Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

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Journal of Experimental Marine Biology and Ecology

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Growth variation in bivalves: New insights into growth, physiology and somaticaneuploidy in the carpet shell Ruditapes decussatus

Joana Teixeira de Sousa a,b, Domitília Matias a, Sandra Joaquim a,Radhouan Ben-Hamadou c, Alexandra Leitão a,⁎a Instituto Nacional de Recursos Biológicos (INRB, I.P.)/L-IPIMAR, Av. 5 de Outubro s/n, P-8700-305 Olhão, Portugalb IBB-CGB/Universidade de Trás-os-Montes e Alto Douro, Vila Real Portugalc Centro de Ciências do Mar (CCMAR), EcoReach Research Group, Universidade do Algarve, Campus de Gambelas, P-8005-139 Faro, Portugal

⁎ Corresponding author. Tel.: +351 281 326951; fax:E-mail address: aleitao@ipimar.pt (A. Leitão).

0022-0981/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.jembe.2011.06.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 September 2010Received in revised form 27 May 2011Accepted 1 June 2011Available online 2 July 2011

Keywords:AneuploidyBivalveChromosomeGrowthPhysiological conditionRuditapes decussatus

The carpet shell, Ruditapes decussatus is a species with a high commercial importance in Portugal and otherSouthern European countries. However, high growth rate variability is a major problem in the aquaculturalproduction of this species. The objective of this work was then to study the possible differences between fast-,medium- and slow-growing individuals at three times of sampling, of a same cohort of R. decussatus in termsof eventual numerical chromosomal changes, aneuploidy, but also the level of physiological capacity, byassessing the condition index and the biochemical composition: proteins, total lipids, neutral lipids,phospholipids, carbohydrates, free reducing sugars, polysaccharides and total energy. Hypoploid cells of2n=37, 36, 35 or 34 were observed in all groups of individuals. The aneuploidy values observed ranging from19% to 79%, were substantially higher than those observed in previous studies for other bivalve species. Thedifferences in the observed aneuploidy between fast- and slow-growing clams were highly significant and anegative correlation was also observed between the level of aneuploidy and length. In general, the synthesisof the evaluated biochemical components showed no significant differences among groups suggesting thatthis synthesis could be related to extrinsic factors or that the analyzed biochemical components were notspecific to detect the lack of certain aminoacids and enzymes involved in growth. The only exception was fortotal lipids, which were significantly lower in the medium and slow growing groups relative to the fastgrowing group. The evaluation of the relationships between all the studied parameters was performedthrough a principal component analysis (PCA). The results of this study demonstrate the existence of anegative correlation between somatic aneuploidy and growth in the carpet shell, as previously reported forother bivalve species. Moreover, for the first time in this study, this negative correlation is confirmed overtime in a same cohort of the clam R. decussatus.

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1. Introduction

The carpet-shell clam Ruditapes decussatus is one of the mostcommercially important bivalve species in Southern Europe. InPortugal, it represents 27% of national aquaculture production and64% of shellfish production (DGPA, 2008). However, in populations ofthis species, it is often observed that there are three groups ofindividuals, the fast-, the medium- and the slow-growers according totheir size. Moreover, a decrease in the maximum size reached by theindividuals, in each production cycle (2 years), has been noticed bythe producers, in the last years.

Aneuploidy is a cytogenetic phenomenon known as an abnormalchromosome number that involves the loss (hypoploidy) or gain

(hyperploidy) of one or more individual chromosomes. This phe-nomenon mainly originates from the non-disjunction of chromo-somes during mitosis or meiosis (Martin and Rademaker, 1990) ordue to Premature Centromere Division, PCD (Major et al., 1998).Recent studies performed in plants, suggest that there is also achromosome loss during interphase, involving the nuclear expulsionaccompanied by post mitotic micronuclei formation (Gernand et al.,2005). This phenomenon is often lethal or associated with growth anddevelopmental retardation in higher animals, like mammals, but theeffects of aneuploidy seem to be better tolerated in plants and loweranimals (Verma, 1990). Genetic factors may be involved in thisphenomenon, indeed in humans and in some plants, evidence existsfor the presence of genes that are responsible for chromosome non-disjunction (e.g. Bond and Chandley, 1983; Garcia-Cruz et al., 2010).

Hypoploidy, the lack of one or two chromosomes in a pair ofhomologues, has often been observed in several populations ofbivalve species (e.g. Dixon, 1982; Leitão et al., 2001a; McCombie et al.,

Fig. 1. Frequency distribution of the size population at the beginning of the experiment.

47J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

2005). Moreover, in the Japanese oyster Crassostrea gigas, a negativerelationship between growth rate and the phenomenon of aneuploidywas demonstrated. In several populations of this species, the fast-growing animals always presented a lower percentage of aneuploidycomparing to the slow-growing ones (Leitão et al., 2001a; Thiriot-Quiévreux, 1986; Thiriot-Quiévreux et al., 1988; Zouros et al., 1996).In this species the aneuploidy phenomenon was also showed to beassociated with genetic (Leitão et al., 2001b) and environmentalfactors (Bouilly et al., 2002).

A recent study on this phenomenon was performed in thePortuguese oyster C. angulata, C. gigas and in the interspecific hybrids(Batista et al., 2007). Both the growth and the survival rate weresignificantly higher in C. gigas than in C. angulata and the hybridsshowed intermediate performances. Significant differences, betweenthese two species, were observed in the proportion of aneuploid cellsand in the proportion of missing chromosomes, with C. angulatashowing the highest values. Intermediate values were observed in thehybrids, suggesting once more that there is a genetic basis underlyinganeuploidy level.

These results are particularly important since high growth ratevariability has been often observed in bivalve populations and is oneof the major problems for the aquaculture production. However,until now, no study on aneuploidy was performed in the speciesR. decussatus.

Indeed, the only cytogenetic studies, performed until now in thisspecies, involved the determination of the chromosome number(Gérard, 1978; Patterson review, 1969), finding a diploid number of38 chromosomes and the characterization of the karyotype, which iscomposed by 6 metacentric, 3 submetacentric and 10 subtelocentricchromosome pairs (Borsa and Thiriot-Quiévreux, 1990). Morerecently, the digestion with restriction endonucleases (REs), whichallows the individual identification of all chromosomal pairs and theestablishment of the precise karyotypes, was also performed in thisspecies (Leitão et al., 2006).

Assessment of the physiological status of bivalve juveniles can becarried out by examining various biochemical parameters or indices ofinstantaneous growth (Fathallah et al., 2010). Changes in biochemicalcomposition may be of great importance concerning energy metab-olism necessary to bivalve's life cycle development, especiallysurvival, growth and reproduction (e.g. Jayabal and Kalyani, 1986;Labarta et al., 1999; Lodeiros et al., 2001; Navarro et al., 1989; Phillips,2006). The bivalve's biochemical composition depends on externalfactors (e.g. temperature and food availability), the gonad conditionand the individual metabolic activity (Ruiz et al., 1990) and internalfactors (Matias et al., 2009). The use of the metabolic reserves beginspreferably with the carbohydrates, with the free reducing sugarsbeing more rapidly metabolized and polysaccharides having anenergy storage function. According to Gabbott (1976), the lipids,especially neutral lipids, represent an important energetic reservebecause of their high caloric value and are mainly used in chronicstress conditions. Phospholipids, also called structural lipids, are themost important lipids of bivalve's growth, since they play animportant role in the cell membranes formation. Proteins, the mainbiochemical component of the organisms may be subject to metabolictransformation, yet this component is not subject to accumulationprocesses like lipids and carbohydrates (Ruiz et al., 1990).

Therefore, the study of biochemical composition can contributenot only to a better understanding of the constraints in the growthsuccess, but also for amore efficient characterization of the organism'scondition and quality (Ruiz et al., 1990). Parallel evaluation of thecondition index allows an assessment of the physiological status ofthe shellfish populations and is recognized as a useful biomarker (e.g.Fernández-Castro and Vido deMattio, 1987;Mann, 1978;Matias et al.,2009).

The main objective of this work was then not only to provide newinsights into growth, physiology and somatic aneuploidy in the carpet

shell R. decussatus, through the determination of the aneuploidy leveland its possible relationship with growth, but also for the first time inbivalves, the study of the evolution of the level of this phenomenonover time. In order to verify whether this phenomenon was reflectedin the physiological condition of this species, biochemical compositionand condition index were also assessed.

2. Materials and methods

2.1. Origin of the studied clams

Juvenile specimens of R. decussatus resulting from a singlespawning of wild parents, with 10 females and 8 males in a total of30 individuals stimulated, with a shell length between 35 and 45 mm,induced in June 2008 in the experimental bivalve hatchery of NationalInstitute of Biological Resources (INRB, I.P./L-IPIMAR) in Tavira,Portugal, were studied. These specimens were obtained from abroodstock of Ria de Aveiro (40º42′N; 08º40′W), constituting a singlecohort.

2.2. Experimental design

After 5 months the size population ranged between 2 and 16 mm(Fig. 1). The juveniles were separated based on their average shelllength and classified in three groups, as fast-growers (9.57±1.29 mm; 0.16±0.07 g), medium-growers (6.58±0.67 mm; 0.05±0.01 g) and slow-growers (3.36±0.59 mm; 0.01±0.00 g). The fast-and medium-growers were composed of 500 individuals while theslow-growing groupwas composed of more than 500 individuals, dueto its minute size. In order to increase the natural mitotic index of theselected individuals, the animals were kept in the optimal conditionsfor its development, particularly in terms of temperature, salinity, pHand food (Falcão, 1997). All the individuals were placed in a sieveprovided with air-lift on one 80 L fiber-glass tank in a close systemcircuit containing natural seawater filtered through a 5 μm mesh.Water salinity was 33‰ with a temperature of 20±1 °C. The juvenileclams were feed with a mixture of the microalgae Isochrysis galbana(clone T-ISO), Skeletonema costatum and Chaetoceros calcitrans, in aproportion of 1:1:1, corresponding to 4% of the drymeat weight in dryweight of algae (Utting and Millican, 1997).

2.3. Biological sampling

The experiment took place over a period of 6 months beginning inNovember 2008. During this period, samples were taken at threedifferent times (day 0 — T0; day 107 — T1 and day 199 — T2) with aninterval of about three months between each sampling, in order tostudy the evolution of the aneuploidy level, condition index andbiochemical composition over time.

48 J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

2.4. Growth

The growth of all the individuals was estimated based on twobiometric parameters: maximum length and live weight.

To evaluate the individual's total growth rate, we applied theGrowth Global Index, in length and weight (IcT), which is acumulative index (Rodiles, 2004):

IcT = Ln At–Ln A0ð Þ= t½ � × 100:

Ln At Neperian logarithm of medium length and weight in t time;Ln A0 Neperian logarithm of medium length andweight in time 0;t Time in days between the beginning and the end of the

experimental period.

2.5. Condition index

Drymeat and shell weights of 30 juvenile clams from each group ofindividuals, at each time, were determined after oven drying at 80 °Cfor 24 h. The condition index (CI) was calculated according to Walne(1976):

CI =dry meat weight ðmgÞdry shell weight ðmgÞ × 100:

2.6. Biochemical composition

The meat of 50 fast-, 50 medium- and 20 slow-growing clams ofeach sampling time was frozen stored at−18 °C for later biochemicalanalysis. The samples were fractionated according to the procedureproposed by Holland and Hannant (1973). For each group, proteinswere determined using the modified Lowry method (Lowry et al.,1951) and carbohydrates and free reducing sugars were determinedusing the Folin and Malmros (1929) method. The levels of poly-saccharides were calculated by the difference between carbohydratesand free reducing sugars. Total and neutral lipids were determined bythe Marsh and Weinstein (1966) method and phospholipids werecalculated by the difference between total and neutral lipids.

Caloric content of total lipids, carbohydrates and proteins in tissueswere calculated using the factors 35.24, 17.16 and 18.00 KJ.g−1,respectively (Beukema and De Bruin, 1979).

2.7. Aneuploidy scoring

Twenty fast-, 20 medium- and 50 slow-growing clams of eachsampling time, were incubated for 8–10 h in seawater containing0.005% colchicine. Then gills were dissected in seawater, treated for30 min in 0.9% sodium citrate and fixed in a freshly prepared mixtureof absolute alcohol-acetic acid (3:1) with three 20 min changes. Slideswere made from one individual gill following the air drying techniqueof Thiriot-Quiévreux and Ayraud (1982). The preparations werestained for 10 min with Giemsa (4%, pH 6.8). Chromosome counts

Table 1Size distribution of the three groups of R. decussatus juveniles, throughout the experimenta

T0 T1

Average Median Minimum Maximum Average

Fast-growers Length 9.20 9.15 6.90 15.20 14.38Weight 0.15 0.13 0.06 0.62 0.66

Medium-growers Length 6.67 6.60 5.20 8.00 10.32Weight 0.06 0.05 0.03 0.09 0.28

Slow-growers Length 3.40 3.30 2.30 5.10 5.29Weight 0.01 0.01 0.00 0.03 0.05

were made directly by microscope observation (Nikon Eclipse 80iwith camera image acquisition incorporated Nikon DS-Fi1) onapparently intact metaphases. The level of aneuploidy was estimatedby counting the total number of aneuploid metaphases over the totalnumber of metaphases counted per individual. To avoid any bias onthe data treatment due to a different number of cells counted perindividual, a pool was performed for each group. The weightedaverage of each animal gave us the final percentage of aneuploidy foreach group of individuals. The counting of chromosomes of all theindividuals was always performed by the same observer to eliminatethe subjectivity associated to different observers in the interpretationof the results.

2.8. Data analysis

Differences in aneuploidy between fast-, medium- and slow-growers were tested by analysis of covariance. In the expressedpercentage data, the statistical comparison was carried out afterprocessing the arcsine transformation (Snedcor and Cochran, 1980).Statistical analyses were computed using Sigma-Stat for Windows.Differences between averages of different growth groups and differentsampling times for thedifferentparameterswere assessedbyanalysis ofvariance (ANOVA) and when assumptions of normality and homosce-dasticity were not fulfilled, even after data transformation, thenonparametric Kruskal–Wallis test was used. Results were regardedas significant for pb0.05.When results suggested significant differencesbetween means, the Tukey honest significant difference (HSD) test, forpost-hoc comparison of means, was applied, the nonparametric Dunn'smultiple comparisons were used for the alternative nonparametricKruskal-Wallis test (Zar, 1984).

In order to investigate discrepancy between clam groups withrespect to all estimated descriptors and to evaluate the relationshipsbetween all the studied parameters, a principal component analysis(PCA) using MATLAB® 7.1 and a Pearson's parametric correlationcoefficient, were carried out.

3. Results

3.1. Growth

The size distribution of the whole population by groups, during theexperimental period, is presented in Table 1. An increase in themaximum length and live weight was observed along the experimentalperiod, with the exception of the slow-growers between T1 (mean of5.29 mm and 0.04 g) and T2 (mean of 4.14 mm and 0.03 g) (Fig. 2).Moreover, as shown in Table 2, the slow-growing clams presented thelowest values of Growth Global Index. The growth assessment showedsignificant differences between groups (p≤0.001) and betweensampling dates (p≤0.001).

3.2. Condition index

The variation of the condition index during the experimentalperiod is presented in Fig. 3. Significant differences between groups

l period.

T2

Median Minimum Maximum Average Median Minimum Maximum

13.54 10.92 21.08 18.42 18.42 12.99 25.420.54 0.29 1.92 1.22 1.15 0.40 2.97

10.41 5.42 15.78 13.40 13.21 7.02 21.250.26 0.04 0.80 0.53 0.42 0.07 1.635.43 2.43 9.02 4.14 3.94 1.85 7.790.04 0.00 0.16 0.03 0.02 0.01 0.14

Fig. 2. Evolution of the mean values (±σ) in length (mm) and weight (g) of the threegroups of Ruditapes decussatus juveniles, throughout the experimental period.

Fig. 3. Variation of the mean values (±σ) of condition index of the three groups ofRuditapes decussatus juveniles, throughout the experimental period.

49J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

and sampling periods (pb0.001) can be observed, with the fast- andslow-growers suffering a statistically significant decrease between T0and T1, contrastingwith themedium-growing clams, which presentedconstant values over time. Unfortunately, it was not possible to assessthe condition index in T2 for slow-growers, due to a high mortalitylevel observed at T2. However, since no mortality was recorded in allother groups and sampling times, this unique and isolated episodewas not taken into consideration in the results interpretation.

3.3. Biochemical composition

Proteinswere thepredominant constituent of juveniles (51.7–60.7%)followed by carbohydrates (21.3–27.6%) and total lipids (15.0–21.1%).The percentage of proteins in the total organic content increasedthroughout the experimental period in all three groups (Table 3). Therewere no significant differences observed between groups (pN0.05) anddifferences were only registered between sampling times (T2≠T0=T1,pb0.05),with thehighest values beingobserved inT2,without, however,any evidence of interaction between groups and time (pN0.05).

For the carbohydrates, a decrease was observed over time in allgroups, except for slow-growers, where an increase was evidencedbetween T0 and T1 followed by a decrease in T2. Significant differenceswere found only between sampling times, T2 differing from T1 and T0(pb0.05) (Table 3). The free reducing sugars were the predominantconstituent of carbohydrates (52.5–71.3%). In the fast- and medium-growers this compound varied similarly through the experimentalperiod, with an increase observed between T0 and T1 followed by adecrease in T2, while the slow-growers suffered a decrease between T0and T1 followed by an increase in T2 (Table 3). There were nosignificant differences observed between groups (pN0.05) either forfree reducing sugars and/or for polysaccharides. However, significantdifferences between T2 and T1 (pb0.05) and a significant interactionbetween sampling times and groups (pb0.05), were recorded. Withinfast- andmedium-growers, differences were found between samplingtimes (pb0.05), contrasting with slow-growers (Table 3).

In total lipids, significant differences were found between groups(pb0.05), with the fast-growers displaying the highest values,

Table 2Growth Global Index in length and weight and respective confidence intervals of thethree groups of R. decussatus juveniles, throughout the experimental period.

Growth global index

Length Weight

Fast-growers 0.35 [±0.01] 1.05 [±0.03]Medium-growers 0.35 [±0.01] 1.14 [±0.03]Slow-growers 0.1 [±0.02] 0.63 [±0.04]

differing from medium- and slow-growers (pb0.05), and betweensampling times (pb0.05), from T0 to T2 (pb0.05) (Table 3). For neutrallipids, the highest constituent of total lipids (49.6–74.0%), an increasewas observed for the slow-growers, during the experimental period,with the lowest percentages being observed in T0 and the highest inT2, while a decrease over time was observed for fast- and medium-growers. Phospholipids (26.1–50.4%) and neutral lipids had anopposite behavior. However, for both components no significantdifferences were observed between groups or sampling periods(pN0.05) (Table 3).

The total energy content remained constant throughout theexperimental period (Table 3).There were no significant differencesbetween groups and sampling times (pN0.05).

3.4. Aneuploidy scoring

Although R. decussatus has a diploid number of 38, hypoploid cellsof 2n=34, 35, 36 or 37 were observed in all the studied groups(Fig. 4).

Looking at the three groups (Table 4), slow-growing clams alwaysshowed the higher levels of aneuploidy, followed by the medium- andfast-growers, respectively. The extreme values of aneuploidy rangedfrom18.7% in T1 for the fast-growers to 78.7% in T0 for the slow-growers.The level of aneuploidy was significantly different between all thegroups (pb0.05).

Concerning the evolution of the level of aneuploidy within groupsover time, there were no significant differences observed in the fast-and medium-growers, however in the slow-growers significantdifferences were observed between T0 and T2 (pb0.05), with 78.7%and 41.4% respectively.

Significant differences were also observed, for both T0 and T1,between groups. These differences were observed between slow-,medium- and fast-growers (pb0.05) for both T0 and T1 samplingtimes. In T2 no significant differences were observed between groups(pN0.05).

A significant negative linear correlation between the percentage ofaneuploidy and total length was observed for T0 (r2=0.4964)(Fig. 5a), T1 (r2=0.4133) (Fig. 5b) and T2 (r2=0.2092) (Fig. 5c).The percentage of aneuploidy was always significantly higher in thejuveniles that presented the lowest growth throughout the experi-mental period.

A Bi-plot Principal Component Analysis was applied to project inthe same reduced dimension space, the 9 clams groups, i.e. the threegroups of individuals (slow-, medium- and fast-growers) at eachsampling time and the 12 studied parameters. The obtained plan wascomposed by the first PC1 and second PC2 principal componentaccounting for 44.5% and 28.1% of the total variance, respectively(Fig. 6). Explaining 72.6% of the total variance, the obtained first plan

Table 3Variationof themeanvalues (±σ) ofproteins, total lipids, carbohydrates, neutral lipids, phospholipids, free reducing sugars, polysaccharides andenergyof the threegroupsofR. decussatusjuveniles for the different sampling periods. The results are expressed as organic weight percentages.

Proteins(% organic content)(p groupsN0.05;p timesb0.05)

Total lipids(% organic content)(p groupsb0.05;p timesb0.05)

Carbohydrates(% organic content)(p groupsN0.05;p timesb0.05)

Composition of lipid classes Composition of carbohydrate classes Energy(KJ.g-1)(p groupsN0.05;p timesN0.05)

Neutral lipids(% total lipids)(p groupsN0.05;p timesN0.05)

Phospholipids(% Total lipids)(p groupsN0.05;p timesN0.05)

Free reducing sugars(% carbohydrates)(p groupsN0.05;p timesb0.05)

Polysaccharides(% carbohydrates)(p groupsN0.05;p timesb0.05)

Fast T0 52.0±3.1 20.5±0.5 26.4±2.7 59.5±7.0 40.5±7.0 58.5±8.8 41.5±8.8 9.7±0.5Medium T0 54.2±1.4 18.2±3.5 27.6±3.6 66.8±15.0 33.2±15.0 63.3±11.4 36.7±11.4 9.8±0.3Slow T0 51.7±4.7 20.4±1.7 26.3±2.8 66.8±13.6 27.4±12.8 61.1±16.9 38.9±16.9 10.5±0.5Fast T1 52.8±3.6 21.1±3.3 25.9±1.9 55.5±15.7 44.5±15.7 69.0±9.2 31.0±9.2 9.9±0.6Medium T1 55.1±4.8 16.4±1.4 25.2±3.8 63.1±13.2 36.9±13.2 71.3±6.4 28.7±6.4 9.1±0.8Slow T1 56.7±4.4 15.0±2.0 27.5±2.6 68.7±4.6 31.3±4.6 53.1±6.6 46.9±6.6 9.2±0.6Fast T2 57.9±2.1 18.2±3.1 24.2±2.4 49.6±16.2 50.4±16.2 52.5±5.4 47.5±5.4 8.4±0.3Medium T2 57.7±5.2 15.3±1.0 24.9±2.2 60.0±3.6 40.1±3.6 56.7±10.0 43.4±10.0 10.2±0.9Slow T2 60.7±4.7 16.1±2.2 21.3±1.9 74.0±16.2 26.1±16.1 58.1±6.0 41.9±6.0 9.6±1.1

Fig. 4. Examples of hypoploid metaphases of Ruditapes decussatus. a) Metaphase cellwith 2n=36 chromosomes. b) Metaphase cell with 2n=37 chromosomes. Scalebar=5 μm.

50 J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

was considered sufficient to explore the main internal structure of theobtained data and to explain its variance. This figure presents the dualprojection of object scores (clam group per time of sampling) andvariable vectors. Vectors pointing in the same direction correspondedto parameters which are highly correlated. Parameters explaining orinducing the discrepancy between clams groups were those wellcorrelated to PC1 and PC2 (i.e. presenting high scores as vectorprojection to the corresponding PC). “Aneuploidy” was highlypositively correlated to PC1 and by opposition negatively correlatedto “Length”, “Weight” and “Condition Index” parameters.

Except for the sampling periods, the groups were clearly separatedinto fast-, medium- and slow-growers, especially along the firstprincipal axis PC1. The second axis discriminated the samplingperiods within the groups, where it's possible to observe the samplingtimes T0 at the top, with fast-grower individuals at T1, while theremaining were at the bottom. “Total Lipids” variable was highlypositively correlated to the PC2 and segregate groups of clams withhigher (mainly T0 sampled individuals) to lower (mainly T2 sampledindividuals) amount of “Total Lipids”.

“Phospholipids” were positively correlated with the “ConditionIndex” and “Length”; however, “Neutral Lipids” were negativelycorrelated with “Length” and “Weight”, but positively correlated withthe “Total Energy”.

4. Discussion

In general, the growth (length and live weight) of the threejuvenile groups of R. decussatus, increased throughout the experi-mental period. The values of growth showed significant differences,both between the studied groups and over time. The Growth GlobalIndex of the fast- and the medium-growers was higher than the oneobserved in the slow-growers. The values observed in the first twogroups were similar to those previously presented for the samespecies with the same age (Matias, 2007).

The condition index, one of the parameters that evaluate thephysiological condition of bivalves, showed significant differencesbetween groups. In all sampling periods, the fast-growers presentedthe highest values, followed by the medium-growers and finally theslow-growers. We can then conclude that fast-growers, as expected,presented the best physiological condition. The high mortality levelsuffered by the slow-growers at T2 further supports the existence ofsubstantial differences at the physiological level between fast- andslow-growers. Differences over sampling periods in condition indexwere not relevant, except for fast- and slow-growers, between T0 andT1.

In this study the proteins and the carbohydrates (consisting of≈60% of reducing sugars and ≈40% of polysaccharides) were thebiochemical constituents that most contributed to the total organiccontent in R. decussatus juveniles, with about 55% and 25%

respectively, while the lipids (consisting of ≈60% of neutral lipidsand ≈40% of phospholipids) were present in smaller amounts,representing around 20%. Similar proportions have already beenwidely observed in the literature for juveniles of this and other bivalvespecies (e.g. Albentosa et al., 1996, 2006; Fathallah et al., 2010;Okumus and Stirling, 1998; Robert et al., 1993).

In general, the synthesis of the evaluated biochemical componentsshowed no significant differences among groups suggesting that thissynthesis could be related to extrinsic factors or that the analyzedbiochemical components were not specific to detect the lack of certainaminoacids and enzymes involved in growth (e.g. Albentosa and

Table 4Biometric measurements and final percentage of aneuploidy of fast-, medium- and slow-growers in T0, T1 and T2.

Samplingtime

Growthcategory

Meanweight(g)

Meanlength(mm)

Number of cells observed with Totalnumber ofobserved cells

Totalnumber ofaneuploid cells

Aneuploidy(%)

2 N=38 2 N=37 2 N=36 2 N=35 2 N=34

T0 Slow 0.01 3.36 13 17 15 8 8 61 48 78.7Medium 0.05 6.58 139 57 27 11 8 242 103 42.6Fast 0.16 9.57 314 24 32 11 10 391 77 19.7

T1 Slow 0.05 5.7 50 24 22 10 8 114 64 56.1Medium 0.36 11.53 110 14 16 10 10 160 50 31.3Fast 1.12 17.55 143 7 12 8 5 175 32 18.7

T2 Slow 0.03 4.27 51 13 10 4 9 87 36 41.4Medium 0.54 13.05 124 13 32 5 10 184 60 37.5Fast 1.34 18.32 280 24 22 13 10 349 69 20.6

51J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

Moyano, 2008; Fathallah et al., 2010; Zhou et al., 2007). The onlyexception was for total lipids, which were significantly lower in themedium and slow growing group relative to the fast growing group.

Fig. 5. a) Correlation between the percentage of aneuploidy and length in T0.b) Correlation between the percentage of aneuploidy and length in T1. c) Correlationbetween the percentage of aneuploidy and length in T2.

These results suggest that medium- and slow-growers do notaccumulate this component in the same way as fast-growers. Giventhe importance of lipids in the energetic balance (Benninger andLucas, 1984), it is possible that the groups with lower growth havechanneled greater lipid content to the metabolic maintenance(Albentosa et al., 1996; Labarta et al., 1999). The evaluation of thelipid class composition in future studies could then contribute to theclarification of this question, since several studies have showed theimportance of the triacylglycerol content in the physiological qualityin bivalves (adults, larvae and juveniles) and that it could be used as abiomarker (e.g. Fernández-Reiriz et al., 1999; Pernet et al., 2003).

All the aneuploid cells observed in this study corresponded tohypoploidy cases. This was not unexpected since hypoploidy seems tobemore frequent in bivalves than hyperploidy (e.g. Batista et al., 2007;Leitão et al., 2001a; McCombie et al., 2005).

Our results suggested the existence of a negative correlationbetween growth and the level of somatic aneuploidy in the clam R.decussatus. Fast-growing individuals always showed significantlylower levels of aneuploidy than slow-growing ones. The same hadbeen previously observed in studies performed in natural andcultivated populations of C. gigas, over several years, where fast-growers had also always showed lower percentages of aneuploidythan the slow-growers (e.g. Leitão et al., 2001a).

Interestingly, in this study the aneuploidy values, varying from 19%to 79%, were substantially higher than those observed in other bivalvespecies in previous studies, particularly for slow-growers (varyingfrom 41% to 79% of aneuploidy). Indeed, for instance, in C. gigas,aneuploidy ranged from 5% to 22% (Leitão et al., 2001a; Thiriot-Quiévreux et al., 1988) and in the Portuguese oyster C. angulata, theproportion of aneuploid cells was about 20% (Batista et al., 2007).

The considerably higher aneuploidy values observed for slow-growers in this study, might suggest a higher aneuploidy level in thisspecies but might also be due to the substantially younger age of theseindividuals when compared to the age of slow-growers of previousstudies (e.g. Leitão et al., 2001a, 2007). In previous studies, individualscorresponding to the slow growing-group in this study, were probablynot viable, not reaching the T0 of those studies, which could have ledto a general lower aneuploidy level.

Moreover, in this work we performed, for the first time, a study ofthe evolution in time of the aneuploidy level in all three groups. Nostatistically significant differences were found between each samplingtime, T0, T1 and T2, in the fast- and medium-growers, however in theslow- growers a decrease between T0 and T2 was observed, suggestingthat the percentage of aneuploidy remained constant throughout theexperimental period, except for the slow growing ones. Thisinteresting finding may be due to the high mortality already reportedbetween T1 and T2 in this group, suggesting that perhaps many of theindividuals that survived had less aneuploidy in their cells. Previousstudies, have indeed, suggested that aneuploid somatic cells would behomozygous for recessive deleterious mutations, resulting in thenegative correlation between growth and somatic aneuploidy, andconsequently death, due to the combined effects of a high load of

Fig. 6. Biplot Principal Component Analysis of the studied data. Asterisks represents clam groups (F0—T0 fast-growers; F1—T1 fast-growers; F2—T2 fast-growers; M0- T0 medium-growers; M1—T1 medium-growers; M2—T2 medium-growers; S0—T0 slow-growers; S1—T1 slow-growers; S2—T2 slow-growers). Vectors indicate variables correlations scores withthe two Principal Components PC1 and PC2.

52 J.T. de Sousa et al. / Journal of Experimental Marine Biology and Ecology 406 (2011) 46–53

recessive mutations and somatic cell aneuploidy (Launey andHedgecock, 2001). However, it must be focus that, in this study, thesampling period had only the duration of 9 months and that it ispossible that changes in the aneuploidy levels could appear in a longersampling period.

When comparing all the studied parameters, it was possible toverify that those with greater influence on the results, discriminatingfast- from slow-growing individuals, were the growth (length andweight) and aneuploidy. In addition, both the growth and conditionindex were negatively correlated with aneuploidy.

It was also noted that phospholipids were positively correlatedwith condition index and length. Since phospholipids are the mainconstituent of the cell membranes (Pernet et al., 2008), it could,indeed, be expected that the individuals with the higher growth,would be the ones with the higher levels of this component. Incontrast, the neutral lipids were negatively correlated with conditionindex and length. Since neutral lipids represent an importantenergetic reserve (Gabbott, 1976), it means that the individualswith the lower growth accumulated more energetic reserves.

The consistency of the negative relationship between growth andaneuploidy in bivalves, observed in this and in previous studies (e.g.Batista et al., 2007; Leitão et al., 2001a), opens new fields for researchin order to have a better understanding of aneuploidy in bivalves andits relationship with growth rate.

One question that needs to be clarified is why some chromosomesare more easily lost than others. The identification of the missingchromosomes in the aneuploid karyotypes of R. decussatus, to verify ifchromosomal loss is random or reflects a differential chromosomalsusceptibility in this species, would help to clarify this question.Indeed, a differential chromosome loss has been previously put inevidence in pairs 1, 5, 9 and 10 in the oysters C. gigas (Leitão et al.,2001c) and C. angulata (Leitão personal communication).

It would also be important to understand the genetic basis of thisphenomenon, already observed in other bivalve species (Batista et al.,2007; Leitão et al., 2001b) in order to prevent selection for slow-growth by bivalve shellfish producers, and instead propagate fast-growers with low levels of aneuploidy in future generations. It wouldalso be interesting to try to establish a link between this genetic basisand the one previously reported for growth (Ward et al., 2000) in

bivalves. Another important approach would be the study of thepossible inheritance of the level of aneuploidy through the study ofprogenies originating from crosses within and between populationswith contrasting levels of aneuploidy.

The aneuploidy phenomenon should indeed be a parameter totake into account in future breeding programs of the carpet shell,R. decussatus.

Acknowledgments

This work was supported by the projects PTDC/MAR/72163/2006:FCOMP-01-0124-FEDER-007384, “Ciência 2007 program” and 0251_ECOAQUA_5_E-Program POCTEP. We are also grateful to MargareteMatias and João Maurício Teixeira for the excellent technical assis-tance. The authors are also grateful to A. Good for revising the English.[RH]

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