Environmental stress and life-stage dependenceon the detection of heterozygosity–fitnesscorrelations in the European eel, Anguilla anguilla

J.M. Pujolar, G.E. Maes, C. Vancoillie, and F.A.M. Volckaert

Abstract: Heterozygosity–fitness correlations (HFCs) have been reported in populations of many species, although HFCscan clearly vary across species, conspecific populations, temporal samples, and sexes. We studied (i) the temporal stabilityof the association between genetic variation and growth rate (length and mass increase) and (ii) the influence of geneticvariability on survival in the European eel (Anguilla anguilla L). HFCs were assessed using genotypes from 10 allozymeand 6 microsatellite markers in 22-month-old experimental individuals. The results were compared with those of a pre-vious study carried out in 12-month-old individuals, in which more heterozygous individuals showed a significantly fastergrowth rate. In contrast, 22-month-old individuals showed no evidence that genetic variability was correlated with growthrate. Additionally, heterozygous individuals did not show a higher survival rate compared with more homozygous individu-als after either handling stress or parasite infection. The decrease in HFCs over time is consistent with the general predic-tion that differences in growth and survival among individuals are maximal early in life and in our case most likely due tothe relaxation of environmental conditions related to population-density effects. Alternatively, the decline in HFCs couldbe attributed to either ontogenetic variance in gene activity between 12- and 22-month-old individuals or differential mor-tality leaving only the largest individuals.

Key words: allozymes, european eel, fitness, heterozygosity, microsatellites, ontogeny, stress, survival.

Resume : Les correlations entre l’heterozygotie et le « fitness » (HFC, pour « heterozygosity-fitness correlations ») ontete rapportees chez des populations de plusieurs especes, bien que les HFC puissent clairement varier entre des especes,populations d’une espece, echantillons pris dans le temps ou sexes. Les auteurs ont etudie (1) la stabilite dans le temps del’association entre la variation genetique et le taux de croissance (augmentation de la longueur et de la masse) et (2) l’in-fluence de la diversite genetique sur la survie chez l’anguille europeenne (Anguilla anguilla L.). Les HFC ont ete mesuressur la base des genotypes a 10 locus alloenzymatiques et 6 locus microsatellites chez des individus experimentaux ages de22 mois. Les resultats ont ete compares a ceux d’une etude anterieure effectuee sur des individus de 12 mois et chez les-quels les individus plus heterozygotes affichaient un taux de croissance significativement superieur. De plus, les individusheterozygotes ne presentaient pas un taux accru de survie par rapport aux individus plus homozygotes suite a un stress demanutention ou d’une infection parasitaire. La diminution du HFC au cours du temps concorde avec la prediction generalevoulant que les differences en matiere de croissance et de survie entre les individus sont maximales tot au cours de la vie.Dans le cas present, cette diminution serait vraisemblablement due a un relachement des conditions environnementalesliees aux effets de densite des populations. Alternativement, on pourrait attribuer le declin des HFC a une variance ontoge-nique dans l’activite genique entre les individus de 12 et de 22 mois, ou encore a une mortalite differentielle laissant seulsles plus gros individus.

Mots cles : allozymes, anguille europeenne, « fitness », heterozygotie, microsatellites, ontogenie, stress, survie.

[Traduit par la Redaction]

Introduction

The empirical observation of heterozygosity–fitness corre-lations (HFCs), the correlation between the degree of indi-vidual genetic heterozygosity and fitness-related traits suchas growth rate, fecundity, survival, and developmentalstability, has been reported in organisms as diverse as plants,marine bivalves, crustaceans, amphibians, salmonids, andmammals (David 1998). Determining the mechanismsunderlying HFCs is of fundamental importance for under-standing the action of natural selection on molecularmarkers in natural populations (Thelen and Allendorf 2001).Studies combining the use of simultaneously functional

Received 22 March 2006. Accepted 9 August 2006. Publishedon the NRC Research Press Web site at http://genome.nrc.ca on25 January 2007.

J.M. Pujolar,1,2 G.E. Maes, and F. Volckaert. KatholiekeUniversiteit Leuven, Laboratory of Aquatic Ecology, Ch. deBeriotstraat 32, B-3000 Leuven, Belgium.C. Vancoillie. Royaal BV, Breedijk 13, 5705 CJ Helmond,Netherlands.

1Corresponding author (e-mail: pujolar@biology.ucsc.edu).2Present address: Department of Ecology and EvolutionaryBiology, Earth & Marine Sciences, University of California,Santa Cruz, CA 95064, USA.

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(e.g., allozymes) and neutral DNA (e.g., microsatellites)markers in the same individuals seem to be the most ad-equate, as they allow for the evaluation of the possiblecauses of HFCs (Pogson and Zouros 1994; Thelen andAllendorf 2001; Borrell et al. 2004; Maes et al. 2005;Pujolar et al. 2005).

Many studies have recognized positive correlations be-tween heterozygosity and growth rate, although thestrength and stability of HFCs can clearly vary across spe-cies, conspecific populations, temporal samples, and sexes(Liskauskas and Ferguson 1991; David 1998). Differentsources may contribute to such variation. (i) HFCs are ex-pected to decrease or even disappear with age, since dif-ferences in growth are maximal early in life (David 1998)and because unfit genotypes are selectively eliminated inageing cohorts (Koehn and Gaffney 1984). It has been sug-gested that energy is mostly directed to growth at early butnot at later stages (David et al. 1995). For instance, in theflat oyster Ostrea edulis, Bierne et al. (1998) recorded sig-nificant multilocus heterozygosity–growth correlations inlarvae and postlarval-stage individuals, which were not ob-served in 1-year-old cohorts (Marsic-Lucic and David2003). In fish, salmonids seem to produce different HFCpatterns at different life stages (Wang et al. 2002). (ii) Onthe other hand, HFCs are not only variable in time but alsohighly dependent on environmental conditions (Danzmannet al. 1998). Also, Lesbarreres et al. (2005) observed thatthe heterozygosity–survival association is most pronouncedin stressful environments (i.e., limited food) in the commonfrog Rana temporaria, suggesting that laboratory-inducedor natural stress may enhance HFCs, although the effectsare generally poorly understood.

In contrast with heterozygosity–growth associations,heterozygosity–viability correlations are much less com-monly documented, and few studies have attempted to cor-relate heterozygosity and survival (David 1998). Positiveassociations have been described mainly in marine bivalves,including clam Macoma balthica (Green et al. 1983), Amer-ican oyster Crassostrea virginica (Zouros et al. 1983), andscallop Placopecten magellanicus (Foltz and Zouros 1984).Inconsistent and negative correlations have also been docu-mented, in blue mussel Mytilus edulis (Gaffney 1990), theEuropean oyster Ostrea edulis (Alvarez et al. 1989), and thesurf clam Spisula ovalis (David and Jarne 1997). Severalstudies have shown a direct relation between genetic varia-bility and parasite infection, the less heterozygous individu-als being more susceptible to parasitism and less likely tosurvive (Coltman et al. 1999; Cassinello et al. 2001). Sev-eral lines of evidence indicate that survival after pathogeninfection is linked to major histocompatibility complex(MHC) loci (Bernatchez and Landry 2003).

The species of interest in our study is the European eel(Anguilla anguilla L.; Anguillidae; Teleostei), a catadro-mous fish species that moves between marine (spawning,larval phase, and maturation) and freshwater (feeding,growth, and partial maturation) environments. Recruitmentof European eel has declined in recent decades beyondsafety limits (Dekker 2003), and thus a better understandingof the genetic factors influencing the survival of the speciesis needed. We previously reported a positive correlation be-tween genetic variability and growth rate at allozyme loci in

European eel raised at an eel farm for 12 months (Pujolar etal. 2005). More heterozygous individuals showed a signifi-cantly greater length and mass increase and an above-averagecondition index in comparison with more homozygous indi-viduals. To a lesser extent, microsatellite loci showed a sim-ilar pattern, with positive but nonsignificant correlationsbetween heterozygosity and growth rate.

Our aim was first to examine the effect of life stage onthe association between genetic variability and growth inthe European eel by testing whether HFCs are consistentover time or tend to disappear with age. Time consistencyof the observed HFCs was tested by relating genetic varia-tion (as assessed from 10 allozyme and 6 microsatellitemarkers) with 2 phenotypic measures of growth (length andmass increase) in 12-month-old (Pujolar et al. 2005) and 22-month-old experimental individuals (this study). We alsotested the influence of population-density effects due tograding (early splitting of individuals into size classes) onthe association between multilocus heterozygosity (MLH)and growth. HFCs may be environmentally dependent, anddensity-related stress may have an effect on the observedHFCs. Our second aim was to compare genetic variabilityin surviving and dead individuals under 2 conditions thatcause high mortality in eel aquaculture: handling stress andparasite infection. Handling stress refers to loss of individu-als during transport from the collecting sites to the aquacul-ture facilities, which causes stress-induced responses,including metabolic disturbance (DeKoning et al. 2004;Morales et al. 2005). Low survival of individuals in aqua-culture is also attributable to a high sensibility to patho-gens, including the swimbladder nematode Anguillicolacrassus and the monogenean trematodes Gyrodactilus andPseudodactylogyrus (Tesch 2003).

Materials and methods

Experimental conditionsIn the experiment, conducted in a closed recirculation sys-

tem at the eel farm of Royaal BV (Helmond, the Nether-lands), glass eels were successfully raised in rearing tanksfrom December 2001 until December 2003. Initial samplesconsisted of wild glass eel individuals collected in themouth of the river Adour (south-western France), with a uni-form size of 70.96 ± 4.04 mm and mass of 0.35 ± 0.09 g.During the first year, samples were kept in 2 separatebatches. A subsample of 100 individuals was collected fromeach batch at the start of the experiment and after 1 year inthe tanks. One batch was further monitored for another10 months, so that individuals could be examined after a to-tal time of 22 months in the tanks. Because of size differen-ces, individuals were graded after the first year and dividedinto 2 batches consisting of small (<40 g; batch 2.1) andlarge (‡40 g; batch 2.2) individuals, respectively. Threemonths after grading, batch 2.1 was graded again, and largeindividuals were removed from the tank. Subsamples of 60individuals from each batch were selected at random at theend of the experiment.

Additionally, the study included (i) a sample of 50 indi-viduals that died during transportation to the eel farm as aresult of handling stress and (ii) a sample of 50 individuals

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that died after 1 month in the tanks following a seriousPseudodactylogyrus (Gyrodactylidae; Monogenea) infection.

Allozyme electrophoresisSamples screened for protein variation using cellulose

acetate gel electrophoresis included initial (n = 200), 12-month-old (n = 250), and 22-month old (n = 120) individu-als, plus individuals that died from handling stress (n = 50)and parasite infection (n = 50). Tissue extraction, electro-phoresis, procedures for visualizing proteins, and the buffersystems used (Tris Glycine (TG) and Tris Malate (TM)) aredescribed in Maes and Volckaert (2002). We examined 7enzymatic systems: aspartate aminotransferase (AAT-1*,AAT-2*, AAT-3*, EC 2.6.1.1, TM), alcohol dehydrogenase(ADH*, EC 1.1.1.1, TG), glucose-6-phosphate isomerase(GPI-1*, GPI-2*, EC 5.3.1.9, TG), isocitrate dehydrogenase(IDHP*, EC 1.1.1.42, TM), malate dehydrogenase (MDH-2*, EC 1.1.1.37, TM), mannose-6-phosphate isomerase(MPI*, EC 5.3.1.8, TG), and phosphoglucomutase (PGM*,EC 5.4.2.2, TG). Genetic nomenclature followed the sugges-tions of Shaklee et al. (1990). Allele assignment was carriedout by comparing the relative mobility with that of the mostcommon allele (*100).

Microsatellite analysisDNA was extracted from the same individuals analysed

with respect to allozymes. DNA purification and polymerasechain reaction (PCR) amplification are described in Pujolaret al. (2005). Genotypes were examined at 6 dinucleotiderepeat microsatellite loci: Aan 01, Aan 03, Aan 05 (Daemenet al. 2001), Aro 063, Aro 095, and Ang 151 (Wirth andBernatchez 2001). PCR products were visualized on an au-tomated sequencer (LI-COR 4200; LI-COR Inc., Lincoln,Nebr.) using a molecular ladder (Westburg, Leusden, Neth-erlands) to quantify allele sizes. Fragment data were ana-lysed using Gene ImagIR v. 4.03 (Scanalytics, BDBiosciences, Rockville, Md.).

Data analysisAll individuals were measured for standard length (L;

mm) and body mass (W; g). Length and mass increase werecalculated as the difference between individual length andmass after 22 months in the tanks and mean length andmass upon arrival at the farm. Differences in morphometricmeasures among groups were tested by an analysis of var-iance (ANOVA). Condition index was not used as a growthestimator since our previous study showed that it was not agood proxy of fitness in aquaculture because of high varia-bility in mass related to optimal feeding conditions (Pujolaret al. 2005).

Within-sample genetic variation was assessed by the ob-served heterozygosity per locus (Ho) and level of polymor-phism (P0.95) using GENETIX v. 4.05 (Belkhir et al. 2004).Additionally, allelic richness (AR) was calculated usingFSTAT 2.9.3. (Goudet 2002). Deviations from Hardy–Weinberg equilibrium, linkage disequilibrium, and differen-ces in allele and genotype frequencies among samples weretested using GENEPOP v. 3.4 (Raymond and Rousset1995). Significance levels for multiple simultaneous com-parisons were adjusted using the sequential Bonferronitechnique (Rice 1989). Individual multilocus heterozygosity(MLH) was calculated as the proportion of loci that wereheterozygous (corrected for nonscored loci). Values ofmean MLH across groups were compared by 1-way facto-rial ANOVA. The distribution of observed MLH was com-pared with expected distribution under random matingwithout subsequent selection. This was done by simulating1000 new individuals from each parental sample usingHYBRIDLAB (Nielsen et al. 2001). HYBRIDLAB gener-ates hybrids between 2 populations by randomly drawingalleles from 2 different samples. If 2 identical input filesare used, this corresponds to sampling from an infinitepopulation by multinomial sampling. The difference be-tween observed (MLH obs) and expected (MLH exp) fre-quencies was computed for each MLH class. Regressionanalysis was performed between individual heterozygosity(MLH) values and growth estimators (length increase,mass increase) to test for possible HFCs. Spearman’s cor-relation was used since variables did not approximate anormal distribution following logarithmic transformation.In the case of survival, we used a binary generalized linearmodel with a log likelihood function and binomial distribu-tion of error variance, with survival as the dependent valueand MLH as the independent variable. All statistical analy-ses were performed in STATISTICA v. 6.0 (Statsoft,

Table 1. Values of observed heterozygosity (Ho),level of polymorphism (P0.95), and allelic richness(AR) at all allozyme and microsatellite loci forsmall (<127 g) and large (‡127 g) Anguilla angu-illa individuals after 22 months.

(a) Allozymes

LocusSmallindividuals

Largeindividuals

AAT-1* 0.086 0.113AAT-2* 0.000 0.014AAT-3* 0.000 0.014ADH* 0.448 0.521GPI-1* 0.207 0.183GPI-2* 0.069 0.113IDHP* 0.035 0.056MDH-2* 0.241 0.239MPI* 0.172 0.099PGM* 0.017 0.014Mean Ho (SD) 0.128 (0.143) 0.136 (0.153)P0.95 0.4 0.5AR 2.7 3

(b) Microsatellites

LocusSmallindividuals

Largeindividuals

Aan 01 0.333 0.600Aan 03 0.205 0.123Aan 05 0.951 0.831Aro 063 0.912 0.914Aro 095 0.762 0.780Ang 151 0.861 0.932Mean Ho(SD) 0.671 (0.320) 0.697 (0.305)P0.95 1.000 1.000AR 12.33 12.33

Note: SD, standard deviation. Asterisks (*) denoteloci.

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Tulsa, Okla.). Significance for all statistical tests was takenas 0.05.

Results

Ontogeny effectIndividuals from batch 2.1 showed an average L of

283.53 ± 76.06 mm and an average W of 56.30 ± 46.73 gafter 22 months on the farm, ranging from 144–429 mmand 3.31–183.50 g. After the same period, individuals frombatch 2.2 were larger (444.75 ± 104.61 mm) and heavier(227.83 ± 201.11 g), with size ranges of 175–750 mm and7.77–933.50 g. As a consequence of grading, the samplingdistribution was bimodal with a median of approximately127 g. Of batch 2.1, 88% belonged to the group of small in-dividuals, and of batch 2.2, 90% belonged to the group oflarge individuals.

Small and large individuals showed similar levels of ge-

netic variation at allozyme and microsatellite loci, with nosignificant differences in Ho, P0.95, or AR (Table 1).

No significant correlations were observed between MLHat allozymes and length increase (r = 0.101; p = 0.271) ormass increase (r = 0.088; p = 0.388) (Fig. 1). Mean MLHwas higher in large individuals (0.135 ± 0.099) than in smallindividuals (0.127 ± 0.106) but not significantly so (p =0.678). Similarly, MLH at microsatellites was not correlatedwith either length increase (r = –0.005; p = 0.959) or massincrease (r = –0.040; p = 0.702) (Fig. 1).

Examination of individual loci showed no significantgreater length or mass increase in heterozygotes than homo-zygotes (Table 2). Significant differences were found only atlocus Aan 01, but the number of homozygotes used in thecomparison was small (N = 10).

When the observed MLH distribution was compared withits simulated distribution under random mating, no differen-ces were found in 22-month-old individuals (Fig. 2). With

Fig. 1. Anguilla anguilla: correlation of multilocus heterozygosity (MLH) at all allozyme and microsatellite loci in all individuals withlength increase (L) and mass increase (W).

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respect to allozymes, low MLH classes characterized smallindividuals as compared with expectations, and among largeindividuals fewer fish within the MLH = 0.10 class were ob-served compared with expectations; however, all compari-

sons were not significant (p = 0.233 for small individuals;p = 0.989 for large individuals). By contrast, small 12-month-old individuals showed significant differences be-tween the observed and expected MLH distribution (p =0.010), with a higher proportion of homozygotes and lowMLH classes than expected at random and a lower propor-tion of high MLH classes. For microsatellites, small andlarge individuals after 12 and 22 months showed a lowerproportion of low MLH classes but also a higher proportionof high MLH classes, although all comparisons were not sig-nificant.

Heterozygosity – survivalOverall tests for Hardy–Weinberg proportions with all

polymorphic loci as well as linkage disequilibrium amongall loci showed no significant departures from expected val-ues. Values of genetic variability at allozyme and microsa-tellite loci were similar when surviving and dead sampleswere compared (Table 3). No significant differences in Ho,P0.95 or AR were found between the handling and parasiteconditions.

Surviving and dead individuals showed similar allele(handling: p = 0.979; parasites: p = 0.291) and genotype(handling: p = 0.952; parasites: p = 0.283) frequencies at al-lozymes. At microsatellites, a significant value was observedonly at locus Aro 063 in the case of parasites, but the signif-icance disappeared upon Bonferroni correction.

No significant differences in mean MLH at allozymes be-tween samples were observed in the case of handling (sur-viving: 0.169 ± 0.105; dead: 0.160 ± 0.081) or parasites(surviving: 0.153 ± 0.104; dead: 0.162 ± 0.120). Similar val-ues of mean MLH at microsatellites were also observed insurviving (0.717 ± 0.177) and dead (0.679 ± 0.213) individ-uals after handling, and surviving (0.735 ± 0.157) and dead(0.724 ± 0.170) individuals after parasite infection. No asso-ciations were observed between survival and MLH using alogistic regression at either allozymes (handling: p = 0.421;parasites: p = 0.840) or microsatellites (handling: p = 0.140;parasites: p = 0.331).

No significant differences were observed when comparingthe observed MLH distribution with its simulated distribu-tion under random survival (Fig. 3). At microsatellites, deadindividuals presented a higher proportion of low MLHclasses but also a higher proportion of high MLH classes,although no comparisons were significant.

Discussion

Reduced HFCs over timeAllozyme and microsatellite data suggest that the associa-

tion between MLH and growth rate (measured as length andmass increase) in farmed European eel is not stable overtime and decreases with age. After 12 months in the tanks,more heterozygous individuals showed a faster growth rateand attained a larger size than did more homozygous indi-viduals (length increase: r = 0.177; p = 0.005, mass in-crease: r = 0.164; p = 0.009; see Pujolar et al. 2005). After22 months (this study), the HFCs disappeared, and no signif-icant correlations were observed between MLH and lengthor mass increase. The significant associations observed after12 months remained when a subsample of 100 individuals

Table 2. Anguilla anguilla: mean length and mean mass ofhomozygotes and heterozygotes at 6 allozyme and 5 micro-satellite loci.

(a) Allozymes

Locus Length (mm) (SD) Mass (g) (SD)

AAT-1*Homozygotes 366.73 (120.95) 143.89 (165.12)Heterozygotes 396.85 (139.20) 185.13 (236.30)D + +

ADH*Homozygotes 366.66 (128.52) 145.97 (175.37)Heterozygotes 372.92 (116.39) 150.15 (170.32)D + +

GPI-1*Homozygotes 368.68 (123.23) 147.76 (172.56)Heterozygotes 374.28 (122.61) 149.22 (177.77)D + +

GPI-2*Homozygotes 366.23 (121.30) 143.29 (166.80)Heterozygotes 407.73 (199.09) 199.09 (232.11)D + +

MDH-2*Homozygotes 362.39 (123.79) 142.52 (169.13)Heterozygotes 393.10 (117.85) 165.50 (186.03)D + +

MPI*Homozygotes 373.84 (112.07) 145.82 (159.64)Heterozygotes 342.94 (179.81) 162.73 (249.04)D – +

(b) Microsatellites

LocusLength (mm)(SD) Mass (g) (SD)

Aan 01Homozygotes 345.32 (101.73) 110.61 (113.34)Heterozygotes 420.67 (153.04) 226.22 (243.55)D + +

Aan 03Homozygotes 392.30 (135.07) 182.34 (213.49)Heterozygotes 325.33 (96.39) 84.39 (64.00)D – –

Aan 05Homozygotes 429.50 (137.70) 208.94 (231.73)Heterozygotes 375.86 (131.59) 162.63 (195.33)D – –

Aro 095Homozygotes 364.33 (127.66) 148.86 (177.91)Heterozygotes 384.41 (132.56) 171.12 (203.58)D + +

Ang 151Homozygotes 356.80 (161.80) 174.75 (224.35)Heterozygotes 381.19 (128.19) 164.44 (195.06)D + –

Note: D indicates the sign of the difference between heterozy-gotes and homozygotes. Asterisks (*) denote loci.

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(length increase: r = 0.240; p = 0.016, mass increase: r =0.230; p = 0.021) was used, ruling out a lack of power inthe present analysis due to smaller samples. The decrease inHFCs over time could be attributed to either ontogeneticvariance in gene activity between 12- and 22-month-old in-dividuals, differential mortality leaving only the largest indi-viduals, or the relaxation of the environmental conditionsrelated to population-density effects.

Most cases of a drop in HFCs with age have been docu-mented in marine bivalves, in which species tend to displaysignificant HFCs a few months rather than a few years aftersettlement (ontogenetic influence). In a population of themussel Mytilus edulis, a positive correlation between MLHand growth observed 2 months after settlement (Koehn andGaffney 1984) vanished when the same population was ex-amined 6 months later (Diehl and Koehn 1985). In the oys-ter Ostrea edulis, a positive association between MLH andsize was observed in 70-day-old individuals (Bierne et al.1998) but not in 1-year-old cohorts (Marsic-Lucic and David2003). In the surf clam Spisula ovalis the correlation be-tween heterozygosity and size was significantly positive dur-ing the first 2 years but approached zero after 3 years(David et al. 1995), which led the authors to suggest that en-

ergy is mostly directed to growth at early but not late lifestages. In our study, homozygote and heterozygote individu-als at GPI* and MPI* showed statistically significant differ-ences in length and mass increase after 12 months but notafter 22 months in the tanks. Both loci are involved in meta-bolic energy pathways that might play a significant role inearly life stages. Using a similar argument, Vaughn et al.(1999) reported that body size in mice is controlled by quan-titative trait loci (QTLs), each having a relative effect on theindividual phenotype. Early growth QTLs mapped to sepa-rate chromosomal locations from those of late growthQTLs, suggesting that early and late growth may be con-trolled by different sets of genes. McElroy and Diehl (2005)showed an ontogenetic component of variation in HFCs inthe earthworm Eisinia andrei but an inconsistent patternamong loci, suggesting different roles of allozymes at vari-ous times in the life history of the species. However, a de-crease in HFCs due to ontogenetic variance in gene activitybetween 12- and 22-month-old individuals does not seemplausible in our study, since we observed a similar growthrate both in length and mass during the 2 years of the study.After an average increase of 176.18 ± 84.97 mm and44.45 ± 44.62 g during the first year, we calculated an aver-

Fig. 2. Anguilla anguilla: distribution of observed and simulated MLH frequencies (MLHobs–MLHexp) in small individuals at all allozymeand microsatellite loci after 12 and 22 months in the tanks (*p < 0.05).

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age increase of 130.97 ± 125.56 mm and 109.48 ± 180.72 gin the following 10 months (until individuals reached22 months old). The increase in size was more apparent inbatch 2.2 (large individuals) with an average increase of207.06 ± 107.46 mm and 192.24 ± 208.50 g. Although 22-month-old individuals seem to correspond to early growth,the positive HFCs observed in 12-month-old individuals stillmight have resulted from the activity of a different set ofgenes (McElroy and Diehl 2005) without affecting the typi-cal growth pattern in eels.

A drop in HFCs can also be explained by selection influ-ence, i.e., sampling a group of individuals after differentialmortality has occurred and eliminating the most unfit indi-viduals (Foltz and Zouros 1984; David et al. 1995). Diehland Koehn (1985) proposed that a reversal in the MLH–growth correlation in a population of Mytilus edulis sampledseveral months apart was caused by selective mortality ofhomozygous individuals early in their life history. Thiscould be the case of European eel in aquaculture, since, de-spite the presence of abundant food, cannibalism on smallindividuals is frequent. Aquaculture experiments haveshown that a faster growth has a clear direct benefit at theindividual level. Competitive struggles among individualsare usually observed in the rearing tanks, in which a few in-dividuals dominate and grow much faster than the others.

The smallest ones in the hierarchy usually die either afterwithering due to their inability to obtain food or after preda-tion by larger individuals. Dominance is more geneticallydetermined and depends less on physiological condition(Tesch 2003). Thus, the elimination of unfit smaller individ-uals could mechanically decrease the MLH–fitness correla-tion and explain the lack of a consistent HFC pattern in 22-month-old individuals. Nevertheless, small individuals wereobserved in all batches; for instance, 29% of batch 2.1 indi-viduals (22 months) showed a smaller L than the mean Lafter 12 months in the same batch (24.13 cm). More impor-tant, individual losses in the tanks were much higher in thefirst year (0–12 months) than in the second, in which mor-tality was negligible (C. Vancoillie, personal communica-tion), suggesting that a decline in HFCs due to differentialmortalities would be less likely.

Population-density effects and the subsequent relaxationof environmental stress after grading might explain the de-crease in HFCs in our study, in which eels were sorted bysize into small and large individuals after 1 year in the tanks(environmental stress influence). Grading is a common prac-tice in eel aquaculture and is aimed at reducing competitiveeffects (Tesch 2003). A consequence of grading is the reduc-tion of environmental stress in the tanks linked to lower pop-ulation densities and less competition for food. Lesbarrereset al. (2005) showed that the levels of expression of HFCsin the common frog (Rana temporaria) were highly environ-mentally dependent, the positive effect of genetic variabilityon survival being more obvious under restricted food, whichsuggested that HFCs are more readily detected under stress-ful than nonstressful environmental conditions. In our study,the relaxation of environmental stress after sorting eels bysize might have resulted in only very subtle fitness differ-ences among individuals of equal or similar size.

Null heterozygote advantage at survival

Under the conditions of this study, genetic variability wasnot significantly correlated with survival in the Europeaneel. Highly heterozygous individuals did not exhibit anabove-average survival under 2 different stressors (handlingstress or parasite infection). Surviving and dead individualsshowed no differences in allelic or genotypic frequencies,suggesting that survival/mortality was not coupled with ge-netic composition, at least not for the markers surveyed inthis study.

Previous studies attempting to correlate survival with allo-zyme or microsatellite heterozygosity have shown mixed re-sults, and because of the difficulties in measuring viabilityand the need of single-cohort analyses (David 1998), it re-mains unclear whether a general pattern exists. In fish, stud-ies have generally shown a positive relation betweenheterozygosity and survival. Mitton and Koehn (1975) re-ported a superior viability in highly heterozygous individu-als when cohorts were compared over successive years inFundulus heteroclitus. Higher heterozygosity has also beenassociated with greater disease resistance and survival inrainbow trout Oncorhynchus mykiss challenged with bacte-rial gill disease (Ferguson and Drahushchak 1990). The lackof correlation between genetic variation and survival ob-served in European eel does not fit the general pattern ob-served in fish. Nevertheless, we should take into account

Table 3. Anguilla anguilla: values of observed heterozygosity(Ho), level of polymorphism (P0.95), and allelic richness (AR) atall allozyme and microsatellite loci for dead and surviving indi-viduals under handling and parasite infection.

Handling Parasites

Locus Dead Survivors Dead Survivors

AAT-1* 0.067 0.150 0.167 0.150AAT-2* 0.000 0.060 0.017 0.040AAT-3* 0.033 0.020 0.014 0.070ADH* 0.567 0.571 0.583 0.474GPI-1* 0.267 0.240 0.233 0.310GPI-2* 0.100 0.120 0.113 0.050IDHP* 0.033 0.100 0.017 0.060MDH-2* 0.400 0.270 0.317 0.263MPI* 0.133 0.160 0.133 0.120PGM* 0.017 0.010 0.017 0.010Mean

Ho(SD)0.160

(0.192)0.170

(0.165)0.162

(0.182)0.155

(0.149)P0.95 0.5 0.7 0.6 0.5AR 2.200 2.678 2.325 2.630

Handling Parasites

Locus Dead Survivors Dead Survivors

Aan 01 0.705 0.706 0.605 0.765Aan 03 0.200 0.208 0.100 0.193Aan 05 0.810 0.773 0.865 0.796Aro 063 0.861 0.942 0.933 0.902Aro 095 0.733 0.840 0.750 0.855Ang 151 0.805 0.855 0.925 0.911Mean

Ho(SD)0.686

(0.244)0.721

(0.264)0.696

(0.317)0.737

(0.273)P0.95 1.000 1.000 1.000 1.000AR 14.666 15.170 13.167 16.500

Note: Asterisks (*) denote loci.

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that negative results are likely to be under-represented in theliterature because of publication bias in favour of significantresults (David et al. 1995).

It has been proposed that the superior viability of moreheterozygous individuals might be related to their low meta-bolic needs, which translates into a surplus of energy to re-sist stressful conditions (Myrand et al. 2002). Most of theallozymes in our study (including GPI, IDHP, MDH, MPI,and PGM) play key roles in metabolic energy pathways, butnone showed a significant heterozygote advantage in sur-vival. Similarly, allele and genotype frequencies at AAT, atransaminase released into the blood as a consequence ofstress (Morales et al. 2005), did not differ significantly be-tween surviving and nonsurviving individuals.

In summary, our study shows an unstable association overtime between individual MLH and growth rate, which wasobserved in 12-month but not in 22-month-old individuals.Our results are consistent with the variation in time andacross populations exhibited by HFCs in previous studiesand with the general prediction that HFCs are maximal earlyin life. The relaxation of environmental conditions aftergrading (by means of lower population densities and lesscompetition for food) appears the most plausible explanationfor the drop in the association between MLH and growthrate. Alternatively, the decline in HFCs could be attributedto ontogenetic differences between groups resulting fromthe activity of different gene sets or, although less likely, adifferential mortality among individuals. On the other hand,the absence of a heterozygote advantage in viability found

in our study suggests that the enzymes studied might havean influence on growth that is related to a metabolic advant-age but show no indications of an effect on survival. Futureadvances in eel reproduction might allow the mapping ofearly/late growth and survival quantitative traits, thus pro-viding new tools for a better understanding ofheterozygosity–fitness correlations in the European eel.

AcknowledgementsWe thank the personnel at Royaal BV for assistance with

sampling. We also thank G. Pogson for his comments on themanuscript and J. Raeymaekers, M. Campero, and X. Agui-lera for suggestions on statistical analysis. Research wasfunded by the EU contract EELREP (Q5RS-2001-01836).J.M. Pujolar received a postdoctoral fellowship from theMinisterio de Educacion, Cultura y Deporte (Spain).G.E. Maes received a PhD fellowship from the I.W.T. (Insti-tute for the Promotion of Innovation by Science and Tech-nology in Flanders) and is now a postdoctoral researcherfunded by the FWO (Scientific Research Fund Flanders).

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