PROBLEMS FACED DURING METAMORPHOSIS AND SETTLEMENT

PROBLEMS FACED DURING METAMORPHOSIS AND SETTLEMENT L. Pérez-Parallé, J.L. Sánchez, A.J. Pazos, C. Mesías-Gansbiller and A. Silva Instituto de Acuicultura Universidad de Santiago de Compostela

Oyster culture in Galicia (Spain) Needs of seed: 40-50 million Disappearance of natural banks and decrease of natural recruitment Increase and appearance of new infections Low production of oyster seed in hatcheries (~ 3 million) Bottlenecks or key events: conditioning and settlement/metamorphosis

Oyster settlement and metamorphosis Response to environmental, chemical or physical cues (light, substrate, larval densities ) Settlement cues metamorphic changes Chemosensory pathways Metamorphosis: transcription de novo cell-cell interactions, hormones

Problems faced during the flat oyster settlement and metamorphosis Variable and low settlement success Lack of synchronised settlement Variable post-settlement growth and survival

Aim To establish an effective and cheap method to induce settlement and metamorphosis of larvae to improve O. edulis production in hatchery all year around

Oyster settlement Does exposure to several compounds (GABA, epinephrine, norepinephrine, L-DOPA and IBMX) improve settlement of oyster larvae?

Materials & Methods Oyster larvae were obtained from adult oysters maintained in a container filled with seawater at 18±2 ºC for 4-6 weeks After the treatment, oysters started spawning and the larvae were released

Materials and Methods Larvae were fed with a mixed diet at 100 equiv. Iso/µl (Isochrysis galbana, Isochrysis galbana tahiti, Monochrysis lutheri, Tetraselmis suecica, Rhodomonas salina, Chaetocerus calcitrans) Veliger larvae were maintained for 12-14 days before harvesting

Materials and Methods Experiments were carried out with competent larvae (>330µm) Settlement assays in the laboratory Settlement assays in industrial tanks

Materials and Methods Settlement assays in the laboratory Triplicate polystyrene 90-mm tissue culture Petri plates 25 ml final volume of U.V.-sterilized, 10 µm filtered FSW 25 veligers of Ostrea edulis approximately

Materials and Methods Larvae were exposed to 10-4, 10-5 or 10-6 of GABA, epinephrine, norepinephrine, L-DOPA and IBMX for 24 and 48 h Each assay included a control without chemical inducers Larvae settlement was monitored after 24 and 48h Larvae mortality was monitored after 24 and 48h

Materials and Methods Settlement assays in industrial tanks Triplicate 100-L polyethylene tanks 200-µm mesh with cockle-shell triturate 5000 larvae/l of Ostrea edulis approximately

Materials and Methods Settlement assays in industrial tanks Larvae were exposed to 10-6 of GABA, 5 L final volume for 4h Larvae were placed in 100-L tanks, open circuit and continuous flow, airlift at 18 ± 1ºC Each assay included control without chemical inducers Larvae settlement was monitored after 4 and 8 days Larvae mortality was monitored after 4 and 15 days Spat size was determine after 15 days

Results Percentage of settlement = total # settled larvae x 100 total # larvae Percentage of mortality = total # dead larvae x 100 total # larvae

Results Settlement assays in the laboratory Percentage of O. edulis larvae induced to settle in presence of sea water or to exposure to GABA, epinephrine, norepinephrine, L-DOPA and IBMX 15.7% 14.4% 11.1% 6.9% mean SE, n=9. * p<0.05 from control

Results Percentage of O. edulis larvae induced to settle in presence of sea water or to exposure to GABA, epinephrine, norepinephrine, L-DOPA and IBMX 64.8% 42.5% 44.7% 36.7% 43.3% 43% 16.1% mean SE, n=9. * p<0.05 from control

Effect of the chemical treatments on the mortality of oyster larvae after 24 and 48 h under laboratory conditions Chemical treatment % Mortality 24h 10-4 10-5 10-6 GABA 1.37 ± 1.94 1.00 ± 1.85 0.75 ± 1.84 Epinephrine 0.83 ± 1.58 1.18 ± 1.97 0.38 ± 0.93 Norepinephrine 1.13 ± 2.33 1.13 ± 2.28 0.93 ± 1.51 IBMX 1.51 ± 1.49 1.33 ± 2.13 1.16 ± 1.98 L-DOPA 0 ± 0 1.57 ± 1.84 0.57 ± 0.97 Control 0.66 ± 0.99 48h GABA 3.96 ± 3.53 4.07 ± 4.57 2.98 ± 3.81 Epinephrine 3.37 ± 3.89 2.70 ± 3.74 3.23 ± 3.01 Norepinephrine 1.31 ± 1.78 1.77 ± 1.84 2.17 ± 2.84 IBMX 4.67 ± 4.12 3.78 ± 5.4 5.04 ± 4.89 L-DOPA 3.15 ± 4.33 4.18 ± 7.19 5.18 ± 8.10 Control 2.64 ± 3.07

Results Settlement assays in industrial tanks Percentage of settlement, mortality and spat size of O. edulis larvae in industrial tanks in presence of sea water or exposed to 10-6 M GABA DAYS AFTER TREATMENT 4 days 8 days 15 days % Settl Mortality % Settl Mortality Spat size GABA 70.7 ± 3.5* 0 % 90.6 ± 3.4 12 ± 1.3* 700 µm Control 38.8 ± 1.7 0% 88 ± 2.0 24.5 ± 1.6 425 µm Data represent the mean SE, n=3. Asterisks indicate significant differences (p<0.05) from control

Conclusions All the chemicals induced larvae to settle at some concentration with low toxicity GABA was the most effective inducer of larval settlement in O. edulis larvae In the hatchery GABA increases the settlement with a higher survival rate GABA also promotes the synchronization or a faster spat growth

Recomendation to Industry 10-6 GABA is recommended to be used as inductor of settlement

Future work GABA exposure in industrial tanks: time, concentration and effect on synchronization and spat growth Use of ions: potassium chloride, calcium chloride. Other neuroactive compounds: choline, acetylcholine and serotonin Biofilms Substrates: cockle shell, oyster shell

Crimgilt Mesías-Gansbiller Verónica Maneiro Antonio J. Pazos José Luís Sánchez Luz Pérez-Parallé Research team Instituto de Acuicultura (USC) Almeja Ría de Arosa SL Arturo Silva EU s Seventh Framework Programme FP7/2007-2013, grant agreement no. 222043 (Project webpage: http://settleproject.com).

Thank you for your attention Bedankt Gracias