Cryopreservation of nauplius larvae of the barnacle, Balanus amphitrite Darwin

The investigation was conducted to develop the prospects for application of cryopreservation technique to nauplius larvae of economically important crustacean, such as Japanese prawns or crabs. Since Balanus amphitrite possesses naupliar stages similar to those of prawn, the nauplius-II larvae of barnacle were applied in this experiment. The larvae were equilibrated for 20 minutes in a cryoprotectant solution containing 1.5 M dimethyl sulfoxide (DMSO) in 29ppt filtered seawater. They were then frozen to -196 degree C by a two-step freezing procedure. The optimum conditions for the cryopreservation of the nauplius-II larvae were: from ice seeding temperature of -12 degree C to the first step cooling temperature of -30 degree C was by a cooling rate of 0.5 degree C/min, then rapidly frozen to -196 degree C. Frozen nauplii were thawed at 50 degree C, and incubated in 29ppt filtered seawater after washing cryoprotectant. The diatom Chaetoceros calcitrans at a concentration of 40 x 10 super(4) cells /ml was provided as food in the rearing of nauplii. After 28 days preservation in liquid nitrogen (-196 degree C), 78% of the larvae recovered and 19.8% of the larvae metamorphosed to cyprid.