Monoculture of the ciliate protozoan Euplotes sp. (Ciliophora; Hypotrichia) fed with different diets

Ciliate protozoa of the genus Euplotes commonly appears contaminating mass cultures of rotifers but also with potential to be used as live food in the larviculture of marine fish. To obtain a monoculture of Euplotes sp., three diets were tested: 1) microalgae Nannochloropsis oculata, 2) commercial diet for rotifers Culture Selco 3000, and 3) baker’s yeast (Saccharomyces cerevisiae). The ciliates were inoculated at 10 ind. mL1. On day 5, protozoa densities in the groups fed the commercial diet (1,911.0 ± 248.7 ind. mL1) and the baker’s yeast (2,600.0 ± 195.3 ind. mL) did not differ, but were higher than the group fed microalgae (2.0 ± 1.4 ind. mL) (p < 0.05). On day 6, the density in the group fed baker’s yeast was higher (15,484.0 ± 1,164.9 ind. mL1) than in the groups fed microalgae (3.0 ± 1.4 ind. mL1) or commercial diet (11,287.0 ± 1,468.0 ind. mL1). An exponential growth curve was observed for the protozoa fed baker’s yeast (R2 = 0.992; p < 0.05) and commercial diet (R2 = 0.979; p < 0.05). The microalgae diet did not result in satisfactory growth of the protozoan. Feeding baker’s yeast or commercial rotifer’s diet to a monoculture of Euplotes sp. can result in very high densities of this protozoan.


Introduction
Ciliate protozoa are ubiquitous in aquatic environments and one of main consumers of bacteria in eutrophic waters (KAMIYAMA, 1994).Because of their abundance and size -similar or even smaller than nauplii of copepods, probably they occupy an important position on aquatic trophic food web.According to Holt and Holt (2000), marine fish larvae in the wild commonly feed on a large array of microzooplankton, including protozoa (tintinnid and ciliates), dinoflagellates, larvae of mollusks and, mainly, eggs and nauplii of copepods.However, there are few studies on their role as source of live food in the early stages of fishes and invertebrates (KRAUL, 2006 apud CÔRTES;TSUZUKI, 2012, OLIVOTTO et al., 2005;THOMPSON et al., 1999).
Food for fish larvae is usually determined by fish mouth size, prey size and fish ability to prey, which suggests that small prey with slow swimming, such as ciliate and dinoflagellate protozoa would be more suited (HUNT VON HERBING;GALLAGER, 2000, HUNT VON HERBING et al., 2001).In the majority of marine fish larvae, especially the ornamental ones, e.g.flame angelfish (Centropyge loricula) (Günther, 1835) TSUZUKI, 2010, KRAUL, 2006 apud CÔRTES;TSUZUKI, 2012, OLIVOTTO et al., 2005), thus they need small sized food, such as Super Small rotifers, copepod nauplii or ciliate protozoa such as Euplotes sp.
In marine fish hatchery, the type of zooplankton to be fed as a first food to fish larvae, i.e., in the transition from endogenous (yolk reserves) to exogenous feeding, is determined not only by those factors mentioned earlier but also by its nutritional quality.To master the techniques to culture prey organisms it is essential to provide constant and controlled production, especially at high densities.Rotifers (Brachionus sp.) and brine shrimp (Artemia sp.) are widely used in marine fish hatcheries.Both organisms meet the required characteristics but they are not always the best option due to their size, nutritional quality or visual attractiveness (STØTTRUP;MCEVOY, 2003).Feeding copepod nauplii as a first live food may be more adequate for many species.Kraul (2006 apud CÔRTES; TSUZUKI, 2012) reported a 10% increase in Almaco jack Seriola rivoliana (Valenciennes, 1833), larvae survival when feeding copepod nauplii.Some copepods are small in the naupliar phase (68 μm, Tisbe cucumarie, Copepoda, Harpacticoida; 65 μm, Acartia spp.) and the nutritional profile meets the marine fish larvae requirements (STØTTRUP; MCEVOY, 2003).However, the drawback of this option lies on the difficulty in rearing copepods intensively under controlled conditions.Nevertheless, Olivotto et al. (2005) reported a 15% increase in survival rates for gobiid larvae Gobiossoma evelynae (Böhlke; Robins, 1968) fed Euplotes sp. with rotifers in comparison to the combination of rotifers and brine shrimp.Unfortunately, little information is available on the culture of the ciliate protozoan.Euplotes sp. is usually considered undesirable contaminant in rotifer cultures (HAGIWARA et al., 2001), however, studies have been trying to demonstrate the importance and potential of these organisms as food source for the larval stages of marine fishes (FIGUEIREDO et al., 2007, OLIVOTTO et al., 2005).
As studies describing protocols to culture ciliate protozoa are scarce, this study aimed to assess the effect of different diets in the monoculture of Euplotes sp.and, thus, determine the diet yielding the best growth performance.Additionally, the potential to make use of Euplotes sp. as first food for ornamental marine fish larvae is also discussed.

Experimental culture conditions
The study was performed at the Laboratório de Piscicultura Marinha (LAPMAR), Santa Catarina Federal University, Florianópolis, Santa Catarina State, Brazil.The ciliate protozoan Euplotes sp. was isolated from a contaminated culture of rotifers Brachionus sp.kept at 25°C water temperature and 25 ppt salinity.First, rotifer culture water was sieved through a 45 μm mesh net, to retain rotifers, and then through an 18 μm mesh net, to retain and isolate Euplotes sp.(Figure 1).Protozoan mean size was 60.0 ± 9.4 μm (mean ± s.d.) in length and 39.0 ± 7.4 μm in width.Baker's yeast and Culture Selco were fed at a concentration of 0.5 g per million protozoa once a day.Throughout the trial, microalgae density was monitored and kept at 2.2 ± 1.7 x 10 6 cells mL -¹.Microalgae were cultured in modified Guillard f/2 medium under constant light and aeration, harvested in the log phase (4.0 x 10 6 cells mL -¹) and supplied to the protozoan culture.
Euplotes sp. was inoculated at a concentration of 10 ind.mL -1 in each of the 2-L glass experimental container.Water was previously chlorinated and dechlorinated with sodium thiosulphate, and the microalgae filtered through a 5-μm mesh net.Experimental cultures were kept under 24h light, 25 ± 1°C (using thermostat-heaters), salinity at 30 ± 2 ppt, constant aeration, and no water renovation.Total ammonia concentration was monitored daily with a commercial test kit (Labcon, Brazil).

Cell counting
Microalgae density was determined with a Neubauer counting chamber.Protozoan density was estimated from a 1-mL sample, after fixation with 5% formalin, using a Sedgewick-Rafter counting chamber at 5 x magnification.The ciliate protozoan (Euplotes sp.) is not always seen in the water column as it prefers to move on the substrate and detrital particles accumulated on the bottom of the aquaria.To ensure reliable counting, each aquarium was homogenized with strong aeration prior to sampling.All counting was performed in duplicate.

Statistical analysis
To determine the diet with best growth performance during the trial, as well as the variation of total ammonia concentration, an analysis of variance was adopted with parcels divided in time.Regression analysis was used to obtain the growth curve.A 5% level of significance was adopted.Data was processed using the software Statistica (STATISTICA, 2009).

Results and discussion
Table 1 shows the increase in abundance of the ciliate protozoan Euplotes sp.fed the three food sources.During the first four days of culture, all groups kept the same density.On day 5, there was no significant difference between the growth of populations in groups fed Culture Selco (1,911.0± 248.7 ind.mL -¹) and baker's yeast (2,600.0± 195.3 ind.mL -1 ), but were statistically higher (p < 0.05) than the group fed microalgae (2.0 ± 1.4 ind.mL -1 ).By the end of the trial (day 6) all groups were statistically different, when the group fed baker's yeast yielded the best result (15,484.5 ± 1,164.9 ind.mL -1 ) (p < 0.05), followed by the group fed Culture Selco (11,287.0 ± 1,468.0 ind.mL -¹).The group fed microalgae presented the lowest result (237.0 ± 468.7 ind.mL -¹).The ciliate culture presented exponential growth curves in the baker's yeast (R² = 0.992; p < 0.05) and Culture Selco (R² = 0.979; p < 0.05) groups.During the trial, total ammonia levels in the water were low until day 3 (0.25 mg L -1 ) but from day 4, levels in groups fed baker's yeast and Culture Selco had increased.On day 6, total ammonia concentration in the baker's yeast group and Culture Selco group had increased to 4 and 3 mg L -1 , respectively.Total ammonia concentration in the microalgae group was lower than the others throughout the trial with values of 0.10 mg L -1 (Figure 2).Studies have demonstrated that protozoa, including Euplotes sp. can feed on bacteria (WILKS; SLEIGHT, 1998).Cheng et al. (2004) reported that ciliates also feed on phytoplankton such as N. oculata and Tetraselmis tetrathele (Butcher, 1959).In the present study, the group fed the microalgae N. oculata did not present significant growth of the Euplotes sp.population.This might indicate that the concentration of microalgae was not sufficient for the protozoan growth or that the protozoan can ingest the algae but cannot digest it well.According to Cheng et al. (2004), these ciliate protozoa do not possess a specialized jaw to grind and digest the hard cell wall of phytoplankton.Baker's yeast was the food source resulting in the best growth but it was also the treatment with higher total ammonia concentration in the water, 4.0 mg L -1 .Although such relatively high concentration could inhibit growth or even kill many aquatic organisms, e.g., rotifers and fish larvae (CHENG et al., 2004;XU et al., 2004), it seems not to have affected the growth of the Euplotes sp.population.Xu et al. (2004) reported that total ammonia concentrations of 100 mg L -1 in the water inhibit growth of Euplotes vannus, and the concentration become lethal from 7,870.5 mg L -1 .In fact, the capacity of these organisms to tolerate such toxic nitrogen compound is extraordinary, they are much more resistant than other live food organisms.However, Euplotes sp.fed only on baker's yeast could not possibly be offered alone to marine fish larvae due to its low nutritional value; it should be enriched mainly with polyunsaturated fatty acids before being offered as live food.Kraul (2006 apud CÔRTES; TSUZUKI, 2012) analyzed the composition of ciliates of the genus Euplotes fed enriched commercial diets and reported that they are capable of incorporating moderate levels of fatty acids (especially n-3 EPA and DHA).Nevertheless, such nutrient incorporation was lower than in rotifers and brine shrimp.
The density of Euplotes sp.fed Culture Selco was satisfactory although it is a product originally designed to feed rotifers.Even with a growth performance lower than the group fed baker's yeast, Culture Selco in the culture of Euplotes sp. may be interesting when the intention is to feed these organisms to fish larvae, as the product is enriched with polyunsaturated fatty acids.
Like other culture organisms, culturing Euplotes sp. has some advantages and disadvantages.Some of the advantages are the small size, fast growth, high culture densities, and resistance to high concentrations of ammonia in the water.The possible disadvantages may be that these protozoa need to be enriched before supplied to fish larvae, and their benthic habit may hinder predation (CHENG et al., 2004;XU et al., 2004).Additionally, the size of young and adult protozoa does not differ, so feeding other live food of larger size, such as rotifers, may be necessary as fish larvae grow.

Conclusion
The present study demonstrates the feasibility of an intensive monoculture of the ciliate protozoan Euplotes sp.Feeding microalgae N. oculata at a density of 2.2 ± 1.7 x 10 6 cells mL -1 to these organisms was not efficient.Enhanced biomass growth may be obtained by feeding baker's yeast or Culture Selco 3000, products that are commonly used in rotifer culture.

Figure 2 .
Figure 2. Total ammonia concentration (mg L -1 ) in the water of the three test groups (baker's yeast , Selco , microalgae Δ) throughout the six-day feeding trial.Points in each day are mean values (n = 4), vertical bars denote standard deviation, and different letters indicate significant different between means (p < 0.05).

Table 1 .
Density of the ciliate protozoan Euplotes sp.fed on baker's yeast, Culture Selco, and microalgae N. oculata during the six-day feeding trial.Values are mean ± standard deviation per treatment group (n = 4).Different letters indicate significant difference between means in the row (p < 0.05).