-------------------
.
.
ICES STATUTORY MEETING 1993
,
, , '
I\fariculture Committee
,
'.,
1993/F:24
.
PERFORMANCE OF TRIPLOID PACIFIC OYSTERS CRASSOSTREA GIGAS
(Thunberg)REARED IN HIGH CARRYING CAPACITY ECOSYSTEM: SURVIVAL,
GRO\VTH, AND PROXIMATE ßIOCIIEMICAL COl\lPOSITION.
by
P. GoulIetquer *, J.P. Joly*, A. Gerard**, E. Le Gagneur*,
J. Morieeau*, J.M. Peignon**, P. Phelipot**.
"
* IFREMER RA, B.P. 32 14520 Port-eri-Bessin (Franee).
** IFREMER URGE, Ronee les Bains, B.P. 133, 17390 La Tremblade.
ABSTRACT: Triploid oysters C. gigas were produeed in 1990 by u-eatlng feitÜized eggs with
cytochalasin B. Triploids, treated diploids, and controls were deployed in early 1991 in a high
carrying capacityeeosystem on the Eastern Coast ofNormandy (Franee). A nionthly monitoring in
1992 showed that triploid yielded signifieantly higher growth rate arid biochemical composition.
However, growth was more heterogeneous. No evidenee was found for abimodal distribution within
triploid groups arter a 26-month rearing cycle. Triploids showed retardation of garnetogenesis.
Carbohydrates content in triploid rcmained almost constant (40%) from Jurie to September. Their
survival rates were significantly lower than diploids, and treated diploids were found more sensitive
to environmental conditions than controls. Therefore~ method of iriduetion should be improved to
maximize triploidy rate. We reeommend further field testings to assess triploid response to stressfulenvironmental eonditions, parueularly in low carrying capaeity'eeosysterri.
Keywords: C. gigas, trlploidy, survivai rate, i>iocheinieai composition.
RESUME: Une production d'huitres japonaises triploIdes C. gigas a 6t6 iiaiisee eri 1990 par
induciion ala cytochalasine B d'oeufs fertilises. TriploIdes, diploIdes refractaires et urie population
temoin ont ete mis en elevage au debut 1991 sur la cote Est de Normandie (France). Le suivi mensuel
moritr6 une meilleure cmissance et composition biochimique des triploIdes avee cependant une
plus forte heterogeneit6 en taille. Un seul mode a ete observe apres 26 mois d'elevage. Les triploIdes
montrent iin retard de gametogenese et une teneür eonstarite en sueres de juin aseptembre. Les taux
de survie sont plus faibles chez les triploIdes~ et les diploIdes traites sont plus sensibles que les
temoins aux eonditions environnementales. La teehnique d'induction doit etre amelioree et la
resistanee des triploIdes testee en eonditioris stressantes.
a
·"
e
MoLS-eies: C. gigas; triploidie, survie, composition biochimique.
Introduction
Extensive literature is now avaiIable regarding ploidy manipulation iri mollusean shellfish (for
review, see Beaumont arid Fairbrother, 1991). Triploidy has been stlldied on several species
includirig the Pacific oyster C. gigas and the Eastem oyster C. virginica (Stanley ei UI., 1981). Ploidy
manipulation is usually considered as a methOd for enhancirig pfoduction. However, few studies
arialyzed oyster production over a full reäririg cycle, arid most of them have focused on yearIings
(Allen and Downirig, 1986; Arashige arid Fushimi, 1992). Since oysterreproductiori effort increases
with age and is arfected by triploidy, it seems ofprirticular interest to study their production over the
entire rearing cycle. Moreover, only few studies eorisidered eiwirorimental constraints on genetically
rnanipulated species (Shpigel et aI., 1992).TIlerefore, triploid oysterpioduetion is almost unpredictable
before field testing.
I
.
1
"
/
Oyster culture is widely distributed along the French coastline, from the English Channel
(Nonnandy) to MCditerranean lagoons. Therefore~ oysters face various environmental conditions
affecting theirphysiological activityarid growthrrite. By way ofexainple, natural spatfall Occurs only
iri the SOlithwest pari of France. Before full-scale cuIture using genetically maniimlated shellfish, it
appeärs Critical to test their physiological capacity and survival rate in various ecosystems. Besides
survival rate, and from a management point ofview, cohort homogeneity is also ofparticular interest
for oyster farmers.
The preserit study was undertaken to rletennine (1) the triploid oyster proouctlon at two leveis
of stocking density in a high carrying capacity ecosystem, and (2) a possible birriOdal distrlbution
resulting from trlploids creatCd during the rrieiosis I arid meiosis 11, and (3) the effect of the
biochemical treatment on diploid. The overall objective is to assess the corrimercial feasibility basCd
upon triploid oyster production.
Materials and Methods
•
, ....
..
~
,
Oysters used in this fieId study were produced as pan of a comprehensive cytogenetic research
progi-am airning to develop new methodologies and improve three species characteristics, the Manila
cIam Tapes philippinarwn, the flat oyster Ostrea edulis arid the Pacific cupped oyster Crassostrea
,
gigas (Dufy and Diter, 1990; Desrosiers et a1., 1993; Gerard et al. 1993).
,C. gigas broodstock was conditioned at IFREMER's research hatchery faciIitY (URGE), Ronceles-Bains (France). Triploids and diploids were prOducCd from the same mass spawn on July 5, 1990.
Triploidy was induced by ti-eating eggs at 25°C with 0.5rng.l- 1 cytochaIasin B (CD) dissolvCd in
DMSO (Img.!-I) for 20mm, beginning 15mm after fertilization. Methodology was derived frorn
Downing and Allen (1987) protocoI. After settlement (day 25th), spat from the CB trei:lted groups
were assayed and found to contain 66% trlploids. Oysters were maintained in nursery and fed with
mixed phytoplankton culture.
"
" "
On 19 March 1991; oysters (triploids averagirig 2.33g arid diploids 2.63g) were randomly
seleetCd and equally deployed on two sites located on the Eastern Coast ofNormandy: Ste Mane du
Mont (site 1) and BayofVeys (site 2), shellfish cultured areas characteriiCd by a low (1,500 metrlc
tons) and high (10,000 tons) stcicking density respectively (Kopp ct aL, 1991; Jeanneret ct aI.~ 1992)
(Fig. i). Moreover, this area is chamcterized by low Summer temperature (T°=17°- 20°C) impedirig
intensive natural spatfall.
,
,
In 1992, experimental populations were sampled on a monthly basis. Length and total weight
were measured individuallyon 30 animals to the nearest inm and o.oi g respeetively. Relative
survival rate was estimated by counting dead animals. Oysters were shucked arid a piece of gill
removed for ploidy control by image cytometrY (Gerard et a1., 1991). Thc remairider of each oyster
was stored at -60°C, then freeze-dried 36 hours before weighing whole diY meat. Proximate
biochemical composition wasestimated using lOmg of freeze dried homogenate. Lipids werc
extracted, ptirlfied and then analyzCd according to Marsh and Weinstein (1966), and aIigh and Dyer
(1959). Carbohydrates and glycogen analysis followCd the proCedure ofDubois et a1. (1956).
Three groups were defined for datll treatment bllsed upon ploirly levels: trlploirls (3N), CR
treated diploids (2N), and diploids (contiul).
Results
Survival
, " . We estini~tCd.th~proportion oftriploids in t~e.spat priorto field deployrrient; 60% ofthe spat
sampled were trIplOId In 1990. After a 26 month reanng cycIe, a sub-sample showed that 53.6% and
70% oftreated rinimals were triploids on site 1 and 2 respectiveIy. A Chi-square test was performed
2
10 km
~ Oysters
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Fig. 1 : Geographie distribution.
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Size dass (mm)
Size class (mm)
Fig. 2 ; Cumulative frequeney (sites 1 & 2).
3
oh data resulting from the 1992 inorithly monitoring on site 1. There was no signÜicarlt (Üfference
in triploid proportion over the course of the experimerit <x2=3.42; d.f.=5, 1»0.05). None of the 62
controls were triploid. Similar tests on the survival rate of the treated and controlled populations
estimated by field counting showed significant differences (2 x 2 contingencyx2 =31.41; P<O.OOOl,
site 1; X2=9.07, P<O.OOl, site 2). Survival rates of controls were sigriificantly higher thari treated
populations (Le., sitel: 68.9% vs 48.6%, site 2: 87.1 % vs 73.8%).
Growth
Distribution fitting
e
."
.
Analysis of total weight frequencies did not allow describing abimodal distribution within the
triploid group, in spite of the 26 months life span. Cumulative frequencies described distributiori
patterns among treated, controlled diploids and triploids (Fig. 2). The lowest slope coefficients were
observed for bothtriploid groups, demonstrating growth heterogeneity. Negative Skewness and
Kurtosis coefficients characterized treated diploidson both sites (Le., site1: -0.15, -1.41; site 2: -0.32,
-1.35) and indicated a distribution trend toward a flat curve with short tails, arid the lower tail longer
than the upper. ControlIed populations showed positive vahies forboth coefficients (Le., Sitel: 0.45,
0.31; Site 2: 0.39, 0.12) describing a steep distribution at the center and upper tail longer than the
lower. Regarding triploids, the distribution tendOO to a flat curve with the upper longer than the lower
tail (Le., site1: 0.69, -0.41; site 2: 0.52, -0.30). However, assumptiori of normal distribution was still
valid in all cases to allow parametric testing.
Site effect
,
,
'
.
;
No significant difference in length and total weight was notOO bCtween sites within each group.
Iri contrast, ANCOVA for the dry meat weight with the total weight as, a covariate estimatOO
significant difference for each group (Le., triploids, F=181.4, P<O.OOOl; treated diploids, F=26.9,
P<O.OOOI; coritrols, F=165.0, P<O.OOOl). Overall growth performance on site 1 was significantly
greater thari those on site 2 (Fig. 5).
Ploidyeffect
,
After completiori of the reanng cycle, total oyster weight and length were significaritly
different among triploids, treated diploids and diploids for both sites (ANOVA: site I, F=3.5, P<O.05;
F=4.4, P<O.02; site2, F=6.35, P<O.Ol; F=7.57, P<O.OO2). Monthly growthdata on site 1demonstrated
that the main difference favoring the triploids occurred during the reproductive period in Summer
(Fig.3)..
"
... ' .
. .
", .
,,
. .
From 18 Februafyto 15Septeml>Cr 1992, overall increasein dry ineat weight was 637% in triploids,
and only 381 % in treated diploids. Following spawning, which oCcurred on site 1 between 3 August
and 15 Septeinber, dry meat weight decreased by 9.1 %, and 14.4% of pre-spawn weight for treated
diploids and diploids respectively. The reproouctive effort for triploids reached 3.9~ of pre-spawn
weight for a standardized oyster (65g totaI weight). Although initiated, triploid spawning Was
probably still in progress at thecompletion ofourstudy. On site 2, spawning was retarded fortriploids
and treated diploids. Dry ineat weight for controls declined by 42.3%.
Ploidy effect was anil1yzOO on Septemberrlry ineat weight daci using ANCOVA with the total
weight as a covariate. Significant difference was observed amorig ploidy level on site 2 (F=1O.7,
P<O.OOOl). Since the spawning was delayed, multiple range an3.1ysis classified treated diploids and
triploids as a homogeneous group. In contrast, ploidyeffect was not significanton site 1 (F=2.76,
P=O.07). This was probably due to the high post-spawning variability (mean±SD: triploid; 4.18±2.07;
treatOO diploid 2.68 ±1.03, diploid 3.04 ±1.27g).
Proximaie Biochemical composition
Triploids, and diploids and treated diploids showed different paiterns of utilization of
carbohydrntes, glycogen and to a lower extent, lipids. cafbohydrates storage occurred imtil June
wheri reaching a record high of 42% for diploids and triploids (FigA). Thereafter a steadily decline
was notOO for controls arid treated diploids until pre-spawning (29.7%). In contrast, carbohydrates
content for triploids remairied constarit around 40%. A similar pattern was n<>ted for glycogen until
4
a
August. Then~ glycogen declined slightly in Septemberwith concomitant lipid increase, indicating
gametogenesis activity. Carbohydrates and glycogen incieased in diploids after spawning while
lipids increasoo and carbohydrates decreased in triploids ~
ANCOVAs with the dry meat weight as a covariate were perfornied on September data. On
site 1~ significant differences were observed among ploidy groups regarding lipids (F=3.25, P<O.05)
and carbohydrates (F=8.1, P<O.OOI). For both variables. treated diploids and controls were
significantly lower than triploids (Fig. 6 and 7). Since glycogen concentration increasoo in postspawning period in diploids and treatoo diploids. and triploids had the opposite trend. no significant
difference was noted among groups (F=1.75. P=O.18). Similar ANCOVAs on site 2 were significant
in all cases (lipids, F=4.03. P<O.OO5; carbohydrates, F=38.1. P<O.OOO1; glycogen, F=40.5. P<O.OOO1).
However. since treatoo diploid spawning was delayoo. average lipid conterit was homogeneous
between treated diploids and triploids (Le.; 3562mg, 41O.2mg) arid significantly higherthan control
mean (i.e.• 156.7mg). Similarly. carbohydrates ilOd glycogen (Fig. 8) contents were significantly
lower in diploids and treated diploids than triploids (351.5mg. 346.5mg, 645.2mg; .311.6mg.
318.2mg. 593.hrig respectively).
Discussion
.
e
•
...
'
. Allen and Downing (1986) have demonstrated an effective gametogenesis and spawning in
tiiploids. Gonadal development in triploids was retarded and resulted in substantial differences in
glycogen coritent during gametogenesis. In our study. triploids showed also a reproductive activity
but ndther carbohydrate and glycogen decline during Summer. Gametogenesis for treated diploids
and controls. induced catabolism of energetic reserves, resulting in carbohydniü~s decline and
concomitant lipids iricrease. The reduced reproductive activity in triploids limited this biochemical
pathway. Only a slightdecline was noted in September. The particularly high food availabilitymight
be responsiblc for this dissimilarity since glycogen content is rcgulated byenvironmental conditions
including tClupcraturc and food level (Gabbott. 1975). Temperature cffects ncurosecretory honllones. which control storage tissues andgerniinal cells (Gaboott. 1975; Lubet arid Mathieu, 1978).
Moreover. Summer temperature in this ecosystem varies around 17°-20°C. the criticallower limit
for C. gigas successful spawning (Herm and Deslous-Paoli; 1991). However diploids did spawn.
Therefore. we can assurne that triploids are urider the same regulatory control as diploids but their
physiological responses might be tied to various thresholds.
..We have shown that triploids weresignificanily largerthan diploids ami treated diploids by the
end ofthe monitoring. This might beexplained by thereduced energetic spending for gametogenesis.
energy preferentially used for growth (Allen and Downirig. 1986). Refererice to heterozygosity has
also often been used to explain growth discrepancies. Triploid oysters created during the release of
the firSt polar bOOy (Meiosis I) are characterized by higher heterozygosity and would yield enhanced
growth. even though this theoretical advantage has not yet convincingly been demonstrated (Stanley
et a1.. 1984. Yamamotoet a1.. 1988). Li eta1. (1992). Jiang eta1. (1993). on the pearl oyster Pinctada
martensii and Mason et a1. (1988) on Mya arenaria were unable to establish a positive correlation
between heterozygosity and growth. In our study; triploids were created at both first and second polar
releases (Meiosis I & 11). CB treatments at first body fonnation may effect laival sUrVival (Downirig
and Allen. 1987). Since no heterozygosity control was carried mit on triploids~ it s6ems difficult to
conclude on anyrelationship. Howeverno expected growth difference within the triploid groups was
observed by the end of the experiment. Individual variability. selective mortality. or lacking effect
of heterozygosity inight be either responsible for this final distribution.
Scveral studies have shown comparable survivai rate between diploid controls arid triploids
(Stanley et al.• 1984; Allen et a1.. 1986; Chaiton and Allen. 1985). Allen and Downirig (1986)
obscrved higher survival during reproduciive activity. arid Akashige and Fushimi (1992) reported
twice as much survival as contrOl during the resting perioo. This study is an exception to these
findings since triploid survivorship is significantly lower. Moreover, no long-terrri negative effects
from cytochalasin B were found on shellfish testing (Downing and Allen, 1987). Since triploidy
percentage remained constant duririg the experiment and survivorship of treated animals lower. we
conclude that treated diploids ure more serisitive. to environmental conditions. Even the recent
evidcnce suggesting there may be some spontaneous chromosome loss in triploids as they age does
not counteract these results (Allen. 1993). This is also ofparticular iriterest forhatchery production
and prompt us to recommend to optimize the methodology to maximize the triploidy rate. Recently.
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Fig. 3 ; comparison of length, total weight, dry meat weight and condition index between
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new inductions yielded 95% of triploid using CB. Besides, the new chemical inducer 6-DMAP has
been alreädy successfully tested, yieldirig to higher larVal suivivorship and 85% of triploid
(Desrosiers et al., 1993; Gerard et al. 1993).
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. The unexpected results of survivorship as weIl asthe higher growth vanability in triploid
demonstrate the need for further testing in vanous ecosystems including low caiTyirig capacity
environment. This would allow better assessment oftriploid sensitivity and performance to stressful
eilVironmental conditions, therefore allowing enhancement of oyster productiori.
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----_. _.' .• _- _.._._-_
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10