Plankton Culture for Feeding Larval Fish and Shellfish LaDon Swann Mississippi-Alabama Sea Grant Consortium Auburn University

Shellfish Larval Culture

Larval Rearing Trocophore D-Hinge or Straight Hinge Size 50-60 microns 6-12 hours post-fertilization D-Hinge or Straight Hinge Size 70-80 microns 24-48 hours post-fertilization Bivalve Shell Velum - Swimming and Feeding organ

Larval Rearing Veliger or Umbo Larvae Pediveliger or “Eyed” Larvae Size 80-300 microns Stage last 8-12 days Well defined umbo Pediveliger or “Eyed” Larvae Size >300 microns Pigmented eye-spot Muscular foot

Phytoplankton Production Feeding Larvae Cell Size 4-8 microns Species Isochrysis galbana Chaetoceros gracilis Nannochloris sp. Chlorella sp. Pavlova lutheri

Pavlova lutheri Morphology Salinity Temperature Culture media Golden brown Spherical with 2 flagella 3-6 µm Salinity 8-32 ppt Temperature 11-26 °C Culture media Guillards f/2 Proximate Analysis 52% Protein 24% Carbs 29% Fat

Isochrysis galbana Morphology Salinity Temperature Culture media Tahiti (T-Iso strain) Golden brown Cells spherical with 2 flagella 5-6 µm length, 2-4 µm wide Salinity 8-32 ppt Temperature 23 - 28°C Culture media Guillards f/2 Proximate Analysis 47% Protein 24% Carbs 17% Fat

Chaetoceros gracilis Morphology Salinity Temperature Culture media Golden brown diatom Medium-size 12 µm wide, 10.5 µm long Cells united in chains Salinity 26 - 32 ppt Temperature 28 - 30°C Culture media Guillards f/2 with Si Proximate Analysis 28% Protein 23% Carbs 9% Fat

Plankton for Shellfish Broodstock and Spat Cell Size 10-24 microns Species Tetraselmis sp. Green Thalassiosra sp. Diatom

Tetraselmis sp. Morphology Salinity Temperature Culture media Ovoid green cells 14 to 23 µm L X 8 µm W 4 flagella Salinity 28-36 ppt Temperature 22-26°C Culture media Guillards f/2 Proximate Analysis 55% Protein 18% Carbs 14% Fat

Thalassiosra sp. Morphology Salinity Temperature Culture media Golden brown diatom Cells united in chains Barrel-shaped Non-motile 4 µm Salinity 26 – 32 ppt Temperature 22-29 °C Culture media Guillards f/2 with Si Other characteristics

Micro Algae Culture Culture Water Sterilization Nutrient Enrichment Inoculation Cell Counts Harvest and Feeding Stock Culture

Culture Water Sources Salinity Seawater Saltwater wells Prepared seawater Salinity 26-32 ppt

Sterilization Methods Heat Pasteurization Autoclave 80 C and cool naturally Autoclave Sodium Hypochlorite (bleach) 0.5 ml/L (10 drops) Neutralize: 10-15 ml sodium thiosulfate (248 g/L) per liter Hydrochloric acid (muriatic) 0.2 ml/L (4 drops) Neutralize: Na2CO3 0.4-0.9 g/L

Nutrient Enrichment Nutrients Conc. (mg/l Seawater) Guillard’s f/2 NaNO3 75 NaH2PO4.H2O 5 Na2SiO3.9H2O 30 Na2C10H14O8N2.H2O (Na2EDTA) 4.36 CoCl2.6H2O 0.01 CuSO4.5H2O FeCl3.6H2O 3.15 MnCl2.4H2O 0.18 Na2MoO4.2H2O 0.006 ZnSO4.7H2O 0.022 Thiamin HCl 0.1 Biotin 0.0005 B12 Guillard’s f/2 Part A and B 0.5 ml/L each part Na2Si03 for diatoms

Inoculation Culture vessels 1,000 ml flask 18.7 L (5 gal.) Carboy (glass) 178 L (47 gal) Transparent Tank Add enough algae to give a strong tint to the water 100,000-200,000/ml Lighting Types Sunlight Fluorescent VHO fluorescent Metal halide Highest Densities: 24/7

Cell Counts Peak Algae Density Hemacytometer I. Galbana T. pseudonana 10-12 million cells/ml 10-14 days 2 wk stability T. pseudonana 4 million cells/ml 3 days 5 day stability Hemacytometer Count total in centermost 1 mm Multiply by 10,000 Product = number/ml Motile cells should be killed

Harvest and Feeding Larvae Density Algae Density Tank cleared in 24hrs 5-10 larvae/ml Algae Density Wk 1 = 50,000 cells/ml Wk 2+ = 100,000 cells/ml Onset of spatting = 200,000/ml Tank cleared in 24hrs Liters to feed = (TD x V)/CD TD = Target Density (1,000s/ml) V = Volume of larval tank (thousands of L) CD = Cell Density (millions/ml)

Harvesting and Feeding Batch Total harvest occurs once or over several days Semi-Continuous Works well with diatoms Part of the algae remains in the vessel New media is added to replenish the algae removed

Stock Culture Purchase pure strain Avoid contamination Holding No aeration Half filed container Redundancy Holding Test tubes Conical flasks Transfer 1 drop/wk for T. pseudonana 1 drop/2 wk for I. galbana

Marine Fish Larval Culture Relies on zooplankton

Marine Rotifer Brachionus plicatilis Culture units 40L plastic bags 40L cone-bottomed tanks Temperature 27-30 C Salinity 26 ppt

Rotifers Laboratory production 100 to 200+ mm size 2-3 week life span small size suitable as first food

Feeding Marine Fish Rotifers Typical first food in hatchery Feed algae or yeast Enrichment needed

Artemia Feeding of older larvae

Use of copepods