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.     

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.     

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.     

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.     

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.     

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     

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.     

Larval growth and settlement of the European oyster (Ostrea edulis) as a function of food quality measured as fatty acid composition

Larval growth rate and settlement of the European flat oyster Ostrea edulis were experimentally studied as a function of the composition of dietary fatty acids. Diets differing in fatty acid composition were composed by mixtures of the microalgae Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans. Fatty acid content in the tissue of the feeding larvae, analyzed by gas chromatography and mass spectrometry, reflected the composition in the diet. Larval growth rate was significantly correlated to the three omega-3 polyunsaturated fatty acids (PUFA) C18:3, C18:4 and C22:6, with minor differences for neutral and polar lipids. No relation between growth rate and the omega-3 PUFA C20:5 was detected, a PUFA often implied as essential for bivalves. It is suggested that naturally occurring variability in fatty acid composition may constrain larval growth. In settlement experiments in both still water and flume flow little substrate selectivity was found for some contrasting substrates. It is concluded that differences in dietary fatty acids may explain as much of settlement success as the variability of substrates. Received: 12 October 1998 / Accepted: 6 April 1999     

Gill morphogenesis in the oyster Ostrea chilensis

Despite the importance of the gills in the acquisition of food by suspension-feeding bivalve mollusks, there is almost no information on gill organogenesis. By means of a series of stereoscan electron micrographs, this paper describes gill development in the Chilean oyster, Ostrea chilensis, from the brooded larval stages to 1-month-old spat. A single gill rudiment was observed on each side of the mantle at a shell length of 320 μm, and the rudiments increased in number and size until the end of the brooding period. During metamorphosis the gill filaments increased in number from 5 or 6 to between 7 and 9. The loss of the velum and the absence of functional gill filaments during metamorphosis are consistent with previous observations of weight loss during this critical period of the life history, because the newly settled juvenile lacks the ability to remove particles from suspension. The end of metamorphosis (100% of spat with dissoconch edge) was reached 36 h after larval settlement, when the gill filaments began to grow cilia, which increased in density and differentiated as the spat developed and acquired the capability of suspension-feeding, accounting for the increase in body weight previously recorded during this stage. The larval rudiments gave rise to the inner demibranchs. The outer demibranchs were observed 10 days after settlement, located between the inner demibranch and the mantle. In 1-month-old spat, the gill did not show differentiation between primary and secondary filaments, indicating that the heterorhabdic condition characteristic of adult oysters had yet to be attained. Received: 11 December 1998 / Accepted: 21 August 2000     

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.     

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.     

Allozyme variation in global populations of striped mullet, Mugil cephalus (Pisces: Mugilidae)

The striped mullet, Mugil cephalus Linnaeus, 1758, is one of the few species of marine shore fish with a worldwide circumtropical distribution. Because of this distribution and the dependency of M. cephalus on coastal waters during various phases of its life cycle, as well as nearshore living habits, questions have been raised regarding levels of genetic divergence and gene flow among transoceanic populations. To cast more light on this, allozyme variation at 27 presumptive gene loci was investigated in ten globally diverse populations. The observed heterozygosity ranged from 0.018 (Hawaii) to 0.081 (Florida), averaging 0.050. The proportion of polymorphic loci showed a similar trend. Several populations were characterised by fixed allelic differences. Estimated gene diversities were very high, the allele frequency variation among populations was found to be 68%; genetic distances reached 0.242, with an average of 0.117. Estimated rates of gene flow were high among Mediterranean populations (Nm = 7.26), and between Mediterranean and East Atlantic populations (Nm= 2.86), but extremely low between non-contiguous populations within the Indian, Pacific and Atlantic Oceans, where Nm ranged from 0.03 to 0.05. Received: 7 April 1997 / Accepted: 4 February 1998     

Seasonal variations in condition,reproductive activity and biochemical composition of the flat oyster,Ostrea edulis,from San Cibran (Galicia,Spain)

The influence of environmental parameters (temperature, salinity and available food) on the condition, reproductive activity and biochemical composition of a native population of Ostrea edulis L. in San Cibran (Galicia, Spain) was studied between September 1988 and December 1989. Histological preparations of gonads showed that gametogenesis started when temperature was at its lowest in winter. The water temperature in San Cibran never fell low enough to interrupt it. Gametogenesis proceeded slowly and spawning took place in May–June, although the predicted time of ripening was early March. Salinity in San Cibran was relatively stable throughout the year; it did not seem to have any influence on gametogenesis. Available food appeared to be a very important factor in controlling gonad growth, once gametogenesis was initiated. The major concentration of suspended organic particulate matter was present in the spring at the time of rapid gonadal maturation. Only one spawning period was observed. Larvae were released when the food in the water was high. Seasonal changes in the main biochemical components of this oyster were determined for a standard individual. Lipids and carbohydrates presented a similar time-course whereas proteins were constant. When food was abundant, energy reserves were built up. Spawning produced a decrease in biochemical constituent levels, and recovery coincided with the phytoplankton bloom. The stored reserves, mainly lipids, were used to overcome a state of energy imbalance in late autumn associated with low food availability. Results show this oyster to be an opportunist organism which concentrates its reproductive effort during a short period of favourable conditions and which is directly dependent on nutritive availability in the environment.     

Allozyme and mitochondrial DNA variation in Cuban populations of the shrimp Farfantepenaeus notialis (Crustacea: Decapoda)

We investigated the genetic diversity among populations of the shrimp Farfantepenaeus notialis, the most abundant penaeid species around Cuba. A total of 25 allozyme loci were analyzed in samples of shrimps from seven localities at the south central platform of the island (Ana María Gulf). Samples from three of these localities and from Batabanó Gulf and Guacanayabo Gulf at the south west and south east platforms of the island, respectively, were also characterized at the mtDNA level through sequence variation of a 2027 bp segment including part of the COI and COIII genes. Of the 25 allozyme loci studied 9 were polymorphic: Akp2, Akp3, AmyB, Est3, Gdh, GP7, and Per1, 2 and 3. In contrast to mtDNA, the pattern of allozyme variation among localities revealed strong population structuring at Ana María Gulf, with significant F _st in all pairwise comparisons. The magnitude of F _st estimates as well as the grouping pattern obtained by a UPGMA analysis based on a distance matrix indicated that the level of differentiation was concordant with the geographical position of the localities and the hydrographic regime. Homogeneity of mtDNA suggested that differentiation of allozyme loci might be due to more recent events rather than historical isolation of the sampled populations. Ana María and Guacanayabo Gulf populations were differentiated by mtDNA from Batabanó Gulf, at the southwestern end of the island. The analysis showed three restriction site differences among them, suggesting genetic isolation of the two regions. The present results also suggest that an artificial introduction of larvae from Tunas de Zaza into Batabanó Gulf, in an effort to repopulate this fishing region, may have been ineffective. Received: 13 December 1999 / Accepted: 2 October 2000     

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.     

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.     

Allozyme variation in populations of the dog-whelkNucella lapillus (Prosobranchia: Muricacea) from the South West peninsula of England

Four populations of the predatory gastropodNucella lapillus were sampled at sites around the South West Peninsula of England in 1986, and analysed for allozyme variation at 18 enzyme loci. Two of these loci, Gpd-1 andHk-1, exhibited sex-specific phenotypes. An absolute locus association was observed between two other loci,Mdh-1 andEst-3. This association was only found at one site (Prawle), and it is suggested that the presence of chromosomal polymorphisms could explain this finding. As a measure of overall similarity, Nei's genetic identity statistic,I, was calculated; the mean for all populations was 0.989, with values ranging from 0.981 to 0.997. Although similar on this gross level, considerable interpopulation variation was evident. Observed mean heterozygosity (per locus) ranged from 0.043 to 0.104 (mean 0.074). Populations differed also in the loci at which significant heterozygote deficits were seen (of the seven deficits recorded only those at thePep-1 locus were consistent across sites) and in the presence of rare alleles undetected elsewhere. The variation observed showed no correlation to shell morphology or geographical distance and confirmed the conclusion that species of the genusNucella show considerable disjunct variation.     

Molecular and morphometric variation in European populations of the articulate brachiopod Terebratulina retusa

Molecular and morphometric variation within and between population samples of the articulate brachiopod Terebratulina spp., collected in 1985–1987 from a Norwegian fjord, sea-lochs and coastal sites in western Scotland, the southern English Channel (Brittany) and the western Mediterranean, were measured by the analysis of variation in the lengths of mitochondrial DNA (mtDNA) fragments produced by digestion with nine restriction endonucleases and by multivariate statistical analysis of six selected morphometric parameters. Nucleotide difference within each population sample was high. Nucleotide difference between population samples from the Scottish sites, both those that are tidally contiguous and those that appear to be geographically isolated, were not significantly different from zero. Nucleotide differences between the population samples from Norway, Brittany, Scotland and the western Mediterranean were also very low. Morphometric analysis confirmed the absence of substantial differentiation.     

Genetic structure of wild European populations of the invasive Pacific oyster Crassostrea gigas due to aquaculture practices

As a result of aquaculture activities, Pacific oysters Crassostrea gigas (Thunberg, 1793) have invaded European coasts. Using seven microsatellites, we found virtually no genetic differentiation between natural populations throughout the European range (from the south of the Wadden Sea (the Netherlands) to the south of France) and French cultivated oysters. The genetic homogeneity of Pacific oyster samples appears to be the result of repeated transfers from same seed stocks made for aquaculture and, to a lesser extent, widespread dispersal due to specific biological traits of this species. The only genetic differentiation of Sylt population in the north of the Wadden Sea (Germany) suggests a stronger, persistent impact of ongoing supply of new genetic material from hatchery production, corresponding to seeds selection made by breeders. All of our genetic data highlighted the importance of aquaculture practices on the genetic structure of the keystone invader C. gigas in Europe.