Effect of zinc on the settlement of the oyster Crassostrea gigas

The influence of zinc upon the pattern and success of settlement was examined in the oyster Crassostrea gigas. Late larvae were more tolerant of zinc than embryos. A delay in settlement was recorded in treatments containing zinc as low as 125 μg/l, and numbers of larvae settling was reduced in the presence of zinc. Larvae subjected to zinc immediately prior to settlement showed evidence of slowing in behavioural development. Those larvae which settled in the presence of zinc, however, when ongrown in clean water were as viable as controls. Zinc at concentrations of 250 and 500 μg/l suppressed spat growth, but recovery was rapid upon subsequent on-growing in clean water conditions.     

Effects of artificial eutrophication on the metabolism of the Japanese oyster Crassostrea gigas

The morphology of the primary tube feet in 15 species of comatulid (unstalked) crinoids from coral reefs in the Palau Islands and Lizard Island, Great Barrier Reef, was investigated using close-up underwater photographs of the tube feet taken in the natural habitat. Measurements of length of the tube feet and their spacing along the pinnule were taken from these photographs. Tube feet of species of the family Comasteridae have a mean length of 0.75 mm and a mean spacing of 6 tube feet mm^-1. Tube feet of non-comasterid species occurring in the same environments have a mean length of 0.55 mm and a mean spacing of about 8 tube feet mm^-1. The relationship between spacing and length of the tube feet is highty significant for both the Palau and Lizard Island samples (P<0.01). Species having longer and more widely spaced tube feet live partly concealed within the infrastructure of the reef and hold the arms and pinnules in a multidirectional posture. Species having shorter and more closely spaced tube feet perch on top of reef pinnacles or alcyonarians and form planar filtration fans normal to unidirectional currents or wave oscillations. Longer and more widely spaced tube feet in species dwelling within the reef infrastructure provide more efficient filtration in the slow, meandering flow prevailing there. Closer spacing of the tube feet in species exposed to near-mainstream flow provides a more efficient filtration mechanism at higher flow velocities. Reduced length of the tube feet in these species may be a consequence of closer spacing of the pinnules. Differentiation of these co-occurring species in spacing and length of the tube feet implies differentiation in food particles captured. This may in some cases constitute resource partitioning. Most species which overlap in living habits are significantly different in spacing and length of the tube feet.     

Induction of metamorphosis in larvae of the oyster Crassostrea gigas using neuroactive compounds

Chemical (neuroactive compounds at different concentrations and exposure times) and physical (water agitation, light) factors with potential effects on the metamorphosis of larvae of the oyster Crassostrea gigas (Thunberg) larvae have been studied. The neurotransmitters l-dihydroxyphenylalanine (DO), epinephrine (EP), norepinephrine (NE), and acetylcholine (AC) have been identified as very active inducers of metamorphosis, whilst serotonin (SE), dopamine (DA) and potassium (K) were less effective inducers. The -aminobutyric acid (GA) and ammonium (AM) were found ineffective at the concentrations tested. Exposure to 10^-4 M EP for 15 min was sufficient to promote >80% metamorphosis within 48 h, whereas NE required 2 h to exert comparable induction. Maximum induction by DO (>50%) was achieved after 2 h exposure to 10^-4 M. However, unlike EP and NE, DO was lethal at that concentration in the long term. Maximum induction by AC (30% metamorphosis) was achieved at a concentration of 10^-4 M. In contrast to other neurotransmitters, AC induced settlement behaviour, cementation and eventual metamorphosis, yielding postlarvae which were all attached to the substratum. EP and NE triggered the morphogenetic changes, by-passing settlement and leading to postlarvae not cemented to the substratum. DO induced mostly attached spat at low concentrations (10^-5 M) and unattached spat at high concentrations (10^-4 M), and a similar pattern was apparent for the weaker inducers SE and DA. Regarding physical factors, a highly reflectant surface significantly increased the percentage of attached spat obtained, compared to a dark bottom. No consistent effect of water current or light was detected on the production of unattached spat. The three different forms of induction are discussed in relation to different regulatory pathways of settlement and metamorphosis.     

Purification and immunolocalization of a vitellin-like protein from the Pacific oyster Crassostrea gigas

A female-specific protein from the hemolymph, and a related ovarian protein were identified in the Pacific oyster Crassostrea gigas using immunological procedures. The latter protein was isolated from ovarian extract by a combination of precipitation in distilled water, ammonium sulfate fractionation, hydroxylapatite chromatography and gel filtration on a Sepharose 6B column, and was identified as a macromolecular glycolipoprotein of 500 kD; sodium dodecylsulfate polyacrylamide gel electrophoresis, under reducing conditions, revealed seven heterogeneous polypeptides. Using a specific antiserum against purified protein, we also examined the immunohistochemical distribution of the protein at light- and electron-microscopic levels. Light microscopy revealed its presence in the oocytes, and electron microscopy identified it in yolk granules and the vitelline coat of the oocytes. These results indicate that the purified protein is a vitellin-like protein. Since no organ other than the ovary reacted with the antiserum, it is probable that the protein is produced inside the ovary, probably autosynthetically by oocytes.     

Effect of zinc on growth and development of larvae of the Pacific oyster Crassostrea gigas

Following the observation of periodic high concentrations of zinc in estuarine waters used in the White Fish Authority's oyster hatchery at Conway, North Wales, two beaker trials were conducted to study the effect of zinc, over the range recorded, on the young stages of larvae of Crassostrea gigas. Zinc, added to sea water both as zinc sulphate and as a natural mine-adit water, was applied for a period of 5 days, after which larvae were maintained for a further 5 days in sea water alone. Increasing concentrations over the range 125 to 500 g/l Zn resulted in decreasing growth, and increasing incidence of abnormality and larval mortality. A second trial with zinc sulphate showed 50 g/l Zn to have little effect on larval development, a progressive decrease in growth at 100 and 150 g/l, and no growth at 200 g/l. It is suggested that the deleterious effect of short-term exposure to zinc may well have contributed to the intermittent failure of larvae and irregular productivity previously recorded at the hatchery. It is also possible that zinc contamination in estuaries may affect natural oyster breeding, and may have to be considered in the future siting of hatcheries for seed production.     

Looking for the clock mechanism responsible for circatidal behavior in the oyster Crassostrea gigas

Valve activity rhythm of the oyster Crassostrea gigas is mainly driven by tides in the field, but in the laboratory, only a circadian clock mechanism has been demonstrated. In an attempt to reconcile these results, the mechanisms underlying the circatidal rhythm were studied in the laboratory under different entrainment or free-running regimes and in the field at Arcachon (44°39′N/1°09′W) in February–April 2011). Results confirm the existence of a circadian clock in C. gigas. Under entrainment regimes (12-h dark/12-h light photoperiod and tidal cycles simulated by a reversing current flow), oysters exhibited both circadian and circatidal cycles. Under free-running conditions (e.g., continuous darkness), the endogenous rhythm appeared to be circadian. There was no experimental evidence for an endogenous circatidal rhythm, even in oysters just transferred from the field, where a clear tidal cycle was expressed. There are two possible mechanisms to explain tidal behavior in C. gigas: an exogenous tidal cue that drives tidal activity and masks the circadian rhythm and an endogenous circatidal clock that is sensitive to tidal zeitgebers and runs at tidal frequency.     

Combined effects of temperature and salinity on fed and starved larvae of the mediterranean mussel Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas

The combined effects of temperature, salinity and nutrition on survival and growth of larvae of the Mediterranean mussel Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas were studied over a period of 7 d in the laboratory. Ripe adults, collected in spring and summer 1987 from natural populations in the Bay of Arcachon, France, were induced to spawn. Larvae of both species were cultured at four temperatures (15°, 20°, 25° and 30°C), four salinities (20, 25, 30 and 35S) per temperature, and two levels of nutrition (fed and unfed) per temperature/salinity combination. The fed larvae received a mixed algal diet of 50 cells each of Isochrysis galbana and Chaetoceros calcitrans forma pumilum per microlitre. In both bivalve species, larvae survived over a wide range of temperature and salinity, with the exception of mussel larvae, which died at 30°C. Statistical analysis indicated that nutrition had the greatest effect on larval development, explaining 64 to 75% of the variance in growth of M. galloprovincialis and 54 to 70% in growth of Crassostrea gigas. Unfed mussel larvae displayed little growth. Compared with temperature, the effect of salinity was very slight. M. galloprovincialis larvae exhibited best growth at 20°C and 35S and C. gigas at 30°C and 30S.     

Physiological basis of extreme growth rate differences in the spat of oyster (Crassostrea gigas)

Juvenile oysters (Crassostrea gigas) (produced in November 2009) reared under uniform hatchery conditions for 4 months were selected for extreme growth rate differences by repeatedly taking larger and smaller individuals to achieve weight differences >30× between fast (F) and slow (S) growers. The physiological basis of differential growth was analyzed in experiments in June 2010, where components of energy gain (clearance and ingestion rates and absorption efficiency), energy loss (metabolic rates) and resulting scope for growth (J h^?1) were compared for groups of F and S oysters fed three different ration levels (≈0.5, 1.5 and 3.0 mg of total particulate matter L^?1). In both F and S oysters, a higher food ration promoted asymptotic increases in energy gain rates through regulatory adjustments to clearance rates, which maintained similar absorption efficiencies across the food concentrations. No significant differences were found between growth groups in mass-specific physiological rates (i.e., per unit of body mass). However, the scaling of these rates to a common size in both groups using allometric coefficients derived for C. gigas revealed higher energy gain rates coupled with lower metabolic costs of growth in fast growers. Thus, appropriate size-standardization is essential in accounting for observed differences in growth rate. Present results are in accordance with previous reports on other bivalve species on the physiological processes underlying endogenous growth differences, suggesting that the same interpretation can be applied to the extremes of these differences.     

Preparation and evaluation of protein microcapsules for a marine suspension-feeder,the Pacific oyster Crassostrea gigas

Little is known of the dietary requirements of marine suspension-feeders, mainly due to difficulties in preparing and delivering microparticulate diets to animals living in an aquatic environment. The food particles must be stable in seawater and digestible by the consumer. A laboratory method for preparing cross-linked, protein-walled capsules is described in this study as a means of delivering dietary protein to marine suspension-feeders such as the oyster Crassostrea gigas. Leakage of ¹⁴C-labelled protein from capsules suspended in seawater for 24 h was reduced to less than 5% by the use of the trifunctional cross-linking agent 1,3,5-benzenetricarbonyl trichloride in wall formation. Invitro experiments indicated that capsules were susceptible to digestion by proteases and by style extracts of oysters. Oysters fed encapsulated ¹⁴C-labelled protein absorbed ¹⁴C with an efficiency of 40.2±5.0% (±1 SE). Biochemical fractionation of capsule-fed oysters indicated breakdown of ingested ¹⁴C-protein into material that was soluble in 7% trichloroacetic acid. Cross-linked, protein-walled capsules can be used successfully to deliver dietary protein to the oyster C. gigas.     

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.     

Comparative effects of indole derivatives as antifouling agents on the growth of two marine diatom species

Antifouling agents, used to prevent biofouling, need to be assessed for their impacts on marine organisms and environment before the application. Diatoms are one of the main components of fouling biofilms, which play important roles in the formation of biofouling. Particularly, diatoms are also the important ingredients of primary production and present interest as ecotoxicological models in marine environment. In this study, two benthic diatoms Nitzschia closterium f. minutissima and Navicula climacospheniae, widely distributed in fouling biofilm, were used as models for screening the activities of potential antifoulants. Nine indole derivatives were tested and CuSO₄ was used as a reference. Indole derivatives showed significant anti-algal activities and the EC₅₀ values of most indole derivatives were lower than that of CuSO₄. Halogen substituent enhanced the anti-algal activities of compounds, and the most efficient compounds for N. closterium f. minutissima were gramine and 7-chloroindole with the EC₅₀ values of 1.94 and 2.1?mg/L, while for N. climacospheniae, 7-chloroindole and 6-bromoindole were the most efficient and the EC₅₀ values were 3.91 and 4.25?mg/L, respectively. In conclusion, indole derivatives would be one of the promising candidates as antifoulants and our results strengthened the need to perform antifouling activity assays and environment-friendly evaluations.     

Settlement behavior and metamorphosis of oyster larvae (Crassostrea gigas) in response to bacterial supernatants

Veliger larvae of the oysterCrassostrea gigas (Thunberg) responded to unknown dissolved chemical inducers found in supernatants of cultures of the bacteriaAlteromonas colwelliana andVibrio cholerae. The response, which was similar to that seen when larvae were exposed to the neurotransmitter precursor L-3,4-dihydroxyphenylalanine (L-DOPA), consisted of an initial settlement phase of swimming with the foot extended and crawling on the substrate. Subsequently larvae attached to the substrate and metamorphosed. The percentage of veligers metamorphosing following inducation of settlement behavior was higher in a group of older larvae, a response similar to that seen with L-DOPA, suggesting that competence to respond to bacterial supernatants is divided into two phases: behavioral competence followed by morphogenetic competence. Following size exclusion chromatography, the molecular weight of the peak containing the activity which induced settlement behavior was determined to be 300 daltons. Autoclaved Marine Broth, which induced low levels of settlement behavior also contained this low molecular weight active peak, suggesting that an oyster settlement inducer is also present in this medium.Contribution # 137 from the Center of Biotechnology, Marine Biotechnology Institute, University of Maryland, USA     

Impact of the supplementation of a docosahexaenoic acid–rich emulsion on the reproductive output of oyster broodstock, Crassostrea gigas

Pacific oyster [Crassostrea gigas (Thunberg)] broodstock were hatchery-conditioned with either a mixed algal diet of Dunaliella tertiolecta, Tetraselmis suecica and Rhodomonas sp. [1:1:1, on a dry weight (DW) basis] (Control), a single diet of D. tertiolecta (Duna) or a single diet of D. tertiolecta supplemented with an emulsion rich in eicosapentaenoic (20:5n-3) and particularly docosahexaenoic acid (22:6n-3) (Duna+Em). Oysters were spawned after a conditioning period of 7 and 8 weeks and larvae were reared for 1 week on an Isochrysis galbana (clone T-Iso) diet. The percentage recovery of D-larvae from eggs of Control-fed oysters and Duna+Em-fed oysters was not significantly different (80% and 63% after 7 weeks of conditioning; 87% and 74% after 8 weeks of conditioning, respectively] but was much higher than from eggs produced by Duna-fed oysters (24% and 41% after 7 and 8 weeks of conditioning, respectively). A reduction in the percentage recovery of D-larvae as a result of temperature or salinity stress was most severe in eggs from Duna-fed oysters. Neither the broodstock diet nor the conditioning period affected the size of eggs or D-larvae, larval performance, or lipid content and lipid class distribution of the eggs. The fatty acid composition of the eggs was modified by the fatty acid composition of the algal diet and the lipid emulsion. The supplementation of the emulsion resulted in a pronounced increase in the percentage of 22:6n-3 in the polar (PL) and neutral lipids (NL) of the eggs (4.0-13.4% and 8.5-14.7% in NL and PL, respectively). The egg lipids (7-8 ng egg^-1) contained about 62% NL and were dominated by triglycerides (TAG), which made up 65% of the NL. The major phospholipids were phosphatidylethanolamine (PE), ceramide aminoethylphosphonate and phosphatidylinositol (CAEP+PI), phosphatidylserine (PS) and phosphatidylcholine (PC). Regardless of the broodstock diet treatment, the PL fraction contained high percentages of non-methylene-interrupted dienes (NMID) and plasmalogens (detected as dimethyl acetals).     

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.     

Effects of temporary starvation on the survival,and on subsequent feeding and growth,of oyster (Crassostrea gigas) larvae

Larvae of oysters, Crassostrea gigas, were maintained without food for 1 to 8 d after fertilization, and fed daily thereafter. There was little difference in survival and growth between controls and larvae kept without food for 2 or 3 d. Survival and growth rates were depressed in larvae starved for 4 or 5 d. For larvae starved for 6 to 8 d, survival was negligible or nil; even those larvae which survived the starvation period died later in the presence of food, apparently because of impaired digestion. Therefore, food availability in the first few days after spawning appears to be of paramount importance to the successful recruitment of Pacific oysters.     

Contrasting pattern of mitochondrial population diversity between an estuarine bivalve, the Kumamoto oyster Crassostrea sikamea, and the closely related Pacific oyster C. gigas

The Kumamoto oyster (Crassostrea sikamea) shows a spatially restricted distribution, favoring estuarine tideland environment. On the other hand, the Pacific oyster (C. gigas) has a broader range of habitat. The present study compared the mitochondrial population structure between the two closely related species. For accurate species identification of oysters sampled from Japanese and East Asian continental coasts, we performed sequencing analysis of the mitochondrial DNA (mtDNA) and PCR-RFLP assay of the first internal transcribed spacer of nuclear rRNA genes. Then, we estimated the extent of population differentiation within each of C. sikamea and C. gigas based on the mtDNA data. Few haplotypes were shared among the sites of sampling in C. sikamea, which contrasted with an extensive haplotype sharing among C. gigas samples. We discuss the mechanisms of elevated population differentiation observed in C. sikamea in light of the ecology and the ancient ocean geography around the present-day habitats.     

Effects of monospecific algal diets of varying biochemical composition on the growth and survival of Pacific oyster (Crassostrea gigas) larvae

Using monospecific diets of Thalassiosira pseudonana cells grown under different steady-state conditions, it was determined that higher growth rates of larval Crassostrea gigas Thunberg were obtained when fed T. pseudonana cells grown under high light. High light grown T. pseudonana cells consistently contained relatively more of the saturated fatty acids 14:0 and 16:0. Considered over three independent experiments, high light grown T. pseudonana cells were lower in protein and higher in carbohydrate than low light grown cells. Higher growth rates of larval C. gigas were obtained on diets with more of the essential fatty acid (EFA) 22:63, and less of the other EFA, 20:53. The relative requirements of C. gigas larvae for the essential fatty acids 20:53 and 22:63 are discussed. Faster growing larvae contained higher percentages of the fatty acids 14:0 and 16:0, and lower percentages of 22:2j. Oyster growth rates were correlated with their content of the fatty acids: 14:0, 16:0 and 22:2j in two experiments utilizing separately spawned batches of larvae. Fatty acid profiles are proposed as a technique for assessing larval condition. C. gigas larvae contained ten times the percent composition of the FAs 16:43, 18:17, 20:17 and 22:2j compared with their diet. Correlation analysis suggests that the dietary source of 18:17, 20:17 and 22:2j was 16:17. It is concluded that T. pseudonana cells grown under high light are a superior diet for C. gigas larvae in comparison with low light grown cells of the same species.