Bivalve hatchery technology: The current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France

The French oyster farming industry relies almost exclusively on juveniles collected from the natural environment; the supply of spat produced by hatcheries is low, about 10 % of the industry's requirements. Development through selective breeding of oyster stocks which are better suited for aquaculture purposes, is likely to reverse this tendency since only hatcheries will be able to supply such animals. Scallop farming, which at present is poorly developed in France, relies exclusively on hatchery produced spat. Although hatchery technology is constantly being improved, significant production problems remain which must be solved before hatcheries become a major supplier of juveniles for the industry. This paper describes the present state of hatchery technology in France based on experimental results obtained with the great scallop Pecten maximus and the Pacific oyster Crassostrea gigas, over the past ten years. Compared to the great scallop, the Pacific oyster has higher D larva yields (60 % for C. gigas vs. 30 % for P. maximus) and a faster larval growth rate (10 μm·d⁻¹ for C. gigas vs. 5 μm·d⁻¹ for P. maximus). However, Pacific oysters have a greater heterogeneity during larval development and higher mortality rates (40 % for C. gigas vs. 25 % for P. maximus) which produce lower overall yields of pediveliger larvae ready-to-set for Pacific oysters (15 %) compared to the great scallop (30 %). Development of continuous larval and post-larval culture methods along with development of continuous phytoplankton technology offers one of the most promising methods to improve molluscan hatchery techniques.     

Towards development of large-scale hatchery cultivation of larvae and spat of the pearl oyster Pinctada mazatlanica in Mexico

The results of a series of pilot-scale runs with P. mazatlanica larvae from 2004 through 2006 are reported. Preliminary runs in 2004 and 2005 used broodstock collected in summer, when massive spawning of wild populations naturally occurs. However, results of larval development were very poor and failed to produce spat in both years. In 2006, ripe broodstock were still collected in summer, but also in the spring time, based on the hypothesis that the gonads in this season were in better reproductive condition that in summer. Three larval runs were conducted in 2006: two in spring and one in summer. Larvae growth and survival greatly increased in both spring runs, ending with two successful productions of spat (∼ 20 × 10³ and ∼ 100 × 10³ juveniles). The summer larval run in 2006 failed again to produce spat. Additionally, the first run of April 2006 refers to an experiment that evaluated two different larval culture conditions: constant temperature (27 °C) and low stocking density (3-4 larvae ml^− 1) versus variable temperature (24-28 °C) and high stocking density (8-9 larvae ml^− 1). The first trial significantly increased larval survival and growth, which in turn resulted in greater numbers of settled spat, in comparison of the second trial, where survival, growth, and settlement of spat were significantly lower. Also in 2006, the quality of seawater used at the hatchery was evaluated with microbiological and chemical tests. The implication of these tests, together with results from all experiments are analyzed and discussed in terms of the potential development of large-scale hatchery cultivation of P. mazatlanica larvae in Mexico.     

Early detection of triploidy in the larvae of Pacific oysters,Crassostrea gigas,by flow cytometry

Triploidy may provide a means to neuter the Pacific oyster, C. gigas, genetically thereby increasing survival and marketability during periods of reproduction. Pressure treatments administered 10 min after fertilization for 10 min duration at 6000–8000 psi consistently produced triploids. The highest proportion obtained was 57%. Triploidy was assayed in all experiments at the larval stage and again as spat (metamorphosed larvae) using flow cytometry. A technique to assay larvae as small as 250 μm is described. Results obtained by flow cytometry were verified by chromosome counts. Analysis of triploidy at the larval stage provides reliable estimates of the proportion of triploids, eliminating several weeks of culture time.     

Embryonic development and influence of egg density on early veliger larvae and effects of dietary microalgae on growth of brown mussel Perna perna (L. 1758) larvae under laboratory conditions

The effect of egg density on embryonic development and larval quality as well as the lipid and fatty acid contents (eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) of cultured microalgae fed to Perna perna larvae was studied under controlled conditions to provide information needed for development of an experimental hatchery. Embryonic development followed the common sequence exhibited by other bivalves. d-larva stage was attained 40–44 h post-fertilisation at 21 ± 1 °C. The umbo-stage was reached in 11 days, and pediveliger larvae were observed 26 days post-fertilisation. Low egg density (range 20–100 eggs cm^?2) produced high proportions of normal d-larvae. Larval development showed two growth phases: 1st—the mixotrophic stage and 2nd—the exotrophic stage where the composition of diets had significant effects on larval growth with higher rates in larvae fed with the mixed microalgae (Isochrysis galbana + Chaetoceros calcitrans, I. galbana + Phaeodactylum tricornutum and I. galbana + Skeletonema costatum) in comparison with the monospecific diet (I. galbana). Fatty acid analysis showed that larval growth and survival were strongly influenced by proportions of dietary DHA and EPA. These results indicate that DHA and EPA are the key factors in determining larval performance, considerably more than the total amount of other fatty acids.     

Ceramic clay reduces the load of organic matter and bacteria in marine fish larval culture tanks

Ceramic clay has been increasingly used to improve contrast and prey detection in tanks for rearing of fish larvae. In contrast to live microalgae or algae pastes, clay increases turbidity without contributing to the organic matter load. In addition, clay may aggregate and sediment organic matter and bacteria, facilitating its removal. Marine larvae are sensitive to infections by opportunistic bacteria. Fish, algae, and live feed increase the microbial carrying capacity of the rearing water which allow exponential growth of bacteria and favor fast-growing opportunists. Reducing substrate levels by replacing microalgae with clay may reduce bacteria proliferation and benefit larvae. We compared the effects of three rearing regimes including live Isochrysis galbana, Nannochloropsis oculata paste, and ceramic clay on the bacterial community, concentration of organic matter, and growth and survival of Atlantic cod larvae (Gadus morhua L.). The application of clay resulted in reduced substrate levels for bacteria in the rearing water compared to the addition of live algae or algae paste. To some extent, clay aggregated and transported organic matter to the bottom of the larval fish tanks, where it could be effectively removed. Fish tanks receiving clay showed a lower abundance of bacteria in the water than tanks added algae paste or live algae. Fish tanks with algae paste showed a higher abundance of bacteria and a higher share of cultivable bacteria and TCBS counts than the other two treatments. Tanks with live algae showed low relative abundances of opportunistic bacteria and TCBS counts in both water and rotifers. Cod larvae in tanks with clay or live algae initiated exponential growth earlier than larvae in tanks with algae paste. Larvae in tanks receiving clay had significantly higher dry weight than larvae in tanks receiving algae paste at day 5 and 20 post hatching. The survival of larvae in the tanks added clay was variable. Two of the three tanks with clay had significantly higher larval survival than the tanks with live algae or algae paste. However, one tank with clay underwent 100% mortality. It is not possible to conclude whether this was related to the use of clay or an incidental development of a harmful microbial community in this tank. The effects of clay addition on larval performance should be studied further. Clay addition appears to be an easy way to reduce bacterial load during early first feeding of marine larvae without compromising the beneficial effects of turbidity.     

The influence of different microalgal diets on European clam (Ruditapes decussatus,Linnaeus, 1758) larvae culture performances

The European clam, Ruditapes decussatus is a species with high commercial importance in Portugal and other Southern European countries. However, the development of R. decussatus culture has been limited by the highly variable patterns of natural recruitment. The development of hatchery technology will provide an alternative source of spat. The effect of six nutritional regimes on the survival, growth and biochemical composition of R. decussatus larvae were evaluated, aiming to provide crucial information on its nutritional requirements. A holistic approach incorporating all physiological response showed that the bispecific diet I. aff galbana and C. calcitrans (60/40 cell μL^?1) was the more adapted throughout larval development. Moreover, the monospecific diet I. aff galbana provided an overall good performance. Larvae cannot use C. calcitrans at early stages of development; however, the inclusion of these microalgae improved late larval development. The results obtained constitute an important first step in the hatchery R. decussatus larval nutrition and a prerequisite for future work on the improvement of larval development and the optimization of feeding practices that will maximize larvae yield and minimize cost in aquaculture hatcheries.     

Metamorphosis of larvae of Crassostrea virginica fed microencapsulated diets

This paper reports the results of two feeding experiments in which oyster larvae (Crassostrea virginica) were grown to metamorphosis on microencapsulated diets, and provides the first successful case of culturing oyster larvae to metamorphosis on an artificial diet. Five to 25% of the larvae fed microencapsulated diets grew to the “eyed” stage in 22–25 days. About 2–20% of these “eyed” larvae metamorphosed and settled and 25–40% of these settled larvae produced spat (> 0.05 mm). Two concentrations of two microencapsulated diets were tested; results indicated that diet concentration affects larval growth and development.Microcapsules with capsule walls made of a lipid mixture containing ethyl cellulose and stearic acid improved the retention of water-soluble components. Capsules were more stable at low temperature (5°C) than at higher temperatures (22 and 28°C). Inclusion of lipid algal extract in gelatin-acacia capsules promoted better growth of larvae than those not containing the algal extract. This suggests that attractants or phagostimulants could play an important role in microencapsulated diets.     

Stocking density evaluation on Catarina scallop (Argopecten ventricosus,Sowerby II, 1842) larvae to improve hatchery production

The Catarina scallop Argopecten ventricosus is a highly valued resource. Although its hatchery spat production has already been reported, the effects of initial larval stocking density have never been reported for production purposes. This study evaluates A. ventricosus growth and survival in triplicate using three stocking densities: low (LD; 2 larvae mL^?1), medium (MD; 4 larvae mL^?1), and high (HD; 6 larvae mL^?1). Three-day old larvae were reared in 18-L plastic carboy at 25.6?±?0.5 °C and fed with a microalgal blend of Isochrysis galbana and Chaetoceros calcitrans (1:1 cell number ratio) for 7 days, equivalent to 10 post-fertilization days (PFD). Higher specific growth rate was recorded at LD (15.8?±?0.2%) after 8 PFD of culture compared to MD (1.6?±?0.5%) and HD (4.1?±?1.8%) densities. The least time required for 60% of the larvae to reach the pediveliger stage was recorded at LD condition (10 PFD). Higher survival was recorded at HD (58.8?±?3.1%) at 8 PFD compared to MD (53.5?±?3.1%) and LD (43.9?±?3.0%). After 8 PFD, stocking density was highly related to larval growth and survival. To increase production and growth, and reduce the time required to reach pediveliger stage, stocking density should start with 6 larvae mL^?1 and be reduced to 2 larvae mL^?1 at 7 PFD.

     

Experiments of the rearing of larval turbot, Scophthalmus maximus L.

Experiments on the live food requirements of cultured turbot larvae are described. A mixture of six species of unicellular algae did not sustain early larvae, but the growth and survival of larvae fed with rotifers were considerably improved when the rotifers were feeding on Isochrysis galbana rather than on Dunaliella tertiolecta. Oyster (Crassostrea gigas) larvae offered alone or with rotifers, did not produce higher larval growth or survival than rotifers alone. Black tanks were found to be more suitable than white tanks for rearing larval turbot.     

Experimental larval culture of the Caribbean scallops Argopecten nucleus and Nodipecten nodosus

Several experiments were performed examining the effect of different culture variables on the growth and survival of Argopecten nucleus and Nodipecten nodosus larvae. Higher growth and survival values were obtained at a low density (1 larvae mL^?1) than at a high density (5 larvae mL^?1), except for N. nodosus's survival, which was not affected by density. Higher growth was obtained when feeding the larvae a diet of Isochrysis galbana, similar results occurred with the diet of Isochrysis galbana+Chaetoceros calcitrans in A. nucleus and with I. galbana combined with the lipid emulsion EmDHA in N. nodosus. Higher survival was obtained when the diet included a mixture of microalgae than when EmDHA was supplemented. Growth and survival of the larvae of A. nucleus were greater when fed continuously, while N. nodosus grew and survived better when food was given discontinuously. Higher growth and survival values were recorded at lower food concentrations ranging from 20 to 40 cells μL^?1 of I. galbana. Higher growth was observed with increasing temperature between 22 and 28 °C, although there was a simultaneous decrease in survival. No significant effects on larval production parameters were found when the culture water was changed totally or partially, but higher larval growth of N. nodosus was verified when a partial renewal of water was performed.     

肾上腺素对岩牡蛎幼虫变态的诱导

为了得到壳型规则、大小均一的单体牡蛎,本研究采用肾上腺素对岩牡蛎单体苗种生产的诱导条件进行研究,阐明了岩牡蛎单体苗种的最适诱导浓度、诱导时间和诱导密度。结果显示,肾上腺素能显著诱导岩牡蛎幼虫的不固着变态,最适诱导浓度为5×10–5 mol/L,最适诱导时间为1 h,提高诱导浓度和延长诱导时间导致岩牡蛎幼虫的不固着变态率、稚贝壳高和存活率显著降低;肾上腺素对低于8个/mL幼虫密度的诱导效果差异不显著,但稚贝壳高和存活率在8个/mL的培育密度下显著低于0.5～4个/mL,研究表明利用肾上腺素诱导岩牡蛎单体时,可大批量处理眼点幼虫,但稚贝充气培养的最适培养密度应不高于4个/mL。     

Effects of stocking density, diet, and water exchange on growth and survival of pearl oyster Pinctada maxima larvae

Three experiments were carried out to separately investigate the effects of stocking density (1.0, 2.0, 4.0 and 8.0 larvae ml^?1), diet (Isochrysis zhanjiangensis, Chlorella spp., Nannochloropsis oculata, 50 % I. zhanjiangensis/50 % Chlorella spp. and 50 % I. zhanjiangensis/50 % N. oculata), and water exchange (25 % once every other day and 25, 50 and 100 % once daily) on growth and survival of pearl oyster Pinctada maxima larvae. Results showed that there existed significant effects of stocking density, diet, and water exchange on the growth of pearl oyster larvae (P < 0.05). Larval survival was significantly affected by stocking density and diet (P < 0.05), but not by water exchange (P > 0.05). Larval growth decreased significantly with increasing stocking density (P < 0.05). A density of 1.0–4.0 larvae ml^?1 was optimal for larval growth of pearl oyster. Chlorella spp. and N. oculata could replace 50 % of I. zhanjiangensis as a food source for the pearl oyster larvae without affecting growth and survival. A water exchange of 25 % once daily provided optimum larval growth and survival.     

Bacterial nutrition of great scallop larvae

Bacterial nutrition of great scallop, Pecten moximus, larvae was investigated using the radioactive tracer technique. The bacterial labelling was studied initially to obtain a high and stable specific radioactivity (¹⁴C) of bacterial cells. A higher bacterial specific ¹⁴C activity was obtained when the tracer (amino acid) was introduced in the culture medium at the beginning of the exponential growth phase. After a 12 h labelling period in a rich nutritive medium, the bacteria were depurated in seawater for 5 h (chase) to prevent further ¹⁴C excretion and then added to larval rearing vessels. The larval labelling was followed for 12 h and then larvae were placed in new vessels without radioactive bacteria. The depuration of larvae was followed for 3 days. Data obtained on ingestion and assimilation efficiency show that bivalve larvae are able to ingest and digest bacteria.     

The food value of Phaeodactylum tricornutum Bohlin to the larvae of Ostrea edulis L. and Crassostrea gigas Thunberg

The food value of three cultures of Phaeodactylum tricornutum composed of three different cell forms was measured for the larvae of the bivalve molluscs Ostrea edulis and Crassostrea gigas. While all three types supported good growth of both species of larvae, there were no significant differences in food value between the three and the Isochrysis galbana controls. Increasing the feeding rate of one type of P. tricornutum culture had no marked effect on the growth rates, survival or settlement of C. gigas larvae in the range equivalent in terms of dry weight to 25–200 cells I. galbana/μl.     

Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas (Thunberg)

The robustness of Pacific oyster, Crassostrea gigas (Thunberg), sperm cryopreservation in the context of selective breeding based on family lines was investigated. Irrespective of egg density, high fertilization success was achieved with cryopreserved sperm when sperm:egg ratios of 1000:1 to 10 000:1 were used. Variation among replicate runs on the same oyster batches was minimal, indicating that cryopreservation and larval rearing procedures were repeatable. Twenty independent single male–female crosses were made to assess the utility of cryopreserved sperm in selective breeding. The fertility of unfrozen sperm was generally a poor predictor of cryopreserved sperm fertility. Based on D‐larval yields, 17 of the 20 crosses were likely to yield adequate spat for selective breeding (>10⁵ D‐larvae from 1 million eggs), two were marginal (5 × 10⁴ D‐larvae) and one was inadequate (4 × 10³ D‐larvae). An alternative fertilization strategy to improve D‐yield from a given number of sperm was then tested. Fertilizing 10 million eggs at a sperm:egg ratio of 200:1 increased the total D‐yield when compared with fertilizing 1 million eggs at a sperm:egg ratio of 2000:1 for the same male–female pair. We conclude that, despite wide variation in fertility, cryopreserved sperm is useful for family production.     

The potential for Chilean oysters, Tiostrea chilensis (Philippi, 1845), from two populations in northern New Zealand as a source of larvae for aquaculture

The use of the Chilean oyster. Tiostrea chilensis (Philippi. 1845), for aquaculture has been hampered by difficulty in obtaining commercial quantities of larvae due to the low fertility of the existing broodstock of this species taken from well-known populations in Chile and the South Island of New Zealand. The larval productions of two wild populations of this oyster in northern New Zealand were assessed as potential alternative sources of broodstock for aquaculture. The study found both populations produced larvae from young, small oysters, and that a much larger proportion of the population was brooding larvae each year than has been reported elsewhere. The mean fertility of one of these populations, at 71 056 ± 3701 (SE) larvae per oyster, was the highest reported for any population of this species. The significantly higher fertility of these populations makes them well suited for further development as broodstock for aquaculture.     

Embryonic development and larval and post‐larval growth of the tropical scallop Nodipecten (= Lyropecten) nodosus (L. 1758) (Mollusca: Pectinidae)

This work describes for the first time the embryonic development of the tropical scallop Nodipecten nodosus. Larval and post‐larval growth parameters and some characteristics of larvae shell morphology were also ascertained. The larvae were obtained from the induced spawning of a group of broodstocks under controlled laboratory conditions. After fertilization, larval cultivation was carried out in conical tanks at a temperature of 26–27 °C. Larval density was controlled as a function of larval growth to give 10, 5 and 3 larvae mL^?1 from days 1, 3 and 8 respectively. The larvae were nourished with a 1:1 mix of Isochrysis galbana (clone T‐ISO) and Chaetoceros gracilis in portions varying between 30 000 and 70 000 cells mL^?1. Expulsion of polar groups was observed 5 and 15 min after fertilization, whereas the first cellular division occurred after 30 min. The first gastrule ciliates and trocophore larvae were noted after 8 and 18 h had elapsed, respectively, whereas prodissoconch I, or D‐larvae, were discerned after 26 h. Subsequently, larvae with prodissoconch II or veliger‐conch appeared at 30 h. Larval development continued for 10–12 days, followed by metamorphosis, at an approximate length of 208–230 µm. The growth of the post‐larvae was evaluated for 9 days. Larval and post‐larval growth corresponded to the linear equations L = 71.85 + 10.85t, r² = 0.99, and L = 44.09 + 17.81t, r² = 0.94 respectively. Accordingly, larval morphology and size disparities are discussed with respect to other tropical pectinids.     

Effect of florfenicol and oxytetracycline treatments on the intensive larval culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819)

The administration of antimicrobials to control bacterial pathologies in Chilean scallop hatcheries is a frequent practice, but their effects on these cultures remained unknown. This study was undertaken to obtain information on the effect of the administration of florfenicol and oxytetracycline on the growth, survival and bacterial content of scallop larvae under farming conditions. Florfenicol‐treated cultures exhibited high survival rates (44% after 17 days of culture), whereas cultures not treated or treated with oxytetracycline collapsed after 11 days of culture. Surprisingly, no significant differences in the heterotrophic (Tukey test; P = 0.226) and Vibrio (Tukey test; P = 0.666) concentrations between the oxytetracycline‐treated and untreated larval cultures were observed. Otherwise, florfenicol administered directly into rearing tanks produced significantly higher larval growth (Tukey test; P = 0.0001) and survival (Tukey test; P = 0.011) than bath treatment. When 2 and 4 mg L^?1 of florfenicol were compared, no significant differences in growth (t‐test; P = 0.4596) and survival (Tukey test; P = 0.057) were observed, suggesting that a concentration of 2 mg L^?1 is sufficient to ensure larval production. The present results demonstrate the efficacy of florfenicol‐based therapy to increase larval survival and growth at commercial scale and prompt the necessity to standardize its use in Chilean scallop hatcheries.     

Use of Ox‐Aquaculture© for disinfection of live prey and meagre larvae,Argyrosomus regius (Asso, 1801)

Live prey used for marine larval fish (rotifers and Artemia) as well as intensive larval rearing conditions are susceptible to the proliferation of bacteria that are the cause for reduced growth and larval mortality. Hydrogen peroxide has been recently proved a good disinfectant in aquaculture, either for eggs, larvae or live prey. In this study the effects of a hydrogen peroxide‐based product, Ox‐Aquaculture^©, on live prey (rotifers and Artemia) and meagre larvae bacterial load, composition and final status have been tested. A 34.6% reduction of total heterotrophic bacteria and 59.7% of Vibrionaceae were obtained when rotifers were exposed for 15 min to 40 mg L^?1 of the product. A 34.3% reduction of total heterotrophic bacteria and 37.7% of Vibrionaceae were obtained when Artemia were exposed for 5 min to 8000 mg L^?1 of the product. More than 95% reduction of total heterotrophic bacteria and 75% of Vibrionaceae were obtained when meagre larvae were exposed for 1 h to 20 mg L^?1 of the product. Furthermore, disinfection of enriched live prey with the product did not change the fatty acid composition and survival of the live prey and improved final larval survival.     

Elimination of the associated microbial community and bioencapsulation of bacteria in the rotifer Brachionus plicatilis

The bioencapsulation of live bacteria in the rotifer Brachionus plicatilis was determined under monoxenic conditions. The first objective was to evaluate the microbiota of the rotifer during intensive production and to obtain sterile rotifer cultures starting from adult females or amictic eggs using PVP-Iodine, Hydrogen peroxide or antibiotic mixtures. In the rotifers, the proportion of vibrios increased significantly during the mass production, displacing other unidentified marine bacteria. Rotifers, in the absence of culturable bacteria were obtained starting from amictic eggs and using Trimetroprim-sulfametoxasole (Bactrim Roche®) at 10 ml l^–1. The effect of members of Vibrionaceae on the survival and growth rate of rotifers was determined under monoxenic conditions. The survival of rotifers was not affected in the presence of different isolates, while amictic egg formation occurred and the populations increased when the strains Vibrio proteolyticus C279 and Aeromonas media C226 were tested. All isolates were successfully incorporated in the rotifers, since there was no significant difference between the numbers of bioencapsulated cells of different strains of isolates. The results show that it is possible to replace the microbial community in rotifer cultures, started from disinfected amictic eggs, with selected bacterial strains. This could be used as a tool for future studies to reveal the role of specific bacteria on first larval stages of marine fish species.