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Biochemical composition and fertilization in the eggs of Mytilus galloprovincialis (Lamarck)
i
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
Journal of Experimental Marine Biology and Ecology 192 (1995) 75-85
ELSEVIER
Biochemical
and fertilization in the eggs of Mytilus galloprovincialis (Lamarck)
F.J. Sedano*,
composition
J.L. Rodriguez,
C. Ruiz, L.O. Garcia-Martin,
J.L. Sgnchez
Departamento de Bioquimica y Biologia Molecular, Facultad de Farrnacia, Universidad de Santiago de Compostela, 15706-Santiago de Compostela, Spain
Received 4 January 1995; revision received 24 March 1995; accepted 4 May 1995
Abstract The biochemical composition of eggs spawned from mussels Mytihs gulZoprovinciuZis (Lamarck) collected from Galician Rias (Spain) is, in general, similar to that indicated for other marine molluscs. Proteins (45% of total dry weight) and lipids (22%) are the main reserves of the egg while carbohydrates (3%) are the lowest constituent. Egg diameter showed a mean value of 61 pm and egg density followed an unimodal distribution of frequencies with a mean value of 1.057 g. cmm3. Fertilization of the eggs of M. galloprovincialis can be delayed for about 4 to 8 h after spawning over the temperature range of lo” to 18°C. After these times, percentage of fertilized eggs decreased to less than 30%. At 18°C the low values in successful fertilization may be due to the degradation of the eggs. A delay in addition of sperm after release of the eggs increases the time in which first cell cleavage appears from the moment of insemination. Variation of the biochemical composition of unfertilized eggs over time was also studied. At 18°C proteins, lipids and carbohydrates decreased by about 45, 46 and 48%, respectively, after 5 h (only 4% of total eggs were fertilized) from spawning. At 10” and 14°C the decreases after 11 and 8 h from spawning, respectively, were lower. Before these times, the decrease of biochemical constituents was not so great and fertilization rates were still high. Keywords: perature
Biochemical
composition;
Egg; Fertilization;
Myths
gdoprovincialis;
Tem-
1. Introduction Bivalve larvae have been reared successfully over many years. However, factors which are of primary importance in the rearing of bivalve larvae are that the eggs * Corresponding author. 0022-0981/95/$09.50 @ 1995 Elsevier Science B.V.All rights reserved SSDI
0022-0981(95)00062-3
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F.J. Sedan0 et al. I .I.
Exp. Mar. Biol. Ecol. 192 (1995) 75-8.5
should be fertilized successfully and that the fertilized eggs should produce a high proportion of normal, healthy larvae. Because of that, it is necessary to ensure that conditions during the fertilization and embryonic development are optimal for the production of normal larvae. Special features of Rias from Galicia made this region the main producer of mussels in the world. Spat and young individuals, used for culture on ropes suspended from rafts, are obtained naturally from the coast. In this environment, mussels are not subjected to stressful conditions like high changes in temperature and salinity or long periods without available food. This fact enables a fast growth of mussels. However, improvement in rearing bivalves requires a deeper knowledge of the whole life-cycle of these organisms. Egg quality and conditions in which fertilization and early development take place are very important factors. A number of studies (Barnes, 1965; Pandian, 1967, 1969, 1970; Holland, 1978; Whyte et al., 1987, 1990) have shown that proteins and lipids are the main constituents of eggs from different species of marine invertebrates (mainly, bivalves and crustaceans). It is also suggested that egg quality varies with the level of lipids stored during vitellogenesis. Bayne et al. (1978) showed that size and content of egg reserves were interrelated, with smaller eggs released from stressed females of Myths edulis containing less lipid and protein than eggs from normal females. Likewise early larval development has been correlated with the lipid content of eggs released from M. edulis (Bayne et al., 1975), Mercenaria mercenaria and Crassostrea virginica (Gallager and Mann, 1986). Other factors to be considered before fertilization are the relationships between gametes and the physical conditions at the moment of fertilization. Some authors have suggested that high sperm concentration and also high concentrations of eggs (Loosanoff & Davis, 1963) may adversely affect rearing success. Despite this, few studies have examined the relationships between sperm and egg concentrations. Loosanoff & Davis (1963) quoted an optimal value of about 30000 eggs/ml in Mercenaria mercenaria. Gruffydd & Beaumont (1970) indicated a concentration of 700 eggs/cm* and low number of sperms (l-100 per egg) in Pecten muximus. Recently, Sprung & Bayne (1984) reported a value of lo4 sperms/ml and not more than 20 eggs/ml in M. edulis. This work was designed to study the following aspects: (1) to determine the physical parameters and biochemical composition in eggs of the mussel, M. gulloprovinciulis (Lamarck) cultured in Rias from Galicia; (2) to know how long fertilization may be delayed, at different conditions of temperature, after release of gametes and, (3) to verify if this delay could involve variations of the initial biochemical content of the unfertilized eggs. The latter fact could have significant consequences on both embryonic development and the initial larval stages.
2. Material and methods
Mature adult mussels of 7-10 cm shell length were collected during the period of March-May, 1989, from Ria de Ares, Galicia, and kept for 48 h in the
F.J. Sedan0
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77
laboratory, without food, at 4°C. Spawning was then induced by vigorous shaking, followed by immersion of each animal in 500-ml tanks with 1 ,um filtered seawater at 20” 2 2°C and 32%, salinity, always under vigorous aeration. After spawning, eggs and sperm were filtered through a 80 and 30 pm sieve, respectively, to remove faeces and other particles of larger size. Number of gametes were determined using a Neubauer chamber for sperm and a Sedgewick rafter counting-cell for eggs. Then, eggs and sperm were kept at a concentration of about 300 per ml and lo5 per ml, respectively, under light aeration. Eggs were kept at lo”, 14” or 18°C depending on the experimental temperature used and sperm at 4°C. Egg samples of the different spawns were taken in order to determine their biochemical composition and physical parameters (diameter, density, dry weight). Previous experiments made in our laboratory gave optimum concentrations of about lo4 sperms per ml and about lo-30 eggs per ml, which are similar to those indicated by Sprung & Bayne (1984) in M. edulis. At different times after spawning eggs and sperm were added to 0.2 pm filtered seawater in a ratio of -10-30 eggs per ml and lo3 sperm per egg. Fertilization was assumed to have been successful when the eggs showed signs of cell cleavage. At different times after addition of sperm, egg samples were taken and immediately examined to determine the time of first cleavage and the percentage of fertilized eggs. The time at which fewer than 30% of eggs were fertilized was chosen as the maximum period over which fertilization remained possible. Mussels treated in a similar way were used for a further experiment. After the eggs were spawned and counted, they were added to 10-l tanks with filtered (0.2 pm) seawater at the same concentration and temperatures as before but without fertilization. At different times, samples of unfertilized eggs were collected for a further analysis of the biochemical composition. All samples collected were rinsed with isoosmotic ammonium formate (3% w/v) to remove salt. Then, those samples used for the analysis of the biochemical composition were frozen, freeze-dried and stored at -80°C until analysis. Egg diameter was determined by using a Nikon Optiphot-2 microscope and egg density by extrapolation from density curves obtained with commercial markers (Pharmacia Fine Chemicals) whose densities were determined previously using a refractometer under the conditions used in our experiments. The silica-sol Percoll (Pharmacia Fine Chemicals) was combined with artificial seawater, made as in Murakami and Machemer (1982), to prepare density gradients isoosmotic with seawater at 32%0 and 18°C by the procedure of Crespo (1989) modified by Sedan0 (1992). Weight data were obtained using a Sartorius 2405 electrobalance (accurate to 0.1 pug). Samples of known number of eggs were placed in preashed, preweighed aluminium boats and dried at 60°C in an oven until constant weight in order to determine total dry weight. Then the samples, in aluminium boats, were combusted in a muffle furnace for 6 h at 450°C and ashed samples were then weighed. The amount of ash-free dry weight is calculated as the difference between the total dry weight and the ash weight. This value is presented as percentage relative to total dry weight (% AFDW).
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F.J. Sedan0 et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
7.~45
For biochemical analysis, 500 ,ul of de-ionized and distilled water was added to 2-3 mg of freeze-dried sample. The sample was then homogenized in a teflonglass homogenizer always under refrigeration with ice. The homogenized material was fractionated according to the procedure of Holland & Gabbott (1971) as modified by Holland & Hannant (1973). Total carbohydrates, total lipids and ribonucleic acid (RNA) were determined by the methods described in Holland & Hannant (1973) and were defined in the same way. Total proteins were defined as material hydrolyzed by 5N NaOH and precipitated proteins as material insoluble in 5% trichloracetic acid (TCA); both were determined by a modification of Lowry’s method (1951) based on the results obtained by Bensadoun & Weinstein (1976) and Hess et al. (1978) using bovine serum albumin as a calibration standard. Statistical treatment of data was according to Holland & Gabbott (1971). Values for the coefficient of variation (C.V.) about the means of the replicate determinations were less than 0.05 with the exception of ribonucleic acid (RNA) which was less than 0.10. The number of replicate samples necessary to obtain repeatable biochemical results, determined by using Student’s f-test as Bartlett (1979) were two. When necessary, data were treated by using one way ANOVA analysis and critical level of significance was taken to be at 5% (p < 0.05).
3. Results and discussion The results indicated a great homogenity among different spawns (Table 1). The dry weight of M. gulloprovincialis eggs varied from 50 to 56 ng/egg with a mean value of 53 ng. Other authors quoted values for M. edulis eggs of 51 ng (Ockelmann, 1965), 52.5 ng (Bayne et al., 1975) 47.3 ng (Thompson, 1979) and 67.4 ng (Sprung, 1983; 1984) which agree with those cited in this paper. As Table 1 shows, mean biochemical composition of eggs from M. galloprovincialis was dominated by protein (45% of total dry weight) and lipid (22%) while carbohydrate (3% ) was a minor component. The range of values for eggs of M. edulis, M. californianus, Astarte sp., Placopecten magellanicus, Spisula solidissima, Mya truncata, Patinopecten yessoensis and Crassadoma gigantea has been reported
as 37-68%, 9-24% and l-8% of the total dry weight for protein, lipid and carbohydrate, respectively (Holland, 1978; Whyte et al., 1987; 1990; Gabbott, 1983). The values quoted for M. gulloprovincialis eggs are within these ranges indicating that they are comparable to those of other marine invertebrates. However, lipid and carbohydrate levels were higher than the respective mean values of 17.1 and 1.9% calculated for M. edulis eggs (Holland, 1978). Protein and lipid contributed with 2.5 and 2.1 kcal/g, respectively, to the total energy content, while carbohydrate (0.2 kcal/g) seemed not to be an important energy reserve. Mean AFDW was 89.3%. This implies an ash content of 10.7% that is similar to the calculated value of 8.9% for M. edulis eggs (Sprung, 1984). Despite this, about 23% of the organic content was unaccounted for by the analytical procedures used, and was presumed to consist of low molecular weight components.
18.5 1.16
24.1 1.09
Mean
SD
18.0 19.9 20.2 17.5 17.6 19.1 16.7 18.6 19.0
24.3 25.8 24.9 23.6 22.5 24.9 22.6 23.9 24.6
1 2 3 4 5 6 7 8 9
TCA proteins
Total proteins
Spawn
7.9 0.64
7.8 8.9 7.3 7.5 7.3 7.6 8.1 7.4 8.9
Neutral lipids
Total
lipids
3.8 0.62
3.9 4.0 3.1 2.9 4.0 3.6 4.3 3.7 5.0
Phospholipids
0.6 0.08
0.5 0.7 0.5 0.7 0.5 0.6 0.5 0.6 0.6 1.2 0.33
1.1 1.3 1.1 1.9 0.8 1.1 1.3 1.0 1.6
Polysaccharides
carbohydrates
Free reducing sugars
Total
3.0 0.53
3.6 3.2 3.6 2.8 2.5 3.0 2.3 2.4 3.6
RNA
1.057 0.0014
1.055 1.056 1.057 1.058 1.059 1.058 1.056 1.058 -
+ + + + + + + +
Density
0.0031 0.0029 0.0015 0.0007 0.0002 0.0020 0.0012 0.0025
61 1.0
61 + 60+ 61 + 60 + 60 + 63 + 61 + 61 + 62 +
0.3 1.2 1.0 1.4 1.5 0.8 2.1 1.2 1.9
Diameter
+ + + + + + + + + 53 1.9
50 55 52 54 52 53 55 55 56
4.8 2.8 4.6 1.0 1.7 3.0 1.5 2.5 3.2
Dry weight
+ + + + + + + + + 89.3 1.97
88.8 86.4 87.9 87.2 90.4 91.3 91.2 88.7 91.9
AFDW
1.18 0.94 1.02 0.63 1.53 0.39 0.54 1.28 0.99
Table 1 Biochemical content (values are mean of two replicate samples) and physical parameters of eggs from M. galloprovincialis. All measurements in pg 10mgind. except diameter (pm), density (g . cm-‘) and AFDW (%). Diameter (n = 30) density (n = 4) dry weight (n = 3) and AFDW (n = 3) are mean +SD. Lower part of table presents mean values +SD from nine different spawns
Y ec:
z ; 2 ? s 2 g k!
$
!? P
5
3 5 2 3 $ z e
80
F.J. Sedan0 et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
75-85
Egg diameter varied from 60 to 63 pm with a mean value of 61.2 ,um which is smaller than those quoted for M. edulis (68-70 pm) by Bayne (1976). Egg density ranged from 1.055 to 1.059 g * cm -’ with a mean value of 1.057. Almost 72% of the eggs from the nine different spawns of M. galloprovincialis banded between 1.056 and 1.058 g. cme3 (Fig. 1). Th ese results indicate that there is a low variation in the values of density which follow an unimodal distribution of frequencies. Gallager & Mann (1986) indicate the important role of the egg density in dispersal of embryonic stages. They also suggest that the high variation of egg densities among species could be a function of evolutionary adaptation of reproductive strategies. Species with a unimodal distribution of egg densities would be at an advantage, compared to species with a multimodal distribution, in stable environments that favour smaller and more dense eggs. The results of our study agree with this hypothesis and, in this way, eggs from M. galloprovincialis, living under conditions present in Rias, are small (61.2 pm) and dense (1.057 g. cm-“). These characteristics confer less hydrodynamic flexibility on the mechanism of dispersal, but permit that eggs may be retained in this environment. The results in Table 2 indicate that there is a long period over which M. gulloprovincialis eggs can be fertilized after spawning for all temperatures tested. Successful fertilization (i.e. >30%) was still possible 4 to 8 h after gamete release and the longest periods of delayed fertilization were tolerated at the lowest temperatures used. The data agree with those quoted for M. edulis by Bayne (1976) which indicate successful fertilization after 4 to 6 h at 18°C and by Sprung & Bayne (1984) which indicate successful fertilization after 6 to 11 h at temperatures of 8” to 16°C. Motility indicates survival of the sperm and the sperm maintained their motility for periods of time longer than those tolerated for fertilization. The limiting factor in successful fertilization may reside in the egg, since the sperm’s capacity for
0’ 1.056
1.054
Egg density
Fig. 1. Mean
density
distribution
of eggs from
1.056
1.060
(g/crn3)
nine spawns
of M. galloprovincialis.
F.J. Sedan0
et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
Table 2 Fertilization success in eggs from Mytilus galloprovincialis and 18°C. Values are mean from three replicate samples Temperature (“C)
Addition time of sperm after spawning (h)
at different
Fertilization Mean
75-85
81
times after spawning
success (% ) SD
Appearance time of first cell cleavage (h)
4.98 0.84 3.62 9.14 11.57 _
2.0 2.0 2.3 2.3 2.3 2.5 _
0 2 4 6 8 10 11
86.0 90.3 92.6 90.3 86.8 22.2
14
0 1 2 3 4 5 6 7 8
82.0 85.1 83.7 83.5 74.0 70.8 77.2 45.7 <30.0
1.07 1.16 0.68 4.60 1.74 0.67 3.11 8.71
1.3 1.4 1.3 1.3 1.4 1.4 1.5 1.5 1.5
18
0 2 2 3 4 5 6
82.5 93.0 95.0 90.0 94.0 4.0 _
3.53 1.63 0.69 2.36 1.04 5.95 _
1.3 1.3 1.5 1.7 1.8 2.0 _
10
at lo”, 14”
fertilization was assumed not to decrease greatly when they are kept at low temperatures. Successful fertilization was still possible within 8, 7 and 4 h after spawning at lo”, 14” and 18°C respectively (Table 2). After these times, percentage of fertilized eggs decreased until values fewer than 30%, with lower fertilization at 18°C than at the other temperatures. The reason for these results may be in the degradation of the eggs which show irregular forms and not the spherical shape presents at the moment of spawning. This event is clearly manifest at 10,8 and 5 h after spawning at lo”, 14” and 18°C respectively. Although there are some fertilized eggs at these times, most of the embryos are not viable showing abnormal cell cleavage or remaining at the stage in which meiosis is reinitiated (extrusion of the first polar body). Possibly, bacteria present either in adult mussels or in water could be responsible of the degradation of eggs. This would be proportional to the holding temperature and its duration. Another significant point is that delay in the addition of sperm delays the appearance of the first cell cleavage increasing when the delay in insemination is bigger (Fig. 2). A number of studies in pelecypods have shown that during the
F.J. Sedan0 et al. I J. Exp. Mar. Biol. Ecol. 192 (199.5) 75-85
l.OJ
0
2 Time
4
of insemination (hours)
6
8
after
Fig. 2. Relationship between delay in the insemination after spawning first cell cleavage after spawning. Regression lines are shown.
10
spawning
and delay in the appearance
of
period of interaction between egg and sperm, referred to as gamete binding (Hylander & Summers, 1977) the acrosomal process interdigitates with microvilli of the egg. Likewise, orientation of microvilli to spermatozoa represents an active process in the association of the egg and sperm (Long0 and Anderson, 1970; Popham, 1975). On the other hand, Dub6 & Dufresne (1990) indicate that metaphase arrest in blue mussel (M. e&&s) oocytes requires the continous synthesis of short-life proteins, the destruction of which is sufficent to induce meiosis resumption with extrusion of the first polar body. The mechanism that induces the inhibition of synthesis of these specific proteins or the increase of some specific proteolytic activities against these short-life proteins is not yet known. Interaction between egg and sperm must have an important role on this mechanism. The delay in the appearance of the first cell cleavage could result from the progressive aging of the egg. The aging, which is clearer at temperatures of WC, may alter the membrane’s properties with respect to egg and sperm binding or the inhibition of protein synthesis related to metaphase arrest. To check if delay in fertilization could involve some effect on the level of the different macronutrients of unfertilized eggs, the variation of the biochemical content over time was determined at the different temperatures used in the previous experiment. The results indicate that a significant (p < 0.05) decrease in energetic reserves of the egg occurred after gamete release at all temperatures tested (Fig. 3). At 18°C proteins decreased about 45% of the initial value in the eggs, lipids 46% and carbohydrates 48% after 5 h from spawning, when fertilization rates are the lowest. At 14°C the decrease was about 22% for
F.J. Sedan0
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Fig. 3. Changes in biochemical content with respect to time after spawning in unfertilized eggs from M. galloprovinciah at three temperatures tested. Values are mean k SD from two replicate samples. TP, total proteins; NL, Neutral lipids; PL, Phospholipids; POL, polysaccharides; FRS, Free reducing sugars; RNA, Ribonucleic acid.
proteins, 16% for lipids and 17% for carbohydrates after 8 h. At 10°C the decrease was about 20% for proteins, 27% for lipids and 34% for carbohydrates after 11 h. We must notice that the great decrease of biochemical components at 18°C may reside in the degradation of eggs after 5 h. However, if we consider the values after 4 h from spawning, the decreases are not so high (14% proteins, 19% lipids and 47% carbohydrates). The same event occurs when we consider the decreases after 10 h and 6 h from spawning at 10” and 14°C respectively. So far, lower rates of fertilization coincide in time with greater decreases of biochemical components. The fact may be explained by the degradation of eggs after those times. However, when we consider the rates of fertilization obtained after 8,7 and 4 h after spawning at lo”, 14” and 18°C and the biochemical content of eggs at these times, we observe success in fertilization but a manifest decrease of the biochemical components. Fertilization can be delayed for long periods of time. Manipulation of gametes during these periods can be possible without a great effect on the rates of fertilization. However, a loss of energetic reserves takes place within these times of delayed fertilization; this could have important effects on the further embryonic development and first larval stages. Therefore, there is an advantage to fertilize soon after gamete release, especially at 18°C a temperature normally used in both laboratory experiments and commercial hatcheries, so that a decrease of biochemical constituents is not a limiting factor for rearing success.
84
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References Barnes, H., 1965. Studies in the biochemistry of cirripede eggs. 1. Changes in the general biochemical composition during development of Balanus balanoides and B. balanus. .I. Mar. Biol. Assoc. U.K., Vol. 45, pp. 321-339. Bartlett, B.R., 1979. Biochemical changes in the Pacific oyster, Crassostrea gigas (Thunberg, 1795) during larval development and metamorphosis. Ph.D. Thesis, Oregon State University. Bayne, B.L., 1976. The biology of mussel larvae. In, Marine mussels: their ecology and physiology, edited by B.L. Bayne, Cambridge University Press, Cambridge, pp. 81-120. Bayne, B.L., P.A. Gabbott & J. Widdows, 1975. Some effects of stress in the adult on the eggs and larvae of Mytilus edulis L. J. Mar. Biol. Assoc. U.K., Vol. 55, pp. 675-689. Bayne, B.L., D.L. Holland, M.N. Moore, D.M. Lowe & J. Widdows, 1978. Further studies on the effects of stress in the adults on the eggs of Mytilus edulis. J. Mar. Biol. Assoc. U.K., Vol. 58, pp. 8277841. Bensadoun, A. & A. Weinstein, 1976. Assay of proteins in the presence of interfering material. Anal. Biochem., Vol. 70, pp. 241-250. Crespo, C.A.. 1989. Histofisiologia de las reservas bioenergeticas del manto de Myfilus galloprovincialis Lmk. Tesis Doctoral, Universidad de Santiago de Compostela (Spain). Dub& F. & L. Dufresne, 1990. Release of metaphase arrest by partial inhibition of protein synthesis in blue mussels oocytes. J. Exp. Zool., Vol. 256, pp. 323-332. Gabbott, P.A., 1983. Developmental and seasonal metabolic activities in marine molluscs. In, The mollusca, environmental biochemistry and physiology, Vol. 2, edited by P.W. Hochachka, Academic Press, New York, pp, 165-217. Gallager, SM. & R. Mann, 1986. Growth and survival of Mercenaria mercenaria (L.) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aquaculture, Vol. 56, pp. 105-121. Gruffydd, L1.D. & A.R. Beaumont, 1970. Determination of the optimun concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusca, Lamellibranchia). Helgol. Wiss. Meeresunters., Vol. 20, pp. 486-497. Hess, H.H., M.B. Lees & J.E. Derr, 1978. A linear Lowry-Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins. Anal. Biochem., Vol. 85, pp. 295-300. Holland, D.L., 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In, Biochemical and biophysical perspectives in marine biology, Vol. 4, edited by D.C. Malius & J.R. Sargent, Academic Press, London, pp. 85-123. Holland, D.L. & P.A. Gabott, 1971. A micro-analytical scheme for the determination of protein, carbohydrate, lipid and RNA levels in marine invertebrate larvae. J. Mar. Biol. Assoc. U.K., Vol. 5 1, pp. 659-668. Holland, D.L. & P.J. Hannant, 1973. Addendum to a microanalytical scheme for the biochemical analysis of marine invertebrate larvae. J. Mar. Biol. Assoc. U.K., Vol. 53, pp. 833-838. 1977. An ultrastructural analysis of the gametes and early Hylander, B.L. & R.G. Summers, fertilization of two bivalve molluscs, Chama macerophylla and Spisula solidissima with special reference to gamete binding. Cell Tissue Res., Vol. 182, pp. 469-489. Longo, F.J. & G. Anderson, 1970. An ultrastructural analysis of fertilization in the surf clam, Spisula sofidissima. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes. J. Ultrastruct. Res., Vol. 33, pp. 515-527. Loosanoff, VL. & C.H. Davis, 1963. Rearing of bivalve molluscs. Adv. Mar. Biol., Vol. 1, pp. 1-136. Lowry, O.H., N.J. Rosebrough, A.L. Farr & R.J. Randall, 1951. Protein measurements with the Folin Phenol Reagent. J. Biol. Chem., Vol. 193, pp. 265-275. Murakami, A. & H. Machemer, 1982. Mechano reception and signal transmission in the lateral ciliated cells on the gill of Mytilus. J. Comp. Physiol.,Vol. 145, pp. 351-362. Ockelmann, K.W.. 1965. Developmental types in marine bivalves and their distribution along the Atlantic coast of Europe. In, Proceedings of the European Malacological Congress, edited by L.R. Cox & J.F. Peake, Malacological Sot. of London, London, Vol. 1, pp. 25535.
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Pandian, T.J., 1967. Changes in chemical compostion and caloric content of developing eggs of the shrimp Crangon crangon. Helgol. Wk. Meeresunters., Vol. 16, pp. 216-224. Pandian, T.J., 1969. Yolk utilization in the gastropod Crepidula fornicata. Mar. Biol., Vol. 3, pp. 117-121. Pandian, T.J.. 1970. Ecophysiological studies on the developing eggs and embryos of the European lobster Homarus gammarus. Mar. Biol., Vol. 5, pp. 154-167. Popham, J.D., 1975. The fine structure of the oocyte of Bankia ausfralis (Teredinidae, Bivalvia) before and after fertilization. Cell Tissue Res.,Vol. 157, pp. 521-534. Sedano, F.J., 1992. Contribution al estudio de1 metabolismo energetic0 durante 10s desarrollos embrionario y larvario en Mytilus galloprovincialis Lmk. Tesis Doctoral. Universidad de Santiago de Compostela (Spain). Sprung, M., 1983. Reproduction and fecundity of the mussel Myrilus eduhs at Helgoland (North Sea). Helgol. Wiss. Meeresunters., Vol. 36, pp. 243-255. Sprung, M., 1984. Physiological energetics of mussel larvae (Mytilus eduhs). I. Shell growth and biomass. Mar. Ecol. Prog. Ser., Vol. 17, pp. 283-293. Sprung, M. & B.L. Bayne, 1984. Some practical aspects of fertilizing the eggs of the mussel Mytilus edulis L. J. Cons. Int. Explor. Mer., Vol. 41, pp. 125-128. Thompson, R.J., 1979. Fecundity and reproductive effort of the blue mussel (Mytilus edulis), the sea urchin (Strongylocentrotus droebachiensis), and the snow crab (Chiomectes opilio) from populations in Nova Scotia and Newfoundland. J. Fish. Res. Board Can., Vol. 36, pp. 955-964 Whyte, J.N.C., N. Bourne & CA. Hodgson, 1987. Assesment of biochemical composition and energy reserves in larvae of the scallop Patinopecten yessoensis. J. Exp. Mar. Biol. Ecol., Vol. 113, pp. 113-124. Whyte, J.N.C., N. Bourne & N.G. Ginther, 1990. Biochemical and energy changes during embryogenesis in the rock scallop Crassadoma gigantea. Mar. Biol., Vol. 106, pp. 239-244.
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
Journal of Experimental Marine Biology and Ecology 192 (1995) 75-85
ELSEVIER
Biochemical
and fertilization in the eggs of Mytilus galloprovincialis (Lamarck)
F.J. Sedano*,
composition
J.L. Rodriguez,
C. Ruiz, L.O. Garcia-Martin,
J.L. Sgnchez
Departamento de Bioquimica y Biologia Molecular, Facultad de Farrnacia, Universidad de Santiago de Compostela, 15706-Santiago de Compostela, Spain
Received 4 January 1995; revision received 24 March 1995; accepted 4 May 1995
Abstract The biochemical composition of eggs spawned from mussels Mytihs gulZoprovinciuZis (Lamarck) collected from Galician Rias (Spain) is, in general, similar to that indicated for other marine molluscs. Proteins (45% of total dry weight) and lipids (22%) are the main reserves of the egg while carbohydrates (3%) are the lowest constituent. Egg diameter showed a mean value of 61 pm and egg density followed an unimodal distribution of frequencies with a mean value of 1.057 g. cmm3. Fertilization of the eggs of M. galloprovincialis can be delayed for about 4 to 8 h after spawning over the temperature range of lo” to 18°C. After these times, percentage of fertilized eggs decreased to less than 30%. At 18°C the low values in successful fertilization may be due to the degradation of the eggs. A delay in addition of sperm after release of the eggs increases the time in which first cell cleavage appears from the moment of insemination. Variation of the biochemical composition of unfertilized eggs over time was also studied. At 18°C proteins, lipids and carbohydrates decreased by about 45, 46 and 48%, respectively, after 5 h (only 4% of total eggs were fertilized) from spawning. At 10” and 14°C the decreases after 11 and 8 h from spawning, respectively, were lower. Before these times, the decrease of biochemical constituents was not so great and fertilization rates were still high. Keywords: perature
Biochemical
composition;
Egg; Fertilization;
Myths
gdoprovincialis;
Tem-
1. Introduction Bivalve larvae have been reared successfully over many years. However, factors which are of primary importance in the rearing of bivalve larvae are that the eggs * Corresponding author. 0022-0981/95/$09.50 @ 1995 Elsevier Science B.V.All rights reserved SSDI
0022-0981(95)00062-3
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should be fertilized successfully and that the fertilized eggs should produce a high proportion of normal, healthy larvae. Because of that, it is necessary to ensure that conditions during the fertilization and embryonic development are optimal for the production of normal larvae. Special features of Rias from Galicia made this region the main producer of mussels in the world. Spat and young individuals, used for culture on ropes suspended from rafts, are obtained naturally from the coast. In this environment, mussels are not subjected to stressful conditions like high changes in temperature and salinity or long periods without available food. This fact enables a fast growth of mussels. However, improvement in rearing bivalves requires a deeper knowledge of the whole life-cycle of these organisms. Egg quality and conditions in which fertilization and early development take place are very important factors. A number of studies (Barnes, 1965; Pandian, 1967, 1969, 1970; Holland, 1978; Whyte et al., 1987, 1990) have shown that proteins and lipids are the main constituents of eggs from different species of marine invertebrates (mainly, bivalves and crustaceans). It is also suggested that egg quality varies with the level of lipids stored during vitellogenesis. Bayne et al. (1978) showed that size and content of egg reserves were interrelated, with smaller eggs released from stressed females of Myths edulis containing less lipid and protein than eggs from normal females. Likewise early larval development has been correlated with the lipid content of eggs released from M. edulis (Bayne et al., 1975), Mercenaria mercenaria and Crassostrea virginica (Gallager and Mann, 1986). Other factors to be considered before fertilization are the relationships between gametes and the physical conditions at the moment of fertilization. Some authors have suggested that high sperm concentration and also high concentrations of eggs (Loosanoff & Davis, 1963) may adversely affect rearing success. Despite this, few studies have examined the relationships between sperm and egg concentrations. Loosanoff & Davis (1963) quoted an optimal value of about 30000 eggs/ml in Mercenaria mercenaria. Gruffydd & Beaumont (1970) indicated a concentration of 700 eggs/cm* and low number of sperms (l-100 per egg) in Pecten muximus. Recently, Sprung & Bayne (1984) reported a value of lo4 sperms/ml and not more than 20 eggs/ml in M. edulis. This work was designed to study the following aspects: (1) to determine the physical parameters and biochemical composition in eggs of the mussel, M. gulloprovinciulis (Lamarck) cultured in Rias from Galicia; (2) to know how long fertilization may be delayed, at different conditions of temperature, after release of gametes and, (3) to verify if this delay could involve variations of the initial biochemical content of the unfertilized eggs. The latter fact could have significant consequences on both embryonic development and the initial larval stages.
2. Material and methods
Mature adult mussels of 7-10 cm shell length were collected during the period of March-May, 1989, from Ria de Ares, Galicia, and kept for 48 h in the
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laboratory, without food, at 4°C. Spawning was then induced by vigorous shaking, followed by immersion of each animal in 500-ml tanks with 1 ,um filtered seawater at 20” 2 2°C and 32%, salinity, always under vigorous aeration. After spawning, eggs and sperm were filtered through a 80 and 30 pm sieve, respectively, to remove faeces and other particles of larger size. Number of gametes were determined using a Neubauer chamber for sperm and a Sedgewick rafter counting-cell for eggs. Then, eggs and sperm were kept at a concentration of about 300 per ml and lo5 per ml, respectively, under light aeration. Eggs were kept at lo”, 14” or 18°C depending on the experimental temperature used and sperm at 4°C. Egg samples of the different spawns were taken in order to determine their biochemical composition and physical parameters (diameter, density, dry weight). Previous experiments made in our laboratory gave optimum concentrations of about lo4 sperms per ml and about lo-30 eggs per ml, which are similar to those indicated by Sprung & Bayne (1984) in M. edulis. At different times after spawning eggs and sperm were added to 0.2 pm filtered seawater in a ratio of -10-30 eggs per ml and lo3 sperm per egg. Fertilization was assumed to have been successful when the eggs showed signs of cell cleavage. At different times after addition of sperm, egg samples were taken and immediately examined to determine the time of first cleavage and the percentage of fertilized eggs. The time at which fewer than 30% of eggs were fertilized was chosen as the maximum period over which fertilization remained possible. Mussels treated in a similar way were used for a further experiment. After the eggs were spawned and counted, they were added to 10-l tanks with filtered (0.2 pm) seawater at the same concentration and temperatures as before but without fertilization. At different times, samples of unfertilized eggs were collected for a further analysis of the biochemical composition. All samples collected were rinsed with isoosmotic ammonium formate (3% w/v) to remove salt. Then, those samples used for the analysis of the biochemical composition were frozen, freeze-dried and stored at -80°C until analysis. Egg diameter was determined by using a Nikon Optiphot-2 microscope and egg density by extrapolation from density curves obtained with commercial markers (Pharmacia Fine Chemicals) whose densities were determined previously using a refractometer under the conditions used in our experiments. The silica-sol Percoll (Pharmacia Fine Chemicals) was combined with artificial seawater, made as in Murakami and Machemer (1982), to prepare density gradients isoosmotic with seawater at 32%0 and 18°C by the procedure of Crespo (1989) modified by Sedan0 (1992). Weight data were obtained using a Sartorius 2405 electrobalance (accurate to 0.1 pug). Samples of known number of eggs were placed in preashed, preweighed aluminium boats and dried at 60°C in an oven until constant weight in order to determine total dry weight. Then the samples, in aluminium boats, were combusted in a muffle furnace for 6 h at 450°C and ashed samples were then weighed. The amount of ash-free dry weight is calculated as the difference between the total dry weight and the ash weight. This value is presented as percentage relative to total dry weight (% AFDW).
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F.J. Sedan0 et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
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For biochemical analysis, 500 ,ul of de-ionized and distilled water was added to 2-3 mg of freeze-dried sample. The sample was then homogenized in a teflonglass homogenizer always under refrigeration with ice. The homogenized material was fractionated according to the procedure of Holland & Gabbott (1971) as modified by Holland & Hannant (1973). Total carbohydrates, total lipids and ribonucleic acid (RNA) were determined by the methods described in Holland & Hannant (1973) and were defined in the same way. Total proteins were defined as material hydrolyzed by 5N NaOH and precipitated proteins as material insoluble in 5% trichloracetic acid (TCA); both were determined by a modification of Lowry’s method (1951) based on the results obtained by Bensadoun & Weinstein (1976) and Hess et al. (1978) using bovine serum albumin as a calibration standard. Statistical treatment of data was according to Holland & Gabbott (1971). Values for the coefficient of variation (C.V.) about the means of the replicate determinations were less than 0.05 with the exception of ribonucleic acid (RNA) which was less than 0.10. The number of replicate samples necessary to obtain repeatable biochemical results, determined by using Student’s f-test as Bartlett (1979) were two. When necessary, data were treated by using one way ANOVA analysis and critical level of significance was taken to be at 5% (p < 0.05).
3. Results and discussion The results indicated a great homogenity among different spawns (Table 1). The dry weight of M. gulloprovincialis eggs varied from 50 to 56 ng/egg with a mean value of 53 ng. Other authors quoted values for M. edulis eggs of 51 ng (Ockelmann, 1965), 52.5 ng (Bayne et al., 1975) 47.3 ng (Thompson, 1979) and 67.4 ng (Sprung, 1983; 1984) which agree with those cited in this paper. As Table 1 shows, mean biochemical composition of eggs from M. galloprovincialis was dominated by protein (45% of total dry weight) and lipid (22%) while carbohydrate (3% ) was a minor component. The range of values for eggs of M. edulis, M. californianus, Astarte sp., Placopecten magellanicus, Spisula solidissima, Mya truncata, Patinopecten yessoensis and Crassadoma gigantea has been reported
as 37-68%, 9-24% and l-8% of the total dry weight for protein, lipid and carbohydrate, respectively (Holland, 1978; Whyte et al., 1987; 1990; Gabbott, 1983). The values quoted for M. gulloprovincialis eggs are within these ranges indicating that they are comparable to those of other marine invertebrates. However, lipid and carbohydrate levels were higher than the respective mean values of 17.1 and 1.9% calculated for M. edulis eggs (Holland, 1978). Protein and lipid contributed with 2.5 and 2.1 kcal/g, respectively, to the total energy content, while carbohydrate (0.2 kcal/g) seemed not to be an important energy reserve. Mean AFDW was 89.3%. This implies an ash content of 10.7% that is similar to the calculated value of 8.9% for M. edulis eggs (Sprung, 1984). Despite this, about 23% of the organic content was unaccounted for by the analytical procedures used, and was presumed to consist of low molecular weight components.
18.5 1.16
24.1 1.09
Mean
SD
18.0 19.9 20.2 17.5 17.6 19.1 16.7 18.6 19.0
24.3 25.8 24.9 23.6 22.5 24.9 22.6 23.9 24.6
1 2 3 4 5 6 7 8 9
TCA proteins
Total proteins
Spawn
7.9 0.64
7.8 8.9 7.3 7.5 7.3 7.6 8.1 7.4 8.9
Neutral lipids
Total
lipids
3.8 0.62
3.9 4.0 3.1 2.9 4.0 3.6 4.3 3.7 5.0
Phospholipids
0.6 0.08
0.5 0.7 0.5 0.7 0.5 0.6 0.5 0.6 0.6 1.2 0.33
1.1 1.3 1.1 1.9 0.8 1.1 1.3 1.0 1.6
Polysaccharides
carbohydrates
Free reducing sugars
Total
3.0 0.53
3.6 3.2 3.6 2.8 2.5 3.0 2.3 2.4 3.6
RNA
1.057 0.0014
1.055 1.056 1.057 1.058 1.059 1.058 1.056 1.058 -
+ + + + + + + +
Density
0.0031 0.0029 0.0015 0.0007 0.0002 0.0020 0.0012 0.0025
61 1.0
61 + 60+ 61 + 60 + 60 + 63 + 61 + 61 + 62 +
0.3 1.2 1.0 1.4 1.5 0.8 2.1 1.2 1.9
Diameter
+ + + + + + + + + 53 1.9
50 55 52 54 52 53 55 55 56
4.8 2.8 4.6 1.0 1.7 3.0 1.5 2.5 3.2
Dry weight
+ + + + + + + + + 89.3 1.97
88.8 86.4 87.9 87.2 90.4 91.3 91.2 88.7 91.9
AFDW
1.18 0.94 1.02 0.63 1.53 0.39 0.54 1.28 0.99
Table 1 Biochemical content (values are mean of two replicate samples) and physical parameters of eggs from M. galloprovincialis. All measurements in pg 10mgind. except diameter (pm), density (g . cm-‘) and AFDW (%). Diameter (n = 30) density (n = 4) dry weight (n = 3) and AFDW (n = 3) are mean +SD. Lower part of table presents mean values +SD from nine different spawns
Y ec:
z ; 2 ? s 2 g k!
$
!? P
5
3 5 2 3 $ z e
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F.J. Sedan0 et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
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Egg diameter varied from 60 to 63 pm with a mean value of 61.2 ,um which is smaller than those quoted for M. edulis (68-70 pm) by Bayne (1976). Egg density ranged from 1.055 to 1.059 g * cm -’ with a mean value of 1.057. Almost 72% of the eggs from the nine different spawns of M. galloprovincialis banded between 1.056 and 1.058 g. cme3 (Fig. 1). Th ese results indicate that there is a low variation in the values of density which follow an unimodal distribution of frequencies. Gallager & Mann (1986) indicate the important role of the egg density in dispersal of embryonic stages. They also suggest that the high variation of egg densities among species could be a function of evolutionary adaptation of reproductive strategies. Species with a unimodal distribution of egg densities would be at an advantage, compared to species with a multimodal distribution, in stable environments that favour smaller and more dense eggs. The results of our study agree with this hypothesis and, in this way, eggs from M. galloprovincialis, living under conditions present in Rias, are small (61.2 pm) and dense (1.057 g. cm-“). These characteristics confer less hydrodynamic flexibility on the mechanism of dispersal, but permit that eggs may be retained in this environment. The results in Table 2 indicate that there is a long period over which M. gulloprovincialis eggs can be fertilized after spawning for all temperatures tested. Successful fertilization (i.e. >30%) was still possible 4 to 8 h after gamete release and the longest periods of delayed fertilization were tolerated at the lowest temperatures used. The data agree with those quoted for M. edulis by Bayne (1976) which indicate successful fertilization after 4 to 6 h at 18°C and by Sprung & Bayne (1984) which indicate successful fertilization after 6 to 11 h at temperatures of 8” to 16°C. Motility indicates survival of the sperm and the sperm maintained their motility for periods of time longer than those tolerated for fertilization. The limiting factor in successful fertilization may reside in the egg, since the sperm’s capacity for
0’ 1.056
1.054
Egg density
Fig. 1. Mean
density
distribution
of eggs from
1.056
1.060
(g/crn3)
nine spawns
of M. galloprovincialis.
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et al. I J. Exp. Mar. Biol. Ecol. 192 (1995)
Table 2 Fertilization success in eggs from Mytilus galloprovincialis and 18°C. Values are mean from three replicate samples Temperature (“C)
Addition time of sperm after spawning (h)
at different
Fertilization Mean
75-85
81
times after spawning
success (% ) SD
Appearance time of first cell cleavage (h)
4.98 0.84 3.62 9.14 11.57 _
2.0 2.0 2.3 2.3 2.3 2.5 _
0 2 4 6 8 10 11
86.0 90.3 92.6 90.3 86.8 22.2
14
0 1 2 3 4 5 6 7 8
82.0 85.1 83.7 83.5 74.0 70.8 77.2 45.7 <30.0
1.07 1.16 0.68 4.60 1.74 0.67 3.11 8.71
1.3 1.4 1.3 1.3 1.4 1.4 1.5 1.5 1.5
18
0 2 2 3 4 5 6
82.5 93.0 95.0 90.0 94.0 4.0 _
3.53 1.63 0.69 2.36 1.04 5.95 _
1.3 1.3 1.5 1.7 1.8 2.0 _
10
at lo”, 14”
fertilization was assumed not to decrease greatly when they are kept at low temperatures. Successful fertilization was still possible within 8, 7 and 4 h after spawning at lo”, 14” and 18°C respectively (Table 2). After these times, percentage of fertilized eggs decreased until values fewer than 30%, with lower fertilization at 18°C than at the other temperatures. The reason for these results may be in the degradation of the eggs which show irregular forms and not the spherical shape presents at the moment of spawning. This event is clearly manifest at 10,8 and 5 h after spawning at lo”, 14” and 18°C respectively. Although there are some fertilized eggs at these times, most of the embryos are not viable showing abnormal cell cleavage or remaining at the stage in which meiosis is reinitiated (extrusion of the first polar body). Possibly, bacteria present either in adult mussels or in water could be responsible of the degradation of eggs. This would be proportional to the holding temperature and its duration. Another significant point is that delay in the addition of sperm delays the appearance of the first cell cleavage increasing when the delay in insemination is bigger (Fig. 2). A number of studies in pelecypods have shown that during the
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l.OJ
0
2 Time
4
of insemination (hours)
6
8
after
Fig. 2. Relationship between delay in the insemination after spawning first cell cleavage after spawning. Regression lines are shown.
10
spawning
and delay in the appearance
of
period of interaction between egg and sperm, referred to as gamete binding (Hylander & Summers, 1977) the acrosomal process interdigitates with microvilli of the egg. Likewise, orientation of microvilli to spermatozoa represents an active process in the association of the egg and sperm (Long0 and Anderson, 1970; Popham, 1975). On the other hand, Dub6 & Dufresne (1990) indicate that metaphase arrest in blue mussel (M. e&&s) oocytes requires the continous synthesis of short-life proteins, the destruction of which is sufficent to induce meiosis resumption with extrusion of the first polar body. The mechanism that induces the inhibition of synthesis of these specific proteins or the increase of some specific proteolytic activities against these short-life proteins is not yet known. Interaction between egg and sperm must have an important role on this mechanism. The delay in the appearance of the first cell cleavage could result from the progressive aging of the egg. The aging, which is clearer at temperatures of WC, may alter the membrane’s properties with respect to egg and sperm binding or the inhibition of protein synthesis related to metaphase arrest. To check if delay in fertilization could involve some effect on the level of the different macronutrients of unfertilized eggs, the variation of the biochemical content over time was determined at the different temperatures used in the previous experiment. The results indicate that a significant (p < 0.05) decrease in energetic reserves of the egg occurred after gamete release at all temperatures tested (Fig. 3). At 18°C proteins decreased about 45% of the initial value in the eggs, lipids 46% and carbohydrates 48% after 5 h from spawning, when fertilization rates are the lowest. At 14°C the decrease was about 22% for
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Fig. 3. Changes in biochemical content with respect to time after spawning in unfertilized eggs from M. galloprovinciah at three temperatures tested. Values are mean k SD from two replicate samples. TP, total proteins; NL, Neutral lipids; PL, Phospholipids; POL, polysaccharides; FRS, Free reducing sugars; RNA, Ribonucleic acid.
proteins, 16% for lipids and 17% for carbohydrates after 8 h. At 10°C the decrease was about 20% for proteins, 27% for lipids and 34% for carbohydrates after 11 h. We must notice that the great decrease of biochemical components at 18°C may reside in the degradation of eggs after 5 h. However, if we consider the values after 4 h from spawning, the decreases are not so high (14% proteins, 19% lipids and 47% carbohydrates). The same event occurs when we consider the decreases after 10 h and 6 h from spawning at 10” and 14°C respectively. So far, lower rates of fertilization coincide in time with greater decreases of biochemical components. The fact may be explained by the degradation of eggs after those times. However, when we consider the rates of fertilization obtained after 8,7 and 4 h after spawning at lo”, 14” and 18°C and the biochemical content of eggs at these times, we observe success in fertilization but a manifest decrease of the biochemical components. Fertilization can be delayed for long periods of time. Manipulation of gametes during these periods can be possible without a great effect on the rates of fertilization. However, a loss of energetic reserves takes place within these times of delayed fertilization; this could have important effects on the further embryonic development and first larval stages. Therefore, there is an advantage to fertilize soon after gamete release, especially at 18°C a temperature normally used in both laboratory experiments and commercial hatcheries, so that a decrease of biochemical constituents is not a limiting factor for rearing success.
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Pandian, T.J., 1967. Changes in chemical compostion and caloric content of developing eggs of the shrimp Crangon crangon. Helgol. Wk. Meeresunters., Vol. 16, pp. 216-224. Pandian, T.J., 1969. Yolk utilization in the gastropod Crepidula fornicata. Mar. Biol., Vol. 3, pp. 117-121. Pandian, T.J.. 1970. Ecophysiological studies on the developing eggs and embryos of the European lobster Homarus gammarus. Mar. Biol., Vol. 5, pp. 154-167. Popham, J.D., 1975. The fine structure of the oocyte of Bankia ausfralis (Teredinidae, Bivalvia) before and after fertilization. Cell Tissue Res.,Vol. 157, pp. 521-534. Sedano, F.J., 1992. Contribution al estudio de1 metabolismo energetic0 durante 10s desarrollos embrionario y larvario en Mytilus galloprovincialis Lmk. Tesis Doctoral. Universidad de Santiago de Compostela (Spain). Sprung, M., 1983. Reproduction and fecundity of the mussel Myrilus eduhs at Helgoland (North Sea). Helgol. Wiss. Meeresunters., Vol. 36, pp. 243-255. Sprung, M., 1984. Physiological energetics of mussel larvae (Mytilus eduhs). I. Shell growth and biomass. Mar. Ecol. Prog. Ser., Vol. 17, pp. 283-293. Sprung, M. & B.L. Bayne, 1984. Some practical aspects of fertilizing the eggs of the mussel Mytilus edulis L. J. Cons. Int. Explor. Mer., Vol. 41, pp. 125-128. Thompson, R.J., 1979. Fecundity and reproductive effort of the blue mussel (Mytilus edulis), the sea urchin (Strongylocentrotus droebachiensis), and the snow crab (Chiomectes opilio) from populations in Nova Scotia and Newfoundland. J. Fish. Res. Board Can., Vol. 36, pp. 955-964 Whyte, J.N.C., N. Bourne & CA. Hodgson, 1987. Assesment of biochemical composition and energy reserves in larvae of the scallop Patinopecten yessoensis. J. Exp. Mar. Biol. Ecol., Vol. 113, pp. 113-124. Whyte, J.N.C., N. Bourne & N.G. Ginther, 1990. Biochemical and energy changes during embryogenesis in the rock scallop Crassadoma gigantea. Mar. Biol., Vol. 106, pp. 239-244.