Role of biochemical energy reserves in the metamorphosis and early juvenile development of the oyster Ostrea chilensis

Metamorphosis in the Chilean oyster Ostrea chilensis was complete 36 h after release of the larvae, when 100% of the individuals showed edge growth of the dissoconch. The size of the larval shell did not change during metamorphosis, although the total dry weight of the larva decreased considerably. During this period, when the gill ciliature was undeveloped and the oyster therefore unable to feed, energy demands were met by biochemical reserves retained from the larval phase. Proteins contributed the largest quantity of energy to the metamorphosing oyster, 69.3% of the total expended, whereas lipids supplied 24.3% and carbohydrates only 6.4%. The process of metamorphosis consumed 64.5% of the energy reserves held by the pediveliger at the time of release. When metamorphosis was complete, growth began and tissue reserves were replenished, protein and carbohydrate accumulating rapidly early in the juvenile stage. Received: 26 December 1997 / Accepted: 8 July 1998     

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.     

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.     

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     

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.     

Gill development, functional and evolutionary implications in the Pacific oyster Crassostrea gigas (Bivalvia: Ostreidae)

Development of the Crassostrea gigas gill was studied in order to better understand the feeding biology of early life stages, identify potentially critical developmental stages which may influence rearing success or recruitment to wild populations, and shed light on the evolution of the basic bivalve gill types. Larvae and juveniles were reared in an experimental hatchery, and larger specimens were obtained from a commercial hatchery. Specimens were relaxed, fixed, dried, and observed using scanning electron microscopy (SEM). The right and left gills developed symmetrically, via a “cavitation–extension” process from the gill buds. The inner demibranchs developed first (V-stage, 0.29–2.70 mm), in a sequential postero-anterior series of homorhabdic filaments. The outer demibranchs developed later (W-stage, from 2.70 mm), also as homorhabdic filaments, synchronously along the gill axis. The principal filaments (PF) developed from the progressive fusion of three ordinary filaments (OF), at a size of 7.50 mm, and the consequent plication was accentuated by the formation of extensive tissue junctions. Effective filament number (number of descending and ascending filaments) showed a marked discontinuity at the transition from the V- to the W- stage of the gill. Filament ciliation showed several important changes: establishment of OF ciliation in the homorhabdic condition (2.70 mm), ciliary de-differentiation of the PF in the heterorhabdic condition (7.50 mm), and establishment of a latero-frontal cirri length gradient from the plical crest to the PF base. Reversal of direction of ciliary beat is also necessary prior to adult functioning of the PF. Three major transitions were identified in C. gigas gill development, each potentially important in rearing success or wild population recruitment: (1) transition from velum to gill at settlement, (2) transition from a V- to a W-shaped gill (2.70 mm), and (3) transition from the homorhabdic to the heterorhabdic condition (7.50 mm). Complete gill development was much more prolonged than in species previously studied. The major ontogenetic differences between the C. gigas heterorhabdic pseudolamellibranch gill and the pectinid heterorhabdic filibranch gill suggest that the heterorhabdic condition evolved independently in these two bivalve families.     

Intra-clonal variation in the red seaweed Gracilaria chilensis

The phenotypic plasticity often found in seaweed populations has been explained only from the perspective of inter-population or inter-individual differences. However, many seaweeds grow and propagate by fragmentation of genetically identical units, each with the capacity to function on its own. If significant differences in performance exist among these supposedly identical units, such differences should be expressed upon the release and growth of these units. In this study we document two such types of variation in the red seaweed Gracilaria chilensis. Populations of sporelings, each grown under similar culture conditions and derived from carpospores shed by the same cystocarp exhibit significant differences in growth. In this species, each cystocarp develops from a simple gametic fusion, and cystocarp fusions occur too infrequently to account for the growth differences observed among recruits. In adult thalli, branches (ramets) derived from the same thallus (genet) and grown under similar conditions exhibit significant variation in growth rates and morphology. These findings have several implications. They suggest that carpospore production is not only an example of zygote amplification but that it also could increase variability among mitotically replicated units. Intra-clonal variability followed by fragmentation and re-attachment may increase intra-population variation which, in species of Gracilaria, is often larger than inter-population variation. In addition, the existence of intra-clonal variability suggests that strain selection in commercially important species may require a more continuous screening of highquality strains because of frequent genotypic or phenotypic changes in the various cultivars.     

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     

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.     

Gill pump characteristics of the soft clamMya arenaria

Pumping rates in the soft clamMya arenaria, collected in June 1987 in the Great Belt, Denmark, were determined as rates at which clams cleared suspensions of algae,Dunaliella marina. The frictional resistance of the siphons to water flow was estimated by studying the effect of excision of the siphons at the base on the rate of water pumping. The frictional resistance was also calculated from the Poiseuille equation. Excision of the siphons had no measurable effect on the pumping rate, and calculations indicated pressure losses ranging between 0.3 to 1.2 mm H₂O. This is consistent with the conception that the capacity of filter feeding bivalves to process large amounts of water depends upon a low resistance to water flow in the siphons.     

Phylogeographic study of the dwarf oyster, Ostreola stentina, from Morocco, Portugal and Tunisia: evidence of a geographic disjunction with the closely related taxa, Ostrea aupouria and Ostreola equestris

Despite the economic importance of oysters due to the high aquaculture production of several species, the current knowledge of oyster phylogeny and systematics is still fragmentary. In Europe, Ostrea edulis, the European flat oyster, and Ostreola stentina, the Provence oyster or dwarf oyster, are both present along the European and African, Atlantic and Mediterranean, coasts. In order to document the relationship not only between O. stentina and O. edulis, but also with the other Ostrea and Ostreola species, we performed a sequence analysis of the 16S mitochondrial fragment (16S rDNA: the large subunit rRNA-coding gene) and the COI fragment (COI: cytochrome oxidase subunit I). Oysters were sampled from populations in Portugal (two populations), Tunisia (two populations) and Morocco (one population), identified as O. stentina on the basis of shell morphological characters. Our data supported a high degree of differentiation between O. stentina and O. edulis and a close relationship between O. stentina and both Ostrea aupouria (from New Zealand) and Ostreola equestris (from Mexico Gulf/Atlantic). The status of this geographic disjunction between these closely related species is discussed. Furthermore, although identified in a separate genus Ostreola by Harry (Veliger 28:121–158, 1985), our molecular data on O. stentina, together with those available for the other two putative congeneric species, O. equestris and Ostreola conchaphila, would favour incorporation of Ostreola in Ostrea. Finally, a PCR-RFLP approach allowed the rapid identification of O. edulis and O. stentina.     

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.     

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.     

An integrative response by Mytilus chilensis to the toxic dinoflagellate Alexandrium catenella

Physiological responses of Mytilus chilensis exposed to the toxic dinoflagellate Alexandrium catenella were measured over 21 days in the laboratory and were compared with control mussels not exposed to the dinoflagellate. Mussels were collected from culturing ropes at Yaldad Bay, southern Chile (43o08′S 73o44′W), in August 2004 and acclimated to laboratory conditions for one week prior to the experiment. After 8 days, the paralytic shellfish poisoning (PSP) toxins (i.e. saxitoxin) in the tissues of exposed mussels exceeded safe levels for human consumption. Clearance rates, ingestion of organic matter, and absorption efficiency of exposed mussels were significantly lower than those of controls on day 0, but this was followed by an increase on day 3. The exposed mussels also increased their excretion rate over time, and this increase was significantly correlated with the accumulation of PSP toxins in their tissues. Oxygen consumption was not affected by the PSP toxins. The scope for growth (SFG) on day 0 was negative in exposed mussels, but it increased during the experiment. Although feeding activity and absorption efficiency were adversely affected during the first few days of exposure to PSP toxins from A. catenella in the laboratory, the M. chilensis cultured in Yaldad Bay may have evolved mechanisms that allow them to exploit the toxic dinoflagellate as a food source.     

Gill lesions in the perch, Anabas testudineus, exposed to monocrotophos

Histopathological effects of sublethal doses of monocrotophos on the gills have been studied by exposing the fish for a period ranging from ten to twenty days. The extent of damage to gills was dependent on the dose and duration of exposure. Histopathological changes in the gills observed were characterized primarily by hemorrhage in the primary and secondary gill lemellae. Degeneration and necrosis of epithelial cells were very prominent. Distortion of the secondary lamellae was very prominent, beginning with disruption of epithelial cells from pillar cells.     

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.