F. salina and D. salina, were collected using a 5-l Van Dorn bottle below the water surface from multi-pond solar salterns located along the Sfax coast (Tunisia, 34°39′N and 10°42′E) (Fig. 1). F. salina was harvested using a plankton net with a 100-μm mesh size, from the first evaporation pond A16 (salinity close to that of seawater: 78.7±8.8p.s.u.) (Table 1). D. salina was collected from the crystallizer pond TS (saturating salt concentrations: 424.5±35.6p.s.u.), in which the phytoplankton community was entirely composed of this Chlorophyceae (Table 2). The second food item consisted of pure cultures of the Prymnesiophyceae I. galbana (Tinamenor S.A. Marisma de Pesués, Cantabria, Spain). Algae and the ciliate were acclimated over three months to continuous illumination (2000 lux) and to Walne's medium (modified from [16], Table 3), respectively.

The commercially available dry yeast, Saccharomyces cerevisiae, was also used as a third food item for Fabrea. Cultures for experimental purposes were maintained in exponential-growth phase by regular transplants to a fresh medium. The cultures were synchronised, 24 h prior to the beginning of the grazing experiments. This was carried out for 6 days in 200-ml pre-sterilised flasks. Two sets of control (algae without Fabrea) and experimental flasks (two replicates each) were used during the experiment in a batch system with a salinity of 50 p.s.u. and maintained at 24 °C in temperature-controlled chambers, and under an illumination of 2000 lux. Ciliates were counted three times in each replicate flask.

The initial concentration of Fabrea was 4 cells ml⁻¹ and the three food items were inoculated separately from each other, in the morning, during the six days of incubation at a density of 4×106cellsml−1, estimated using a Burcker haemocytometer. The food density was maintained during the experiments by a daily cell enumeration. To stop the experiment, a glutaraldehyde solution (Sigma grade I, final concentration 1%) was added to inhibit protozoan motion [17]. A Sedgwick-Rafter counting cell (Graticules LTD, Tonbridge, Kent, UK), mounted on a Type Leica DM LS2 microscope (20X magnification), was used to estimate the average daily changes in Fabrea numbers; Fabrea length and width were estimated using a micrometer and the biovolume of Fabrea was measured according to [18].

The specific growth rate was calculated using the following formula:

2.1 Statistics

3.1 Fabrea density and growth rate

The density of Fabrea fed with D. salina did not exhibit significant changes from the start until the third day of the experiment. The growth rate was low (μ=0.12day−1) and cell numbers did not exceed 2.5 (±1.41) cells ml⁻¹ (Fig. 2a). However, from day 4 until the end of incubation, Fabrea cell numbers increased rapidly (μ=0.94day−1), reaching 17 ± 2.12 cells ml⁻¹ on the 6th day (Fig. 2a). When cultured with I. galbana, the density of Fabrea increased until day 3 (μ=0.50day−1, 18.25±1.77indml−1) and then collapsed (0.5±0.71cellsml−1). From the 4th day until the end of the experiment, Fabrea grew strongly (μ=1.31day−1) up to 25.75±6.01cellsml−1 (Fig. 2b). A similar pattern in the distribution of Fabrea was recorded when the protozoan was fed with the yeast S. cerevisia. However, no latency time on the fourth day was observed (Fig. 2c). Indeed, Fabrea grew (μ=0.18day−1) until the second day (7±0.71cellsml−1), then its density decreased until the fourth day (1.75±1.77cellsml−1). From day 4 onwards, the ciliate growth rate increased again (μ=0.31day−1), yielding a cell density of 4.5±2.83cellsml−1. From the first day until the end of each grazing experiment, the specific growth rates (μ) of Fabrea fed with Isochrysis, Dunaliella and Saccharomyces were 0.3, 0.24 and 0.02, respectively (Fig. 3).

3.2 Length and biovolume of Fabrea

When fed with dry yeast, the length of Fabrea ranged between 140±20μm and 177.8±5μm (mean±SD=160.49±22.63μm), corresponding to a mean biovolume of 5.33±1.81×105μm3. Conversely to the temporal distribution of the prey-predator couples Fabrea–Isochrysis and Fabrea–Dunaliella, the average length of Fabrea cultured with yeast increased throughout the experiment (Figs. 4 and 5).

The results indicate that under controlled experimental conditions, F. salina was able to grow when its diet consisted of D. salina, I. galbana and S. cerevisiae. Indeed, continuous light, a temperature of 24 °C, a salinity of 50 p.s.u. and a small water volume (200 ml) were sufficient to yield optimal growth of Fabrea. This is consistent with the findings of several authors, who showed that Fabrea can develop with various nutritional items [13]. Moreover, Rattan et al. [20] indicated that Fabrea can even grow with fermented wheat and rice grains. However, Repak [6] indicated that this ciliate does not grow in the presence of the Cyanobacteria Synechococcus spp. Many other protists have been successfully cultured, such as Favella sp., Uronema sp., Gymnodinium sp. [21,22]. A high growth rate (r=0.60day−1) was also recorded when the Tintinnid Favella sp. was fed with the Prymnesiophyceae Prymnesium parvum [22]. One should also bear in mind that it is of fundamental importance to keep growth and ingestion constant to evaluate growth yield in ciliates feeding different preys [23–25]. Although yield is constant, absolute biomass production may be higher in one prey than in another. Both high density and growth rates of F. salina were recorded when the diet consisted of I. galbana (26 cells ml⁻¹, r=1.31day−1). Indeed, the peak density recorded on the third day indicated a short-generation period of Fabrea, which may suggest that I. galbana had a high nutritional value, as already reported by Brown [26]. To our knowledge, the culture of Fabrea with Isochrysis has never been reported, so we were unable to compare our findings with those of others. The only available data concerned the genera Strobilidium and Strombidium, which exhibit a maximum growth rate of 2.2 day⁻¹ when fed with Isochrysis [27]. Although the effects of this flagellate on Fabrea development were remarkable, both the length and biovolume of the ciliate were smaller than those recorded when the Fabrea diet consisted of D. salina. This discrepancy may suggest that Fabrea underwent reproduction, as evidenced by the existence of two peaks of Fabrea density recorded on the third and sixth days (Fig. 2b), coinciding with a decrease in cell dimensions on the sixth day (Fig. 4). In support of this, we also found the weakest correlations between the length and biovolume of Fabrea fed with I. galbana (r=0.45, d.f.=27, p<0.01), versus D. salina (r=0.77, d.f.=17, p<0.01) and the yeast (r=0.95, d.f.=10, p<0.01). The highest densities of Fabrea were found when the latter was fed with D. salina and I. galbana. Pandey and Yeragi [13], who worked under similar experimental conditions, reported similar results with D. salina, but they did not consider I. galbana as a food item. They recorded cell densities of 44 and 64 Fabrea ml⁻¹ when grown in 1 and 5-l containers, respectively. Concerning the yeast S. cerevisiae, our results indicate that it was not a good food item for Fabrea, because the ciliate induced the lowest growth rate, cell length, and biovolume. The ANOVA analysis shows that both the density (F=18, d.f.=57), length (F=33, d.f.=57) and biovolume (F=19, d.f.=57) of Fabrea fed yeast differed significantly (p<0.001) from those of Fabrea fed both with D. salina and I. galbana (Tables 5 and 6). This difference may be explained by the deterioration of the medium throughout the experiment induced by yeast, as already suggested by [20]. Indeed, the high Fabrea density recorded on the second day implies a very rapid growth generation period which was strongly stimulated by S. cerevisiae at the beginning of the experiments. Yeast is widely used as a dietary supplement to support the growth of several species such as rotiferans and the anostracan Artemia. Pandey and Yeragi [13] found a density of 20 Fabrea ml⁻¹ when fed with yeast at concentrations of 5 mg l⁻¹. Overall, our results are slightly lower than those reported by Repak [6,7] and Pandey and Yeragi [13]. The maximal length of Fabrea recorded in this study (207 μm) is similar to that recorded by Dolapsakis et al. [28], who cultured F. salina obtained from a Greek saltern in which the salinity varied from 60 to 144 p.s.u. Furthermore, both the length and biovolume of Fabrea recorded in this study were higher than those found in the Sfax saltern. The values reported by [14] from this ecosystem did not exceed 111 μm and 180.1×103μm3, for salinities ranging between 70 and 170 p.s.u., respectively.

In conclusion, our study indicates that the ciliate Fabrea salina was able to grow with different feeds, and may be a good candidate species as food source for the Tunisian aquaculture industry. To improve our overall understanding of the various preys–Fabrea relationships, we are currently investigating the ecophysiological responses of Fabrea to changes in light, temperature, salinity, together with its fatty acid composition.