Production of Eggs and Nauplii

System and Equipment Preparation

1. The copepod culture tank is shown in Figure 5. If starting with a brand new tank, wash the interior of the 300 L Fiberglas tank with a low-residue laboratory detergent (e.g. Alconox or Sparkleen) and water. Rinse thoroughly. Then treat with 100% muriatic acid (HCl) solution outdoors. Rinse thoroughly.
2. Whenever working with acids, observe all appropriate safety precautions, review acid safety protocols, maintain flowing freshwater within reach at all times. Be very careful and wear proper protective gear. (Do not use muriatic acid indoors: chlorine gas (Cl₂) may be generated.)
3. Leach the tank three times (24 h each time) to remove all water-soluble remnants of the manufacturing process.
4. If using an aged tank, wash thoroughly with low-residue laboratory detergent (e.g. Alconox or Sparkleen) and water. Clean with 100% muriatic acid, as needed (see precautions: E.1.1). Rinse thoroughly.
5. Initiate hatching of A. tonsa eggs 48 h prior to projected stocking time. Hatch approximately 50,000 eggs per liter in a clean 20L carboy.
6. Assemble all needed items: tank, cover and brace, air line and stone, harvest siphon, stacked sieves, sieve holder, drain line, wash bottles, beakers, egg storage tubes, dual 40W Cool-White fluorescent fixture, light timer, digital temperature controller, aquarium heater(s).
7. Prior to chlorination of the system, place aquarium heaters (dial heater to hottest settng or to “always on”), digital controller thermal probe, airline with airstone mounted, and harvest squeegee in the tank for chemical sanitary treatment of all immersed plumbing and wiring.
8. Fill the tank to the rim with filtered, UV-treated seawater, adjust the salinity as needed, and chlorinate the tank with 60 ml (0.2 ml/L) commercial 10% Hypochlorite solution per liter. This yields ~1 ppt free chlorine. Do not start aeration.
9. Filling the tank to 200 L working depth prior to chlorination is unacceptable, the freeboard sidewalls and cover underside will not be sanitized.
10. Allow treated system to stand for 24 h. Thereafter, de-chlorinate with 60 ml of stock Thiosulfate solution. Start vigorous aeration.
11. After an hour, dip a ‘free chlorine’ test strip and read to verify zero ‘free chlorine’ remaining. Aerate the treated seawater to at least 6 mg/L DO.

Figure 5. 300L copepod production tank.

Start-up Protocol
Once the equipment is clean, sanitized, and ready to use, complete the following steps:

1. Plug in the digital thermal controller and set to 25° C (or other selected temperature), check that the thermal probe for the controller is located 40 cm away from heater. Plug aquarium heater(s) into the digital controller.
2. Plug airline into 0.2 µm air filter, attach airline from pump. Turn on pump and adjust air to vigorously agitate the tank (while no copepods are present).
3. Allow tank to come to stable 25° C temperature. In continuously hot conditions, replace the heater with a chiller; the stable temperature concept is the same.
4. Plug in a timer for the fluorescent lights set to 14 h light /10 h dark (summer). Lights are mounted directly above the tanks to maximize light intensity for prolonged alga survival. Remember that light intensity drops by the square of the distance from the source.
5. Count cell density of algae in production using a hemocytometer. Follow hemocytometer instructions. Count three samples for each species of feed, calculate averages, and compute cells/ml by container. Record cells/ml by species of algae, container, and date in an Algae Log Book.
6. Compute volume of each species of algae required to feed a 200 L tank at a concentration of 15,000 cells/ml for each algal species (naupliar feed density). Add each required feed volume to obtain total volume of feed needed to add to the tank to achieve proper feed concentrations.
7. Carefully fold back tank cover (using alcohol sanitized hands). Drain tank to 200 L working depth; continue to drain below 200 L by total volume of feed to be added.
8. Minimize aeration and align air stone and heater in tank center.
9. Add the correct volume of each algal species and stock hatched nauplii at one per ml (i.e., for 200 L this requires 200,000 nauplii).
10. Cover tank. Verify that lights are on and timer is set to “automatic”. Verify that the digital controller is on and set to the proper temperature.
11. Nauplii develop to copepodites in less than a week at 25° C, and copepodites mature into adults in less than a week. Inspect the population’s developmental stage regularly and record appropriate data (see Weekly Protocol for sampling technique).

Daily Culture Protocol

1. Count algae concentrations and compute required feed volumes to be added.
2. Siphon detritus from tank bottom daily.
3. Remove sufficient water volume to allow addition of new feed volume(s), while maintaining 200 L total volume. As the population matures to stage C-III and C-IV, this volume can be increased to 300 L. Adjust feed computations accordingly.
4. Connect siphon drain hose to siphon and clamp hose to stacked sieves.
5. Discharge siphon drain hose into submerged stacked sieves (150 µm on top, 53 µm on bottom). Mesh screens on stacked sieves must remain submerged during the entire collecting cycle.
6. Discharge effluent from culture system into filtration or appropriate treatment system.
7. 150 µm upper sieve will retain feces, advanced copepodites, and adults. Rinse upper sieve gently over lower to pass eggs through, then into the beaker marked “adult”.
8. 53 µm lower sieve will retain feces, nauplii, and eggs. Rinse sieve gently into separate collection beaker marked “nauplii”.
9. Beakers are allowed to stand for 10 minutes to allow settling.
10. Adults and nauplii are then attracted to the upper layer of the beaker with light and decanted for return to the tank.
11. Eggs are washed free of feces as thoroughly as possible and then processed as appropriate for immediate hatching or storage for future hatching.
12. The collection from the 48 μm Nitex harvest sieve (the lower one) is first rinsed through an 100 μm Nitex sieve into a beaker to help pulverize the feces and detritus present in this fraction. This collection is then rinsed thoroughly on a 70 μm Nitex sieve, until virtually all feces are removed. Eggs to be stored should be counted, and placed in 30 ml seawater in clearly labeled 50 ml Falcon centrifuge tubes and held at 1ºC.
13. Count a subset of eggs and allow them to hatch for 48 h. The nauplii hatching in 48 h divided by the eggs collected is used to determine the hatch ratio for this cohort of eggs.
14. Rinse sieves vigorously in hot water, submerge in a 10% muriatic acid bath for 30 minutes, rinse again, and allow to air dry.

Weekly Culture Protocol

1. Population counts should be done weekly for feeding adjustments.
2. Population counts are obtained by thoroughly mixing the tank after siphoning eggs for the day and feeding the tank (for return to full 200 L working volume).
3. Remove three 250 ml samples from the thoroughly mixed tank.
4. Sieve each sample individually through 53 µm mesh, rinse into a 50 ml beaker, bring to 10 ml volume, stain with Lugol’s solution and count nauplii, copepodites, and adults. Determine sex of adults for male/female counts. Pool the three counts and average. Compute total population (average/250 ml x 4 x tank volume). Record result.
5. As the population matures (and biomass and tank volume increase), increase feeding concentration to 25,000 cells/ml for each species of algae.
6. The tank should retain a minimum red “cast” to the tank culture water (if using Rhodomonas spp.) after 24 h to imply moderate excess feed availability. If not, increase feeding frequency and/or feed concentrations above 25,000 cells/ml.
7. Excess feeding is unnecessary and unwise. Serious water quality problems can occur. Monitoring must be ever more vigilant and cleaning more thorough at increased feeding concentrations. Additionally, mature algal cultures often display elevated pH (in the 9 – 10 pH range). Adding large volumes of algae culture can cause dramatic pH shifts in the tank, with potentially disastrous results.
8. As the tank approaches senescence, hatching ratios will drop below 75%, irrespective of egg production. Males live shorter lives than females (Parrish and Wilson, 1978), thus females can produce non-viable eggs once the males die out and the attached spermatophores are depleted. Terminate the tank when hatch ratios drop below 65%.
9. If continuous culture of Acartia is desired, alternating tanks must be started on a staggered schedule to allow one population to be maturing, while the other population is providing peak production. Do not wait until one tank is senescent prior to starting the next or you will have a week or longer refractory period with no egg production.




click on chart for a larger view

Figure 6. Typical Production Cycle. July 6 – August 8. Tank stocked with N3 – C1 A. tonsa at 1 per ml on July 6th. One week later egg production started. Two weeks later egg production began to peak. Three weeks later, egg production remained high, but the hatch % was declining. After four weeks of batch culture, hatch was below 25%.