storage

Quantity :vesicular cell

number

Fecundity?+?

Oocytes

Fecundity?+?

Mitosis

Oogonia

Low High

+?

+?

Temperature and photoperiod Food

Small storageQuantity :vesicular cell

number

Oocytes

Quality :EPA, DHA,

AA, Vitamines...Emulsion

Vitellogenesisrate?

Conclusions...

The reproductive cycle of C.gigas is well characterized in Marennes Oleron on cellular and biochemical aspects.

Gonial mitosis should be re-initiated at fall by external factors as temperature-photoperiod. Regulations are understudy.

But storage control is not yet documented

...Conclusions ...Deficiencies in essential nutriments can be

evidenced comparing natural and hatchery processes : PUFAs, Sterols, Vitamins

Promising results were obtained using emulsions :» At spring, deficiencies of monospecific algal

diet can be compensated, but problems of doses and nutriments should be solved

» At fall, emulsion stimulates vitellogenesisrate, but control of storage is not yet solved

...Conclusions :Other particules as spraybeads or liposomes

encapsulating hydrosoluble compounds are understudy (vitamins).

These techniques are promising for a better understanding of essential nutriments for reproduction of bivalves all over the year, and using a low cost algal diet.

But other non nutritional factors affecting storage, gonial mitosis should be investigated and needmore cooperative researches.

Contributions :

» Experiments : IFREMER Brest and La Tremblade» Histology : J.Espinosa, O.Garcia, Univ. Santiago de

Compostella, Espagne, M.Mathieu IBBA Caen» Biochemistry of essential nutriments : IFREMER Brest,

Y.Marty, UMR CNRS 6521 UBO Brest, P.Sorgeloos, P.Coutteau ARC Gent, Belgique

» Glycogen metabolism : M.Mathieu et al. Univ.Caen» Supplementation technics : P.Sorgeloos, P.Coutteau ARC

Gent, Belgique, M.Cansell, Univ.Bordeaux, France, C.Langdon, Oregon Univ. USA

» Hatcheries : SATMAR, Guernesey Sea Farm