Broodstock management and larvi-culture in marine species reared

in Polynesia and New Caledonia : genetic and health approach

Aquacop, G. Le MoullacIfremer, BP 7004,

98719, Taravao Tahiti

Aquaculture productions

• Shrimp : Penaeus stylirostris– New Caledonia and French Polynesia

• Pearl oyster : Pinctada margaritifera– French Polynesia,

• Fish : Lates calcarifer– French Polynesia

Shrimp : Historic• 1970-1980

– Selection of species for their reproductive ability in captivity and growth performances.

• 1980-1990– Broodstock maturation : nutritional improvement – Larval rearing :

• replacement of live food by microparticulate diet, • water management (biological filter)

– Sanitary status : genetic resistance to IHHNV (1987)• 1990-2000 :

– Starting of a genetic program• characterisation and improvement of strains domesticated in

Polynesia and Caledonia• development of tools : molecular markers and method of gametes

conservation

Main French shrimp farming areas

4 hatcheries50 km.Noumea

10 farms

Ifremer-SASV

New-Caledonia

0

500

1000

1500

2000

1980 1985 1990 1995 2000

Yiel

d (to

ns)

New Caledonia

French Polynesia

Ifremer-COP

3 farms1 hatchery

French PolynesiaPapeete

P. stylirostris shrimp : Broodstock management• Maturation

– unilateral eyestalk ablation in females– males kept at 25-26°C – improved feeding – artificial insemination (2 males X 1 female)

• Gamete preservation I – refrigeration at 4°C in conservation medium at pH 7 containing

antibiotic allow to use sperm during 6 days – transfer from Ecuador to Tahiti (2 to 6 days from collection to mating),

165 wild males X 87 domesticated females (17% success)

• Gamete preservation II – cryo-preservation in liquid nitrogen : P. vannamei nauplii obtained in

1991 and 1996, non reproducible probably due to breeder and gamete of insufficient quality, no results obtained with P. stylirostris

P. stylirostris shrimp : Genetic improvement• Resistance to disease

– spontaneous resistance to IHHNV• Selection for growth

– an improvement of 34% of weight at the sixth generation

-15%

-10%

-5%

0%

5%

10%

15%

20%

25%

30%

35%

0 1 2 3 4 5 6 7

Generations

Gen

etic

impr

ovem

ent o

f gr

owth

rate

(% o

f con

trol

)

first experiment

second experiment

P. stylirostris shrimp : Genetic resources

• Micro-satellite and intronic markers have allowed to characterise the strain and found :

– a positive relation between heterozygocity and growth– the loss of genetic variability during the domestication process (20

generations) compared to the wild stock from Ecuador• These markers allow to check shrimp pedigree and now to implement a

strategy to preserve residual variability

– maintenance of several independent populations– structuring of each population into 2 sub-populations

• Importation of genetic variability – Using refrigerated spermatophores :15 F1 families were produced in

quarantine facility

P. stylirostris shrimp : Genetic resourcesEvidence of genetic variability loss during the domestication process (20 generations) compare to the wild stock from Ecuador

Checking of pedigrees

P. stylirostris Allelic Heterozygocitystrains number /

locuslevel

Tahiti 1-3 0.1New Caledonia 0.2Wild Ecuador 14-25 0.9

P. stylirostris shrimp : Sanitary managementSanitary status of breeders

– Except the presence of IHHNV, Polynesia and Caledonia territories keep a favourable sanitary status probably related to the use of closed broodstock

– perspectives• Health state management (SPF)• Immune criteria for health status determination in relation with

environmental, nutritional and genetic state

Control of disease in larval rearing

– antibiotherapy : erythromycine, oxytetracycline (furazolidonereplacement)

– alternative : probiotic (Pseudoalteromonas piscicida)

Pearl oyster industry in French PolynesiaHistoric

- First episode : mother-of-pearl era in XIXth century- mother-of-pearl was exploited to supply the button industry - appearance of synthetic button is the death of this industry in

the middle of XXth century

- Second episode : fast-growing of pearl oyster industry- 1960 : research on pearl production withthe help of Japanese experts especially on spat collection and graft trials

- 1980 : start of pearl oyster industry

- 1999 : 6 metric tons of pearl, 200 million US$, 1076 farms-7000 employment

- 2000 : difficulties due to overproduction,

French Polynesia

Annual environmental change in French Polynesia lagoons according to the latitude

North. Tuamotu

21.522.523.524.525.526.527.528.529.5

09/0

4/98

07/0

7/98

04/1

0/98

01/0

1/99

31/0

3/99

28/0

6/99

25/0

9/99

23/1

2/99

21/0

3/00

18/6

/00

15/0

9/00

13/1

2/00

12/0

3/01

tem

pera

ture

(°C

)

GambierTahiti (Vairao)

00:0000:2800:5701:2601:5502:2402:5203:21

01/0

1/00

05/0

2/00

11/0

3/00

15/0

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2/00

Day

leng

ht(h

)

GambierNorth. TuamotuTahiti (Vairao)

Hydrobiological characterisation of lagoons

Organicmatter (mg.L-1)

Chlorophyll a(µg.L-1)

Makemo 0.35 0.21Manihi 0.50 0.46Takaroa 0.48 0.30Takapoto 0.35 0.40Raiatea 0.42 0.54Vairao 0.50 0.30

Pearl oyster : sex and reproductive cycle

Sex and reproductive cycle are evaluated with

Spawning (induced with thermal shock)

biopsy

histology

gonadal weight changes

Sex ratio and gametogenesis

0%

20%

40%

60%

80%

100%

80-8

9

90-9

9

100-

109

110-

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149

150-

159

160-

189

size class (mm)

prop

ortio

n of

sex

es

HermaphroditeUndeterminedFemaleMale

7 23 39 36 39 42 56 26 27

Gametogenesis in situGonadal weight and gonadal index

(northern Tuamotu) (Pouvreau, 1999)Spawning rate related to the temperature

change (Vairao)

0102030405060708090

100

1 5 9 13 17 21 25 29 33 37 41 45 49

week

spaw

ning

fem

ale

rate

(%)

26.00

26.50

27.00

27.50

28.00

28.50

29.00

29.50

0102030405060708090

100

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52

week

spaw

ning

rate

(%)

26.00

26.50

27.00

27.50

28.00

28.50

29.00

29.50

tem

pera

ture

(°C

)te

mpe

ratu

re(°

C)

Spat collection

Genetic resources of P. margaritifera

Genetic resources of P. margaritiferaGenetic markers

DALP Method (Direct Amplification of Length Polymorphism)CC AC CC AE BD AB AA AC

EPIC Method (Exon Primers Intron Crossing)use of primers defined in exonic sequence (amylase, aldolase) Amplification of intronic sequence

3 markers :➥ pin 1 : 6 alleles➥ pin 2 : 5 alleles : A, B, C, D, E➥ pin 3 : 5 alleles

AA AC AA AA AD AB AA

2 markers: ➥ U4 : 6 alleles➥ aldo : 5 alleles

Development of hatcherySupply spat when the collecting is insufficient

Produce genetically improved spat (triploïd, selected for growth, pearl colour)

Supply spat to restock some atolls by respecting the genetic structure of the natural populations

Broodstock conditioning

synchronism of breedersto control crossbreedingcontrol of sex ratio (increase female percentage)

improve larval qualitywith breeder feeding

Objective

Method

feedingwater management

disease control

Larval rearing

Sanitary rules and zoo-sanitary surveillance network

• 1985 : mass mortality (60% of oysters)• Context still favourable to the emergence of disease

(uncontrolled transfert)• outbreak in Japan (Akoya virus)

• Zoo-sanitary regulations– banning of importation of P. margaritifera from other areas and

new pearl oyster species– surveillance network to prevent outbreak of disease

ConclusionThe industrial rearing of marine species in French

Polynesia and New Caledonia are the subject of genetic and sanitary follow-up

Preservation of the genetic resources

Genetic improvement and creation of selected lines

Saving of the sanitary state with the enforcement of zoo-sanitary regulations

Suppression of antibiotic use