The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:238-248
Selection Against Blue Mussels (Mytilus edulis L.) Homozygotes Under Various Stressful Conditions
From the Station Technologique Maricole des Îles-de-la-Madeleine, Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec, Cap-aux-Meules, Québec, Canada G0B 1B0 (Myrand); Centre aquicole marin-Université du Québec à Rimouski, Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec, Grande-Rivière, Québec, Canada G0C 1V0 (Tremblay); and Ministère des Pêches et des Océans, Institut Maurice-Lamontagne, Mont-Joli, Québec, Canada G5H 3Z4 (Sévigny).
Address correspondence to Bruno Myrand at the address above, or e-mail: bruno.myrand{at}agr.gouv.qc.ca.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionHow Selection Possibly Acted...Selection: An Explanation for...Overdominance or AssociativeReferences |
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Three mussel groups differing in mean multilocus heterozygosity (MLH) were used to examine the MLH-fitness relationship. Mussels were submitted to aerobic and anaerobic stressful conditions in the laboratory, and their LT50 was measured. Mortality was not random in two of the three groups and affected the homozygous individuals more. This selective mortality caused a significant increase in the mean MLH of the survivors, but only for the two groups characterized by the lowest initial MLH and significant deficits in heterozygotes at the onset of the experiments. While these experiments were ongoing, the same two groups also suffered a 40% mortality rate in lantern nets under field conditions. This mortality also increased the mean MLH in survivors. All groups showed strong inverse relationships between MLH and standard metabolism. Our results suggest that the higher resistance of more heterozygous individuals is related to their lower metabolic needs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionHow Selection Possibly Acted...Selection: An Explanation for...Overdominance or AssociativeReferences |
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Parameters associated with fitness, such as developmental stability, growth, fecundity, and survival, seem to be correlated with multilocus heterozygosity (MLH) in several organisms (reviewed in Mitton 1993). However, the MLH-fitness relationship is usually weak and explains less than 10% of the variance in growth and survival in marine bivalves (Bayne and Hawkins 1997; Pecon Slattery et al. 1993). As the underlying mechanism is poorly understood (Hedgecock et al. 1996; Zouros and Pogson 1994), the existence of this relationship has been questioned. For example, Jorgensen (1992) suggests it is only an apparent relationship based on spurious correlations, while Britten (1996) finds little grounds for its existence when applying meta-analyses to data from several studies using shell increment as a surrogate for bivalve fitness. However, shell growth may not be a good surrogate for bivalve fitness since shell and tissue growths are uncoupled (Hilbish 1986; Mallet and Carver 1993). Further, shell length is not always highly correlated with tissue mass, since shell is not shrinking during starvation or spawning, in contrast to tissue mass.
The relationship between MLH and survival is not as well documented as that relating MLH and growth (David 1998; Saavedra and Guerra 1996). For example, only a few studies have examined metabolism, genetic variability, and survival concurrently (e.g., Hawkins et al. 1989; Tremblay et al. 1998d). Maintenance metabolism is known to be negatively correlated with MLH (Hawkins and Day 1996; Tremblay et al. 1998d), so that heterozygous individuals have more energy for growth, reproduction, and survival (Hawkins and Bayne 1992). The lower-maintenance metabolism in heterozygotes results from a lower protein turnover (Hawkins et al. 1986).
The MLH-fitness correlation, or heterosis, is more obvious when metabolism is exacerbated by stressful conditions (Gentili and Beaumont 1988; Koehn 1991; Scott and Koehn 1990). Important modifications of the metabolism are required to maintain internal environment as constant as possible under stressful conditions (Hoffman and Parsons 1991). Such adjustments result in a higher protein turnover caused by the synthesis of proteins such as heat-shock proteins and by the reparation/denaturation of damaged proteins (Hawkins 1991; Hoffman and Somero 1995). Thus the maintenance metabolism is greatly increased under these conditions (Koehn and Bayne 1989) and differences between homozygotes and heterozygotes for growth and survival are amplified (Koehn and Bayne 1989; Mitton 1993).
The aim of this study was to establish a relationship between MLH and fitness using survival under various stressful conditions as a surrogate for fitness and to propose an explanatory mechanism of selection against homozygotes based on their metabolic requirements. Mussels from the Magdalen Islands (Gulf of St. Lawrence, Canada; Figure 1) are very appropriate for testing the effect of MLH on survival, as they are characterized by important differences in mean MLH (Tremblay et al. 1998c), a condition which helps to increase the power of such studies (Zouros and Foltz 1987). Previous studies have shown that, in this area, the mussels with the highest mean MLH also have a higher fitness as suggested by their higher scope for growth, higher oxygen : nitrogen ratio, lower standard metabolic rate, shorter period of destabilization of lysosomal membranes, lower thermosensitivity of metabolism, lower level of stress measured from oxidative processes, and lower sensitivity to summer mortalities (Myrand and Gaudreault 1995; Pellerin-Massicotte 1997; Tremblay et al. 1998a,b,d). All these results may be associated with the measured inverse relationship between MLH and the metabolic requirements of these mussels (Tremblay et al. 1998d).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionHow Selection Possibly Acted...Selection: An Explanation for...Overdominance or AssociativeReferences |
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This study is based on the comparison of mean MLH in survivors to three stressful conditions with that of controls (T0). The effects of one aerobic and one anaerobic stress on MLH were studied under laboratory conditions, and we also took advantage of an undefined stress that occurred under field conditions and caused approximately 40% mortality in some mussel groups under study.
Three groups of mussels from the Magdalen Islands, Gulf of St. Lawrence (Figure 1), were used in the experiments. The first two, Amherst Basin (AB) and House Harbour (HH), were wild mussels characterized by high and low mean MLH, respectively (Tremblay et al. 1998c). The third group was formed with suspension-cultured mussels (SC) that were sleeved (Mallet and Myrand 1995) as spat 4–5 months after their settlement on artificial collectors left in Amherst Basin and then transferred to grow-out sites in the House Harbour lagoon. A decrease in mean MLH occurs after sleeving, so that SC mussels show intermediate values between HH and AB (Tremblay et al. 1998c).
Laboratory experiments were performed twice: August and September. Mussels were collected 1 week before the experiments. First, 75 mussels (34–85 mm) from each group were frozen and later used to establish the allometric relationships between shell length and dry tissue mass. The dry mass of each mussel at the beginning of a given experiment was estimated using these specific relationships. Second, approximately 500 individuals (50–60 mm in shell length) from each group were kept in lantern nets in the House Harbour lagoon until needed.
The day before the beginning of the experiments, 250 individuals from each group were brought to the laboratory while the others were left in the lantern nets in the lagoon. They were kept overnight in sand-filtered seawater at field temperatures (18–20°C) to allow gut clearance. The mussels from each group were then separated into three subgroups: the first was used to characterize the relationship between MLH and metabolism and the other two were subjected to aerobic or anaerobic stressful conditions. The subgroup used to describe the MLH-metabolism relationship was considered as the T0 control.
Metabolism of Mussels Used as Controls (Subgroup 1)
Fifty mussels from each group were sent by air to Quebec City for metabolic measurements and subsequent genetic characterization. They were kept in an insulated icebox for this 4-h transportation period. Upon arrival, mussels were placed in individual 9 mm plastic mesh cages kept in 200 L tanks (50 cages per tank) filled with recirculated artificial seawater at 20°C and 30 ppm, and under natural photoperiod. Each day 40–50% of the water was replaced. The individual cages allowed the transfer of mussels to respirometry chambers without severing their byssus.
Mussels were first starved for 4 days and then fed to satiety during 6 days with a mixture of Monochrysis lutherie, Thalassiosira pseudomona, and Isochrysis galbana provided daily at 0.083 mg dry mass or approximately 10,000 cells/ml/individual. Production of pseudofeces confirmed that ingestion rate was maximal. Mussels reached their maximum metabolism under this feeding regime (Tremblay et al. 1998c) and their oxygen consumption was measured. Mussels were then starved for 8 days before new oxygen consumption measurements were made to quantify their standard (maintenance) metabolism.
Each mussel was acclimated to a respirometry chamber (700 ml) for 2 h during which circulating O2-saturated water was supplied. Then the chamber was tightly closed for at least 1 h for individual measurements. Partial O2 pressure was greater than 100 torr and water was kept well-mixed using a magnetic stirrer. Every 15 min, 2 ml of water was sampled to measure dissolved O2 concentration using a polarographic electrode (YSI, model 5331) coupled to an oxymeter (Cameron Instruments Cie, model OM400). Measurements taken in a chamber containing a mesh cage with only an empty shell were used as controls. Measurements from mussels that spawned in the chambers were discarded and repeated later the same day. Eight respirometry chambers were used in parallel.
Following measurement of its standard and maximal metabolisms, a given mussel was divided into two sections, which were weighed. One section, including a piece of the digestive gland (
100 mg), was frozen at -80°C for further electrophoretic analyses. The other was dried at 65–70°C for 72 h and the total dry mass of tissues was estimated from the measurements of the relative wet mass of the two sections. Oxygen consumption was standardized for a 2 g mussel using the allometric relationship of Tremblay et al. (1998d):
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Aerobic Stress Experiments (Subgroup 2)
The aerobic stress was induced at high temperature and low food levels in well-oxygenated water. One hundred mussels from each group were placed in a tank (length 2.50 m x width 0.45 m x height 0.22 m) filled with 200 L of sand-filtered seawater kept at 26–27°C with a Polyscience heater (Model 210). To reduce bacterial development, the water was recirculated continuously through two Gelman filters (5 and 2 µm) and through an ultraviolet sterilizer at a rate of 20 L/min. Also, half of the water was renewed twice a day (early morning and evening) using a head tank filled with sand-filtered seawater warmed to approximately 26°C with another Polyscience heater. Oxygenation was provided with air stones. Dissolved oxygen was measured (YSI model 58) daily before water renewal and was consistently greater than 90% of saturation. No food was added, so the mussels could only feed on developing bacteria and particles that passed through the sand filter. The small quantity of feces produced throughout the experiments suggested that the mussels were food limited.
Mussels were glued with cyanoacrylate gel onto 30 Plexiglas crosspieces (10 mussels per crosspiece of 45 cm x 1.5 cm). The crosspieces were suspended 6 cm above the bottom. At least three mussels from each group (HH, AB, and SC) were distributed randomly on each crosspiece to minimize spatial variability. Mussels from each group were examined twice each day until 50% of the individuals were dead (LT50). A mussel was considered dead when the valves gaped and did not close in response to three consecutive stimuli (pressure from the experimenter's fingers) applied to provoke closure.
The AB mussels showed a high survival to temperature stress. In order to reduce the time needed to reach the 50% mortality level, the temperature was raised from 26.6 ± 0.2°C to 27.7 ± 0.2°C during the August experiment, when they were the only group still in the raceway. However, the August and September experiments were both stopped after 25–26 days, although the AB mussels still had a survival rate higher than 50%.
Anaerobic Stress Experiments (Subgroup 3)
The anaerobic stress was caused by prolonged exposure to air (Viarengo et al. 1995). One hundred mussels from each group were exposed to air in a room maintained at 17–19°C and at a relative humidity of approximately 100%. The mussels from the three groups were placed in alternate positions and examined twice a day until 50% of the individuals were dead (LT50) using the same criterion as the one used for aerobic stress.
Undefined Stress in Field Conditions
While the August and September experiments were ongoing, about 40% of the mussels that were not used in the laboratory experiments but were left in the lantern net set in the House Harbour lagoon died. We took advantage of this mortality event to examine the consequence of an undefined stress in field conditions. Genetic analyses were carried out on 50 mussels among the survivors.
Determination of Genetic Variability
The mussels used for metabolic measurements from each group and the survivors from each experiment were frozen at -80°C pending genetic analyses. The electrophoretic procedure detailed in Tremblay et al. (1998c) was used for the following polymorphic enzymes: glucose phosphate isomerase (GPI, EC 5.3.1.9), mannose phosphate isomerase (MPI, EC 5.3.1.8), phosphoglucomutase (PGM, EC 2.7.5.1), octopine dehydrogenase (ODH, EC 1.5.1.11), leucine aminopeptidase (LAP, EC 3.4.11), and two esterases (EST, EC 3.1.1.1). Allelic frequencies, observed (Ho) and expected (He) heterozygosities under the assumption of Hardy–Weinberg equilibrium, as well as heterozygote deficiency index (D) were calculated for each locus using the Biosys-1 program of Swofford and Selander (1989). The number of heterozygous loci per mussel (0–7) was used to characterize individual multilocus heterozygosity (MLH).
The MPI* locus was used to discriminate Mytilus trossulus from Mytilus edulis (Mallet and Carver 1995; McDonald et al. 1991). A proportion of only 2.2% of the experimental mussels were identified as M. trossulus, which is in agreement with previous observations (Tremblay et al. 1998c,d). They were discarded from the analyses.
Statistical Analyses
Statistical tests were performed with the SAS package, version 6.12 (SAS 1982), unless stated otherwise. Log10-transformed data were used for the shell length-tissue dry mass allometric relationships, which were compared with ANCOVAs. The relationships between MLH and metabolism were also compared with ANCOVAs.
For each group (AB, HH, and SC) in the August and September experiments, the frequency distribution of alleles at each locus observed in mussels used for metabolic measurements (controls at T0) was compared to that of survivors from each experimental stressful conditions through Monte Carlo simulations. Frequency distributions of the number of heterozygous loci (MLH) were compared with one-tailed Wilcoxon tests to verify the hypothesis that survivors' MLH was higher than that of the T0 group. Only mussels with a complete dataset for all seven loci were used in these tests. For multiple comparisons, sequential Bonferroni tests were used to keep the type I error at the overall level of 
= 0.05 (Rice 1989).
Allelic distributions for each locus were compared with chi-square Monte Carlo simulations (Roff and Bentzen 1989) using the REAP program (McElroy et al. 1991) corrected with sequential Bonferroni tests.
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Results |
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Genetic Variability of the Controls (T0)
The genetic characteristics of the mussels from the three groups that were used as controls in the August and September experiments are described in Table 1. There were no differences in allelic frequency distribution between T0 mussels for the two periods. These six groups showed similar distributions of alleles for each locus, except for PGM* (
2 = 18.31, df = 6, P < .0001). Post hoc multiple comparisons at this locus showed that AB mussels in August differed from HH mussels in August (2 = 18.41, P < .0001), from HH mussels in September (2 = 22.12, P < .0001), and also from AB mussels in September (2 = 12.98, P = .004).
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For the HH mussels used in the August experiment, significant heterozygote deficits (D) were observed for five of the seven loci (EST-1*, EST-2*, LAP*, MPI*, and PGM*; Table 1). Significant D values were also observed for the SC mussels at four loci (EST-1*, EST-2*, GPI*, and PGM*) and at only one locus (MPI*) for the AB mussels. Mean observed heterozygosity (Ho; over all loci) for the AB mussels was 0.575, which is not different from the mean He (0.591). In contrast, the mean Ho was 0.393 (He = 0.564) and 0.350 (He = 0.542) for the SC and HH mussels, respectively.
For the controls of the September experiment, the difference between Ho and He decreased in all groups compared to the controls of the August experiment. The HH mussels showed significant D at only three loci (EST-1*, EST-2*, and LAP*) and the SC mussels at one locus (EST-1*). No significant D was observed in the AB mussels (Table 1). Again, the mean Ho was higher for the AB mussels (0.542) than for the SC (0.444) and the HH (0.389) mussels. The mean Ho of the AB mussels was similar to the He (0.542 and 0.552, respectively).
The mean MLH of controls in the August and September experiments was significantly higher in the AB mussels than in the two other groups, which had similar values (Table 2).
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Metabolism of the Controls (T0)
The standard metabolism (Table 2) of the HH mussels was 2.2 times that of the AB mussels in August (1.16 versus 0.52 mg O2/individual/h) and 1.5 times that in September (0.94 versus 0.64 mg O2/individual/h). The SC mussels showed intermediate values during both experiments. The maximum metabolism was usually similar for the three groups in a given experiment, except for the AB mussels, which had a significantly lower value than the other two in August.
Even though their mean shell length was comparable at the onset of each experiment, the three groups of mussels had different tissue masses (P < .0001) and the AB mussels had the lowest values on both occasions (Tables 2 and 3).
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Relationship Between Heterozygosity and Metabolism in Controls (T0)
The three groups of mussels showed significant inverse relationships between MLH and metabolism at the beginning of both experiments; the only exception was for the maximum metabolism in the HH mussels in August (Figure 2 and Table 4). The relationships were stronger for MLH versus standard metabolism (r2 = 0.44–0.75) than for MLH versus maximum metabolism (r2 = 0.02–0.42). In August, the comparison of the linear relationships showed differences for the slopes among the three groups for the standard (P < .0001) and the maximum (P = .001) metabolisms. In September, there were no differences among the three groups for the standard (comparison of the slopes: P = .10 and Y-intercepts: P = .49) and the maximum (comparison of the slopes: P = .23 and Y-intercepts: P = .26) metabolisms. There were no relationships between MLH and tissue dry mass in each of the three groups at the onset of both experiments (all r2
0.02 and P 
.33).
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Survival Under Aerobic, Anaerobic, and Field Stress Conditions
The AB mussels showed the highest LT50 values under aerobic and anaerobic stressful conditions, while the HH mussels had the lowest (Figure 3). The SC mussels had a much shorter survival than their AB counterparts under both conditions. Further, an important mortality (39%) was observed for the HH and SC mussels left in the lantern net in the lagoon in August, but not the AB mussels. In September, such mortality (41%) was observed only among the HH mussels.
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The distribution of alleles at each locus for survivors to the stressful conditions were compared to those for their corresponding control (T0). Only 12 of the 42 paired comparisons (3 groups x 7 alleles x 2 experiments) with corresponding controls showed significant differences at P < .05. Of these 12 differences, 8 were observed at the LAP* and PGM* loci.
In each group, the MLH of the survivors to stressful conditions was compared to the MLH measured at T0 (Figure 4). The mean MLH of HH and SC survivors was systematically higher than for the T0 controls. These two groups of mussels showed the same pattern under anaerobic and aerobic stresses in the laboratory, as well as under natural stressful conditions. In contrast, the mean MLH of the AB survivors was always similar to the T0 controls in both experiments. There were no significant differences between the mean MLH of the T0 controls of each group measured at the onset of both the August and September experiments (HH: P = .19; AB: P = .49; SC: P = .09).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionHow Selection Possibly Acted...Selection: An Explanation for...Overdominance or AssociativeReferences |
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In this study, selection acted in the laboratory under aerobic and anaerobic stressful conditions, as well as under field conditions. This selection increased the mean number of heterozygous loci (MLH) by the selective mortality of the more homozygous individuals. Consequently a clear MLH-fitness relationship was observed through a better survival of more heterozygous individuals under the stressful conditions of this experiment. However, this selection acted only on the groups characterized by the lowest observed heterozygosity values, Ho, and by initial deficits in heterozygotes at the beginning of the experiments.
Strong inverse relationships between MLH and standard metabolism were observed for all groups. In agreement with previous observations (Tremblay et al. 1998d), metabolic requirements varied greatly among the groups of mussels as a result of their different mean MLH. The HH mussels had the highest standard metabolism and the lowest survival under all stressful conditions, while the AB mussels had the lowest standard metabolism and the longest survival. The SC mussels showed intermediate values. The longer survival of the AB mussels could not be attributed to the higher initial reserves, since these mussels had the lowest tissue masses at the onset of both experiments.
The observed heterozygosity (Ho) of the wild HH mussels increased substantially between the August and September experiments. This increase was probably caused by a selective mortality of homozygotes in the mussel beds, as a massive, although not quantified, mortality was observed between the two experiments. However, this mortality in the mussel beds did not result in an MLH increase as important as the one observed under laboratory conditions or in the lantern nets. Possibly factors other than individual energetic balance were involved at the mussel bed level (e.g., predation).
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How Selection Possibly Acted Against Homozygotes |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionHow Selection Possibly Acted...Selection: An Explanation for...Overdominance or AssociativeReferences |
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The same pattern was observed between MLH and survival under aerobic (laboratory and field) and anaerobic stressful conditions, but distinct mechanisms may have been involved in the selective mortality of homozygotes according to their metabolic pathway. As all groups showed strong inverse relationships between MLH and standard (maintenance) metabolism, the differential levels of metabolic requirements may explain the variable survival of mussels under aerobic stressful conditions. This kind of MLH-metabolism relationship has been observed previously, but was much weaker (Diehl et al. 1986; Hawkins et al. 1986; Koehn and Shumway 1982; Tremblay et al. 1998d). A general response to aerobic stressful conditions is a sharp increase in maintenance metabolism to maintain a stable internal environment (Hoffman and Parsons 1991). Further, energy requirements under stressful conditions are amplified for mussels with higher maintenance metabolisms (Hawkins and Bayne 1992). In this context, homozygotes, with their higher standard metabolism, must rely more extensively on their reserves to maintain vital functions and support physiological responses to stress. In contrast, heterozygotes, with their lower energetic requirements, have a surplus of energy with which to resist against stressful conditions (Hawkins 1991; Koehn and Bayne 1989). However, Hawkins et al. (1987) observed that the lower survival of stressed mussels (starved at 28.5°C) was related not to their low energetic reserves, but to their high protein turnover. Hawkins et al. (1986) showed that mussels with a low MLH have a high protein turnover, which may help to explain why the HH mussels, with the lowest mean MLH, experienced the lowest survival, despite having the highest initial tissue mass.
Under anaerobic conditions, mussels depress their basal metabolism (Shick and Widdows 1981) to increase survival (Famme et al. 1981). Homozygotes may have a reduced capacity to depress their metabolism, which could explain their selective mortality under anaerobic stressful conditions. Furthermore, it can be hypothesized that an inverse relationship may also exist to some extent between MLH and the capacity to depress basal metabolism in the blue mussel (Mytilus edulis).
Mechanisms similar to those presented in this study may explain the better survival of heterozygotes under stressful conditions reported for various organisms such as bivalves, gastropods, and fishes (Beaumont and Toro 1996; Borsa et al. 1992; Nevo et al. 1986; Pecon Slattery et al. 1993; Shikano et al. 2000; Tanguy et al. 1999). However, it should be noted that Alvarez et al. (1989), Gaffney (1990), and Hummell et al. (1995) found no such patterns.
In contrast to the HH and SC groups, there was no selection against homozygous mussels and thus no increase in the mean MLH of AB survivors in both the August and September experiments. Further, the AB mussels suffered no mortality under field conditions. The major genetic differences between the experimental groups was that the HH and SC mussels showed low heterozygosity (Ho) and marked heterozygote deficiency at the onset of the experiments, while the AB mussels had a high Ho and no deficit in heterozygotes. Such an apparent association between heterozygote deficits and MLH-fitness relationships has already been observed (Houle 1989; Zouros 1987).
The observed association between heterozygote deficits and MLH-fitness relationships is possibly the result of variable proportions of homozygotes in the experimental populations. For each group in this study, mussels were arbitrarily defined according to their number of heterozygous loci: those with zero to three heterozygous loci were labeled as "low MLH," while "high MLH" described those with four to seven heterozygous loci. At the onset of the August and September experiments, only 33–41% of the AB mussels had a "low MLH," compared to 69–78% for the HH and SC mussels (Figure 5). Only 28–57% of HH and SC survivors of stressful conditions had a "low MLH." Thus the proportion of "low MLH" mussels in the HH and SC groups dropped to values similar to the AB group at the onset of the experiments. As selection appears to act mainly against homozygotes, its effect would be more important and thus more obvious in the HH and SC groups because they have high numbers of "low MLH" mussels. The proportion of "low MLH" mussels in the AB group was possibly too low to bring about such a decrease in homozygotes. This would explain why stressful conditions did not translate into an increase in mean heterozygosity for the AB group (see below). However, Gaffney (1990) suggested that excess of homozygotes may result from inbred individuals and/or heterozygotes with null alleles and aneuploids. Selection against these individuals would give the impression of a heterozygote superiority, so that once they have been eliminated there would be no more heterozygote deficits and the relationship between heterozygosity and fitness would disappear.
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Our results suggest that beyond a certain limit, factors other than MLH become more important in determining survival under stressful conditions. This is exemplified by the AB mussels, which suffered high mortalities (35–50%) without significant changes in MLH.
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Selection: An Explanation for Heterozygote Deficits? |
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Restricted gene flow between populations and subsequent inbreeding could be responsible for heterozygote deficits (Gaffney et al. 1990; Zouros and Foltz 1984a). However, Tremblay et al. (1998c) observed that gene flow was strong enough between mussel beds from the Magdalen Islands to prevent genetic drift. Further, there are no differences in allelic frequencies between mussels from the Magdalen Islands and Prince Edward Island (unpublished data), which suggests an important dispersal of larvae between these two locations approximately 150 km from each other. Finally, the deficits are not uniform across loci (Tremblay et al. 1998c; this study) as it would be expected under inbreeding (David et al. 1997; Gaffney et al. 1990). These results suggest that inbreeding is seemingly not the main factor explaining the heterozygote deficits observed in the mussels from the Magdalen Islands lagoons (HH mussels in the present study). Zouros and Foltz (1984a) also provided convincing arguments against inbreeding. However, partial inbreeding could be a nonnegligible factor, as suggested by David et al. (1997).
Most likely, there is no single cause to explain deficits in heterozygotes (Gaffney et al. 1990; Raymond et al. 1997), but two main causes have been proposed: Wahlund effects (Bierne et al. 1998; Borsa et al. 1991; Gaffney et al. 1990; Raymond et al. 1997; Zouros and Foltz 1984b) and selection (Beaumont 1991; Fairbrother and Beaumont 1993; Mallet et al. 1985; Zouros and Foltz 1984a). Wahlund effects were not observed in the Magdalen Islands (Tremblay et al. 1998c), but selection could not be rejected. If acting, selection would affect early life stages (larval or postlarval phase), as Tremblay et al. (1998c) observed similar Ho for spat (4–5 months after settlement) and adults for the same experimental groups as those used in this study. Other observations support such an early selection hypothesis (Fairbrother and Beaumont 1993; Mallet et al. 1985), which probably occurs after settlement (Beaumont 1991).
In the Magdalen Islands, mussels from the lagoons exhibit a heterozygote deficit comparable to that observed in many other marine bivalves (Beaumont 1991; Bierne et al. 1998; Raymond et al. 1997; Zouros and Foltz 1984b), but not the AB mussels, which have a high Ho and no deficits. This study was not designed to identify factors causing heterozygote deficits and thus we cannot elucidate this question. However, we can hypothesize that the AB site was simply not subjected to factors causing heterozygote deficits or the AB mussels initially had a deficit in heterozygotes, as do many other bivalve populations, but experienced selection against an excess of homozygotes early (up to 4–5 months) after settlement. If the second hypothesis is true, water temperature could be suggested as a possible agent of selection, as temperatures greater than 20°C are stressful for blue mussels (Tremblay et al. 1998a; Widdows 1973), including young stages like larvae (Lutz and Kennish 1992). The AB mussels are confronted with daily temperatures greater than 20°C between June and August, with peaks of 22–23°C (Myrand B, unpublished data). In contrast, the HH mussels are rarely subjected to temperatures greater than 20°C, and when it happens, peaks of 20–21°C last for only a few days (Myrand et al. 2000). Thus the early stages of AB mussels could suffer higher mortalities than HH mussels and the energetically more demanding homozygotes could be decimated in much higher proportions than heterozygotes. The elimination of a large proportion of homozygotes during the early life stages in the AB group most likely explains the absence of selective effects of stressful conditions in the present study.
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Overdominance or Associative |
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Overdominance Hypothesis
Two main hypotheses have been proposed to explain the MLH-fitness relationship: the associative overdominance hypothesis states that the scored loci act as neutral markers reflecting the level of homozygosity of an organism for recessive deleterious genes; the overdominance hypothesis implies that the scored loci themselves are directly responsible for the MLH-fitness correlation (e.g., Zouros and Foltz 1987). Our results do not provide clear information favoring one particular hypothesis.
The results with ODH*, the only locus acting in the anaerobic metabolism, appear to be in accordance with the overdominance hypothesis. During the August and September experiments, heterozygosity increased at this locus, but only in survivors to anaerobic stress. No changes were observed in survivors to the aerobic stressful conditions. These results are in accordance with other studies invoking overdominance to explain polymorphism of ODH* in organisms like Metridium senile (Walsh 1981) and some marine bivalves (Perez et al. 2000; Sarver et al. 1992; Volckaert and Zouros 1989).
For other loci, an increase in heterozygosity was usually observed under both aerobic and anaerobic stressful conditions, an observation which is not in accordance with the overdominance hypothesis. Standard metabolism variation, which is considered to be the main physiological explanation for the MLH-fitness relationship, is largely explained (up to 75%) by the number of heterozygous loci, not by specific ones. These results seem to support the associative overdominance hypothesis.
Relevance to Summer Mortalities
In the Magdalen Islands, susceptibility to summer mortalities seems to be related to metabolic needs and heterozygosity. Mussels from the lagoons are highly susceptible to summer mortalities and are characterized by high metabolic requirements and low MLH, while those from Amherst Basin are highly resistant and have low metabolic requirements and high MLH (Myrand and Gaudreault 1995; Tremblay et al. 1998c). The direct relationship between low MLH, high standard metabolism, and low survival observed in this study provides support for the hypothesis that susceptibility to summer mortality is related to differences in metabolic needs, and thus ultimately to heterozygosity (Myrand et al. 2000; Tremblay et al. 1998d).
In the present study, mussels used for both experiments were collected in early and late August and thus were in postspawning condition (Myrand et al. 2000). Further, the experimental mussels succumbed to stressful conditions in the lagoon during the usual period of summer mortality in the Magdalen Islands. As summer mortality is thought to decimate postspawning mussels facing stressful (high temperature and low food quality) conditions in August (Myrand et al. 2000; Tremblay et al. 1998d), we suggest that the results from the present study, particularly those obtained under natural conditions (lagoon), could be extrapolated to explain this phenomenon.
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Acknowledgments |
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We would like to thank the technical staff of the Station Technologique Maricole des Iles-de-la-Madeleine (STMIM) du ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ), whose entire collaboration was essential to the success of this project. We are also grateful to Michel Fournier (Moules de culture des Iles) and Marcel Roussy (STMIM-MAPAQ), who provided us with the suspension-cultured mussels and the wild mussels from Amherst Basin, respectively. The algae used to feed the experimental mussels were provided by Francis Coulombe (Centre Aquicole Marin de Grande-Rivière, MAPAQ). This study was supported by funds from the Direction de l'Innovation et des Technologies (MAPAQ). Finally, we greatly appreciated all the valuable comments on this article from Dr. E. Zouros and anonymous referees.
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Footnotes |
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Corresponding Editor: Philip Hedrick
Received June 29, 2001
Accepted March 29, 2002
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