OBTAINING THE NECESSARY LIVING FOOD FOR REARING THE TURBOT (Psetta maxima maeotica) IN ITS EARLY LIFE

STAGES
VICTOR NI1, TANIA ZAHARIA2, VALODEA MAXIMOV2, CARMEN NICOLAE1 1 University of Agronomical Sciences and Veterinary Medicine Bucharest 2 National Institute for Marine Research and Development Grigore Antipa Constanta Keywords: turbot, living food, aquaculture SUMMARY
Ensuring the food necessary for a planet that becomes over-populated remains a major challenge in the third millennium, although humanity confronts with vital problems, incurable diseases, accidental pollutions. Aquaculture can be an alternative and an infinite food source, while also giving a chance to endangered species conservation, by compensating the losses due to overfishing and supplying 20% of the consumable aquatic products at global scale. The turbot (Psetta maxima maeotica) is a demersal species that populates the Romanian Black Seas continental shelf and is an important segment of the regional aquaculture potential under the aspect of market demand, both on the national and international level. The paper hereby presents some aspects concerning the aquaculture of green algae, rotifers and Artemia, very important living food products that are successfully used in the Black Sea turbot rearing, in its first stages of development.

1. MATERIAL AND METHOD

The paper hereby is based on the consultation of an abundant bibliographic material, as well as on the results of the research and experiences carried out within NIMRD between 1980 and 2011. The experiments were recently reproduced, using new technologies and sources of biological material. Unlike tens of years before, we used pure algal strains purchased from laboratories in U.S.A. For starting the rotifer cultures we used resting eggs, purchased also from U.S.A. We used the experimental base of the Institute, comprising both exterior concrete basins and laboratories, equipped with all the necessary facilities.
2. RESULTS AND DISSCUSIONS

In Romania, the ecological state of the Black Sea and the diminished fish catches produced catastrophic changes in the natural exploitable resources productivity. Today, the marine aquaculture is a relatively recent field of activity, which started to develop scientifically, both for economical and ecological reasons. Aquatic organisms are easily affected by environmental conditions. Their maximum sensibility manifests during ontogenesis and metamorphosis. The chance to survive and develop normally depends a lot on the quality and naturalness of the ingested food. Through its composition, the living food essentially contributes at covering the entire necessities specific for fish larvae, concerning the composition in proteins, lipids, glucose, vitamins, oligo-elements, hormones and enzymes, ensuring the energetic support in living, growing and development.

ALGAL CULTURES

Within turbot reproduction, the phytoplankton comprise anyone or all the following functions: (a) food for the rotifers, (b) enriches the nutritive value of the rotifers and Artemia, (c) detoxifies the water in which the turbot larvae are raised, by assimilating or neutralizing the toxic substances such as ammonia and pesticides. The growing model has five phases: the moderate growing, the fast growing, the slowing of growing, the stationary and the collapse one. At the beginning of the experiment, pure strains of Nannochloropsis sp. and Tetraselmis sp. were purchased from a specialized laboratory in U.S.A., in sealed plastic recipients. Stock culture. In this small scale culture phase, the stock culture is maintained in testtubes in laboratory. A separate room is used, in order to avoid contamination with rotifers or other algae. The stocking room for the algae must be sterilized weekly. The room must have air conditioner, for keeping the temperature between 18 - 22C, the proper temperature for the algae. The photoperiod is controlled to 12 hours light 12 hours dark, by using fluorescent lamps. In this stage, the glass test-tubes and recipients are recommended. The sea water is filtered through fiberglass filters and kept in recipients for a few months before use. The old water is put in culture recipients and sterilized.

Photos 1 and 2: Stock cultures of Nannochloropsis sp. and Tetraselmis sp. kept in controlled environment Test-tubes culture. Two stock culture drops must be inoculated in every one of the 20 test-tubes used at this phase, by using sterilized pipettes. The test tubes contain 10 ml of nutritive culturing solution. A clean, uncontaminated stock culture is recognized by its transparency. Also, a uniformity of the cell dimensions, observed at the microscope, is a good indicator. The cultures are let to grow for two three weeks, and after that they can be transferred in bigger recipients. The test-tubes must be daily agitated, for preventing the accumulation of the cells at the bottom. 5 l culture. The recipients must have air evacuation tubes and cotton wool plugs. A strong aeration (6 l/min.) must be ensured for homogenizing the culture. A single uncontaminated test-tube is inoculated in a 5 l recipient, together with 3.5 l of nutritive

solution. The density for Nannochloropsis sp. is of 80 100*106 cells/ml at 4 7 days after inoculation. 100 l culture. Transparent polycarbonate tanks will be filled with water and chlorinated with 300 ppm liquid chlorine for one night before using. In the morning, the water will be neutralized with sodium thyosulphate for a few minutes. The aeration must be adjusted to 8 l/min. Agricultural fertilizers will be added into tanks, and the 5 l starter culture will be added also. The density will reach 40*106 cells/ml in one week. Now, the cultivated algae will be used as living food for rotifers.

Photos 3 and 4: 5 and 100 l cultures of Nannochloropsis sp. and Tetraselmis sp. obtained in controlled environment Cell counting. The density of each culture should be daily recorded. The counting of algal cells is made by using an inverted microscope and a hemacytometer.

Photos 5 and 6: Inverted microscope and hemacytometer counting detail Measures for avoiding contamination. Visual inspection of tanks will be made daily and microscopic observations also. If green balloons at water surface or dirt on tank walls exist, a rotifer contamination can be suspected. A sample should be analyzed at the

microscope. If rotifers will be found, chlorine solution at 15 ppm will be added in the tank for 30 minutes and then it will be neutralized. Because rotifer eggs might survive this treatment, the procedure will be redone the next day.
ROTIFER CULTURES

This is a small group of aquatic invertebrate organisms, comprising about 2000 species. In turbot breeding it is essential to use rotifers as living food for the early life stages, because of the mouth dimensions. Brachionus plicatilis is the most cultivated. Small scale culture: at the beginning of the experiment, B. plicatilis was purchased from a specialized laboratory in U.S.A. as resting eggs, 1000 3000 per each sealed glass recipient. The eggs were placed in sterilized marine water, and in about 48 hours they began to hatch.

Photos 7 and 8: Rotifer resting eggs, delivered in sealed glass recipients, and their filling with sea water for hatching 200 ml culture. Before putting the freshly hatched rotifers from one recipient in the 200 ml Erlenmeyer glass, 20 ml of fresh Tetraselmis sp. culture will be placed in. The algal density will be adjusted to 2*106 cells/ml. After one week, rotifer density will be 150 rotifers/ml. One part will be transferred into test tubes for stock culture, and other will be inoculated in bigger recipients as following. 5 l culture. The rotifers from the 200 ml recipient will be poured in the 5 l one, by tilting the smaller recipient. The rotifers will be fed with 200 ml fresh Tetraselmis sp. culture. Every two days, 350 ml of algal culture will be added to ensure food, until the culture volume reaches 4 l (about 150 rotifers/ml). Aeration is still not necessary. Mass culture. There are two standard cultivation systems. In mixed culture system, rotifers in the tank will be totally harvested and a part of them will serve for inoculating other tanks. In exploitation culture system, rotifers are only partially harvested, and the volume difference in the tank will be filled with algal culture.

Photos 9 and 10: 5 liter and mass culture of rotifer Brachionus plicatilis Rotifer counting. Samples from tanks will be analyzed at stereomicroscope to determine the density. A drop of Lugol solution is used for killing the organisms. The number of ovigerous rotifers will be divided to the total number, for obtaining the fertility rate. A culture having a fertility rate below 10% has regeneration problems.

Photos 11 and 12: Counting the rotifer B. plicatilis at stereomicroscope Harvesting. The drainage valve will be opened and the rotifers will be retained in a net having the mesh eye of 80 m. During harvesting, we must avoid the collision of rotifers with air bubbles, because it causes a high mortality.
ARTEMIA NAUPLII

In turbot cultures, Artemia sp. nauplii are the second living food, which follows the rotifer diet. In our experiments, Artemia sp. eggs were purchased from specialized stores, and they began to hatch at 24 hours from their placement in marine water. Cylindrical tanks are filled with sea water and aerated. The eggs are placed in these tanks, in a concentration of 1 g/l. They are put to hatch for 22 hours, with strong aeration.

Before harvesting, the aeration is stopped and the tank is let for 10 minutes, so the eggs that have not hatched to depose at the bottom. Then, the hatched ones will be harvest using a net with the mesh eye of 114 m, and rinsed with salt water.

Photos 13 15: Cultivation of Artemia sp. (photos made by L. Alexandrov)

3. CONCLUSIONS

The turbot is an important segment of the national marine aquaculture potential under the aspect of market demand. Its chance to survive and develop normally in aquaculture farms depends on the quality and naturalness of the ingested food. Through its composition, the living food essentially contributes at covering the fish larvae necessities, by its composition in proteins, lipids, glucose, vitamins, oligo-elements, hormones and enzymes. In the past few years we used pure algal strains bought from specialized laboratories in U.S.A and rotifer starter-kits, bought also from U.S.A. The experiments showed that using this kind of sources for the biological material is valid, the food chain (phytoplankton zooplankton turbot larvae) being well reproduced within our experimental base. Acknowledgements: the results of the present paper are part of the first authors Ph.D. thesis, which is supported within POSDRU/107/1.5/S/76888 program.
REFERENCES
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