Doi:10.1016/j.jembe.2007.07.009

Journal of Experimental Marine Biology and Ecology 352 (2007) 226 – 233 Individual relationship between aneuploidy of gill cells and growth rate in the cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids Frederico M. Batista a,b,1, Alexandra Leitão a,c,1, Vera G. Fonseca d,e, Radhouan Ben-Hamadou e, Francisco Ruano f, Maria A. Henriques b, Henrique Guedes-Pinto c, Pierre Boudry d,⁎ a Instituto Nacional de Investigação Agrária e das Pescas (INRB/IPIMAR), CRIPSul, Av. 5 de Outubro, 8700-305 Olhão, Portugal b Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal c Departamento de Genética e Biotecnologia, Centro de Genética e Biotecnologia da Universidade de Trás-os-Montes e Alto Douro (IBB/CGB), P-5000-911 Vila Real, Portugal d Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France e Centro de Ciências do Mar do Algarve (CCMAR), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal f Instituto Nacional de Investigação Agrária e das Pescas (INIAP/IPIMAR), Departamento de Aquicultura, Av. de Brasília, 1449-006 Lisboa, Portugal Received 1 May 2007; received in revised form 18 July 2007; accepted 24 July 2007 The Portuguese oyster, Crassostrea angulata, is taxonomically close to the Pacific oyster, C. gigas, but there are clear genetic and phenotypic differences between these taxa. Among those differences, the faster growth of C. gigas compared with C. angulata has oftenbeen observed in the field. Crosses between C. angulata and C. gigas were performed to investigate the relationship between growthvariation and somatic aneuploidy at the individual level in the two taxa and their reciprocal hybrids. The different progenies were rearedin Ria Formosa (Portugal) under standard farming conditions. Growth rate and survival were significantly higher in C. gigas than in C.
angulata, and the hybrids showed intermediate performances. Significant differences were also observed in the proportion of aneuploidcells (PAC) and of missing chromosomes (PMC) between the two taxa, C. angulata showing the highest values. Intermediate values ofPAC and PMC were observed in the hybrids, supporting additive genetic bases of these parameters. Our results also confirm the negativecorrelation between somatic aneuploidy and growth rate at the individual level, as previously reported in C. gigas.
2007 Elsevier B.V. All rights reserved.
Keywords: Aneuploidy; Crassostrea angulata; Crassostrea gigas; Hybrids; Growth variation Somatic aneuploidy of an organism can be defined as ⁎ Corresponding author.
an alteration in the number of chromosomes in a proportion E-mail address: (P. Boudry).
1 Authors contributed equally to this paper.
of the somatic cells due to abnormalities that arise during 0022-0981/$ - see front matter 2007 Elsevier B.V. All rights reserved.
F.M. Batista et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 226–233 mitosis. This phenomenon has been documented in several have also been reported, including differences in species and some of the mechanisms responsible for the production yield thought to be mainly due to the fast appearance and cellular surveillance of aneuploidy have growth rate of C. gigas ( been demonstrated ( ). Some ecophysiological para- ). In bivalve molluscs, aneuploid gill cells have been meters (valve activity: oxygen consumption: observed in the Pacific oyster Crassostrea gigas ; feeding and respiratory time ), the Antipodean flat oyster Ostrea activities: ) were found to account for angasi ) and mussels of the the differences in growth between these taxa. However, genus Mytilus ). In C.
aneuploidy had never been investigated in C. angulata gigas, which has a normal diploid chromosome number of or compared with the levels observed in C. gigas.
2n = 20, a high proportion of hypodiploid cells, with The known negative relationship between aneuploi- 2n = 19, 18 or 17, has been observed in several natural and dy and growth in C. gigas and the growth difference hatchery-produced populations between C. gigas and C. angulata led us to examine Further studies in this species showed that monosomies survival, growth and somatic aneuploidy in these two were observed in only 4 of the 10 chromosome pairs and taxa and their reciprocal hybrids. The main objective of no nullisomy has ever been observed this work was to establish if the individual relationship ). These findings clearly demonstrate between growth and aneuploidy, as previously observed that chromosome loss in C. gigas was not random or an within C. gigas, could also be observed in these two artefact of the air drying technique taxa, both at the individual and the taxon levels.
). A similar phenomenon can be observed intetraploid C. gigas, at higher frequency and level than in 2. Materials and methods The causal factors of somatic aneuploidy remain 2.1. Parental populations unknown in oysters. However, negative correlationbetween the degree of somatic aneuploidy and growth Ripe Crassostrea angulata were collected from a wild rate within and among families of C. gigas has been population in Monte-da-Pedra in Sado estuary (Portugal).
reported in several previous studies ( C. gigas adults were collected from the naturalized popula- tion in the Seudre estuary (Marennes-Oléron area, France).
Additionally, pollutants such as herbicides (e.g. ), aromatic hydrocarbons ) and 2.2. Crosses and larval rearing heavy metals (significantly increaseaneuploidy and affect the same chromosome pairs Crosses and larval rearing of the different progenies In oysters, however, most studies were performed in the Shellfish Hatchery of IFREMER were performed on C. gigas and the relevance of this Station in La Tremblade (France). Gametes were stripped phenomenon in other oyster taxa remained to be directly from gonads ).
investigated ).
Oocytes from 15 C. angulata and 10 C. gigas females The Portuguese oyster C. angulata and C. gigas are were pooled for each taxon and distributed in beakers with two commercially important species ).
filtered seawater. The same procedure was used with the Based on larval shell morphology (), spermatozoa of 5 C. angulata and 8 C. gigas males.
experimental hybridization (e.g., Crosses between C. angulata and C. gigas were produced and electrophoretic studies of enzyme following a 2 × 2 factorial mating to obtain the following polymorphism (several authors groups: C. angulata (AA); C. gigas (GG); hybrids derived have considered C. angulata and C. gigas to be the same from C. angulata females and C. gigas males (AG); and species. Moreover, there is also evidence that the two hybrids derived from C. gigas females and C. angulata taxa can hybridize in the wild ).
males (GA). Fertilization, larval rearing and settlement Studies on the mitochondrial cytochrome oxidase C were performed as described by subunit I (COI) gene (have shown that these two taxa are of Asian 2.3. Juvenile rearing and grow-out phase origin and closely related, although there are cleargenetic () and cytogenetic (e.g. Spat with 49 days post-fertilization (DPF) was differences between them. In addition, transferred to the Tavira Shellfish Hatchery Station of the significant phenotypic differences between the two taxa Portuguese Institute for Fisheries and Sea Research F.M. Batista et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 226–233 (INRB/IPIMAR). Oysters were reared as described by and growth rate) were preserved in absolute ethanol.
At day 204 after fertilization, 4 fine mesh Total DNA was extracted from gill tissue using a bags with 50 oysters each were prepared for each of the modification of a phenol/chloroform method described four groups. Twenty five individuals in each bag were by . The primers COI3 (5′- tagged with a non-toxic epoxy resin. Hence, 100 in- GTATTTGGATTTTGAGCTGT-3′) and COI4 (5′- dividuals for each group were individually labelled. The GAGGTATTAAAATGACGATC-3′) were used to am- bags were then deployed in the experimental site at Cacela- plify a 584 bp fragment of the mitochondrial gene Velha in Ria Formosa Lagoon (Portugal). The position of cytochrome oxidase C subunit I (COI). PCR reactions the bags on the tables was randomly changed every month.
were performed as described by .
PCR products were digested with Msp I as described by 2.4. Growth and survival the manufacture (Promega) and separated by electro-phoresis in agarose gels.
The live weight of each tagged individual from the 4 groups was recorded monthly during the grow-out phase.
2.7. Statistical analyses The animals were first measured at the beginning of theexperiment on 3 March 2004, subsequently, on 6 April, 4 Survival of oysters from the different groups was May, 2 June, 1 July, 29 July, 31 August, 30 September, examined using the Kaplan–Meier method. The 27 October and finally on 26 November 2004. Only the Mantel–Cox test was used to compare the survival data from oysters that were alive at end of the trends between groups at the 0.05 level after correction experimental period and that were scored for chromo- for multiple testing using the sequential Bonferroni some loss were used for growth rate analysis (i.e. 120 method (). Since no significant difference individuals). Individual growth rates (GR) were calcu- (Kruskal–Wallis test; p N 0.05) in live weight was lated using the slope of the linear regression of live observed among bags within each group, the effect of weight against time as described by bags was not considered for analysis of individual Mean Pearson r2 correlation of live weight against time growth rate. The suitability of data for parametric for each group (n = 30) ranged from 0.935 to 0.959, analysis was evaluated prior to examination using the indicating that it is a very good estimator of individual Kolmogorov–Smirnov test for goodness of fit (nor- growth in the present experiment (the lowest individual mality) and the Cochran test (heteroscedasticity).
value being 0.839). Survival was assessed monthly.
Growth rate (GR) differences among groups were anal-ysed by one-way analysis of variance (ANOVA). Fol- 2.5. Aneuploidy scoring lowing the ANOVA, multiple comparisons betweengroups were performed using Tukey honest significant At the end of the grow-out phase, 30 animals of each (HSD) tests. Non-parametric tests were used to identify group were incubated for 10 h in seawater containing differences among groups in the proportion of aneu- 0.005% colchicine. Their gills were then dissected in ploid cells (PAC) and proportion of missing chromo- seawater, treated for 30 min in 0.9% sodium citrate and some (PMC). When significant differences (p b 0.05) fixed in a freshly prepared solution of absolute ethanol- were identified with Kruskal–Wallis tests, multiple acetic acid (3:1) with three 20 min changes. Slides were comparisons were performed using Nemenyi tests. In performed from one individual gill following the air drying order to determine if PAC and live weight or GR of technique developed by the individuals analysed were correlated Kendall's non- . Chromosome counts were made directly, by parametric rank correlation coefficient was used. Data microscope observation (Nikon microscope), on 30 well- analysis was carried out using Statistica 5.1 and spread metaphases per individual. Two different measures of somatic aneuploidy described by were used: (1) the proportion of aneuploid gill cells (out of 30) (PAC) and (2) the proportion of missingchromosomes from a random sample of 30 cells (PMC).
All oysters progenitors (n = 38) and progenies (n = 120) analysed with the mitochondrial marker 2.6. DNA isolation, amplification and PCR-RFLP analysis showed the expected RFLP patterns. This allowed theconfirmation of the taxa of the sampled parental oysters Gill fragments from parental oysters and studied and the partial genetic confirmation of their progenies progenies analysed (i.e. scored for somatic aneuploid (i.e., maternal origin).

F.M. Batista et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 226–233 Fig. 1. Survival rate of C. angulata (AA), C. gigas (GG) and the reciprocal hybrids AG and GA using the Kaplan–Meier method. The female originin the crosses is listed first.
3.1. Survival and growth . These two parameters are well correlated(Kendall's τ within each group ranging from 0.63 and A low, but not unusual in the study site, survival was 0.85, p b 0.001) and consequently provide rather similar observed over the study period (40.0 to 54.5%).
results. A significant difference in PAC was observed Survival rates of C. angulata and C. gigas progenies among the four groups (Kruskal–Wallis test; H = 9.34; as well as their hybrids, using the Kaplan–Meier p = 0.02). A posteriori Nemenyi tests failed to distin- method, are presented in . The highest and lowest guish hybrids from both parental lines, but revealed survival rates were observed for C. gigas and C.
significant differences (p b 0.05) between C. angulata angulata progenies, respectively, with significant differ- (PAC = 20.0%) and C. gigas (PAC = 16.7%) progenies.
ences between the two groups (Mantel–Cox = 7.09; Similarly, PMC also differed significantly among the p b 0.05). No significant differences in survival wereobserved among the other groups (Mantel–Cox test;p N 0.05).
Live weight of labelled oysters from the four groups increased through time (Growth rate (GR) weresignificantly different among groups (ANOVA,p b 0.01). The GR of GG group (mean of 0.098 andstandard deviation of 0.031) was significantly higher(Tukey's HSD, p b 0.01) than AA group (0.071 ± 0.029),but not from AG (0.089 ± 0.027) and GA (0.097 ± 0.031)groups. No significant differences were observedbetween AG group and any of the other groups (Tukey'sHSD, p N 0.05). The GR of GA group was onlysignificantly higher than the AA group (Tukey's HSD,p b 0.01).
3.2. Somatic aneuploidy Fig. 2. Mean (the error bars represent standard error) live weigh of C.
angulata (AA), C. gigas (GG) and the reciprocal hybrids AG and GA The proportion of aneuploid cells (PAC) and missing (30 individuals per group) during the experimental period. The female chromosomes (PMC) for the four groups is presented in origin in the crosses is listed first.
F.M. Batista et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 226–233 four groups (Kruskal–Wallis test; H = 8.55; p = 0.04), with significant differences observed only between C.
Kendall's non-parametric correlation coefficient between theproportion of aneuploid cells (PAC) and final live weight as well as angulata (PMC = 1.67%) and C. gigas (PMC = 0.83%) growth rate for C. angulata (AA), C. gigas (GG) and the reciprocal progenies (Nemenyi Test; p b 0.05).
hybrids AG and GA. The female origin in the crosses in listed first Kendall's correlation coefficients between PAC and Final live weight final live weight and RG within each 4 group arepresented in . Relationships between final live weight and somatic aneuploidy are illustrated in Fig. 3 (at the individual level within each group) and (between groups).
⁎pb0.1; ⁎⁎pb0.05; ⁎⁎⁎pb0.01.
and reared under similar conditions after approximately 3 years. The faster growth of C. gigas relatively to C.
angulata was also observed by The mortality of C. gigas was lower than that of C.
and on comparative field studies angulata supporting results previously obtained by where hatchery-produced animals were reared in different sites on the coast of France.
The differences observed between the two taxa mayhave been due to different environmental requirements 4.3. Growth rate of the hybrids (e.g., emersion rate, temperature, salinity) but, to ourknowledge, the available ecophysiological studies do The reciprocal hybrids showed an intermediate not provide evidence for such different requirements.
growth rate between C. angulata and C. gigas The reciprocal hybrids showed an intermediate progenies. This observation is consistent with the results survival between C. gigas and C. angulata progenies and no significant differences were obtained between that did not also observe hybrid vigour nor out- them and the other groups. This is consistent with the breeding depression in crosses between the two taxa.
results obtained by and Differences in growth rate between reciprocal hybrids and supported by the additive genetic basis produced by crosses have been reported amongst inbred of the observed variation for survival in C. gigas lines of C. gigas (as well as in inter-specific hybrids between Mytilus edulis and M.
galloprovincialis (). Maternal 4.2. Growth rate of C. angulata and C. gigas effects can usually be detected at early stages and areexpected to dissipate with age of the offspring and are The growth rate results obtained in the present study therefore often neglected ( support previous findings that showed that C. gigas They can also be caused by mitochondrial genes.
grow faster than C. angulata. observed that Although a maternal effect for growth rate has been the total weight of C. gigas individuals was 2 times observed in reciprocal hybrids of C. angulata and C.
higher than C. angulata individuals with the same age gigas ), thiswas not detected in our study nor in the ecophysiologicalstudy by .
Table 1Proportion of aneuploid cells (PAC) and missing chromosomes (PMC) 4.4. Somatic aneuploidy in adult oysters of C. angulata (AA), C. gigas (GG) and the reciprocalhybrids AG and GA at the end of the experimental period (n = 120).
The female origin in the crosses is listed first In our study, a significant higher proportion of aneuploid cells (PAC) and missing chromosomes (PMC) were observed in the C. angulata progeny comparatively to the C. gigas progeny. These results suggest the existence of a different susceptibility to chromosome loss between the two taxa and therefore support the hypothesis of a genetic basis for somatic F.M. Batista et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 226–233 Fig. 3. Linear regression lines of live weight (at the end of the experimental period) against the percentage of aneuploid cells (PAC) in C. angulata(AA), C. gigas (GG) and the reciprocal hybrids AG and GA (30 individuals per group). The female origin in the crosses is listed first.
An intermediate level of somatic aneuploidy, although not . Interestingly, this relationship is also observed significantly different from parental lines, was observed in between taxa, the slower growing taxon showing the the reciprocal hybrids, suggesting the additive nature of highest level of aneuploidy So the present study such genetic variation. also did not find confirms that a negative correlation between the degree any heterotic effect for somatic aneuploidy in individuals of somatic aneuploidy and growth rate exists in oysters, produced by controlled crosses amongst inbred lines of C.
both at the individual () and the taxon levels gigas, although hybrids showed considerable heterosis for Our results also support the existence of a negative correlation between the degree of somatic aneuploidyand growth rate in oysters, as previously observed in C.
gigas (see for review ), but not in C.
virginica (). Most previousstudies compared "slow" and "fast" growing oysters(e.g. ) and only one study examined the individualrelationship between growth rate during a 8 monthperiod in 36 individual of a hatchery bred progenyIn this last case, highly significantlinear negative correlations were observed betweenaneuploidy and total weight in 12-, 15-and 20-month-old oysters r2 = 0.40, 0.53 and 0.58, respectively. In ourstudy, such correlations are lower, but still significant.
This might be due to the fact that, in our study, rearing of Fig. 4. Relationships between the median live weight (at the end of theexperimental period) and the median percentage of aneuploid cells oysters was performed in a more natural and conse- (PAC) in C. angulata (AA), C. gigas (GG) and the reciprocal hybrids quently less controlled (and therefore less homoge- AG and GA (30 individuals per group). The female origin in the neous) environment than in the study of crosses is listed first. Error bars represents SE.
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Source: http://www.mun.ca/biology/dinnes/Papers/Batista_et_al_2007.pdf