X-ray diffraction study of calcification processes in embryos and larvae of the brooding oyster Ostrea edulis

X-ray powder diffraction was used to study shell calcifications of the oyster Ostrea edulis, sampled in the Limski Kanal, Istria (Adriatic Sea), in May 1992. All the developmental stages were followed, from the embryonic stage through the transition between the trochophore and veliger larva (prodissoconch I and II) and later, after swarming, the pelagic free-swimming larval stages, up to their settlement and attachment (from the D-shaped to the fully formed pediveliger larva), and finally during metamorphosis and juvenile stages (dissoconch). In the first gastrula stage, only an amorphous tissue is present (a periostracum and organic matrix). The beginning of shell formation (at the end of gastrulation) in early trochophores is manifested by the appearance of calcite (up to 1–7% of total volume) and then aragonite (about 1%). In the later stage of the veliger larva the fraction of calcite decreases as well as the amorphous fraction, while the fraction of aragonite rapidly increases. In the prodissoconch II stage and during the whole pelagic period aragonite is dominant, accompanied by a very small amorphous fraction and traces of calcite. The shell mineral composition does not change until metamorphosis, whereupon the fraction of calcite rapidly increases and the fraction of aragonite decreases. The postmetamorphic valves of the juvenile and adult oyster consist mainly of calcite, except the resilium and myostracum which remain aragonitic, possibly as a continuation of the inner layer of the larval shell. Received: 28 May 1997 / Accepted: 1 July 1997     

Transport of naphthalene in the oyster Ostrea edulis

In small oysters (Ostrea edulis), transport of naphthalene between tissues is primarily by diffusion and not via the circulatory system. In intact oysters, accumulation in the adductor muscle and body followed accumulation in the gills after a large lag-time. In isolated tissues with no shell to impede water flux over the body and adductor muscle, there was no lag-time. The molecular diffusivity (D) of naphthalene in oyster tissue, estimated by Fick's second law of diffusion is D=8x10^-8 cm² s^-1, a value similar to D determined for lateral diffusion of lipophilic compounds in lipid membrane systems.     

Biochemical correlates of dissolved mercury uptake by the oyster Ostrea edulis

From previous work, the equilibrium concentration factor for dissolved mercury in the digestive gland of Ostrea edulis Linnaeus was found to be three to four times higher than that in the gills. In the present study, an analysis of soluble protein revealed values of 49.3±14.2 mg g wet tissue^-1 for the digestive gland and 0.7 ±0.1 mg g wet tissue^-1 for the gills. Starvation significantly reduces the soluble protein level of the digestive gland to 31.1±6.4 mg g^-1 and that of the gills to below the limit of detection. These results suggest that the difference in concentration factors between the gills and digestive gland may be based on a quantitative difference in macromolecular binding sites. However, the uptake of dissolved mercury over a period of 48 h was considerably greater in the gills, so that although the soluble protein content of the tissue may influence the final concentration factor, it does not appear to affect the rate at which this equilibrium is achieved. A more detailed investigation of the mechanism of dissolved mercury uptake by oyster gills has been carried out using isolated tissues. The process is inhibited by 5mM 2–4 dinitrophenol, by the absence of a readily metabolizable substrate (dextrose) in the uptake medium, and by 30mM K⁺. The effect of K⁺ necessitated further investigation with a specific inhibitor of K⁺ transport. Strophanthin G (ouabain), at a concentration of 0.01 mM, caused a significant increase in mercury uptake.     

Energy metabolism of newly settledOstrea edulis spat during metamorphosis

An energy budget for metamorphosis in oyster spatOstrea edulis L., collected in the Instituto Español de Oceanografía during March–April 1988, was determined. The energy cost of metamorphosis was calculated from measurements of biochemical components which form the principal reserves of energy and which are used significantly during metamorphosis. The results indicated that protein supplied most of the energy inO. edulis spat. The idea that neutral lipids were used selectively as an energy reserve during the metamorphosis of this bivalve marine larvae was questioned.     

Allozyme variation in European populations of the oyster Ostrea edulis

Variations at 22 enzyme coding loci were surveyed in 11 populations of the oyster Ostrea edulis L., which were sampled between 1988 and 1990 along the Atlantic and Mediterranean coasts of Europe. Atlantic oyster beds suffered a steady decline during the last century, and restocking of beds with oysters of foreign origin has probably resulted in a high degree of interbreeding of natural oyster stocks from all Atlantic Europe. Our study confirms the low levels of genetic variability previously reported for the oyster populations from the Atlantic coasts, and extends it to the Mediterranean coasts. The locus arginine-kinase (ARK ^*) exhibited a high degree of interpopulation differentiation (F _ST=0.289), resulting from extensive variation in gene frequencies along a geographical cline. However, the overall genetic differentiation between populations was slight, and similar to that reported for other local populations of bivalves (mean genetic distance between populations is 0.010, mean F _ST=0.062). A general pattern of increasing differentiation along the coastline in an Atlantic-mediterranean direction emerged; but genetic differentiation among the Atlantic populations was not significantly lower than that observed among the Mediterranean populations. This and other results suggest that the effects of extensive transplantation of oysters among various areas in Europe are detectable only in some particular localities. The geographical distribution of low-frequency alleles suggests a restriction to gene flow outwards from the Mediterranean Sea, across the Straits of Gibraltar.     

Uptake and accumulation of naphthalene by the oyster Ostrea edulis,in a flow-through system

A flow-through system was used to follow naphthalene and naphthalene metabolite accumulation in the seawater and in the tissue of the oyster Ostrea edulis. After 72 h, 82.5% of the naphthalene carbon was recovered from the system. Glucose was added to seawater to stimulate the pathways of glucose metabolism in the oysters. Streptomycin (100 ppm) reduced microbial oxidation of naphthalene and glucose, and reduced bacterial growth. However, even in the presence of streptomycin, microbial oxidation of naphthalene was considerable. The main oxidation product recovered from seawater was ¹⁴CO₂. Radioactivity was also associated with compounds which separated by TLC with 2- and 1- naphthol. The pattern of naphthalene uptake and accumulation in oyster tissues was relatively constant after only a few hours of exposure to naphthalene. The potential of tissues to accumulate naphthalene was shown to be a function of multiple variables such as nutritional state, lipid concentration, length of exposure to naphthalene, and the external naphthalene concentration. Carbon-14-labeled metabolites derived from ¹⁴C-naphthalene were consistently recovered from digests of the oyster tissues. Non-CO₂ alkaline-soluble substances were the primary metabolites. Hexane-extractable substances, which separated by TLC with known standards of 2- and 1- naphthol, were consistently recovered from seawater and tissue digests. It was not possible to conclude that these metabolites were a result of naphthalene metabolism by oyster enzyme systems.     

The nature of zinc and copper complexes in the oyster Ostrea edulis

The zinc and copper associated with the soft tissues of the oyster Ostrea edulis Linnaeus have been separated into a soluble component and a tissue-residue, cell-debris bound component. In the case of zinc, the tissue-bound component was found to contain at least two species of complex; a firmly-bound species, exchangeable with ⁶⁵Zn²⁺ and a less-firmly, reversibly-bound species, exchangeable with ⁶⁵Zn²⁺. The soluble component, which constitutes some 40% of the total zinc and copper, was fractionated on Sephadex G-25 and the zinc and copper shown to be weakly-complexed to the small molecular weight compounds, taurine, lysine, ATP and possibly homarine (N-methyl--picolinic acid) and to be fully exchangeable with ⁶⁵Zn²⁺. These soluble complexes can act as a freely available mobile reserve of metal to ensure a constant saturation of metal-dependent enzyme systems operating under adverse environments. Sephadex G-25 acts as a weak ion-exchange resin, which can cause a translocation of zinc and copper from its soluble weak complexes and result in the spurious association of the metals with other compounds.     

X-ray diffraction study of the first larval shell of Ostrea edulis

X-ray powder diffraction was used to study the calcification of the first larval shell of Ostrea edulis (sampled in Limski kanal, Istria, Adriatic Sea in April 1986) from the trochophore stage to the veliger larvae (prodissoconch I), and development of the latter up to several days postfertilization (prodissoconch II). In the first stage, only the amorphous component is present (periostracum and organic matrix). The beginning of shell formation is manifested by the appearance of calcite (up to 1–4% of the total vol.) and then aragonite (2 to 7%). In a later stage of the veliger larvae the fraction of calcite decreases, as well as the fraction of the amorphous component, while the fraction of aragonite rapidly increases. In the prodissoconch II stage, aragonite is dominant, with a very small amount of amorphous component and traces of calcite. In contrast, the valves of the adult O. edulis are composed mainly of calcite, with traces of aragonite.     

Genetics of larvae and spat growth rate in the oyster Crassostrea virginica

The heritability of oyster (Crassostrea virginica) larval growth rate was estimated to be in the range of 0.25 to 0.50 and a significant part of this genetic variation is of the additive type. Larval growth rate and spat growth rate were found to be highly correlated. These results suggest that a selection program for faster growing larvae and spat would be successful.     

Intracellular digestion in two sublittoral populations of Ostrea edulis (Lamellibranchia)

The rhythmicity of intracellular digestion was examined in two sublittoral populations of Ostrea edulis L. On the West coast of Ireland. During 12 h cycles at each station, 20 oysters were collected each hour, grouped as sub-samples of 5, facing each of the 4 cardinal points of the compass. A segment of digestive diverticula from each oyster was examined histologically and classified according to the digestive phases of the tubules. Oysters at both stations exhibited fluctuations in digestive activity which were not correlated with tidal ebb and flow nor with orientation to tidal currents. A relationship between variations in suspended particulate matter concentration in the water body and digestion is proposed. It is suggested that increases in the levels of particulate matter, by stimulating feeding, cause a significant increase in the proportion of absorptive-phase tubules 4 to 6 h later.     

Effects of cadmium exposure on metal-containing amoebocytes of the oyster Ostrea edulis

Oysters, Ostrea edulis, were exposed to cadmium (0.1 mg l^-1) for up to 110 d (in 1982) under laboratory conditions in order to determine the effect of Cd exposure on blood amoebocytes. The results demonstrate that Cd-accumulation does not alter the total Cu and Zn concentrations in gill tissue. There was a decrease in the numbers of metal-containing amoebocytes, and electron microprobe analysis showed that this was largely due to a reduction in numbers of the mixed Cu/Zn-containing cells rather than in Cu-or Zn-containing cells. It is postulated that this response, which may involve the release of metals from amoebocytes into gill tissue, is a generalised stress response of this oyster. No evidence was found for the presence of a specific Cd-containing blood cell or Cd-binding protein in blood cells.     

The tidal rhythm of extracellular digestion and the response to feeding in Ostrea edulis

Under natural conditions, on the shore, there is a tidal rhythm for changes in pH, length, and protein and amylase content of the crystalline style of Ostrea edulis L. When oysters were kept immersed and fed continuously for 2 weeks, in the laboratory, the rhythm of extracellular digestion was lost. Oysters were fed discontinuously for 2 weeks, in the laboratory, with a 6 h-on, 6 h-off feeding regime. During the feeding period, the changes in pH, size, and protein content of the style were similar to the changes observed in the field over the period of high tide. It is our hypothesis that the tidal rhythm of extracellular digestion in Ostrea is not endogenous, but is controlled by feeding activity.     

Cadmium kinetics in oysters — a comparative study of Crassostrea gigas and Ostrea edulis

The accumulation of cadmium was investigated in two species of oysters [Crassostrea gigas (L.) and Ostrea edulis (L.)] from the same environment and in oysters of the same species (O. edulis) from two different environments (contaminated and uncontaminated), under controlled laboratory conditions (33‰ salinity, 10°C, 100 μg Cd l^-1) for up to 111 d in 1982. C. gigas accumulated cadmium twice as fast as O. edulis (1.07 vs 0.52 μg Cd g^-1 wet wt d^-1). Furthermore, O. edulis from an uncontaminated environment accumulated cadmium faster than O. edulis from a metal-contaminated environment (0.52 vs 0.34 μg Cd g^-1 wet wt d^-1). There was no effect of cadmium exposure on total soft-tissue copper and zinc concentrations. Investigation of cytosolic metal-binding using Sephadex G-75 gel-permeation chromatography indicated that binding to very low molecular weight ligands (MW<1000) accounted for>70% of the cytosolic zinc in all oysters and>40% of the cytosolic cadmium in all oysters except O. edulis from Conwy at 83 d. In copper-contaminated oysters, excess copper was also associated with very low molecular weight ligands. Intermediate molecular weight cadmium/copper-binding proteins (similar to metallothionein in molecular weight) were observed in the cytosol and were shown to differ between species in terms of their behavior on Sephadex G-75. Finally, the distribution of accumulated cytosolic cadmium in O. edulis from the contaminated environment was shown to have a unique distribution, i.e., there was no cadmium associated with high molecular weight cytosolic macromolecules. The data indicate that both genetic and environmental factors influence cadmium accumulation in oysters.     

Geographical patterns of variability at allozyme loci in the European oyster Ostrea edulis

The variability of 14 enzyme-coding genes has been analysed in samples from 19 populations of the oyster Ostrea edulis L., collected along the Atlantic and Mediterranean coasts of Europe. We found an abundance of clines, which appeared at 8 loci, including the most polymorphic (AP-2 ^*, ARK ^*, EST-4 ^*, MDH-2 ^*, ME-1 ^*, 6PGH ^*, PGI ^* and PGM ^*). Another 6 loci (ALDH ^*, EST-3 ^*, EST-5 ^*, IDH-2 ^*, MDH-1 ^*, ME-2 ^*) exhibited V-shaped patterns of gene-frequency variation, with clines at one or both sides of the Straits of Gibraltar. The observation of coincident clines at many loci can be explained by a model of secondary intergradation. The geographical location of the midpoints of the clines and V-shaped patterns suggests the existence of two ancient Atlantic and Mediterranean oyster stocks which became differentiated in allopatry and subsequently merged. Clines observed along Atlantic and/or Mediterranean coasts at the loci with V-shaped patterns must have arisen independently. The large heterogeneity observed in the levels of gene differentiation (G _ST ) across loci (G _ST ranged from 0.008 to 0.290) and important differences in estimates of gene flow obtained by different methods suggest that the populations of O. edulis are not in genetic equilibrium. Lack of population equilibrium can be due to natural selection and/or restrictions to gene flow. The average among-population variability was higher than in other oyster species that do not show incubatory habits, and represented 8.8% of the total heterozygosity. Levels of intrapopulation variability were lowest in populations from the North Atlantic, suggesting low population sizes in that area.     

Observations on the behaviour of the pediveliger of Ostrea edulis during attachment and cementing

The behaviour of pediveligers of Ostrea edulis L. during attachment can be divided into 5 clearly defined phases. These behavioural phases are generally successive, and may be a hierarchy of fixed motor patterns which terminates in the consummatory act of cementing. During attachment, the larva utilises two different mechanisms of movement: the first, a fast, smooth, gliding action is probably due to cilia only; the second, a jerky, muscular method which becomes progressively slower as cementing is approached, is related to the burrowing movements of adult lamellibranchs. The byssus thread, which is discharged during the period of muscular locomotion, was the only secretion seen, but the site of its discharge could not be determined by direct observation.     

Influence of algal concentration and temperature on the filtration rate of Mytilus edulis

The filtration rates of Mytilus edilis (=galloprovincialis; 40 mm) were determined in relation to food concentration and temperature, using pure suspensions of the unicellular alga Platymonas suecica in concentrations ranging from 3x10⁵ cells/l to 1.5x10⁸ cells/l. The rate of filtration (ml/h/mussel) generally decreased as cell concentrations increased, and dropped to low values when concentrations above 5x10⁷ cells/l were supplied. The amount of water swept clear varied continuously, and noticeable differences in the filtration activity of M. edulis were observed over short time intervals (5 min). Fluctuations of filtered volumes per unit time were greater with lower than with higher concentrations of algae. The influence of temperature on filtration activity was highest between 5°–15°C and 25°–30°C. A temperature increase from 15° to 25°C resulted in only a slight increase in filtration rate. At 5° and 30°C, filtration dropped to very low values, namely 350 and 100 ml/h, respectively. The temperature coefficients for the filtration rates of M. edulis were determined as: Q₁₀ (5° to 15°C)=4.96; Q₁₀ (10° to 20°C)=1.22. The amount of algae cells ingested per mussel per hour is directly related to food concentration. The maximum number of cells filtered/mussel/h in an algal suspension of 70x10⁶ cells/l was 21.5x10⁵ cells/h. Cell concentrations of up to 40x10⁶ cells/l were swept clear without producing pseudofaeces. The critical cell density for M. edulis was reached at algal concentrations of 70 to 80x10⁶ cells/l. Above these concentrations no normal filtration activity was observed.     

Food-particle size and selection by bivalve larvae in a temperate embayment

Epifluorescence microscopy was used to analyze the stomach contents of bivalve larvae collected in the Baie des Chaleurs (western Gulf of St. Lawrence, Canada) in order to document food-particle sizes, compare feeding among taxa, and compare the diet with the in situ phytoplankton community. Stomach contents were mainly composed of small autotrophic flagellates (<5 μm) and cyanobacteria (<2 μm), reflecting the microbial food web which characterizes these waters. More than half (55%) of all veligers examined contained algal cells of 5 to 15 μm, whereas only 3% had cells of 15 to 25 μm. Differences in the size ranges of ingested algal cells among similar-sized larvae of different species suggests that veligers actively selected food particles. Among the smallest veligers (185 to 260 μm), scallops (Placopecten magellicanus) and mussels (Mytilus edulis) ingested more <5 μm and 5 to 15 μm algae than clams (Mya arenaria). Among larger veligers (261 to 405 μm), clams contained significantly more <5 μm cells than mussels, whereas mussels contained significantly more 5 to 15 μm algae than clams. Algal cells of 15 to 25 μm were preferentially ingested by mussel veligers. Feeding also differed between different-sized veligers within taxa, i.e. the smallest clam veligers ingested fewer of 5 to 15 μm algae than the larger size classes. Mussel veligers ingested significantly more 15 to 25 μm and fewer <5 μm cells as their size increased. The dominance of ultraplankton in the nearshore waters of Baie des Chaleurs and in the stomach contents suggests that veliger larvae may be an important export path for carbon produced by small phytoplankton. Received: 17 July 1996 / Accepted: 20 September 1996     

The distribution of zinc in the oyster Ostrea edulis and its relation to enzymic activity and to other metals

The role of zinc in the oyster Ostrea edulis Linnaeus has been studied in its relation to the zinc-dependent enzymes present and in relation to the copper, calcium, magnesium, sodium, potassium and phosphate contents. Only carbonic anhydrase, alkaline phosphatase, carboxypeptidase A and malic dehydrogenase zinc metalloenzyme activities could be detected. -D-mannosidase, a zinc-dependent enzyme hitherto not reported for the oyster, was also detected. After tissue dissection into muscle, palps, gills, mantle and digestive mass and subcellular fractionation of these tissues, analysis indicated that no single tissue concentrates zinc or the zinc-dependent enzymes. The total amount of zinc found is far in excess of the amount of zinc contributed by the zinc-dependent enzymes, but the amount of non-dialysable zinc is of the same order of magnitude. It is suggested that this apparent excess of dialysable zinc is a consequence of the high levels of calcium found in the tissues, demonstrating a competition between calcium and zinc in their uptake, as is well documented in many other phyla.     

Energy budgest,growth and filtration rates in Mytilus edulis at different algal concentrations

Growth of Mytilus edulis L. was measured in aquaria with through-flowing sea water at different levels of constant algal concentrations. The amount of food and oxygen consumed by the mussels were measured over given periods as well as the changes in dry organic weight during the same periods. From these parameters it was possible to make simple energy budgets and to compare the estimated growth with actual growth, and, further, to determine growth efficiences at different food levels. Energy budgets were made for mussels grown at algal concentrations of 0, 1.6×10³, 3.0×10³ and 26.0×10³ Phaeodactylum tricornutum cells x ml^-1. The estimated growth was found to be close to actual growth at algal concentrations above maintenance level and the net growth efficiency was found to be between 18% (3.0×10³ cells x ml^-1) and 61% (26×10³ cells x ml^-1). It has been shown that the filtration rate is independent of algal concentrations between about 1.5×10³ to 30×10³ P. tricornutum cells x ml^-1. Outside this range a decrease in filtration rate was noticed.     

A study of the morphology,ultrastructure, and histochemistry of the food of the pediveliger of Ostrea edulis

During the settlement of Ostrea edulis L., the foot of the pediveliger implements temporary attachment and cementing. The morphology of the foot has been re-examined to try to clarify its role during this period. The foot has a highly developed nervous system and musculature, but mainly consists of sub-epidermal gland cells of 9 different types. Cells of the first gland contain acid mucopolysaccharide, and open over the ventral surface of the foot. The second type of gland contains a neutral glycoprotein, and opens at the tip of the foot. The third gland opens mid-ventrally half way down the foot, and largely contains an aromatic protein. The fourth type of gland contains mainly proteoglycan, and the cells open mid-ventrally behind the third gland. The fifth gland contains acid mucopolysaccharide, and the cells open onto the heel of the foot. The sixth, seventh, eighth and ninth glands contain a variety of acid mucopolysaccharides, proteoglycans and possibly a neutral glycoprotein; their cells open into the byssus duct, which discharges at the base of the heel. The grouping of the glands, position of the openings of the cells, and histochemical properties of the secretions suggest that they may be involved in localised and general adhesion of the foot during temporary attachment, as well as in cementing of the larva.