**2. Materials and methods**

All the larval rearing experiments reported here took place at the Mote Marine Laboratory facilities, located in Sarasota, Florida. During the first two years experiments were carried out in aquaculture systems located at Mote Marine Laboratory. The studies carried out in the later two years were conducted in the new aquaculture systems located at Mote Aquaculture Research Park (MAP).

Artificial seawater (Instant Ocean ®) was used at both locations; however, conditions at Mote Marine Laboratory were not ideal because the systems were located under a building that had poor ventilation, limited lighting, and a lack of temperature control. At MAP, experiments were conducted in a variety of tank systems equipped with state-of-art filtration, and in isolated and temperature controlled experimental rooms.

#### **2.1 Live culture**

The suitability of three live food types (microalgae, rotifers, copepods) were investigated in parallel. Each live culture was maintained in separate rooms using water from a different reservoir, in order to avoid any possibility of cross-contamination.

#### **2.1.1 Microalgae**

The main microalgae used in the snook trials was *Nannochloropsis occulata;* this is non motile, green coloured cell with no flagella. It is a small, elliptical cell, 4-6 µm in diameter, with few distinguishing features. The chloroplast usually occupies much of the cell. Cells tend to float in culture and stay in suspension without aeration. This organism is placed in a separate division from Nannochloris because of its lacks of chlorophyll b. These algae are a popular food source for rotifers and filter feeders.

*N. occulata* was used to feed the rotifer cultures and to create a green water environment in the larval systems. The procedure used for its culture was the classical batch culture method, which consists of inoculating culture tubes with low density of algae cells. After two weeks, test tube cultures were transferred into 250 ml flasks and later (1 week) into a larger 19 L carboy culture vessels. After a week, a 100-liter cone shape transparent tank was inoculated with a full carboy. The culture was kept running with four 200 L transparent fiberglass tanks during the experiment's duration to ensure reliable microalgae production. All cultures were exposed to 24 hour white light condition (1000 lux), water temperature was kept at 29 ºC, and had constant aeration.

*N. oculata* paste was also obtained from Reed Mariculture. This paste is a highly concentrated media (68 billion/ml) of *N. oculata* that was kept frozen until the day before it was used. The paste was used to reduce the time involved in batch culture of live algae and to test the difference between live and frozen paste algae as a food source for rotifers and for creating a green water environment.

#### **2.1.2 Rotifers**

192 Aquaculture

Commission (Ager *et al*., 1978; Shafland and Koehl, 1980, Chapman et al., 1978). These studies provided information on the lower lethal temperature (15°C) for juveniles (Howells *et al*., 1990) and preliminary developmental results for laboratory reared larvae and juveniles (Lau and Shafland, 1982). These studies described basic common snook biology and the principles for captive rearing. The early studies on this species in Florida and Texas conducted in the 1970's, 1980's and 1990's, were unable to identify the appropriate culture

The objectives of the series of experiments reported here were to improve larval survival of

requirements to support captive spawning and larval rearing of common snook.

2. Establish the effect of egg stocking density on larval survival and growth

4. Investigate the effect of the green water technique on larval survival and growth

6. Investigate effect of alternative live food species on larval survival and growth

All the larval rearing experiments reported here took place at the Mote Marine Laboratory facilities, located in Sarasota, Florida. During the first two years experiments were carried out in aquaculture systems located at Mote Marine Laboratory. The studies carried out in the later two years were conducted in the new aquaculture systems located at Mote

Artificial seawater (Instant Ocean ®) was used at both locations; however, conditions at Mote Marine Laboratory were not ideal because the systems were located under a building that had poor ventilation, limited lighting, and a lack of temperature control. At MAP, experiments were conducted in a variety of tank systems equipped with state-of-art

The suitability of three live food types (microalgae, rotifers, copepods) were investigated in parallel. Each live culture was maintained in separate rooms using water from a different

The main microalgae used in the snook trials was *Nannochloropsis occulata;* this is non motile, green coloured cell with no flagella. It is a small, elliptical cell, 4-6 µm in diameter, with few distinguishing features. The chloroplast usually occupies much of the cell. Cells tend to float in culture and stay in suspension without aeration. This organism is placed in a separate

filtration, and in isolated and temperature controlled experimental rooms.

reservoir, in order to avoid any possibility of cross-contamination.

common snook during the first 14 days after hatching.

1. Investigate the influence of temperature on hatch rate

3. Determine the influence of flow rate on larval survival

5. Determine the influence of rotifer density on larval survival

7. Evaluate the acceptance of micro-diet feeding by larval snook

**1.2.6 Objectives** 

The main aims were to:

**2. Materials and methods** 

Aquaculture Research Park (MAP).

**2.1 Live culture** 

**2.1.1 Microalgae** 

Four different types of rotifers were used in the snook larval rearing trials. *Brachionus rotundiformis* or small (S-type) rotifers and *B. plicatilis* or large (L-type) rotifers, which can be clearly distinguished by their morphological characteristics: the lorica length of the L-type ranges from 130 to 340 µm (average 239 µm), and in the S-type ranges from 100 to 210 µm (average 160 µm). Moreover, the lorica of the S-type has pointed spines, while the L-type has obtuse angled spines. Two other types of rotifers were used. The SS type rotifer (Super small rotifers) ranges between 100-120 µm, which are preferred for the first feeding of fish larvae with small mouth openings (rabbitfish, groupers, and other fish with mouth openings less than 100 µm at first feeding). Those rotifers, however, are not genetically isolated from Sstrains, but are smaller than common S-strains (Person Le Ruyet, *et al*., 1993). The last strain was an SS rotifers from the University of Ghent, Belgium, that were genetically modified to resist warmer temperatures (above 30 ºC).

All the rotifers strains were cultured using a batch culture method. Batch cultivation, due to its simplicity, is probably the most common type of rotifer production in marine fish hatcheries (Fukusho, 1989; Nagata and Hirata, 1986; Snell, 1991). The culture strategy consists of either the maintenance of a constant culture volume with an increasing rotifer density or the maintenance of a constant rotifer density by increasing the culture volume. In batch culture, a total harvest of the rotifers is done with part of the rotifers being used as food for fish larvae and part used as inoculum for the next culture (Hirata, 1980; Lubzens, 1987). All the rotifers were fed *N. oculata*.

#### **2.1.3 Copepods**

The calanoid copepod *Acartia tonsa* (*Acanthacartia*) was cultured for some of the feeding experiments. This species was chosen due its small size (80-100µm), nutritional value and availability. The copepods were cultured at Florida State University (Tallahassee) and eggs were sent every two weeks on ice (4°C) in 100 ml flasks. Once in the lab, eggs were refrigerated at 4°C until they were needed. Copepod eggs were taken out the 100 ml flask and placed in a 500 ml transparent flask with seawater at 35 ppt and 28ºC under a 12 hours light:dark period. No aeration was needed during the 48 hours hatching period. After hatch, *A. tonsa* were fed to the snook larvae. Feeding densities varied depending on the experiment.

#### **2.2 Larval rearing systems**

All the experiments were conducted using two independent experimental systems: small and large microcosm systems.
