6.3.1. Introduction
6.3.2. Production of trochophora larvae
6.3.3. Quality control of the produced trochophora larvae
6.3.4. Cryopreservation
Figure 6.3. General scheme of a trochophora larva.
For some marine fish species (i.e. siganids, groupers, snappers) very small zoo-plankton, such as trochophora larvae (Fig. 6.3.) need to be used as a starter feed, since the commonly used rotifers are too big. Trochophora larvae of the Pacific oyster Crassostrea gigas are 50 µm in size and free-swimming (slow circular swimming pattern) ciliated organisms which have a high nutritional value for marine fish larvae. For example, trochophora larvae may contain up to 15% (of total fatty acid) of both EPA and DHA.
6.3.2.1. Mussel larvae
6.3.2.2. Pacific oyster and Manila clam larvae
Unripe mussels are brought in acclimation tanks with flowing seawater, after the removal of excess epifauna. The temperature is kept at 10-12°C for a minimum period of two weeks. During the acclimation period the mussels are fed on algal suspensions of Dunaliella tertiolecta and/or Chlamydomonas coccoides. The spawning of the animals is induced by bringing the conditioned mussels in a plastic bucket and shaking them violently for 2 to 3 min. After returning the stimulated mussels to the spawning tanks (lightly aerated static seawater at 14-15°C) spawning takes place within 12 h. The trochophora larvae can be harvested after 24-48 h by concentrating them on a 25 µm sieve. After 10 weeks the broodstock should be replaced, since the gametes are reabsorbed as a result of temperature stress and inadequate food supply.
Broodstock acclimation systems consist of 150-200 l fibre glass tanks, each stocked with 50 broodstock animals of 20-25 g each. The broodstock tanks are continuously provided with preheated unfiltered natural seawater at a minimum rate of 1 l.min-1. Algae (Tetraselmis sueccica, Skeletonema costatum and Thalassiosira pseudonana) are continuously added to the seawater by means of a peristaltic pump. In the case of clams a substrate of sand and/or gravel can be used, but this is not essential. Under controlled temperature conditions gametogenesis and gamete maturation can be induced year round by submitting the bivalves to a sudden temperature shock (increasing the temperature 2 to 4°C). Spawning will take place within 15 min. and the gametes are released into the tank. During this period the water flow must be stopped in order to allow fertilization. A gentle aeration can be used to keep the gametes in suspension.
Monitoring during the development is necessary to estimate the time of harvesting of the trochophora larvae, which generally takes place after a few hours. The trochophores are harvested from the incubation suspension by pouring the content of the incubation tank on a submerged 35 µm sieve. After washing with pure preheated seawater the trochophora larvae can be fed to the fish or shrimp larval tanks.
Obtaining good quality trochophores with good swimming behaviour and a high nutritional value is important. Firstly, the broodstock must be fed with algae with a high nutritional value. Secondly, spawning must be synchronized, as there is rapid loss in sperm fertility. Thus, when males start spawning before the females, the males must be removed from the container and left out of the water, so as to stop the male spawning; the males are put back in the water when a sufficient number of females start to spawn. At no time should sperm older than 30 minutes be used.
To have a better control over the quality of the trochophores, one can divide the broodstock animals after the spawning shock over individual containers. After spawning is completed the females should be taken out so as to let the eggs settle on the bottom. Clumps of eggs must be separated to obtain good fertilization and this is achieved by pouring the content of the dishes or beakers through a 60 µm mesh screen and collecting the individual eggs on a 15 µm mesh sieve. The eggs are then washed with clear seawater, screened on their quality (eggs must hydrate within 10 min. in seawater and must have a uniformly dense, granular appearance), and pooled. Sperm from various males is pooled to ensure a good genetic mix in offspring. Fertilization is carried out by gently mixing 2 ml of a dense sperm suspension to 1 l of egg suspension, after which the suspension is allowed to stand for several hours. Within this period the fertilized eggs start to divide. However, densities of developing embryos should not exceed 80,000.l-1.
Bivalve larvae can be cryopreserved at -196°C and used as live feed for later use. Cryopreservation has been successfully achieved with trochophora larvae of Crassostrea gigas and Tapes philippinarum. The larvae are equilibrated in a seawater solution of 2 M dimethylsulfoxide (DMSO) with 0.06 M trehalose (cryo-protectans) for 10 minutes at 25°C and are then sealed into polyethylene straws at a density of 15 and 50 million trochophores each. The straws are then rapidly cooled from room temperature to 0°C and then from 0°C to -12°C at a freezing rate of -1°C.min-1. The straws are then held at -12°C for 5 to 15 minutes allowing equilibration of the temperature of the biomass. Finally, the trochophores are slowly cooled at -2°C.min-1 to -35°C, after which they are allowed to equilibrate for 10 to 20 minutes before being submerged in liquid nitrogen (-196°C) (Chao et al., 1995). Before use the content of the straws is rapidly defrozen in a seawater bath at 28°C and after 1 h the actively swimming trochophores can be administered to the fish larvae. Cryopreserved trochophores are also commercially available as Trochofeed (Cryofeeds Ltd., Canada). They are produced from certified disease-free broodstock oysters of selected genetic strains.
