PROBLEMS FACED DURING
METAMORPHOSIS AND SETTLEMENT
L. Pérez-Parallé, J.L. Sánchez, A.J. Pazos, C. Mesías-Gansbiller and A. Silva
Instituto de Acuicultura
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
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
6.9%
15.7% 14.4%
11.1%
Results
31/1/2013
Percentage of O. edulis larvae induced to settle in presence of sea water or to exposure to GABA, epinephrine, norepinephrine, L-DOPA and IBMX
16.1% 64.8% 42.5%
44.7% 36.7%
43.3% 43%
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
Effect of the chemical treatments on the mortality of oyster larvae after 24 and 48 h under laboratory conditions
Results
Settlement assays in industrial tanks
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
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
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
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
Research team
Crimgilt Mesías-Gansbiller Verónica Maneiro
Antonio J. Pazos José Luís Sánchez Luz Pérez-Parallé
Almeja Ría de Arosa SL
Arturo Silva
31/1/2013
Instituto de Acuicultura (USC)
EU s Seventh Framework Programme FP7/2007-2013, grant agreement no. 222043 (Project webpage: http://settleproject.com).