Bioenergetic and genetic parameters in relation to susceptibility of blue mussels, Mytilus edulis (L.) to summer mortality

Journal of Experimental Marine Biology and Ecology, 221 (1998) 27–58

L

Bioenergetic and genetic parameters in relation to susceptibility of blue mussels, Mytilus edulis (L.) to summer mortality a b c d ´ Rejean Tremblay , Bruno Myrand , Jean-Marie Sevigny , Pierre Blier , a, Helga Guderley * a

b

´ ´ , P.Q., Canada G1 K 7 P4 Departement de Biologie, Universite´ Laval, Quebec ˆ , Direction des Innovations et des Technologies, Station Technologique Maricole des Iles-de-la-Madeleine ` de l’ Agriculture, des Pecheries ˆ ´ , 190 Rue Principale, Ministere et de l’ Alimentation du Quebec ˆ , P.Q., Canada GOB 1 B0 Case-Postale 658, Iles-de-la-Madeleine c ´ ´ et de la Biologie Experimentale ´ ` des Direction des Invertebres , Institut Maurice-Lamontagne, Ministere ˆ ´ , 850 Route de la Mer, Mont-Joli, P.Q., Canada G5 H 3 Z4 Peches et des Oceans d ´ ´ Departement de Biologie, Universite´ du Quebec a` Rimouski, Rimouski, P.Q. Canada G1 V 3 V4 Received 8 October 1996; received in revised form 26 March 1997; accepted 15 April 1997

Abstract Our study examined whether the differences in susceptibility to summer mass mortality of two ´ stocks of mussels from the Magdalen Islands (Quebec, Canada) are related to bioenergetic and / or genetic factors. The relative importance of maintenance and maximal metabolic rates, scope for growth (SFG) and the O:N ratio were followed over time to assess whether the increased incidence of mortality in late summer reflects a decrease in bioenergetic status at this period. The stock of mussels which was more susceptible to summer mortality had higher values of VO 2 . Furthermore this stock had a more negative scope for growth and lower O / N ratio in early August. These parameters are likely to reflect unfavourable environmental conditions, which led the mussels to rely upon protein catabolism. We also observed a negative correlation between multiple-locus heterozygosity and standard VO 2 . The more susceptible stock of mussels had a lower degree of multiple-locus heterozygosity. Thus, we suggest that the periodic, but irregular, outbreaks of summer mortality are the result of a synergistic interaction involving dietary deficiencies, temperature, a possible post-spawning stress and the genetic characteristics of the stock. The higher metabolic demand associated with a reduced degree of heterozygosity will impose a supplementary stress and render such stocks more vulnerable to summer mortality. The results are in agreement with the hypothesis that high levels of heterozygosity are related with lower costs of maintenance.  1998 Elsevier Science B.V. Keywords: Cytochrome C oxidase; Heterozygosity; Mytilus edulis; Scope for growth; Summer mortality; Oxygen consumption *Corresponding author. 0022-0981 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0022-0981( 97 )00114-7

28

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

1. Introduction Summer mass mortality is a significant problem in Canadian and American populations of Mytilus edulis (Incze et al., 1980; Mallet et al., 1990; Myrand and Gaudreault, 1995). Since the culture industry started in the Magdalen Islands (Gulf of St. Lawrence), mortalities eliminating up to 80% of the cultured mussels have occurred frequently, albeit sporadically (Myrand and Gaudreault, 1995). Following the experiments of Dickie et al. (1984), several workers have studied the variations of mortality and growth rates when different populations of mussels were transferred to various growing sites (Mallet et al., 1990; Tedengren et al., 1990; Fuentes et al., 1992; Okumus and Stirling, 1994; Myrand and Gaudreault, 1995). These studies have reached the common conclusion that stock origin explains most of the variance in mortality, while environmental conditions are more important in explaining the variance in growth. In the Magdalen Islands, mussels from the major cultivation site, Great Entry lagoon, have a higher mortality rate and also a lower growth rate than those from Amherst Basin (Fig. 1; Myrand and Gaudreault, 1995). The mortality differences are maintained when the stocks are transferred between the sites suggesting that the variation in mortality among stocks is due to genetic differences rather to environmental factors. Indeed, the stock factor alone explains 92% of the variance of the cumulative survival rate. However as Myrand and Gaudreault (1995) transferred the stocks among sites 10 months after settlement, it remains possible that conditioning during their initial growth influences the subsequent susceptibility to mortality. Allozyme analysis shows significant differences in genetic constitution among mussels from different sites in the North-Eastern Atlantic (Gartner-Kepkay et al., 1980, 1983). Furthermore, two mussel species, Mytilus edulis and Mytilus trossulus, are endemic in the Canadian Atlantic coast and coexist in numerous localities (Varvio et al., 1988; Koehn, 1991; McDonald et al., 1991). Hilbish et al. (1994) have recently demonstrated significant differences between M. edulis and M. galloprovincialis in physiological and growth traits associated with the allele frequency at the esterase (EST*) and octopine dehydrogenase (ODH*) loci. It is plausible that population differences in susceptibility to summer mortality in the Canadian Atlantic coast reflect differences in species composition. Furthermore, the two species coexist regularly in the areas in which summer mortalities occur. Mytilus edulis and M. trossulus are difficult to separate by morphological criteria but can be distinguished by one diagnostic electrophoretic locus, the mannose phosphate isomerase (MPI*) (Varvio et al., 1988; McDonald et al., 1991; Gosling, 1992; Carver and Mallet, 1995). Differential mortality between mussel stocks may be also related to bioenergetic differences reflecting the degree of heterozygosity. For several species of bivalve molluscs, varying degrees of genetic heterozygosity are correlated with differences in growth rates, fecundity and bioenergetic properties (Zouros and Foltz, 1987; Volckaert and Zouros, 1989; Koehn, 1990; Gosling, 1992; Mitton, 1993). More heterozygous mussels show higher growth rates due to lower maintenance metabolic rates (Diehl et al., 1986) and greater efficiencies of protein synthesis (Hawkins et al., 1986). These individuals should more easily survive stressful periods than less heterozygous mussels. In fact, numerous studies have shown that heterozygotes appear to be generally

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

29

Fig. 1. Map of the study area: the Magdalen Islands in the Gulf of St. Lawrence showing Great Entry lagoon (origin of the mussels which are susceptible to summer mortality) and Amherst Basin (origin of the mussels which are resistant to summer mortality).

advantaged under stressful conditions (Rodhouse and Gaffney, 1984; Gentili and Beaumont, 1988; Scott and Koehn, 1990; Borsa et al., 1992). Such genetically based metabolic differences could explain population differences in resistance to summer mortality. Our study examined whether differences in susceptibility to summer mortality of the

30

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

mussel stocks at the Magdalen Islands are related to bioenergetic and / or genetic differences between the mussels from these populations. We compared the properties of mussels from the stock which is susceptible to summer mortality (from Great Entry lagoon) to those of mussels from the stock which is resistant to summer mortality (from Amherst Basin). The mussel spat were collected at the two sites, socks containing the two stocks were produced a few months after settlement and the mussels were reared in one environment, Great Entry lagoon. We examined whether these differences in susceptibility to summer mortality are related to differing degrees of heterozygosity, of infiltration by Mytilus trossulus or to bioenergetic differences between the populations. In examining the metabolic rates we supposed that mussels from the resistant stock allocate a smaller proportion of their aerobic capacity to maintenance requirements than mussels from the susceptible stock. Individuals with lower maintenance metabolism would be able to allocate more energy to production and reproduction (Hawkins and Bayne, 1992). We chose to examine the metabolic requirements at high temperatures, since in a more stressful environment, the effect of differences in maintenance efficiency upon tolerance of stress should be accentuated (Koehn and Bayne, 1989). The correlation between genetic variability and physiological performance should also be more apparent under conditions of potential stress than under more optimal conditions for growth and survival. As a complementary indicator of maximal organismal aerobic capacity, we compared the total activity of cytochrome C oxidase (CCO), an integral component of the inner mitochondrial membrane and a controlling step in mitochondrial respiration (Groen et al., 1982) between the two stocks. We also examined the scope for growth of these mussels to establish whether the mussels from the susceptible and resistant stocks differ in their capacity to exploit the food available in their habitat. Determination of scope for growth integrates estimates of basic physiological processes (feeding, food absorption, respiration and excretion) into an index of the energy available for growth and reproduction (Warren and Davis, 1967). We also examined the O:N ratio, the rate of oxygen consumed to nitrogen excreted. This ratio reflects the relative importance of protein in energy metabolism. A O:N ratio under 25–30 indicates that a high proportion of protein is being catabolized (Widdows, 1978). To summarize, we have examined whether the differential mortality of two stocks of mussels is associated with the standard and maximum metabolism, with cytochrome C oxidase activity, with scope for growth, with the O:N ratio and with multiple locus heterozygosity.

2. Materials and methods

2.1. Sampling and field observations From 1990 to 1992, mussel spat were obtained from collectors placed in June in Amherst Basin, where mussels are resistant to summer mortality, and in Great Entry lagoon, where mussels are susceptible to summer mortality (Fig. 1). Spat at each site were retrieved in October and passed through a commercial declumper–grader. Spat were placed in numbered stocks and suspended on several long lines at the commercial

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

31

culture site in Great Entry lagoon. The long lines were kept at least 2-m below the surface to avoid ice damage during the winter. Unfortunately, the lines containing two-year-old mussels from Amherst Basin were damaged in winter 1992–1993 leading to the loss of these mussels. For VO 2 studies, one-year-old mussels were sampled in late July 1991 and in the middle of each month between May and September 1992. In 1993, one and two-year-old mussels were sampled in mid-July, in early August and in mid-August. In 1994, two-year-old mussels were sampled at these dates. For each stock, a section of sock was sampled by SCUBA to avoid the emersion of the other mussels in the sock. After harvesting, mussels were transported in insulated containers to the laboratory at Cap-aux-Meules (transport time less than 1 h) for VO 2 measurements. In ´ 1991 and 1994 mussels were placed on ice in insulated containers and flown to Quebec (transport time less than 6 h) for VO 2 measurements. At each sampling time, the length of the mussels was chosen to minimize intra and inter-stock variation (Table 1). Mortality was measured in numbered plastic cages each containing 50 mussels following the experimental design of Myrand and Gaudreault (1995). In November, spat were placed in pearl-nets and, then the following June, the mussels were transferred into cages. The cages were changed on a monthly basis between June to November. As the use of cages permits the retention of shells from dead mussels, they provide a precise measure of survival rate. In 1991, mortality was also determined in the socks. One-year-old mussels were harvested six times between June and November 1991. Each time, four or five socks were sampled. The number of living and dead mussels larger than 5 mm were divided: ,30 mm and .30 mm. Approximately 5000 mussels were examined for each stock at

Table 1 Length (mm) of mussels (n524) used in VO 2 measurements between 1991 to 1994 for one-year-old (1 1 ) and two-year-old (2 1 ) mussels. The susceptible stock comes from Great Entry Lagoon and the resistant stock from Amherst Basin. Mean6SD Length (mm) according to age and origin Stock origin

Great Entry

Age

1

1

Amherst Basin 2

1

11

1991

August

39.866.1

40.966.1

1992

May June July August September

29.962.0 34.962.1 34.862.2 42.763.3 45.463.4

35.762.3 40.362.6 44.063.3 47.863.1 50.163.2

1993

July Early August Mid-August

30.163.2 34.662.1 39.263.0

1994

a

Lost in a storm.

July Early August Mid-August

49.863.5 55.963.4 58.766.1 44.762.2 54.963.4 56.464.2

36.663.3 39.063.0 42.462.2

21

a a a

50.864.0 55.063.2 59.864.1

32

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

each harvest. Many dead mussels are lost from socks, making this measure of mortality less exact than those obtained in cages.

2.2. Animal and holding conditions Upon arrival at the laboratory, 24 mussels from Amherst Basin and 24 mussels from ´ Great Entry were placed in recirculating artificial seawater in 1991 and 1994 at Quebec and in filtered seawater in 1992 and 1990 (0.45 mm) at Cap-Aux-Meules. The salinity was 30 ppm (as in the lagoon), the natural photoperiod was followed and the temperature was maintained at 208C. Mussels were fed with a mixed culture of Isochrysis gulbana, Monochrysis lutherie and Thalassiosira pseudonana. Algal concentrations were measured with a haemocytometer in 1991 and with a Coulter Counter (TAII with a probe of 100 mm) from 1992 to 1994. Each algal species was grown in batch culture. The algae were mixed before their addition to the main aquarium.

2.3. Metabolic rate determinations: general aspects Our strategy was to approximate maximal VO 2 values by feeding the mussels at a maximal rates and standard metabolic rates by starving the mussels, at constant temperature of 208C for each month and year of sampling. For maximal VO 2 values, mussels were fed daily with 10 000 cells per ml per individual or 0.083 mg dry mass per ml per individual. Pseudofaeces production was observed at maximal feeding levels, indicating that the mussels’ filtration system was saturated and that maximal feeding rates were obtained (Thompson and Bayne, 1974; Foster-Smith, 1975). Nevertheless, this algal concentration did not suppress filtration rate and assimilation efficiency. Widdows et al. (1979) determined that the algal concentrations that suppress filtration rate are 0.22 mg?ml 21 and 0.33 mg?ml 21 for mussels of 3 and 7 cm in length, respectively. Assimilation efficiency is suppressed at 25 000 cells per ml (Thompson and Bayne, 1974). After oxygen uptake, each set of mussels was frozen at 2808C for later determination of genotypes, activity of cytochrome C oxidase, dry mass and sex. Dry mass was determined by drying at 708C for 72 h. Oxygen uptake for one-year-old mussels was expressed as the rate (ml?g dry mass 21 ?h 21 ) expected for a standard mussel with 1 g dry flesh mass by applying the allometric correction (VO 2 5(1 g per dry flesh mass) 0?65 VO 2 measured) (Tremblay et al., in press). For two year-old mussels we expressed metabolic rate for a standard mussel of 2 g dry flesh mass. CCO activity was expressed for a standard one year-old mussel of 1 g and a standard two year-old mussel of 2 g wet flesh mass by an allometric correction. The allometric equations obtained for VO 2 and cytochrome C oxidase activities for mussels from Great Entry lagoon and mussels from Amherst Basin did not differ between stocks. Oxygen solubility tables were used to convert pO 2 values to oxygen concentration in ml?l 21 .

2.4. Metabolic rate determinations: specific aspects 2.4.1. Determinations in 1991 Approximately 20 mussels were placed in individual 4 l aquaria filled with artificial seawater aquaria and fed for 2 d to establish maximal VO 2 . Maintenance VO 2 values

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

33

(standard VO 2 ) were approximated by starving the mussels for 4 d (Bayne, 1973). In all conditions, the water in which the mussels were held was changed daily. In 1991, oxygen uptake was measured for individual mussels in 500-ml flasks. Maximum VO 2 and standard VO 2 values were evaluated by taking mussels which had been feeding or starved at the above conditions and placing them in individual flasks filled with artificial seawater without algae. After an equilibration period of 2 h, a sample of water (20 ml) was taken, the flasks were closed and subsequent samples were taken at hourly intervals. Samples were taken without introducing air and oxygen saturation levels did not fall below 100 torr. Oxygen concentrations in the samples were determined using a micromodification of the Winkler technique (Aminot and Chaussepied, 1983).

2.4.2. Determinations in 1992 – 1994 For each stock, 24 mussels and a control (empty shells) were placed in individual numbered cages constructed of Vexar (plastic netting with large mesh size) and suspended in the main aquarium. This avoided breaking the byssus during the transfer of mussels into the respirometry chambers. The seawater was oxygenated. Half the seawater was changed daily without exposing the mussels to air. Before the measures of oxygen uptake, mussels were starved for 2 d. Then, maximal VO 2 values were approximated by feeding mussels at maximal levels for 4 d. Maintenance metabolism (standard VO 2 ) was approximated by starving the mussels for 8 d in filtered seawater (0.45 mm; Bayne, 1973). Finally the mussels were fed another 4 d to verify whether maximal VO 2 values were restored. During these periods of food supply and starvation, oxygen uptake was measured every 2 d. Oxygen uptake was measured for each mussel and control in a closed respirometer (580 ml) for a minimum of 1 h and at O 2 partial pressures .100 torr. Water was circulated in the closed respirometer with a magnetic stirrer. The oxygen saturation level was measured by a polarographic electrode (YSI, model 5331) coupled to an oxymeter (Cameron Instruments Company, model OM400), and a chart recorder (Linear, model 0585). Before the beginning of measurements, mussels were kept in the respirometer, with continued circulation of fresh aerated water, for 2 h to habituate to their new environment (Bayne et al., 1976). When mussels spawned during the VO 2 determinations, the data were eliminated. 2.5. Scope for growth and O: N ratio Scope for growth is measured by subtracting the energy respired and excreted from the energy absorbed from the food as follows (Widdows and Johnson, 1988; Gilek et al., 1992): P 5 A 2 (R 1 U ) P5energy incorporated into somatic growth and gamete production; A5energy absorbed from the food5C 3 Ab; C5clearance rate (l?g 21 ?h 21 )3POM (mg l 21 )323 J?mg 21 ash-free dry mass; POM5particulate organic matter; Ab5absorption efficiency;

34

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

R5energy catabolized5VO 2 (ml O 2 ?g 21 ?h 21 )320.33 J?ml 21 O 2 ; U 5energy excreted5mg NH 4 ?g 21 ?h 21 319.4 J?mg NH 421 . O:N ratio was calculated in atomic equivalents according to the formula (Widdows and Johnson, 1988): O:N 5 (mg O 2 ? h 21 / 16) /(mg NH 4 ? h 21 / 14) The physiological rates needed for the calculation of these indices were measured weekly – near the culture site in Great Entry lagoon during July and August 1993. All physiological rates were determined individually on 12 susceptible mussels and 12 resistant mussels. As the mussels can filter and remove all suspended particles between 4 and 90 mm, with a retention efficiency that is close to 100% (Lucas et al., 1987; ˚ Riisgard, 1988), we prefiltered water from the lagoon at 105 mm. The rates of oxygen uptake, clearance rate, absorption efficiency and ammonia excretion were measured for each mussel. We separated the flesh mass into two halves each containing half of the organs present. One half was dried for 72 h at 658C to assess mussel dry mass. The other halves were placed in individual containers and immediately frozen at –808C for later measurements of CCO activity. As our mass exponent for the allometric equation for oxygen uptake was similar to the value of Widdows and Johnson (1988), we used the values of these authors in the allometric equations for clearance rate (0.4) and ammonia excretion (0.65) to estimate mass-specific rates for mussels of 1 g dry mass. Oxygen uptake was measured in a closed experimental chamber (500 ml) filled with air-saturated filtered seawater (0.45 mm) held in a temperature controlled water bath. Oxygen uptake was measured with the same set-up as in VO 2 measurements in 1992–1994. Ammonia excretion was determined by placing mussels in 200 ml of air-saturated filtered seawater held in a temperature-controlled water bath. Following the 3-h incubation period, water samples were taken from each experimental chamber, and from a control chamber with two empty valves. NH 4 analysis was performed in duplicate by using the phenol hypochlorite method of Solorzano (1969) and expressed in terms of mg excreted per h?g 21 . Clearance rate is defined as the volume of water cleared of suspended particles per unit time and biomass (Widdows and Johnson, 1988). Mussels were placed in an experimental chamber (500 ml) with a magnetic stirrer. The flow-rate in the chamber was maintained constant at 150 ml?min 21 . The inflow into the chamber was adjacent to the mussel’s inhalant mantle edge, and the outflow occurred via an overflow at the top, thereby helping to minimize recirculation of water by the mussel. Before the beginning of measurements of particle concentrations, mussels were left undisturbed for at least 60 min to allow their valves to open, and feeding to resume. Food particles were counted from the outflow of all containers including the control (an empty experimental chamber) every 15 min during 45 min (Gilek et al., 1992), with a Zm Coulter Counter using a 200-mm orifice tube. Three replicates were made for each determination. Only particles

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

35

over mm were counted. Clearance rate was calculated as follows (Widdows and Johnson, 1988): C 5 FI 3 (C1 2 C0 ) /C1 C5clearance rate; FI5flow rate; C1 5inflow concentration (water sampled from the control chamber); C0 5outflow concentration (water sampled from the outflow of each experimental chamber). Absorption efficiency was measured by the ratio of Conover (1966) which indicates the efficiency with which organic material is absorbed from the ingested food. This ratio is particularly convenient for use with bivalves feeding on natural seston (Navarro and Thompson, 1994). Ab 5 (F 2 E) /((1 2 E) ? F ) Ab5absorption efficiency; F 5ash-free dry mass / dry mass ratio of the food; E5ashfree dry mass / dry mass ratio of the faeces. Seston concentration (mg?l 21 ) in the inflowing water was sampled in triplicate every 3 h. Faeces were collected by pipette from the chambers used for measurements of clearance-rates. Pseudofaeces were eliminated. Mussels were left between 6 to 12 h to allow the harvest of a maximum amount of faeces. Faeces were treated as seston (see below) to determine dry, mass and ash-free dry mass.

2.6. Seston characteristics Two litres of inflowing seawater were filtered in triplicate through washed, ashed and pre-weighed GFC filters. Salt was washed out with 10 ml of isotonic ammonium formate (3%). Filters were dried at 708C for 24 h and weighed to obtain the total particulate matter (TPM). Then, they were ashed for 4 h at 4508C in a muffle furnace and reweighed to obtain the particulate inorganic matter (PIM). Particulate organic matter (POM) was calculated by subtracting the PIM from the TPM. Chlorophyll-a was also determined in inflowing seawater as an index of phytoplankton biomass. A 500-ml volume of seawater was filtered in triplicate under vacuum through a Whatman GFC filter and filters were treated by the method described by Strickland and Parsons (1972). To compute the total volume of particulate matter (ml?l 21 ), the number and size distribution of particles in seawater were determined in ten samples using a Coulter Counter. Bayne et al. (1987) and Navarro et al. (1991) concluded that both food quantity and quality are fundamental parameters influencing the scope for growth. Thus, we used two different parameters to define the nutritional value of seston: percent organic matter (POM: TPM ratio) and ‘quality’, defined by Bayne et al. (1987) as the organic content per unit volume of particles (mg organic matter?ml 21 ).

2.7. Cytochrome C oxidase Mussels used in measurements of VO 2 in 1992, 1993 and 1994 were also used to quantify the activity of cytochrome C oxidase by the method of Goolish and Adelman

36

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

(1987). The activity of cytochrome C oxidase was determined in a half individual, homogenized in 5 volumes of 10 mM phosphate buffer (pH 7.6), and expressed as mmol of cytochrome C oxidized per min (U) per g of wet tissue. A mM extinction coefficient ´ of 19.1 was used. Cytochrome C (Boehringer Mannheim, Montreal, Canada) was reduced with ascorbic acid (10 mM) that was subsequently removed by dialysis. Reduced cytochrome C was stored frozen under a nitrogen atmosphere until needed. The reaction followed the decline in absorbance at 550 nm using 1 mM reduced cytohrome C. Blanks were composed of 1 mM cytochrome C oxidized with 0.03% (W/ V) potassium ferricyanide.

2.8. Genetic analysis Frozen mussels (2808C) used in the VO 2 measurements in 1992 and 1993 were used for electrophoretic study of enzymes. A small piece of digestive gland was homogenized in an approximately equal volume of homogenization buffer (0.2 M Tris-HCl, pH 8.0, with 30% sucrose, 1% polyvinyl–polypyrrolidone, 0.1% NAD, 5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride). Samples were centrifuged at 15 000 g for 30 min at 48C and the supernatant applied to a horizontal cellulose acetate gel (Hebert and Beaton, 1989). The enzymes studied were glucose phosphate isomerase (GPI*, EC 5.3.1.9), octopine dehydrogenase (ODH*, EC 1.5.1.11) and mannosephosphate isomerase (MPI*, EC 5.3.1.8). Esterases (EST-1 *, EST-2 *, EC 3.1.1.1) were scored after migration on vertical discontinuous polyacrylamide slab gels (Ornstein, 1964). Enzymes were stained according to Harris and Hopkinson (1976) and Hebert and Beaton (1989). On each gel a standard comprised of a mix of individuals of different genotypes was applied. Thus, each gel contained a standard with all possible genotypes to facilitate scoring. Genetic data were used to estimate allele frequencies and the heterozygote deficiency index (Pasteur et al., 1989): D 5 (Ho 2 He ) /He D5heterozygote deficiency; Ho 5observed heterozygosity; He 5expected heterozygosity under the assumption of Hardy–Weinberg equilibrium. The MPI* data were used to evaluate the infiltration of M. trossulus into M. edulis populations. M. edulis and M. trossulus both have three alleles at the MPI* locus with the intermediary allele predominating. All MPI* alleles of M. trossulus migrate more slowly than those of M. edulis. Thus, there is no correspondence in migration distance between alleles of M. edulis and alleles of M. trossulus. The MPI* allozyme can be therefore considered diagnostic between M. edulis and M. trossulus as noted by Gosling (1992); Carver and Mallet (1995); McDonald et al. (1991); Varvio et al. (1988). Standards with the three alleles for both species were applied on each gel of MPI* to assure the exact identification of each individual. Individuals identified as M. trossulus were discarded from further data analysis.

2.9. Sex determination The sex of the mussels was determined by the method of Jabbar and Davies (1987). Briefly, a small piece of the mantle was placed in a test tube with 2 ml of 20% (w / v)

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

37

trichloroacetic acid and some antibumping granules, followed by addition of 0.5 ml freshly prepared 0.75% (w / v) thiobarbituric acid reagent. The mixture was placed in a boiling water bath for 20 min. Males develop a yellow colour, whereas females develop a pink colour.

2.10. Statistical analysis Physiological data were log-transformed to normalize the values. Measurements of VO 2 , scope for growth and cytochrome C oxidase activity were analyzed by ANOVA’s followed by Tukey a posteriori tests to identify specific differences among sampling periods. Factorial ANOVA’s were used to determine the effect of independent variables (sex, age and stock). The X 2 -test was used to examine whether the genotype frequencies observed were in accordance with those expected under the Hardy-Weinberg equilibrium (goodness of fit) as well as to evaluate differences in allelic frequencies between mussels from Amherst Basin and Great Entry lagoon (heterogeneity). For these analyses, uncommon alleles were pooled into a single class as recommended when expected frequencies of some classes are low (Sokal and Rohlf, 1981). Regression analyses were used out to evaluate the relationship between the degree of heterozygosity and VO 2 or cytochrome C oxidase activity. All these tests were carried out with Systat (Systat Inc.) except the analysis of genetic data which was done partly with Biosys-1 (Swofford and Selander, 1989).

3. Results

3.1. Mortality curves In 1991, mussels from the susceptible stock (from Great Entry lagoon) had a significantly higher percent mortality than mussels from the resistant stock (from Amherst Basin) both in cages (78 vs 10%; G-test p,0.001; Fig. 2) and in socks (G-test p,0.01; Fig. 3). Thus cages do not seem to modify relative mortality rates. During 1991, for both stocks in socks, mortality was negligible until early August and reached a peak in early September, after which it declined. Small and large mussels showed similar changes of mortality, indicating that it was not size-selective. The difference in estimates of mortality rate between cages and socks reflects the fact that dead mussels may have fallen off the socks during culture. From 1992 to 1994, no summer mortalities were noted for either stock of mussels (Fig. 2).

3.2. Metabolic rates Throughout the study, one-year-old (Fig. 4) and two-year-old mussels from the resistant stock (Fig. 5) tended to have lower mass specific rates of oxygen uptake than their counterparts from the susceptible stock, both after food deprivation (standard VO 2 , DF 5372, p,0.001) and after feeding to satiety (maximal VO 2 ; F 555, DF 5374, p,0.001). For mussels from the resistant stock, the standard and active VO 2 values did

38

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 2. Mortality rates from June to November between 1991 to 1994 (n550) of mussels from Amherst Basin and Great Entry lagoon, transplanted in cages to the commercial culture site of Great Entry lagoon.

not differ among months, except for one-year-old mussels sampled in May 1992. In this period the maximal VO 2 was higher than in other months (Tukey: p,0.01). One and two-year-old mussels from the susceptible stock generally showed an increase in the standard VO 2 , in mid-August (Tukey: p,0.05). The proportion of the maximal VO 2 required for maintenance requirements was estimated from the ratio of standard metabolic rate to maximal VO 2 , for each mussel measured between 1992 and 1994 (Table 2). As in 1991 the standard and the maximal VO 2 were determined for different individuals and this ratio was calculated from the

Fig. 3. Mortality rate in stocks of mussels from Amherst Basin and Great Entry lagoon for small (5–30 mm) and large (.30 mm) individuals between May to December 1991, transplanted to the commercial culture site of Great Entry lagoon.

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

39

Fig. 4. Variation of standard and maximum VO 2 at 208C for the one-year-old mussels from Amherst Basin (n524) and Great Entry (n524) sampled in differents months from 1991 to 1993. Significant differences among sampling dates (Tukey test) are indicated by *( p,0.05) and **( p,O.01). Mean6SE.

means of the metabolic rates. This ratio did not differ among months or between mussels from the susceptible and resistant stock, except in mid-August. At this period, one and two-year-old mussels from the susceptible stock showed a significantly higher ratio of standard to maximal VO 2 compared with the other sampling periods or compared with mussels from the resistant stock ( p,0.001). In mid-August, for mussels from the

40

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 5. Variation of standard and maximum VO 2 at 208C for the two year old mussels from Amherst Basin (n524) and Great Entry (n524) sampled in differents months from 1993 to 1994. Significant differences between sampling dates (Tukey test) are indicated by *( p,0.05) and **( p,0.01). Mean6SE.

susceptible stock the standard VO 2 represented between 46 and 52% of the maximal VO 2 , while it accounted for only 28 to 36% of the maximal VO 2 at the other sampling times as well as for mussels from the resistant stock. The increased ratios in mid-August were due to an increase of standard VO 2 of the mussels from the susceptible stock (Figs. 4 and 5). The increase of maximal VO 2 in May 1992 for mussels from the resistant stock (Fig. 4) significantly decreased the ratio of standard: maximal VO 2 (Tukey: p,0.05). In May 1992, the standard VO 2 represented only 17% of the maximal VO 2 , compared to 27–31% during the other months (June to September).

3.3. Seston characteristics There was a net increase of chlorophyll-a in mid-July 1993 (Tukey: p,0.01)

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

41

Table 2 The proportion of the maximal VO 2 required for maintenance metabolism at 208C estimated from the ratio of standard to maximal VO 2 rates (%) between 1991 to 1994 for one-year-old (1 1 ) and two-year-old (2 1 ) mussels Stock origin

Great Entry

Age

1

1

Amherst Basin 2

1

11

1991

August

64

59

1992

May June July August September

3162 3162 3462 4663 b 3663

1762 2762 2861 3162 2862

1993

July Early August Mid-August

3363 3662 5265 b

1994

July Early August Mid-August

3262 3662 4864 3662 4262 4862 b

3162 3062 3562

21

a a a

3561 3662 3662

The susceptible stock comes from Great Entry (n524 at each sampling) lagoon and the resistant stock from Amherst Basin (n524 at each sampling). Mean6SE. a Lost in a storm. b p,0.05, Tukey–Significantly different from other sampling periods and from other stock.

followed by more stable values in August (Fig. 6a). TPM generally followed the same pattern as POM with a significant increase at the end of August (Tukey: p,0.05; Fig. 6b). The unit volume of particles decreased markedly in mid-July (Tukey: p,0.01) and increased in mid-August (Tukey: p,0.05; Fig. 6c). The nutritional value of seston was evaluated by its percent organic matter and quality. Both varied significantly with time (% organic matter: DF 514 and 6, F 54, p,0.01; quality: DF 514 and 6, F 560, p,0.001; Fig. 7). On August 9 these two indices of nutritional value of seston decreased (Tukey: p,0.05). The quality of seston showed peak values on July 19 and August 23 (Tukey: p,0.05).

3.4. Scope for growth ( SFG) Mussels from the susceptible stock had higher routine VO 2 (rate between standard and maximum VO 2 ) values than those from the resistant stock (F 513.8, DF 51 and 74, p,0.001; Fig. 8). In mid-August the routine VO 2 values of the mussels from the susceptible stock declined to reach those of the mussels from the resistant stock (t-test: p.0.05). The routine VO 2 values of the mussels from the resistant stock changed little during the summer. Mussels from the two stocks exhibited the same rate of ammonia excretion until August 2, when mussels from the susceptible stock showed higher rates for two weeks (Fig. 8; t-test: p,0.001). A small increase of ammonia excretion in mussels from the resistant stock was detected during one week in mid-August (Tukey: p,0.05), but it was much lower than that shown by the mussels from the susceptible

42

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 6. Weekly variation of seston characteristics at the culture site of Great Entry lagoon between July to August 1993: (A) Chlorophyll-a, (B) total particulate matter (TPM) and particulate organic matter (POM), (C) unit volume of particulate matter. Significant differences between sampling dates (Tukey test) are indicated by *( p,0.05) and **( p,0.01). Mean6SE.

stock (t-test: p,0.001). Clearance rates and absorption efficiency of mussels from Amherst Basin and Great Entry did not differ significantly (Fig. 8). No significant variation of clearance rates was observed throughout the summer for either stock. On August 9, the absorption efficiency reached a minimum of 20% (Tukey: p,0.001) immediately followed by a rapid increase reaching values around 50% for the two stocks (Tukey: p,0.05). Absorption efficiency of both stocks was correlated with the quality of seston (Amherst Basin: r 2 50.56, p,0.01; Great Entry: r 2 50.64, p,0.01). Changes in the O:N ratio, the SFG and CCO activity for the two stocks of mussels are shown in Fig. 9. The two stocks had highest O:N ratios in July, indicating the use of lipid and carbohydrate as substrates. On August 9, O:N ratios in both stocks decreased to

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

43

Fig. 7. Weekly variation of the nutritional value of seston at the culture site in Great Entry lagoon between July to August 1993: (A) Percent organic mass and (B) ‘quality’ defined as organic mass per unit volume of particulate matter. Significant differences between sampling dates (Tukey test) are indicated by *( p,0.05) and **( p,0.01). Mean6SE.

values under 26 (Tukey: p,0.05), indicating an increased use of proteins. At this time mussels from the susceptible stock had significantly lower values (1463) compared to mussels from the resistant stock (2663; t-test: p,0.01). On August 16, the O:N ratios of mussels from the resistant stock suggested their recovery from a stressful period with value over 30 (Tukey: p,0.05), whereas mussels from the susceptible stock continued to have low values (1065; t-test: p,0.05) suggesting continued depletion of protein. In July, SFG of mussels from both stocks was very low with total production near zero. SFG fell below zero (Tukey: p,0.05) on August 2 for both stocks with lower values for mussels from the susceptible stock (t-test: p,0.05). The lowest values were 220.665.1 J?g -1 ?h 21 for mussels from the susceptible stock compared to 22.763.4 J?g 21 ?h 21 for mussels from the resistant stock. The negative values confirm that mussels from the two stocks were stressed and utilising their body reserves. After this difficult period in early August, both stocks showed marked increases of SFG by the end of August. On August 23, mussels from the resistant stock showed significantly higher values than mussels from the susceptible stock (t-test: p,0.001).

3.5. Cytochrome C oxidase activities One and two-year-old mussels from Great Entry lagoon always had higher total

44

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 8. Weekly variation of the components of scope for growth for mussels from the resistant stock (Amherst Basin, n512) and from the susceptible stock (Great Entry, n512) from July to August 1993. Significant differences among sampling dates (Tukey test) are indicated by *( p,0.05) and **( p,0.01). Mean6SE.

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

45

Fig. 9. Weekly variation of O:N ratio, scope for growth and cytochrome C oxidase levels for mussels from the resistant stock (Amherst Basin, n512) and from the susceptible stock (Great Entry, n512) between July to August 1993. Significant differences between sampling dates (Tukey test) are indicated by *( p,0.05), **( p,0.01) and ***( p,0.001). Mean6SE.

46

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 10. Cytochrome C oxidase activity of the individuals used in VO 2 measurements from 1992 to 1994 (n524 at each sampling). Mean6SE.

activities of cytochrome C oxidase than mussels from Amherst Basin (Figs. 9 and 10; ANOVA with three factors: p,0.01). There were no significant relations between CCO activity and physiological parameters such as VO 2 . Also, no differences were observed between males and females.

3.6. Genetic analysis With the use of an electrophoretic tool, the MPI* locus, only four Mytilus trossulus

Table 3 Allele percentages at 5 enzyme loci for one- (1 1 ) and two-year-old (2 1 ) animals from the resistant (from Amherst Basin) and susceptible stocks (from Great Entry lagoon) Resistant mussels

Susceptible mussels

EST-1*

EST-2*

GPI*

A B C D E F n Ho HE D

20 20 46 14 0 0 27 0.667 0.686 20.046

29 48 23 0 0 0 28 0.611 0.632 20.112

4 15 29 33 11 8 36 0.611 0.762 20.209 a

19 77 4 0 0 0 34 0.412 0.377 0.077

12 85 3 0 0 0 34 0.235 0.258 20.101

21 A B C D E F n Ho HE D

29 8 46 17 0 0 24 0.583 0.588 20.028

30 50 20 0 0 0 28 0.571 0.619 20.094

7 13 20 43 7 10 28 0.679 0.744 20.105 a

15 78 7 0 0 0 27 0.333 0.368 20.011

4 84 10 2 0 0 28 0.286 0.283 20.007

1

MPI*

ODH

All loci

EST-1*

EST-2*

GPI*

MPI*

ODH*

All loci

0.499 0.552 20.074

19 17 58 6 0 0 55 0.545 0.592 20.087

25 57 18 0 0 0 53 0.509 0.577 20.125

3 10 27 42 11 7 68 0.513 0.739 20.311 a

21 74 5 0 0 0 68 0.346 0.409 20.159 a

10 83 7 0 0 0 68 0.221 0.295 20.257 a

0.221 0.425 20.188 a

0.491 0.531 20.069

32 9 40 19 0 0 29 0.399 0.692 20.441 a

37 55 8 0 0 0 30 0.401 0.556 20.299 a

0 18 20 47 10 5 31 0.387 0.695 20.452 a

11 84 5 0 0 0 31 0.258 0.281 20.098

5 85 8 2 0 0 31 0.226 0.261 20.146 a

0.331 0.505 20.291 a

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

1

Ho The observed proportion of heterozygotes per locus, HE 5Hardy–Weinberg proportions of heterozygotes. D5coefficient for heterozygote deficiency or excess and n5sample size. a p,0.05. 47

48

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

were identified from the mussels used in physiological measures between 1992 to 1994, and they were discarded from the analysis. Chi-square tests showed no differences in allele frequencies at any locus between mussels from the susceptible and resistant stocks and between one and two-year-old mussels (Table 3). However mussels from the resistant stock were significantly more heterozygous (mean degree of heterozygosity of 3.4) than mussels from the susceptible stock (mean degree of heterozygosity of 1.8) (Wilcoxon: p,0.001). There were no differences between the mean degrees of heterozygosity of one and two-year-old mussels from each stock (susceptible: 1 1 51.82 and 2 1 51.79; resistant: 1 1 53.40 and 2 1 53.48). The coefficients for heterozygote deficiency or excess demonstrated that resistant mussels do not significantly deviate from the Hardy–Weinberg equilibrium, except for GPI* in one-year-old mussels which showed a heterozygote deficiency (Table 3). In mussels from the susceptible stock, many loci showed deficiencies. Three loci are significantly deficient in heterozygotes (GPI*, MPI* and ODH* ) in one-year-old, and four out of five loci were deficient (EST.1*, EST-2*, GPI* and ODH* ) in two-year-old mussels. The GPI* locus showed a greater deviation from the Hardy–Weinberg equilibrium than the other loci for both ages. The loci for esterases showed heterozygosity deficiencies only in two-year-old mussels and that for MPI* only in one-yearold mussels. Finally, we observed an increase of heterozygote deficiency with age in mussels from the susceptible stock but not in those from the resistant stock. For both stocks, there were significant relationships between multiple-locus heterozygosty and VO 2 but not with CCO activity. Both standard VO 2 and the ratio of standard to maximal VO 2 decreased as heterozygosity increased, and this for one- and two-year-old mussels from both stocks (Figs. 11 and 12). Overall, mussels from the susceptible stock showed higher energy requirements for maintenance and a lower degree of heterozygosity than mussels from the resistant stock.

4. Discussion The reciprocal transplant experiments of Myrand and Gaudreault (1995) showed that mussels from Great Entry and Amherst Basin maintained their differential survival rates in cages maintained at the different growing sites. Thus, mussels from Great Entry showed very low survival compared to mussels from Amherst Basin in either Great Entry lagoon or Amherst Basin. In our study, mussels in cages and in socks showed the same results in 1991 as found by Myrand and Gaudreault (1995), with markedly lower survival of mussels from the susceptible stock (from Great Entry lagoon) compared to those from the resistant stock (from Amherst Basin) even though they lived in the same environmental conditions (Great Entry lagoon) during their first year of growth. These results are in agreement with previous findings (Mallet et al., 1990; Tedengren et al., 1990; Fuentes et al., 1992; Myrand and Gaudreault, 1995) suggesting that mortality rates are probably largely based on genetic differences. Mussels from the resistant stock had different bioenergetic properties than those from

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

49

Fig. 11. The relationships between the degree of heterozygosity and VO 2 for one-year-old mussels sampled in 1992 from each stock. Standard metabolic rate (A) and the ratio of standard to maximum metabolic rate (B) of each mussel are plotted as a function of the number of loci for which the animal is heterozygous (degree of heterozygosity). The regression equation of each curve is noted with its significance. Individuals for which all 5 loci could not be studied were excluded from this analysis.

the susceptible stock. Both for the standard and maximum VO 2 values, mussels from the susceptible stock tended to show higher values. This suggests that the energy expenditure for maintenance, food ingestion and absorption was higher for the susceptible stock. Furthermore, we observed an increase of the proportion of the

50

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Fig. 12. The relationships between the degree of heterozygosity and VO 2 for two-year-old mussels sampled in 1993 from each stock. Standard metabolic rate (A) and the ratio of standard to maximum metabolic rate (B) of each mussel are plotted as a function of the number of loci for which the animal is heterozygous (degree of heterozygosity). The regression equation of each curve is noted with its significance. Individuals for which all 5 loci could not be studied were excluded from this analysis.

maximal VO 2 represented by the standard VO 2 in mussels from the susceptible stock in mid-August, indicating a greater maintenance demand. This high metabolic expenditure could have a negative impact upon the survival of mussels from the susceptible stock during this period. The metabolic requirements of mussels from the resistant stock

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

51

remain low in mid-August. The differences in mortality rates and bioenergetic properties seemed unrelated to the presence of Mytilus trossulus, since almost all the experimental mussels (over 98%) from the two stocks were identified as Mytilus edulis, with the use of MPI* locus. Presently, MPI* is the most diagnostic locus for separating M. trossulus and M. edulis in eastern Canadian coast populations (McDonald et al., 1991; Carver and Mallet, 1995). Heath et al. (1996) have described a PCR marker, but it only separates M. californianus from the three other Mytilus species (M. edulus, M. trossulus and M. galloprovincialis). Differences in maintenance metabolism can have a major impact on energy status (Reeds et al., 1985; Van Steenbergen, 1987; Waterlow, 1988). A simulation presented by Hawkins and Bayne (1992) demonstrates that a 20% reduction in energy requirements for maintenance in Mytilus edulis should double the cumulative production over 10 years (larger somatic size and higher reproduction output). However, in our data, increased maintenance demands (the proportion of the maximal VO 2 , required for maintenance requirements) in the susceptible stock were only observed in mid-August. Thus, resistant mussels should be bioenergetically favoured primarily during this period. Accordingly, differences in ‘monthly’ growth rate between mussels from the susceptible (Great Entry) and resistant stocks (Amherst Basin), grown together in Great Entry lagoon, were observed only in late August–late September (Myrand and Gaudreault, 1995). Many results indicate unfavourable environmental conditions in the Magdalen Islands in August, even when summer mortalities of mussels do not occur. In 1993, we observed a negative scope for growth in early August combined with low values of the O:N ratio (,25) for both stocks, indicating that the mussels were using protein to sustain their maintenance requirements. Proteins are generally used in mussels only as a last recourse. Nevertheless, intrinsic factors, particularly the seasonal gametogenic cycle, alter the O:N ratio (Bayne et al., 1985). The main feature of the seasonal variation is a marked decline of O:N during and immediately after spawning period, reflecting the poor condition of the animals at a time when the mantle is undergoing tissue autolysis, reorganisation and regeneration following spawning. The negative scope for growth indicated that environment did not supply all the energy needed to maintain a positive energy balance and the maintenance requirements had to be met with energy reserves (Bayne and Newell, 1983; Griffiths and Griffiths, 1987). While this pattern was the same for the two stocks, the negative energy balance and the extent of protein catabolism were more pronounced in mussels from the susceptible stock. Again the patterns of metabolic organization seem to favour the mussels from Amherst Basin. In addition, water temperature regularly exceeds 208C during this period (Myrand, 1991), a temperature generally considered stressful for Mytilus edulis (Bayne and Newell, 1983). Finally, we noted also a decrease in food quality in Great Entry lagoon in mid-August 1993, even though quantity remained high. We observed a negative correlation between multiple-locus heterozygosity and standard VO 2 as found by Diehl et al. (1986). Mussels from the susceptible stock were generally more homozygous than mussels from the resistant stock. The heterozygote deficiency of the susceptible stock was related to a more pronounced reaction to stress and to slower recuperation from the stressful period in early August. Subsequently, in periods of positive energy balance (late-August) mussels from the susceptible stock had

52

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

a lower scope for growth than mussels from the resistant stock. These results agree with the model suggested by Volckaert and Zouros (1989), stating that energy savings due to heterozygosity are used to maximize fitness. For reasons that are still unknown, heterozygosity reduces the levels of protein synthesis required for basal metabolism and, therefore, reduces the costs (in terms of ATP) of maintenance. The surplus energy is channelled into somatic growth (or facilitates the response to stress) and into increased gamete production (Zouros et al., 1980; Garton et al., 1984; Koehn and Gaffney, 1984; Rodhouse et al., 1986; Gosling, 1992; Mitton, 1993). Ours result do not agree with the literature on heterozygosity in one aspect. Many authors present data consistent with the model where heterozygote inferiority (underdominance) at the presettlement stage is compensated by heterozygote superiority (overdominance) in adults (see review of Gosling, 1992). According to this model, the heterozygote deficits should diminish with increasing age. Mussels from the resistant stock in our study (Amherst Basin) follow this model. However, in mussels from the susceptible stock, heterozygote deficits increased with age. Even though the two stocks were in the same environment (Great Entry lagoon), selection seemed to act differently for each stock. Contrary to mussels from Amherst Basin, a selective mortality of heterozygotes seemed to occur with age in mussels from the susceptible stock (Great Entry). This result may explain the higher susceptibility to mortality of two-year-old compared to one-year-old mussels from Great Entry lagoon (Myrand, 1991). Toro and Vergara (1995) have also observed an increased heterozygote deficiency with age in the oyster, Ostrea chilensis. Thus, the fragility of mussels from Great Entry lagoon to summer mortality can be related to an increased heterozygote deficiency and subsequently higher maintenance demands. Summer mortality of cultured mussels is a widespread phenomenon, particularly in North America (Incze et al., 1980; Worrall and Widdows, 1984; Mallet et al., 1990; Emmett et al., 1987). These mortalities may have many possible causes including pathologies, high temperatures (.208C), post-spawning weakness, excess population density, and limited food availability. Examination of mussel specimens during the summer mortality in 1991 provided no evidence that any pathogen caused this mortality (S. McGladdery, Dept. Fisheries and Oceans, Moncton, pers. comm.). Furthermore, Mayzaud and Souchu (1991) demonstrated that the mussel biomass cultivated in Great Entry lagoon does not exceed the support capacity of the lagoon. Our results indicated that the decrease in food quality is related to an increase in the unit volume of seston particles. These results agree with Mayzaud and Souchu (1991) and Myrand (1991), who observed changes in the size-spectrum of phytoplankton in Great Entry Lagoon such that large particles became relatively more abundant during the period of summer mortality. Roy et al. (1991) observed that phytoplankton in Great Entry Lagoon in summer 1987 was primarily constituted of small particles (2–20 mm), and a second population of 50 mm appeared in early August. We also observed a decrease of percent organic matter in early August. The presence of inorganic particles in suspension (at concentrations below the pseudofaeces threshold) may have diluted the organic matter present and reduced absorption efficiency (Widdows et al., 1979). The decrease in food quality expressed as energy per unit volume of particles seemed to affect the two stocks, as reflected by the marked decreases of absorption efficiency

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

53

(,15% for mussels from Great Entry and ,20% for mussels from Amherst Basin). A similar relationship was found by Bayne et al. (1987) in a laboratory study using a mix of cultured plankton and silt and by Navarro et al. (1991) in a field study using natural seston. A decreased food availability at temperatures over .208C could precipitate the periods of summer mortality (Incze et al., 1980). Chlorophyll-a levels and particulate organic matter do not decrease during the period of summer mortality (August). However, during a period of one to two weeks in August, we observed an increase in the unit volume of the phytoplankton which could possibly lead to dietary deficiencies. Our physiological measurements support the hypothesis of a decreased availability of appropriate food during August as we found very low measures of absorption efficiency and negative values of scope for growth at this time. For mussels from the British Isles, thermal stress, particularly exposure to temperatures over 208C, decreases energetic reserves and induces cellular autophagy (Lowe et al., 1982). Clearly, such stress could decrease the survival of mussels. In Great Entry Lagoon, summer mortality generally occurs when water temperatures are at their summer peaks, but between 1992 and 1994, no summer mortalities occurred with water temperatures over 208C. In other years summer mortalities have been observed with temperatures under 208C (Myrand and Gaudreault, 1995). These results make it difficult to link summer mortalities in the Magdalen Islands solely with the occurrence of temperatures over 208C. We observed that thermal sensitivities of metabolism were higher in mussels susceptible to mortality (from Great Entry) comparatively to those from resistant stock (from Amherst Basin), but only in August at temperatures over 208C (Tremblay et al., in press). Hawkins et al. (1987) and Hawkins (1995) demonstrated that metabolic sensitivity to temperature varied positively with the proportion of protein synthesis required for protein turnover and thus with maintenance requirements. During times of food shortage, protein breakdown increases dramatically to mobilise amino acids preferentially for catabolism (Hawkins, 1991). In our results, the O:N ratios were lowest and maintenance costs highest in mussels from susceptible stock at the time where mortality generally occured. These results suggest that protein turnover was most intense in the susceptible stock during this period. Thus, is it possible that the mortality of susceptible stock observed in 1991, may have stemmed from exhausted reserves, associated with elevated metabolic demands that reflect both the increased turnover that was required to mobilise protein reserves and the resulting higher thermal sensitivity at temperature of 208C. But in this context, why was no mortality observed in 1992 when sea temperature exceeded 208C and food quality values were low between the beginning and the middle of August? Various authors cite post-spawning weakness as a probable cause of mortality (Bayne et al., 1978; Worrall and Widdows, 1984; Emmett et al., 1987; Newell and Lutz, 1991). Mature mussels use a major portion of their energetic reserves for gametogenesis (Bayne et al., 1982). After spawning their reserves are at very low levels and thus, mussels are more sensitive to environmental stress. In the Magdalen Islands the main spawning of mussels usually occurs in late June (Myrand, 1991) and a second spawning may happen in late July (Myrand and Gaudreault, 1995). Thus, mussels may be in a post-spawning condition in August and should be restructuring mantle tissue. As energetic reserves are low, mussels would normally require food intake (Gabbott and Peek, 1991). Therefore,

54

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

it is possible that the absence of a post-spawning weakness (no second spawning in late July), is the factor determining the absence of mortality. The high metabolic rate of mussels from the susceptible stock (Great Entry) could intensify the effect of postspawning weakness and increase the propensity towards mortality. If spawning leads to an equivalent weakening of the two stocks of mussels, mussels from the susceptible stock should require more time to repair the spawning-induced tissue damage. This could make them less resistant to subsequent periods of environmental stress like high temperatures or dietary deficiencies. Nevertheless, presently we have no data to confirm differences in reproductive cycle and / or in reproductive effort between the mussels from the susceptible and resistant stocks. Maybe the observed differences among the stocks could be explained by differential investment in reproduction, mussels from susceptible stocks channeling more energy in their reproduction. Differential mortality among stocks could arise from distinct spawning periods. Rios et al. (1996) showed that in a wild scallop population, Pecten jacobaeus, the more heterozygous individuals tended to spawn earlier. As mussels from the resistant stocks (Amherst Basin) were more heterozygous than mussels from susceptible stock (Great Entry), it is possible than the resistant mussels spawned earlier and had more time to recuperate after spawning and before the period of summer mortality. Our results seem to demonstrate that summer mortality is the result of more than one factor. We suggest that a synergistic interaction among dietary deficiency, increased temperature and possible post-spawning stress is responsible for the outbreaks of summer mortality.

Acknowledgements ˆ This work was supported by funds from CORPAQ (Conseil des Recherches en Peche ˆ ´ ` de l’Agriculture des Peches et en Agro-alimentaire du Quebec), MAPAQ (Ministere et ´ de l’Alimentation du Quebec) and DFO (Department of Fisheries and Oceans). The authors would like to thank Pierre Bergeron from BIOREX Inc., Eric Parent from Maurice-Lamontagne Institute (DFO) and all the staff of the Station Technologique Maricole des ˆIles of MAPAQ for their assistance. Special thanks to Sonia Gareau, ´ Lepine ´ Marie-Josee and Karl Arsenault for technical help.

References Aminot, A., M. Chaussepied, 1983. Manuel des Analyses Chimiques en Milieu Marin. CNEXO, Brest, pp. 75–92. Bayne, B.L., 1973. Aspects of the metabolism of Mytilus edulis during starvation. Neth. J. Sea Res. 7, 399–410. Bayne, B.L., R.C. Newell, 1983. Physiological energetics of Marine Molluscs. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, vol. 4, Physiology, Part 1, Academic Press, Toronto, pp. 407–515. Bayne, B.L., Bayne, C.J., Carefoot, T.C., Thompson, R.J., 1976. The physiological ecology of Mytilus californianus Conrad. Metabolism and energy balance,. Oecologia 22, 211–228.

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

55

Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Livingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing A.R.D., Widdows, J., 1985. The Effects of Stress and Pollution on Marine Animals. Praeger Publishers, New York. Bayne, B.L., Bubel, A., Gabbott, P.A., Livingstone, D.R., Lowe, D.M., Moore, M.N., 1982. Glycogen utilisation and gametogenesis in Mytilus edulis L. Mar. Biol. 3, 89–105. Bayne, B.L., Hawkins, A.J.S., Navarro, E., 1987. Feeding and digestion by the mussel Mytilus edulis L. (Bivalvia: Mollusca) in a mixtures of silt and algal cells at low concentrations. J. Exp. Mar. Biol. Ecol. 111, 1–22. Bayne, B.L., Holland, D.L., Moore, M.N., Lowe, D.M., Widdows, J., 1978. Further studies on the effects of stress in the adult and on the eggs of Mytilus edulis. J. Mar. Biol. Ass. 58, 825–841. Borsa, P., Tousselin, Y., Delay, B., 1992. Relationships between allozyme heterozygosity, body size and survival to natural anoxic stress in the palourde Ruditapes decussatus L. (Bivalvia: Veneridae). J. Exp. Mar. Biol. Ecol. 155, 169–181. Carver, E.A., Mallet, A.I., 1995. Comparative growth and survival patterns of Mytilus trossulus and Mytilus edulis in Atlantic Canada. Can. J. Fish. Aquat. Sci. 52, 1873–1880. Conover, R.J., 1966. Assimilation of organic matter by zooplankton. Limnol. Oceanogr. 11, 338–354. Dickie, L.M., Boudreau, P.R., Freeman, K.R., 1984. Influences of stock and site on growth and mortality in the blue mussel (Mytilus edulis). Can. J. Fish. Aquat. Sci. 41, 134–140. Diehl, W.J., Gaffney, P.M., Koehn, R.K., 1986. Physiological and genetic aspects of growth in the mussel Mytilus edulis. I. Oxygen consumption, growth, and weight loss. Physiol. Zool. 59 (2), 210–211. Emmett, B., Thompson, K., Popham, J.D., 1987. The reproductive and energy storage cycles of two populations of Mytilus edulis L. from British Columbia. J. Shell. Res. 6 (1), 29–36. Foster-Smith, R.L., 1975. The effect of concentration of suspension on the filtration rates and pseudofaecal production for Mytilus edulis L., Cerastoderma edule (L.) and Venerupis pullastra. J. Exp. Mar. Biol. Ecol. 17, 1–22. Fuentes, J., Reyero, I., Zapata, C., Alvarez, G., 1992. Influence of stock and culture site on growth rate and mortality of mussels (Mytilus galloprovincialis Lmk.) in Galicia, Spain . Aquaculture 105, 131–142. Gartner-Kepkay, K.E., Dickie, L.M., Freeman, K.R., Zouros, E., 1980. Genetic differences and environments of mussel populations in the Maritime Provinces. Can. J. Fish. Aquat. Sci. 37, 775–782. Gartner-Kepkay, K.E., Zouros, E., Dickie, L.M., Freeman, K.R., 1983. Genetic differentiation in the face of gene flow: a study of mussel populations from a single Nova Scotia embayment. Can. J. Fish. Aquat. Sci. 40, 443–451. Gabbott, P.A., Peek, K., 1991. Cellular biochemistry of the mantle tissue of the mussel Mytilus edulis L. Aquaculture 94, 165–176. Garton, D.W., Koehn, R.K., Scott, T.M., 1984. Multiple-locus heterozygosity and physiological energetics of growth in the coot clam Mulinia lateralis, from a natural population. Genetics 108, 445–455. Gentili, M.R., Beaumont, A.R., 1988. Environmental stress, heterozygosity and growth rate in Mytilus edulis. J. Exp. Mar. Biol. Ecol. 120, 145–153. Gilek, M., Tedengren, M., Kautsky, N., 1992. Physiological performance and general histology, of the blue mussel, Mytilus edulis., from the Baltic and North Seas. Neth. J. Sea. Res. 30, 11–21. Goolish, E.M., Adelman, I.R., 1987. Tissue-specific cytochrome oxidase activity in largemouth bass: the metabolic costs of feeding and growth. Physiol. Zool. 60 (4), 454–464. Gosling, E., 1992. Genetics of Mytilus. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture. Elsevier Science Publishers B.V., Amsterdam, pp. 309-382. Griffiths, C.L., Griffiths, R.J., 1987. Bivalvia. In: Pandia, T.J., Vernberg, F.J. (Eds.), Animal Energetics vol. 2, Bivalvia through Reptilia. Academic Press, New York, pp. 1–88. Groen, A., Wanders, R.J.A., Westerhoff, H.S., Vander Meer, R., Tagers, J.M., 1982. Quantification of the contribution of various steps to the control of mitochondrial respiration. J. Biol. Chem. 257, 2754–2757. Harris, H., Hopkinson, D.A., 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North Holland, Amsterdam. Hawkins, A.J.S., 1991. Protein turnover: a functional appraisal. Func. Ecol. 5, 222–233. Hawkins, A.J.S., 1995. Effect of temperature change on ectotherm metabolism and evolution: metabolic and physiological interrelations underlying the superiority of multi-locus heterozygotes in heterogeneous environments. J. Therm. Biol. 20, 23–33.

56

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Hawkins, A.J.S., Bayne, B.L., 1992. Physiological interrelations, and the regulation of production. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture, Elsevier Science Publishers B.V., Amsterdam, pp. 171–222. Hawkins, A.J.S., Bayne, B.L., Day, A.J., 1986. Protein turnover, physiological energetics and heterozygosity in the blue mussel, Mytilus edulis: the basis of variable age-specific growth. Proc. R. Soc. Lond. B229, 161–176. Hawkins, A.J.S., Wilson, I.A., Bayne, B.L., 1987. Thermal responses reflect protein turnover in Mytilus edulis. Funct. Ecol. 1, 339–351. Hebert, D.P.N., Beaton, M.J., 1989. Methodologies for Allozyme Analysis Cellulose Acetate Electrophoresis. Helena Laboratories, Texas, 32 pp. Heath, D.D., Hatcher, D.R., Hilbish, T.J., 1996. Ecological interaction between sympatric Mytilus species on the west coast of Canada investigated using PCR markers. Mol. Ecol. 5, 443–447. Hilbish, T.J., Bayne, B.L., Day, A., 1994. Geneties of physiological differentiation, within the marine mussel genus Mytilus. Evolution 48, 267–286. Incze, L.S., Lutz, R.A., Watling, L., 1980. Relationships between effects of environmental temperature and seston on growth and mortality on Mytilus edulis in a temperate Northern Estuary. Mar. Biol. 57, 147–156. Jabbar, A., Davies, I., 1987. A simple and convenient biochemical method for sex identification in the marine mussel, Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 107, 39–44. Koehn, R.K., 1990. Heterozygosity and growth in marine bivalves: comments on the paper by Zouros, Romero-Dorey, and Mallet (1988). Evolution 44, 213–216. Koehn, R.K., 1991. The cost of enzyme synthesis in the genetics of energy balance and physiological performance. Biol. J. Linn. Soc. 44, 231–247. Koehn, R.K., Bayne, B.L., 1989. Towards a physiological and genetical understanding of the energetics of the stress response. Biol. J. Linn. Soc. 37, 157–171. Koehn, R.K., Gaffney, P.M., 1984. Genetic heterozygosity and growth rate in Mytilus edulis. Mar. Biol. 82, 1–7. Lowe, D.M., Moore, M.N., Bayne, B.L., 1982. Aspects of gametogenesis in the marine mussel Mytilus edulis L. J. Mar. Biol. Ass. U.K. 62, 133–145. Lucas, M.I., Newell, R.C., Shumway, S.E., Seiderer, L.J., Bally, R., 1987. Particle clearance and yield in relation to resource availability in estuarine and open coast populations of the mussel Mytilus edulis L. Mar. Ecol. Prog. Ser. 87, 215–224. Mallet, A.L., Carver, C.E.A., Freeman, K.R., 1990. Summer mortality of the blue mussel in eastern Canada: spatial, temporal, stock and age variation. Mar. Ecol. Prog. Ser. 67, 35–41. ´ Mayzaud, P., Souchu, P., 1991. Environnement trophique et bilan metabolicque des populations de moules ˆ ´ ´ des Iles-de-la-Madeleine (Quebec, ´ cultivees Canada). In: Conseil de l’Aquiculture et des Peches du Quebec ´ (Ed.), Atelier de Travail sur la Mortalite´ Estivale des Moules aux Iles-de-la-Madeleine, Quebec, pp. 61–74. McDonald, J.H., Seed, R., Koehn, R.K., 1991. Allozymes and morphometric characters of three species of Mytilus in the northern and southern hemispheres. Mar. Biol. 111, 323–333. Mitton, J.B., 1993. Enzyme heterozygosity, metabolism, and developmental stability. Genetica 89, 47–65. ´ Myrand, B., 1991. Conditions environnementales dans les lagunes des Iles-de-la-Madeleine et parametres ˆ ´ biologiques de la moule bleue. In: Conseil de l’Aquiculture et des Peches du Quebee (Ed.), Atelier de ´ Travail sur la Mortalite´ Estivale des Moules aux Iles-de-la-Madeleine, Quebec, pp. 47–58. Myrand, B., Gaudreault, J., 1995. Summer mortality of blue mussels (Mytilus edulis Linneaus, 1758) in the Magdalen Islands (southern Gulf of St Lawrence Canada . J. Shellfish Res. 14, 395–404. Navarro, E., Iglesias, J.I.P., Camacho, A.P., Labarta, U., Beiras, R., 1991. The physiological energetics of mussels (Mytilus galloprovincialis Lmk) from different cultivation rafts in the Rib de Arosa (Galicia N.W. Spain). Aquaculture 94, 197–212. Navarro, E., Thompson, R.J., 1994. Comparison and evaluation of different techniques for measuring absorption efficieney in suspension feeders. Limnol. Oceanogr. 39, 159–164. Newell, C.R., Lutz, R.A., 1991. Growth and survival of cultivated mussels in Maine. In: Conseil de ˆ ´ l’Aquiculture et des Peches du Quebec (Ed.), Atelier de travuil sur la morlalite´ estivale des moules aux ´ Iles-de-lu-Madeleine, Quebec, pp. 131–138. Okumus, I., Stirling, H.P., 1994. Physiological energetics of cultivated mussel (Mytilus edulis) populations in two Scottish west coast sea lochs. Mar. Biol. 119, 125–131.

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

57

Ornstein, L., 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci. 121, 321–349. ` ` ` Pasteur, P., Pasteur, G., Bonhomme, F., Catalan, J., 1989. Manuel Technique de benetique par electrophorese ` des probeines. Tec et Doc Lavoisia, Paris, 205 p. Reeds, P.J., Muller M.F., Nicholson, B.A., 1985. Metabolic basis of energy expenditure with particular reference to protein. In: Garrow J.S., Halliday, D. (Eds.), Substrate and Energy, Metabolism in Man, Libbey, London, pp. 46–57. ˚ Riisgard, H.U., 1988. Efficiency of particle retention and filtration rate in 6 species of Northeast American bivalves. Mar. Ecol. Prog. Ser. 45, 217–223. ˜ J.B., 1996. Genotype-dependent spawning evidence from a wild population of Rios, C., Canales, J., Pena, Pecten jacobaeus (L.) (Bivalvia: Pectinidae). J. Shellfish Res. 15, 645–651. Rodhouse, P.G., Gaffney, P.M., 1984. Effect of heterozygosity on metabolism during starvation in the American oyster Crassostrea virginica. Mar. Biol. 80, 179–187. Rodhouse, P.G., McDonald, J.H., Newell, R.I.E., Koehn, R.K., 1986. Gamete production, somatic growth and multiple-locus enzyme heterozygosity in Mytilus edulis. Mar. Biol. 90, 209–214. Roy, S. Mayzaud, P. Souchu, P., 1991. Environnement physico–chimique et trophique d’un site mytilicole aux ˆIles-de-la-Madeleine (Quebec): ´ ` particulaire, composition biochimique et productivite´ primaire. II-Matiere ´ ou Grand Estuaire? Can. J. Fish. Aquat. Sci. In: Therriault, J.-C., Le Golfe du Saint-Laurent: Petit Ocean 113, 219–230. Scott, T.M., Koehn, R.K., 1990. The effect of environmental stress on the relationship of heterozygosity to growth rate in the coot clam Mulinia lateralis (Say). J. Exp. Mar. Biol. Ecol. 135, 109–116. Sokal, R.R., Rohlf, F.J., 1981. Biometry. W.H. Freeman and Co., San Francisco, 2nd ed., 776 pp. Solorzano, L., 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14, 799–801. Strickland, J.D.H., Parsons, T.R., 1972. A practical handbook of seawater analysis. J. Fish. Res. Board Can. 167, 311. Swofford, D.L., Selander, R.B., 1989. A computer program for the analysis of allelic variation in populution genetics and biochemical systematics (Release 1.7). David L. Swolford, lllinois Natural History Survey, 43 pp. Tedengren, M., Andre, C., Johannesson, K., Kautsky, N., 1990. Genotypic and phenotypic differences between Baltic and North Sea populations of Mytilus edulis evaluated through reciprocal transplantations III. Physiology,. Mar. Ecol. Prog. Ser. 59, 221–227. Thompson, R.J., Bayne, B.L., 1974. Some relationships between growth, metabolism and food in the mussel Mytilus edulis. Mar. Biol. 27, 317–326. Tremblay, R., Myrand B., Guderley. H. Thermal sensitivity of organismal and mitochondrial VO 2 in relation to susceptibility of blue mussels, Mytilus edulis (L.), to summer mortality. J. Shellfish Res. (in press). Toro, J.E., Vergara, A.M., 1995. Evidence for selection against heterozygotes: Post settlement excess of allozyme homozygosity in a cohort of the chiliean oyster, Ostrea chilensis Philippi, 1845. Biol. Bull. 188, 117–119. Varvio, S.-L., Koehn, R.K., Vainola, R., 1988. Evolutionary genetics of the Mytilus edulis complex in the North Atlantic region. Mar. Biol. 5, 51–60. Van Steenbergen, E.J., 1987. Genetic variation of energy metabolism in mice. In: Verstegen M.W.A., Henken, A.M. (Eds.), Martinus Kijhoff Energy Metabolism in Farm Animals, Boston, pp. 467-477. Volckaert, F., Zouros, E., 1989. Allozyme and physiological variation in the scallop Placopecten magellanicus and a general model for the effects of heterozygosity on fitness in marine molluscs. Mar. Biol. 103, 51–61. Warren, C.E., Davis, G.E., 1967. Laboratory studies on the feeding bioenergetics and growth of fish. In: Gerking, S.D. (Ed.), The Biological Basis of Freshwater Fish Production, Blackwell Scientific, Oxford, pp. 175–214. Waterlow, J.C., 1988. The variability of energy metabolism in man. In: Blaxter, K., McDonald, I. (Eds.) Comparative Nutrition, Libbey, London, pp. 133–139. Widdows, J., 1978. Combined effects of body size, food concentration and season on the physiology of Mytilus edulis. J. Mar. Biol. Ass. U.K. 58, 109–124. Widdows, J., Johnson, D., 1988. Physiological energetics of Mytilus edulis: Scope for growth. Mar. Ecol. Prog. Ser. 46, 113–121.

58

R. Tremblay et al. / J. Exp. Mar. Biol. Ecol. 221 (1998) 27 – 58

Widdows, J., Bayne, B.L., Livingstone, D.R., Newell, R.I.E., Donkin, P., 1979. Physiological and biochemical responses of bivalve molluscs to exposure to air. Comp. Biochem. Physiol. 62A, 301–308. Worrall, C.M., Widdows, J., 1984. Investigation of factors influencing mortality in Mytilus edulis L. Mar. Biol. Lett. 5, 85–97. Zouros, E., Foltz, D.W., 1987. The use of allelic isozyme variation for the study of heterosis. Isozyme 13, 1–59. Zouros, E., Singh, S.M., Miles, H.E., 1980. Growth rate in oysters: An overdominant phenotype and its possible explanation. Evolution 34, 856–867.