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Competition between the rotifer Brachionus rotundiformis and the ciliate Euplotes vannus fed on two different algae
Aquaculture 241 (2004) 331 – 343 www.elsevier.com/locate/aqua-online
Competition between the rotifer Brachionus rotundiformis and the ciliate Euplotes vannus fed on two different algae Shin-Hong Chenga, Shigeru Aokib,*, Masachika Maedac, Akinori Hinob a
Biotechnology Division, Taiwan Fisheries Research Institute, Tungkang, Pingtung 928, Taiwan b Department of Ecosystem Studies, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 1136587, Japan c Department of Biological Production and Environmental Science, Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai Nishi 1-1, Miyazaki 8892192, Japan Received 30 March 2004; received in revised form 2 August 2004; accepted 4 August 2004
Abstract The ciliate Euplotes vannus is a common protozoan in mass cultures of rotifers, and rapid reproduction of E. vannus occasionally reduces the rotifer yield. We performed a competition experiment between the rotifer Brachionus rotundiformis and the ciliate E. vannus. The ciliate was inoculated at three stages of the rotifer growth curve: the lag phase, the logarithmic growth phase and the stationary phase. When feeding on the alga Tetraselmis tetrathele, the ciliate increased and the rotifer growth was suppressed. The interference with rotifer growth was stronger in the lag and stationary phases than in the logarithmic growth phase. In contrast, when feeding on the alga Nannochloropsis oculata, the ciliate did not increase and the rotifer growth was similar to the control. In addition to the competition experiments, 15N incorporation experiments were performed on the ciliate to investigate its food preference. The nitrogen incorporation rates (% of body nitrogen h1) of the ciliate were 0.7% through dead algae and 1.9% through rotifer feces when using T. tetrathele, and 0.4% through dead algae and 3.4% through rotifer feces when using N. oculata. The incorporation rate through bacteria-free rotifer feces was about one-third that of normal rotifer feces. These results indicated that the ciliate did not incorporate algal nitrogen
* Corresponding author. Tel.: +81 3 5841 8931; fax: +81 3 5841 8921. E-mail address: [email protected] (S. Aoki). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.006
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directly, but rather through the microbial food chain, from phytoplankton to rotifer feces to bacteria. E. vannus incorporated the nitrogen of N. oculata and grew for a short time but the growth was soon suppressed. This growth inhibition may be due to allelopathy of N. oculata for E. vannus. Consequently, we propose that periodic addition of N. oculata should control E. vannus, and that this technique of applying allelopathy should be more sustainable than conventional treatments. D 2004 Elsevier B.V. All rights reserved. Keywords: Rotifer; Ciliate; Competition;
15
N; Nannochloropsis oculata
1. Introduction The brackish rotifers Brachionus rotundiformis and Brachionus plicatilis are cultured as a live food for marine and freshwater fish larvae all over the world. At the end of 1980s, a basic mass batch culture system for rotifers was established (Fukusho, 1989a,b), and thereafter a high density mass culture system (Yoshimura et al., 1997; Suantika et al., 2003) and a chemostat culture system (James and Abu-Razeq, 1989; Fu et al., 1997) have been introduced into hatcheries as developmental techniques. However, we still occasionally encounter serious issues, for instance, unexpected death or suppressed rotifer growth. The factors affecting rotifer cultures consist of both biotic and abiotic factors. Abiotic factors include instability of the temperature or salinity, and accumulation of excretory substances, for example, ammonia. At present, these abiotic factors are reduced as much as possible by using an indoor tank with a regulated temperature and performing early renewal of rotifer cultures. The major biotic factor is microbial interaction. In mass culture tanks, various species of microbes including protozoans and bacteria coexist, and may have some effects on the rotifers. In some cases, the mechanisms of the effects have already been clarified in some cases, for example, certain bacteria provide a source of the essential vitamin, B12, and stimulate the growth of rotifers (Yu et al., 1989), while a heliozoon protozoa has direct lethality on rotifers (Cheng et al., 1997). Ciliates are the most common protozoan in rotifer tanks. Reguera (1984) reported that high propagation of the ciliate Euplotes vannus during mass cultures of rotifers resulted in considerably reduced rotifer yields. Maeda and Hino (1991) investigated the fauna of protozoans in rotifer cultures, and proved that the ciliates Uronema and Euplotes were the major species. Furthermore, they isolated a bacteria strain from a Euplotes sp. culture that strongly inhibited rotifer growth, and demonstrated that protozoan interference in rotifer cultures is closely related to the bacterial flora. Subsequently, Hagiwara et al. (1995) compared the population growths of mono-cultures and mixed-cultures, and concluded that B. rotundiformis did not affect the growth of Euplotes, whereas the Euplotes sp. population interfered with the growth of B. rotundiformis. However, the competition experiment was only run under one set of conditions. The environmental conditions of rotifer cultures change rapidly during the course of the culture, and thus the results of a competition experiment are thought to depend on the environmental conditions at the start of the experiment.
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Under natural conditions, a large biodiversity of plankton is observed and multiple species can coexist sympatrically. This concept, against a simple competition model, was proposed as the dparadox of the planktonT by Hutchinson (1961). The coexistence of plankton may depend on non-equilibrium conditions, which are derived from an external noise (e.g., seasonal or weather-driven fluctuations), spatial heterogeneity or intrinsic oscillation of the community (reviewed in Scheffer et al., 2003). As compared with nature, only dominant species often survive through competition in laboratory microcosms. In general, competition is divided into interference and exploitation. A major example of interference competition for rotifers is that large cladocerans directly affect rotifer feeding (reviewed in Gilbert, 1988). An example of exploitation competition is the mechanistic resource competition theory (Tilman, 1977) between sibling rotifers (Rothhaupt, 1988; Ciros-Perez et al., 2001). As mentioned above, various protozoa coexist in rotifer mass cultures and it is impossible to completely eradicate them. A skillful rotifer culture is one that continuously makes the rotifers dominant under equilibrium conditions. In this study, we inoculated the ciliate E. vannus at three stages of the rotifer growth curve with feeding on two different algae, and compared the competition results between the initial experimental conditions. In addition, 15N incorporation experiments were performed to investigate the food preference of the ciliate. We clarified the mechanism of the competition by introducing some ecological concepts (e.g., non-equilibrium conditions, exploitation and interference). Finally, an improved method for achieving more stable rotifer cultures is proposed.
2. Materials and methods 2.1. Organisms The algae Tetraselmis tetrathele and Nannochloropsis oculata were propagated in modified Hirata medium (Aoki et al., 1995) under constant light conditions. The algae were harvested at the end of their logarithmic growth phases, and condensed by centrifugation before use for feeding zooplankton. The rotifer B. rotundiformis Tamano strain was obtained from the Tamano Branch of the National Center for Stock Enhancement, and individuals were about 220 Am in lorica length. A ciliated protozoan was isolated from the rotifer culture water and identified as E. vannus. The sizes ranged from 75 to 110 Am. Stock-cultures of zooplankton and all experiments were conducted in seawater of 20 PSU at 25 8C in total darkness. 2.2. Counting Algal densities were counted using a hematocytometer. The rotifer and ciliate densities were estimated from a 1-ml sample under a stereo-microscope after fixation with Lugol’s solution. E. vannus do not always inhabit pelagic water, and prefer to move on detrital particles accumulated on the bottom rather than swim. To ensure that the ciliate density was assessed correctly, the culture medium was therefore mixed well before each sampling. All counting was performed in triplicate.
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2.3. Growing experiments 2.3.1. Single-species cultures Rotifers and ciliates were cultured on fresh T. tetrathele or N. oculata. The culture vessels were 2 l beakers for B. rotundiformis and 500 ml beakers for E. vannus, and the cultures were aerated gently to maintain uniform oxygenation. Both B. rotundiformis and E. vannus were added at an initial density of 10 ml1 and the number was counted every day for 21 days for rotifers and 13 days for E. vannus. The feeding densities of the alga were 5105 cells ml1 for T. tetrathele and 5106 cells ml1 for N. oculata, and the diet was provided every day for B. rotundiformis and once at the beginning of the culture for E. vannus. All food alga was perfectly consumed by rotifers every day, in contrast, uneaten alga was left in ciliate cultures until the end of the experiment. In addition to the fresh algae, the food-quality of dead algae and rotifer feces were also tested for the ciliate only. Fresh algae frozen at 80 8C for 1 week were thawed and defined as the dead algae. The rotifer feces were from rotifer cultures (500 individuals ml1) feeding on alga at 1107 cells ml1 for N. oculata or 1106 cells ml1 for T. tetrathele. After 1 day, the culture medium was filtered through an 80 Am mesh to remove the rotifers. All growing experiments were conducted in duplicate. 2.3.2. Competition cultures E. vannus were added to B. rotundiformis single-species cultures. Inoculation was performed at three stage of the rotifer growth curve: the lag phase, the logarithmic growth phase and the stationary phase (Fig. 1). A stock-culture of E. vannus was filtered through a 63 Am mesh to remove large particles, and then the E. vannus were washed thoroughly
Fig. 1. Rotifer growth curves of single cultures fed on T. tetrathele (open circles) and N. oculata (closed circles). The arrows indicate the inoculation timing of E. vannus for the competition cultures: a, lag phase; b, logarithmic growth phase; and c, stationary phase.
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using a 25 Am mesh and seawater filtered through a 0.2 Am Millipore filter. The purified E. vannus were inoculated into 500 ml rotifer cultures obtained from 2 l single-species cultures of the rotifer. The numbers of B. rotundiformis and E. vannus were measured every day for 21 days. 2.4. Feeding experiments using a stable isotope The incorporation of food nitrogen by E. vannus was measured using 15N as a tracer. The algae N. oculata and T. tetrathele were labeled with 15N following the method described in Aoki et al. (1995). The food nitrogen was supplied in dead alga and rotifer feces. After labeling one of these, the two were mixed and fed to the ciliates, such that one diet was labeled and the other was unlabeled in each ciliate culture. The dead alga and rotifer feces were prepared as described in Section 2.3.1. In addition to the rotifer feces, bacteria-free rotifer feces were also evaluated as a nitrogen source for the ciliate. Consequently, we prepared three treatments: labeled alga plus nonlabeled feces, nonlabeled alga plus labeled feces, and nonlabeled alga plus labeled bacteria-free feces. In making the bacteria-free rotifer feces, antibiotics (100 mg ml1 kanamycin and 100 mg ml1 ampicillin) were added together with the algae. Bacteria-free was not checked by any methods. Experiments were conducted in 500 ml beakers, and 400 ml of the filtered medium including rotifer feces and dead algae were added such that the nitrogen amounts of the rotifer feces and the dead algae were equal. The concentrations of dead algae in the medium were 5106 cells ml1 for N. oculata and 5105 cells ml1 for T. tetrathele. Each experiment was started by the addition of E. vannus to a concentration of 1000 cells ml1. The number of E. vannus was counted at 0, 3 and 6 h after the beginning of the experiment. Thirty milliliters of culture medium was sampled at several time points until 7 h after the beginning of the experiment. The medium samples were filtered through a 53 Am mesh to remove larger particles. The ciliates were then captured on a 25 Am mesh and washed thoroughly with seawater filtered through a 0.2 Am Millipore filter. The purified E. vannus were collected on a GF/C-filter, which had previously been combusted at 400 8C for 2 h. After freeze-drying, the 15N ratio of E. vannus was measured using a continuous flow mass spectrometer (Tracermass; Europa Scientific) equipped with a CN analyzer (Roboprep-CN; Europa Scientific) following the method described in Kanda et al. (1998). All samples were measured in triplicate. The incorporation ratio was calculated using the ciliate 15N ratio, the diet 15N ratio and the following equation. Incorporation ratio ¼ ðciliate
15
N ratio 0:37Þ= diet
15
N ratio 0:37Þ 100
This incorporation ratio is the ratio (%) of nitrogen incorporation through each food source to the ciliate nitrogen, since 0.37% is the natural 15N ratio. 2.5. Analysis The specific growth rate (r, day1) was estimated by: r=(ln N t ln N t1), where N t was the organism density at t days after the start of the experiment. The average density (D t )
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was calculated as the geometric average: D t =(N t N t1)1/2. To examine the relationships between the growth and the density, multiple regression analysis was performed using the computer program StatView (version 5.0; SAS Institute).
3. Results 3.1. Single-species cultures The growth curves of single cultures are shown in Fig. 1 for B. rotundiformis and Fig. 2 for E. vannus. B. rotundiformis grew well with when fed either N. oculata or T. tetrathele. The logarithmic growth phase was maintained for 10 days soon after a 1-day lag phase, and thereafter the stationary phase continued until the end of the culture. At the stationary phase, r (specific growth rate) was close to zero, and therefore the maximum density was calculated as the r=0 density (Snell et al., 2001) using the regression and constants obtained in Table 1. The r=0 densities were about 1400 rotifers ml1 with feeding on T. tetrathele and about 1300 rotifers ml1 with feeding on N. oculata. The growth patterns of E. vannus were similar with feeding on both dead algae and rotifer feces regardless of the algae species. After 4 days of the logarithmic growth phase, the stationary phase lasted until the end of the experiment. The food qualities of the algae were quite different between N. oculata and T. tetrathele, and the r=0 density of 2400 cells ml1 with feeding on T. tetrathele was much higher than the 150 cells ml1 with feeding on N. oculata. On the other hand, fresh T. tetrathele delayed the onset of the logarithmic growth phase in E. vannus by 3 days. E. vannus did not grow on fresh N. oculata.
Fig. 2. Ciliate growth curves of single cultures fed on N. oculata and T. tetrathele, respectively. Three food types were prepared: fresh algae (crosses), dead algae (open circles) and rotifer feces (closed circles).
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Table 1 Regression analysis between growth rate and density Culture type and diet alga
Competiotion culture T. tetrathele N. oculata
Single culture Rotifer-T. tetrathele Rotifer-N. oculata Ciliate-T. tetrathele Ciliate-N. oculata
Organism
n
R2
F
Constant l max
a
b
rotifer ciliate rotifer ciliate
52 52 51 51
0.53 0.08 0.44 0.01
25.9** 2.2ns 18.5** 0.15ns
0.95F0.12 – 0.57F0.18 –
0.49F0.07 – 0.59F0.10 –
0.12F0.03 – 0.01F0.03 –
rotifer rotifer ciliate ciliate
17 16 20 20
0.65 0.69 0.40 0.38
27.4** 31.0** 12.2* 11.1*
0.92F0.14 0.71F0.09 1.48F0.34 1.14F0.29
0.65F0.12 0.54F0.10 0.61F0.17 7.6F2.3
– – – –
Regression formula is r=l maxaD obD c; r, specific growth rate (day1); l max, potential maximum growth rate (day1); a, coefficiency of intraspecific competition; b, coefficiency of interspecific competition; D o and D c, own density and competitors density, respectively (103 numbers ml1). ns; not significant ( pN0.05), presented constant is valueF1 S.D. * pb0.01. ** pb0.001.
3.2. Competition cultures Time series density profiles of the rotifer and the ciliate in the competition experiments are shown in Fig. 3. When feeding on T. tetrathele, E. vannus added at the lag phase or the
Fig. 3. Rotifer–ciliate density profiles in the competition experiments. The ciliate was inoculated at the lag phase (a), the logarithmic growth phase (b) and the stationary phase (c) of the rotifer culture. Each character indicates rotifer–ciliate density of duplicate experiments, and the bold characters were the starting point of each experiment. Each dashed line is the approximated growth curve by free-hand.
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stationary phase increased rapidly and the growth of B. rotundiformis was suppressed. On the other hand, E. vannus added at the logarithmic growth phase decreased temporally from 1000 to 300 cells ml1 and the rotifer grew as well as the control culture for the first 5 days, but thereafter the E. vannus began to increase and the rotifer–ciliate density profiles followed the pattern of the stationary phase addition. When feeding on N. oculata, E. vannus did not increase regardless of the inoculation time, and the rotifer grew as well as the control, except for when the ciliate was inoculated during the stationary phase. At the stationary phase, although E. vannus did not increase, the rotifer density was significantly less than the control. The density-dependent effects on growth are shown in Table 1. When feeding on T. tetrathele, the coefficiency of interspecific competition from the ciliate to the rotifer was about one-fifth the coefficiency of intraspecific competition of the rotifer, and the rotifer growth rate reduced to 0 at the ciliate density of 8000 cells ml1 (The r=0 densities were calculated following the method described in Section 3.1). Regardless of the algal species, the ciliate growth had no density-dependent effect in the competition cultures. 3.3. Feeding experiments using a stable isotope The labeling ratios (15N at.%) of the food source in the feeding experiments were 89% for N. oculata, 86% for T. tetrathele, 59% for rotifer feces feeding on N. oculata, and 50% for rotifer feces feeding on T. tetrathele. The specific growth rate of E. vannus populations during the experiments was 0.82 day1 (=0.034 h1) with feeding on N. oculata and 0.58 day1 (=0.024 h1) with feeding on T. tetrathele. These growth rates were lower than the
Fig. 4. Nitrogen incorporation ratios (% of body nitrogen) of E. vannus. The nitrogen was incorporated through dead algae (open bars) or rotifer feces (closed bars). Solid line is the steady nitrogen incorporation rate (% of body nitrogen h1) by E. vannus.
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Table 2 Nitrogen incorporation rate (% of body nitrogen h1) of E. vannus from each food source Algae
T. tetrathele N. oculata
Food source Dead algae
Rotifer feces
Bacteria-free rotifer feces
0.7 0.4
1.9 3.4
0.7 0.8
The food sources and feeding regimes are explained in Materials and methods.
potential maximum growth rate of the single-species cultures (Table 1), especially at feeding on T. tetrathele. 15 N was rapidly incorporated in E. vannus and the incorporation ratio reached 12% during the first 30 min with feeding on both N. oculata and T. tetrathele (Fig. 4). Later, the incorporation ratio grew steadily until the end of the experiment with feeding on N. oculata. Meanwhile, with feeding on T. tetrathele, the incorporation stopped temporarily between 30 and 90 min and then resumed until 300 min. The steady incorporation rates were 3.8% h1 on N. oculata and 2.5% h1 on T. tetrathele, calculated from the incorporation ratios between 90 and 300 min. These incorporation rates agreed well with the specific growth rate of E. vannus. Almost all the nitrogen was incorporated through the rotifer feces (90% on N. oculata and 74% on T. tetrathele). When feeding on the bacteriafree rotifer feces, the incorporation rate dropped to 24% of the normal rotifer feces on N. oculata and 39% on T. tetrathele (Table 2).
4. Discussion 4.1. What affects ciliate growth? 4.1.1. Food It has been believed that many protozoans, also including E. vannus, are bacterivorous. Wilks and Sleigh (1998) performed grazing experiments using fluorescent latex microspheres of various sizes in E. mutabilis, which is closely related to and approximately the same size as E. vannus. They showed that E. mutabilis can ingest particles of sizes between 0.57 and 10.0 Am in diameter and the maximum volume ingested was obtained for 5.85 Am diameter microspheres. These results demonstrate that Euplotes spp. can efficiently ingest not only bacteria but also phytoplankton. We actually observed that the ciliate ingested both N. oculata and T. tetrathele under a fluorescence microscope. In the tracer experiment, nitrogen incorporated into E. vannus through algae reached the maximum value until 30 min, and was maintained at a constant level thereafter (Fig. 4). This means that E. vannus can ingest algae, but does not digest it well. Rotifers have a complex chitinous jaw composed of a dental mill, and the jaw functions to grind the firm phytoplankton cells. Ciliates do not possess a specialized jaw and may not be able to digest the hard cell wall of the phytoplankton. In contrast, nitrogen was incorporated through the rotifer feces until the end of the experiments. The incorporation rate was fast during the first hour but then fell during the next 3 h (Fig. 4). This trend was consistent
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with the results of Zubkov and Sleigh (1996), and may be an error due to the experimental procedure in which the ciliates could be starved for a short time just before the experiment, indeed, the specific growth rate during the feeding experiments was lower than the potential maximum growth rate (see Section 3.3). They measured the 14C accumulation in Euplotes mutabilis feeding on bacteria labeled with 14C-leucine, and demonstrated that the ingestion of bacteria and accumulation of 14C by normal Euplotes were linear in the interval between 1 and 4 h. Thus, to calculate the steady incorporation rate, we used time points between 90 and 300 min, rather than during the first hour. The observation that almost all the nitrogen (74–90%) was incorporated through the rotifer feces with bacteria shows that the ciliate prefers rotifer feces to plain algae. When supplied with N. oculata, the rotifer egests 70% of the ingested nitrogen as feces (Aoki et al., 1995). Thus, rotifer feces are rich in nitrogen and have numerous bacteria attached to the surface. The incorporation rate with feeding on the feces without bacteria was about one-third of the rate on feces with bacteria. This difference in the results depended on the existence of bacteria, and thus cell wall breakage by the rotifers contributes one-third of the reason why the protozoan can digest rotifer feces, while the microbial food chain through phytoplankton to rotifer feces to bacteria contributes the remainder. In these experiments, E. vannus also mainly incorporated bacterial nitrogen. 4.1.2. Allelopathy Although E. vannus actively incorporated feces from rotifers fed N. oculata (Fig. 4), the maximum density (r=0 density) of E. vannus with feeding on N. oculata was much lower than that with feeding on T. tetrathele. The instantaneous growth rates during the logarithmic growth phase, constant l max in Table 1, did not differ significantly between ciliate-T. tetrathele and ciliate-N. oculata ( pb0.01). This suggests that E. vannus can incorporate the nitrogen of N. oculata through bacteria and consequently grow, but the growth was soon suppressed by some factors involved with N. oculata. Moreover, E. vannus prolonged the lag phase when fed on both fresh algae (Fig. 2). This algal inhibition of the ciliate growth may possibly be explained by an allelopathic interaction. A similar phenomenon was observed for the interactions between the green algae Chlorella and the ciliate Colpidium, and chlorellin seems to be a harmful substance (Hulot et al., 2001). However, no chemicals suppressing the ciliate growth have yet been isolated from N. oculata or fresh T. tetrathele. Therefore, this allelopathic interaction may not be a direct effect, but rather an indirect effect with a change in the bacterial flora. Further studies are required to obtain direct proof of an allelopathic interaction between algae and ciliates. Furthermore, the interactions between N. oculata and other ciliates, e.g., Uronema sp, have not been clarified and remain the next issue to be investigated. 4.2. Interspecific and intraspecific competition In a single culture of the rotifer, density-dependent growth suppression was observed (Table 1). This growth suppression was thought to be a result of intraspecific competition. The mechanisms of intraspecific competition include accumulation of metabolites, food limitation, interference and so on. The maximum rotifer density of 1400 rotifer ml1 (Fig. 1) is a normal density for rotifer batch culture in a hatchery, although it is well-known fact
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that B. rotundiformis can reach 10000 rotifer ml1 if sufficient food and oxygen are supplied. Thus, this intraspecific competition of rotifers is due to food limitation rather than an autotoxin, as suggested by Snell et al. (2001). It has long been known that some ciliates inhibit rotifer growth in rotifer mass cultures (Reguera, 1984). In this paper, we have shown that the path of rotifer–ciliate density depended on the inoculation timing of the ciliate, and finally reached ciliate dominance when feeding on T. tetrathele (Fig. 3). B. rotundiformis and E. vannus show interspecific competition and cannot coexist in the same space for a long time. With inoculation at the lag phase or the stationary phase, the rotifer–ciliate competition easily progresses to ciliate dominance, and this phenomenon corresponds to the trend that a ciliate plague often occurs just after rotifer inoculation or when using an old rotifer culture tank. The environment of a rotifer culture tank is non-equilibrium in time series and it is expected that a change in the environment is related to changes in interspecific competition. The major environmental factors are food, space and accumulation of excretory substances. The rotifer and the ciliate utilize different foods from each other. The rotifer mainly grazes on microalgae, whereas the ciliate mainly eats and digests bacteria as mentioned above. When food algae are abundant, resource competition will definitely not exist between these two species. Regarding space utilization, these two species have different preferences. The healthy rotifer swims well using cilia as a propeller. The ciliate does not always inhabit pelagic water, but prefers to move on detrital particles accumulated on the bottom rather than swim. The two species are therefore segregated in a rotifer culture tank. Food and space were thus excluded as environmental factors that influence the interspecific competition. Except for exploitation and direct interference in the space utilization, indirect interference is listed as the mechanism of the interspecific competition. Bacterial flora may possibly be related to the indirect interference. Indeed, Maeda and Hino (1991) isolated a bacteria strain from a Euplotes sp. culture that strongly inhibited rotifer growth. Recently, it was demonstrated that the bacterial community structure is regulated by protozoan-selective predation (Langenheder and Jurgens, 2001). In rotifer cultures, excretory substances accumulate day by day and therefore the bacterial flora is also in non-equilibrium (Maeda and Hino, 1991). This non-equilibrium of the bacterial flora may explain the different results for the rotifer–ciliate dynamics due to the timing of the ciliate inoculation. In contrast, when feeding on N. oculata, the ciliate did not grow and the rotifer grew as well as the control. This result is inconsistent with the results of Hagiwara et al. (1995) in which Euplotes sp. populations interfered with the growth of B. rotundiformis when feeding on N. oculata. However, after re-examining their results, the maximum ciliate density was less than 100 cells ml1 and the interference was statistically significant but too weak. Thus, it is concluded that their results showed almost the same trend as our results. 4.3. A new technique for rotifer culture In a latest intensive rearing system like a recirculation system (Suantika et al., 2003) or a chemostat system (Fu et al., 1997), the water quality is well managed and a ciliate explosion is rare. However, in a conventional extensive culture like as a batch or a thinning culture, competition between rotifers and ciliates is inevitable, and suppression of ciliate
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growth is necessary for a stable rotifer culture. In a rotifer tank, much of the particulate organic matter originates from the accumulation of rotifer feces (Aoki and Hino, 1996), and this debris supplies protozoans with food and habitats. We proved that the ciliate E. vannus actively incorporated nitrogen through the rotifer feces and grew well. The accumulation of detritus therefore increases the risk of a protozoan explosion. An adhesive mat is commonly used to remove debris in rotifer tanks and has some benefits in suppressing protozoa propagation. The competition between B. rotundiformis and E. vannus may be related to the bacterial flora, and thus control of the bacterial flora should be a useful technique for minimizing the ciliate growth. Indeed, Yu et al. (1989) succeeded in controlling the bacterial flora for a stable rotifer culture by using a vitamin B12 producing bacteria, but maintenance of the bacteria control has not yet been tested and it may be difficult in the non-equilibrium environment of a rotifer batch culture. At present, condensed freshwater Chlorella enriched with vitamin B12 (Maruyama and Hirayama, 1993) is the most popular microalga for rotifer culture in Japan rather than N. oculata. When using freshwater Chlorella, protozoans grow well and sometimes occupy a rotifer tank. Periodic addition of N. oculata with freshwater Chlorella is expected to control E. vannus, and this technique of applying alleropathy is expected to be more sustainable than bacterial flora control.
Acknowledgements The authors would like to express their gratitude to Dr. Isao Koike, Ocean Research Institute of the University of Tokyo, for providing the use of a mass spectrometer. The authors are grateful for a scholarship from the National Science Council, Republic of China, and the support of Dr. I-Chiu Liao, director of the Taiwan Fisheries Research Institute (T.F.R.I.).
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Hagiwara, A., Jung, M.M., Sato, T., Hirayama, K., 1995. Interspecific relations between the marine rotifer Brachionus plicatilis and zooplankton species contaminating in the rotifer mass culture tank. Fish. Sci. 61, 623 – 627. Hulot, F.D., Morin, M.M., Loreau, M., 2001. Interactions between algae and the microbial loop in experimental microcosms. Oikos 95, 231 – 238. Hutchinson, G.E., 1961. The paradox of the plankton. Am. Nat. 95, 137 – 145. James, C.M., Abu-Razeq, T.A., 1989. An intensive chemostat culture system for the production of rotifers for aquaculture. Aquaculture 81, 291 – 301. Kanda, J., Sada, K., Koike, I., Yokouchi, K., 1998. Application of an automated carbon–nitrogen analyzer for elemental and isotopic analysis of samples retained on glass-fiber filters. Int. J. Environ. Anal. Chem. 72, 163 – 171. Langenheder, S., Jurgens, K., 2001. Regulation of bacterial biomass and community structure by metazoan and protozoan predation. Limnol. Oceanogr. 46, 121 – 134. Maruyama, I., Hirayama, K., 1993. The culture of the rotifer Brachionus plicatilis with Chlorella vulgaris containing vitamin B12 in its cells. J. World Aquac. Soc. 24, 194 – 198. Maeda, M., Hino, A., 1991. Environmental management for mass culture of rotifer, Brachionus plicatilis. In: Fulks, W., Main, K.L. (Eds.), Rotifer and microalgae culture systems, Proceedings of US–Asia Workshop. The Oceanic Institute, Honolulu, HI, pp. 125 – 133. Reguera, B., 1984. The effect of ciliate contamination in mass cultures of the rotifer, Brachionus plicatilis O.F. Muller. Aquaculture 40, 103 – 108. Rothhaupt, K.O., 1988. Mechanistic resource competition theory applied to laboratory experiments with zooplankton. Nature 333, 660 – 662. Scheffer, M., Rinaldi, S., Huisman, J., Weissing, F.J., 2003. Why plankton communities have no equilibrium: solutions to the paradox. Hydrobiologia 491, 9 – 18. Snell, T.W., Dingmann, B., Serra, M., 2001. Density-dependent regulation of natural and laboratory rotifer populations. Hydrobiologia 446/447, 39 – 44. Suantika, G., Dhert, P., Sweetman, E., O’Brien, E., Sorgeloos, P., 2003. Technical and economical feasibility of a rotifer recirculation system. Aquaculture 227, 173 – 189. Tilman, D., 1977. Resource competition between planktonic algae: an experimental and theoretical approach. Ecology 58, 338 – 348. Wilks, S.A., Sleigh, M.A., 1998. Grazing rates in Euplotes mutabilis: relationship between particle size and concentrations. Microb. Ecol. 36, 165 – 174. Yoshimura, K., Usuki, K., Yoshimatsu, T., Kitajima, C., Hagiwara, A., 1997. Recent development of high density mass culture system of the rotifer Brachionus rotundiformis. Hydrobiologia 358, 139 – 144. Yu, J.P., Hino, A., Ushiro, M., Maeda, M., 1989. Function of bacteria as vitamin B12-producers during mass culture of the rotifer Brachionus plicatilis. Nippon Suisan Gakkaishi 55, 1799 – 1806. Zubkov, M.V., Sleigh, M.A., 1996. Bacterivory by the ciliate Euplotes in different states of hunger. FEMS Microbiol. Ecol. 20, 137 – 147.
Competition between the rotifer Brachionus rotundiformis and the ciliate Euplotes vannus fed on two different algae Shin-Hong Chenga, Shigeru Aokib,*, Masachika Maedac, Akinori Hinob a
Biotechnology Division, Taiwan Fisheries Research Institute, Tungkang, Pingtung 928, Taiwan b Department of Ecosystem Studies, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 1136587, Japan c Department of Biological Production and Environmental Science, Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai Nishi 1-1, Miyazaki 8892192, Japan Received 30 March 2004; received in revised form 2 August 2004; accepted 4 August 2004
Abstract The ciliate Euplotes vannus is a common protozoan in mass cultures of rotifers, and rapid reproduction of E. vannus occasionally reduces the rotifer yield. We performed a competition experiment between the rotifer Brachionus rotundiformis and the ciliate E. vannus. The ciliate was inoculated at three stages of the rotifer growth curve: the lag phase, the logarithmic growth phase and the stationary phase. When feeding on the alga Tetraselmis tetrathele, the ciliate increased and the rotifer growth was suppressed. The interference with rotifer growth was stronger in the lag and stationary phases than in the logarithmic growth phase. In contrast, when feeding on the alga Nannochloropsis oculata, the ciliate did not increase and the rotifer growth was similar to the control. In addition to the competition experiments, 15N incorporation experiments were performed on the ciliate to investigate its food preference. The nitrogen incorporation rates (% of body nitrogen h1) of the ciliate were 0.7% through dead algae and 1.9% through rotifer feces when using T. tetrathele, and 0.4% through dead algae and 3.4% through rotifer feces when using N. oculata. The incorporation rate through bacteria-free rotifer feces was about one-third that of normal rotifer feces. These results indicated that the ciliate did not incorporate algal nitrogen
* Corresponding author. Tel.: +81 3 5841 8931; fax: +81 3 5841 8921. E-mail address: [email protected] (S. Aoki). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.006
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directly, but rather through the microbial food chain, from phytoplankton to rotifer feces to bacteria. E. vannus incorporated the nitrogen of N. oculata and grew for a short time but the growth was soon suppressed. This growth inhibition may be due to allelopathy of N. oculata for E. vannus. Consequently, we propose that periodic addition of N. oculata should control E. vannus, and that this technique of applying allelopathy should be more sustainable than conventional treatments. D 2004 Elsevier B.V. All rights reserved. Keywords: Rotifer; Ciliate; Competition;
15
N; Nannochloropsis oculata
1. Introduction The brackish rotifers Brachionus rotundiformis and Brachionus plicatilis are cultured as a live food for marine and freshwater fish larvae all over the world. At the end of 1980s, a basic mass batch culture system for rotifers was established (Fukusho, 1989a,b), and thereafter a high density mass culture system (Yoshimura et al., 1997; Suantika et al., 2003) and a chemostat culture system (James and Abu-Razeq, 1989; Fu et al., 1997) have been introduced into hatcheries as developmental techniques. However, we still occasionally encounter serious issues, for instance, unexpected death or suppressed rotifer growth. The factors affecting rotifer cultures consist of both biotic and abiotic factors. Abiotic factors include instability of the temperature or salinity, and accumulation of excretory substances, for example, ammonia. At present, these abiotic factors are reduced as much as possible by using an indoor tank with a regulated temperature and performing early renewal of rotifer cultures. The major biotic factor is microbial interaction. In mass culture tanks, various species of microbes including protozoans and bacteria coexist, and may have some effects on the rotifers. In some cases, the mechanisms of the effects have already been clarified in some cases, for example, certain bacteria provide a source of the essential vitamin, B12, and stimulate the growth of rotifers (Yu et al., 1989), while a heliozoon protozoa has direct lethality on rotifers (Cheng et al., 1997). Ciliates are the most common protozoan in rotifer tanks. Reguera (1984) reported that high propagation of the ciliate Euplotes vannus during mass cultures of rotifers resulted in considerably reduced rotifer yields. Maeda and Hino (1991) investigated the fauna of protozoans in rotifer cultures, and proved that the ciliates Uronema and Euplotes were the major species. Furthermore, they isolated a bacteria strain from a Euplotes sp. culture that strongly inhibited rotifer growth, and demonstrated that protozoan interference in rotifer cultures is closely related to the bacterial flora. Subsequently, Hagiwara et al. (1995) compared the population growths of mono-cultures and mixed-cultures, and concluded that B. rotundiformis did not affect the growth of Euplotes, whereas the Euplotes sp. population interfered with the growth of B. rotundiformis. However, the competition experiment was only run under one set of conditions. The environmental conditions of rotifer cultures change rapidly during the course of the culture, and thus the results of a competition experiment are thought to depend on the environmental conditions at the start of the experiment.
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Under natural conditions, a large biodiversity of plankton is observed and multiple species can coexist sympatrically. This concept, against a simple competition model, was proposed as the dparadox of the planktonT by Hutchinson (1961). The coexistence of plankton may depend on non-equilibrium conditions, which are derived from an external noise (e.g., seasonal or weather-driven fluctuations), spatial heterogeneity or intrinsic oscillation of the community (reviewed in Scheffer et al., 2003). As compared with nature, only dominant species often survive through competition in laboratory microcosms. In general, competition is divided into interference and exploitation. A major example of interference competition for rotifers is that large cladocerans directly affect rotifer feeding (reviewed in Gilbert, 1988). An example of exploitation competition is the mechanistic resource competition theory (Tilman, 1977) between sibling rotifers (Rothhaupt, 1988; Ciros-Perez et al., 2001). As mentioned above, various protozoa coexist in rotifer mass cultures and it is impossible to completely eradicate them. A skillful rotifer culture is one that continuously makes the rotifers dominant under equilibrium conditions. In this study, we inoculated the ciliate E. vannus at three stages of the rotifer growth curve with feeding on two different algae, and compared the competition results between the initial experimental conditions. In addition, 15N incorporation experiments were performed to investigate the food preference of the ciliate. We clarified the mechanism of the competition by introducing some ecological concepts (e.g., non-equilibrium conditions, exploitation and interference). Finally, an improved method for achieving more stable rotifer cultures is proposed.
2. Materials and methods 2.1. Organisms The algae Tetraselmis tetrathele and Nannochloropsis oculata were propagated in modified Hirata medium (Aoki et al., 1995) under constant light conditions. The algae were harvested at the end of their logarithmic growth phases, and condensed by centrifugation before use for feeding zooplankton. The rotifer B. rotundiformis Tamano strain was obtained from the Tamano Branch of the National Center for Stock Enhancement, and individuals were about 220 Am in lorica length. A ciliated protozoan was isolated from the rotifer culture water and identified as E. vannus. The sizes ranged from 75 to 110 Am. Stock-cultures of zooplankton and all experiments were conducted in seawater of 20 PSU at 25 8C in total darkness. 2.2. Counting Algal densities were counted using a hematocytometer. The rotifer and ciliate densities were estimated from a 1-ml sample under a stereo-microscope after fixation with Lugol’s solution. E. vannus do not always inhabit pelagic water, and prefer to move on detrital particles accumulated on the bottom rather than swim. To ensure that the ciliate density was assessed correctly, the culture medium was therefore mixed well before each sampling. All counting was performed in triplicate.
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2.3. Growing experiments 2.3.1. Single-species cultures Rotifers and ciliates were cultured on fresh T. tetrathele or N. oculata. The culture vessels were 2 l beakers for B. rotundiformis and 500 ml beakers for E. vannus, and the cultures were aerated gently to maintain uniform oxygenation. Both B. rotundiformis and E. vannus were added at an initial density of 10 ml1 and the number was counted every day for 21 days for rotifers and 13 days for E. vannus. The feeding densities of the alga were 5105 cells ml1 for T. tetrathele and 5106 cells ml1 for N. oculata, and the diet was provided every day for B. rotundiformis and once at the beginning of the culture for E. vannus. All food alga was perfectly consumed by rotifers every day, in contrast, uneaten alga was left in ciliate cultures until the end of the experiment. In addition to the fresh algae, the food-quality of dead algae and rotifer feces were also tested for the ciliate only. Fresh algae frozen at 80 8C for 1 week were thawed and defined as the dead algae. The rotifer feces were from rotifer cultures (500 individuals ml1) feeding on alga at 1107 cells ml1 for N. oculata or 1106 cells ml1 for T. tetrathele. After 1 day, the culture medium was filtered through an 80 Am mesh to remove the rotifers. All growing experiments were conducted in duplicate. 2.3.2. Competition cultures E. vannus were added to B. rotundiformis single-species cultures. Inoculation was performed at three stage of the rotifer growth curve: the lag phase, the logarithmic growth phase and the stationary phase (Fig. 1). A stock-culture of E. vannus was filtered through a 63 Am mesh to remove large particles, and then the E. vannus were washed thoroughly
Fig. 1. Rotifer growth curves of single cultures fed on T. tetrathele (open circles) and N. oculata (closed circles). The arrows indicate the inoculation timing of E. vannus for the competition cultures: a, lag phase; b, logarithmic growth phase; and c, stationary phase.
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using a 25 Am mesh and seawater filtered through a 0.2 Am Millipore filter. The purified E. vannus were inoculated into 500 ml rotifer cultures obtained from 2 l single-species cultures of the rotifer. The numbers of B. rotundiformis and E. vannus were measured every day for 21 days. 2.4. Feeding experiments using a stable isotope The incorporation of food nitrogen by E. vannus was measured using 15N as a tracer. The algae N. oculata and T. tetrathele were labeled with 15N following the method described in Aoki et al. (1995). The food nitrogen was supplied in dead alga and rotifer feces. After labeling one of these, the two were mixed and fed to the ciliates, such that one diet was labeled and the other was unlabeled in each ciliate culture. The dead alga and rotifer feces were prepared as described in Section 2.3.1. In addition to the rotifer feces, bacteria-free rotifer feces were also evaluated as a nitrogen source for the ciliate. Consequently, we prepared three treatments: labeled alga plus nonlabeled feces, nonlabeled alga plus labeled feces, and nonlabeled alga plus labeled bacteria-free feces. In making the bacteria-free rotifer feces, antibiotics (100 mg ml1 kanamycin and 100 mg ml1 ampicillin) were added together with the algae. Bacteria-free was not checked by any methods. Experiments were conducted in 500 ml beakers, and 400 ml of the filtered medium including rotifer feces and dead algae were added such that the nitrogen amounts of the rotifer feces and the dead algae were equal. The concentrations of dead algae in the medium were 5106 cells ml1 for N. oculata and 5105 cells ml1 for T. tetrathele. Each experiment was started by the addition of E. vannus to a concentration of 1000 cells ml1. The number of E. vannus was counted at 0, 3 and 6 h after the beginning of the experiment. Thirty milliliters of culture medium was sampled at several time points until 7 h after the beginning of the experiment. The medium samples were filtered through a 53 Am mesh to remove larger particles. The ciliates were then captured on a 25 Am mesh and washed thoroughly with seawater filtered through a 0.2 Am Millipore filter. The purified E. vannus were collected on a GF/C-filter, which had previously been combusted at 400 8C for 2 h. After freeze-drying, the 15N ratio of E. vannus was measured using a continuous flow mass spectrometer (Tracermass; Europa Scientific) equipped with a CN analyzer (Roboprep-CN; Europa Scientific) following the method described in Kanda et al. (1998). All samples were measured in triplicate. The incorporation ratio was calculated using the ciliate 15N ratio, the diet 15N ratio and the following equation. Incorporation ratio ¼ ðciliate
15
N ratio 0:37Þ= diet
15
N ratio 0:37Þ 100
This incorporation ratio is the ratio (%) of nitrogen incorporation through each food source to the ciliate nitrogen, since 0.37% is the natural 15N ratio. 2.5. Analysis The specific growth rate (r, day1) was estimated by: r=(ln N t ln N t1), where N t was the organism density at t days after the start of the experiment. The average density (D t )
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was calculated as the geometric average: D t =(N t N t1)1/2. To examine the relationships between the growth and the density, multiple regression analysis was performed using the computer program StatView (version 5.0; SAS Institute).
3. Results 3.1. Single-species cultures The growth curves of single cultures are shown in Fig. 1 for B. rotundiformis and Fig. 2 for E. vannus. B. rotundiformis grew well with when fed either N. oculata or T. tetrathele. The logarithmic growth phase was maintained for 10 days soon after a 1-day lag phase, and thereafter the stationary phase continued until the end of the culture. At the stationary phase, r (specific growth rate) was close to zero, and therefore the maximum density was calculated as the r=0 density (Snell et al., 2001) using the regression and constants obtained in Table 1. The r=0 densities were about 1400 rotifers ml1 with feeding on T. tetrathele and about 1300 rotifers ml1 with feeding on N. oculata. The growth patterns of E. vannus were similar with feeding on both dead algae and rotifer feces regardless of the algae species. After 4 days of the logarithmic growth phase, the stationary phase lasted until the end of the experiment. The food qualities of the algae were quite different between N. oculata and T. tetrathele, and the r=0 density of 2400 cells ml1 with feeding on T. tetrathele was much higher than the 150 cells ml1 with feeding on N. oculata. On the other hand, fresh T. tetrathele delayed the onset of the logarithmic growth phase in E. vannus by 3 days. E. vannus did not grow on fresh N. oculata.
Fig. 2. Ciliate growth curves of single cultures fed on N. oculata and T. tetrathele, respectively. Three food types were prepared: fresh algae (crosses), dead algae (open circles) and rotifer feces (closed circles).
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Table 1 Regression analysis between growth rate and density Culture type and diet alga
Competiotion culture T. tetrathele N. oculata
Single culture Rotifer-T. tetrathele Rotifer-N. oculata Ciliate-T. tetrathele Ciliate-N. oculata
Organism
n
R2
F
Constant l max
a
b
rotifer ciliate rotifer ciliate
52 52 51 51
0.53 0.08 0.44 0.01
25.9** 2.2ns 18.5** 0.15ns
0.95F0.12 – 0.57F0.18 –
0.49F0.07 – 0.59F0.10 –
0.12F0.03 – 0.01F0.03 –
rotifer rotifer ciliate ciliate
17 16 20 20
0.65 0.69 0.40 0.38
27.4** 31.0** 12.2* 11.1*
0.92F0.14 0.71F0.09 1.48F0.34 1.14F0.29
0.65F0.12 0.54F0.10 0.61F0.17 7.6F2.3
– – – –
Regression formula is r=l maxaD obD c; r, specific growth rate (day1); l max, potential maximum growth rate (day1); a, coefficiency of intraspecific competition; b, coefficiency of interspecific competition; D o and D c, own density and competitors density, respectively (103 numbers ml1). ns; not significant ( pN0.05), presented constant is valueF1 S.D. * pb0.01. ** pb0.001.
3.2. Competition cultures Time series density profiles of the rotifer and the ciliate in the competition experiments are shown in Fig. 3. When feeding on T. tetrathele, E. vannus added at the lag phase or the
Fig. 3. Rotifer–ciliate density profiles in the competition experiments. The ciliate was inoculated at the lag phase (a), the logarithmic growth phase (b) and the stationary phase (c) of the rotifer culture. Each character indicates rotifer–ciliate density of duplicate experiments, and the bold characters were the starting point of each experiment. Each dashed line is the approximated growth curve by free-hand.
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stationary phase increased rapidly and the growth of B. rotundiformis was suppressed. On the other hand, E. vannus added at the logarithmic growth phase decreased temporally from 1000 to 300 cells ml1 and the rotifer grew as well as the control culture for the first 5 days, but thereafter the E. vannus began to increase and the rotifer–ciliate density profiles followed the pattern of the stationary phase addition. When feeding on N. oculata, E. vannus did not increase regardless of the inoculation time, and the rotifer grew as well as the control, except for when the ciliate was inoculated during the stationary phase. At the stationary phase, although E. vannus did not increase, the rotifer density was significantly less than the control. The density-dependent effects on growth are shown in Table 1. When feeding on T. tetrathele, the coefficiency of interspecific competition from the ciliate to the rotifer was about one-fifth the coefficiency of intraspecific competition of the rotifer, and the rotifer growth rate reduced to 0 at the ciliate density of 8000 cells ml1 (The r=0 densities were calculated following the method described in Section 3.1). Regardless of the algal species, the ciliate growth had no density-dependent effect in the competition cultures. 3.3. Feeding experiments using a stable isotope The labeling ratios (15N at.%) of the food source in the feeding experiments were 89% for N. oculata, 86% for T. tetrathele, 59% for rotifer feces feeding on N. oculata, and 50% for rotifer feces feeding on T. tetrathele. The specific growth rate of E. vannus populations during the experiments was 0.82 day1 (=0.034 h1) with feeding on N. oculata and 0.58 day1 (=0.024 h1) with feeding on T. tetrathele. These growth rates were lower than the
Fig. 4. Nitrogen incorporation ratios (% of body nitrogen) of E. vannus. The nitrogen was incorporated through dead algae (open bars) or rotifer feces (closed bars). Solid line is the steady nitrogen incorporation rate (% of body nitrogen h1) by E. vannus.
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Table 2 Nitrogen incorporation rate (% of body nitrogen h1) of E. vannus from each food source Algae
T. tetrathele N. oculata
Food source Dead algae
Rotifer feces
Bacteria-free rotifer feces
0.7 0.4
1.9 3.4
0.7 0.8
The food sources and feeding regimes are explained in Materials and methods.
potential maximum growth rate of the single-species cultures (Table 1), especially at feeding on T. tetrathele. 15 N was rapidly incorporated in E. vannus and the incorporation ratio reached 12% during the first 30 min with feeding on both N. oculata and T. tetrathele (Fig. 4). Later, the incorporation ratio grew steadily until the end of the experiment with feeding on N. oculata. Meanwhile, with feeding on T. tetrathele, the incorporation stopped temporarily between 30 and 90 min and then resumed until 300 min. The steady incorporation rates were 3.8% h1 on N. oculata and 2.5% h1 on T. tetrathele, calculated from the incorporation ratios between 90 and 300 min. These incorporation rates agreed well with the specific growth rate of E. vannus. Almost all the nitrogen was incorporated through the rotifer feces (90% on N. oculata and 74% on T. tetrathele). When feeding on the bacteriafree rotifer feces, the incorporation rate dropped to 24% of the normal rotifer feces on N. oculata and 39% on T. tetrathele (Table 2).
4. Discussion 4.1. What affects ciliate growth? 4.1.1. Food It has been believed that many protozoans, also including E. vannus, are bacterivorous. Wilks and Sleigh (1998) performed grazing experiments using fluorescent latex microspheres of various sizes in E. mutabilis, which is closely related to and approximately the same size as E. vannus. They showed that E. mutabilis can ingest particles of sizes between 0.57 and 10.0 Am in diameter and the maximum volume ingested was obtained for 5.85 Am diameter microspheres. These results demonstrate that Euplotes spp. can efficiently ingest not only bacteria but also phytoplankton. We actually observed that the ciliate ingested both N. oculata and T. tetrathele under a fluorescence microscope. In the tracer experiment, nitrogen incorporated into E. vannus through algae reached the maximum value until 30 min, and was maintained at a constant level thereafter (Fig. 4). This means that E. vannus can ingest algae, but does not digest it well. Rotifers have a complex chitinous jaw composed of a dental mill, and the jaw functions to grind the firm phytoplankton cells. Ciliates do not possess a specialized jaw and may not be able to digest the hard cell wall of the phytoplankton. In contrast, nitrogen was incorporated through the rotifer feces until the end of the experiments. The incorporation rate was fast during the first hour but then fell during the next 3 h (Fig. 4). This trend was consistent
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with the results of Zubkov and Sleigh (1996), and may be an error due to the experimental procedure in which the ciliates could be starved for a short time just before the experiment, indeed, the specific growth rate during the feeding experiments was lower than the potential maximum growth rate (see Section 3.3). They measured the 14C accumulation in Euplotes mutabilis feeding on bacteria labeled with 14C-leucine, and demonstrated that the ingestion of bacteria and accumulation of 14C by normal Euplotes were linear in the interval between 1 and 4 h. Thus, to calculate the steady incorporation rate, we used time points between 90 and 300 min, rather than during the first hour. The observation that almost all the nitrogen (74–90%) was incorporated through the rotifer feces with bacteria shows that the ciliate prefers rotifer feces to plain algae. When supplied with N. oculata, the rotifer egests 70% of the ingested nitrogen as feces (Aoki et al., 1995). Thus, rotifer feces are rich in nitrogen and have numerous bacteria attached to the surface. The incorporation rate with feeding on the feces without bacteria was about one-third of the rate on feces with bacteria. This difference in the results depended on the existence of bacteria, and thus cell wall breakage by the rotifers contributes one-third of the reason why the protozoan can digest rotifer feces, while the microbial food chain through phytoplankton to rotifer feces to bacteria contributes the remainder. In these experiments, E. vannus also mainly incorporated bacterial nitrogen. 4.1.2. Allelopathy Although E. vannus actively incorporated feces from rotifers fed N. oculata (Fig. 4), the maximum density (r=0 density) of E. vannus with feeding on N. oculata was much lower than that with feeding on T. tetrathele. The instantaneous growth rates during the logarithmic growth phase, constant l max in Table 1, did not differ significantly between ciliate-T. tetrathele and ciliate-N. oculata ( pb0.01). This suggests that E. vannus can incorporate the nitrogen of N. oculata through bacteria and consequently grow, but the growth was soon suppressed by some factors involved with N. oculata. Moreover, E. vannus prolonged the lag phase when fed on both fresh algae (Fig. 2). This algal inhibition of the ciliate growth may possibly be explained by an allelopathic interaction. A similar phenomenon was observed for the interactions between the green algae Chlorella and the ciliate Colpidium, and chlorellin seems to be a harmful substance (Hulot et al., 2001). However, no chemicals suppressing the ciliate growth have yet been isolated from N. oculata or fresh T. tetrathele. Therefore, this allelopathic interaction may not be a direct effect, but rather an indirect effect with a change in the bacterial flora. Further studies are required to obtain direct proof of an allelopathic interaction between algae and ciliates. Furthermore, the interactions between N. oculata and other ciliates, e.g., Uronema sp, have not been clarified and remain the next issue to be investigated. 4.2. Interspecific and intraspecific competition In a single culture of the rotifer, density-dependent growth suppression was observed (Table 1). This growth suppression was thought to be a result of intraspecific competition. The mechanisms of intraspecific competition include accumulation of metabolites, food limitation, interference and so on. The maximum rotifer density of 1400 rotifer ml1 (Fig. 1) is a normal density for rotifer batch culture in a hatchery, although it is well-known fact
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that B. rotundiformis can reach 10000 rotifer ml1 if sufficient food and oxygen are supplied. Thus, this intraspecific competition of rotifers is due to food limitation rather than an autotoxin, as suggested by Snell et al. (2001). It has long been known that some ciliates inhibit rotifer growth in rotifer mass cultures (Reguera, 1984). In this paper, we have shown that the path of rotifer–ciliate density depended on the inoculation timing of the ciliate, and finally reached ciliate dominance when feeding on T. tetrathele (Fig. 3). B. rotundiformis and E. vannus show interspecific competition and cannot coexist in the same space for a long time. With inoculation at the lag phase or the stationary phase, the rotifer–ciliate competition easily progresses to ciliate dominance, and this phenomenon corresponds to the trend that a ciliate plague often occurs just after rotifer inoculation or when using an old rotifer culture tank. The environment of a rotifer culture tank is non-equilibrium in time series and it is expected that a change in the environment is related to changes in interspecific competition. The major environmental factors are food, space and accumulation of excretory substances. The rotifer and the ciliate utilize different foods from each other. The rotifer mainly grazes on microalgae, whereas the ciliate mainly eats and digests bacteria as mentioned above. When food algae are abundant, resource competition will definitely not exist between these two species. Regarding space utilization, these two species have different preferences. The healthy rotifer swims well using cilia as a propeller. The ciliate does not always inhabit pelagic water, but prefers to move on detrital particles accumulated on the bottom rather than swim. The two species are therefore segregated in a rotifer culture tank. Food and space were thus excluded as environmental factors that influence the interspecific competition. Except for exploitation and direct interference in the space utilization, indirect interference is listed as the mechanism of the interspecific competition. Bacterial flora may possibly be related to the indirect interference. Indeed, Maeda and Hino (1991) isolated a bacteria strain from a Euplotes sp. culture that strongly inhibited rotifer growth. Recently, it was demonstrated that the bacterial community structure is regulated by protozoan-selective predation (Langenheder and Jurgens, 2001). In rotifer cultures, excretory substances accumulate day by day and therefore the bacterial flora is also in non-equilibrium (Maeda and Hino, 1991). This non-equilibrium of the bacterial flora may explain the different results for the rotifer–ciliate dynamics due to the timing of the ciliate inoculation. In contrast, when feeding on N. oculata, the ciliate did not grow and the rotifer grew as well as the control. This result is inconsistent with the results of Hagiwara et al. (1995) in which Euplotes sp. populations interfered with the growth of B. rotundiformis when feeding on N. oculata. However, after re-examining their results, the maximum ciliate density was less than 100 cells ml1 and the interference was statistically significant but too weak. Thus, it is concluded that their results showed almost the same trend as our results. 4.3. A new technique for rotifer culture In a latest intensive rearing system like a recirculation system (Suantika et al., 2003) or a chemostat system (Fu et al., 1997), the water quality is well managed and a ciliate explosion is rare. However, in a conventional extensive culture like as a batch or a thinning culture, competition between rotifers and ciliates is inevitable, and suppression of ciliate
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growth is necessary for a stable rotifer culture. In a rotifer tank, much of the particulate organic matter originates from the accumulation of rotifer feces (Aoki and Hino, 1996), and this debris supplies protozoans with food and habitats. We proved that the ciliate E. vannus actively incorporated nitrogen through the rotifer feces and grew well. The accumulation of detritus therefore increases the risk of a protozoan explosion. An adhesive mat is commonly used to remove debris in rotifer tanks and has some benefits in suppressing protozoa propagation. The competition between B. rotundiformis and E. vannus may be related to the bacterial flora, and thus control of the bacterial flora should be a useful technique for minimizing the ciliate growth. Indeed, Yu et al. (1989) succeeded in controlling the bacterial flora for a stable rotifer culture by using a vitamin B12 producing bacteria, but maintenance of the bacteria control has not yet been tested and it may be difficult in the non-equilibrium environment of a rotifer batch culture. At present, condensed freshwater Chlorella enriched with vitamin B12 (Maruyama and Hirayama, 1993) is the most popular microalga for rotifer culture in Japan rather than N. oculata. When using freshwater Chlorella, protozoans grow well and sometimes occupy a rotifer tank. Periodic addition of N. oculata with freshwater Chlorella is expected to control E. vannus, and this technique of applying alleropathy is expected to be more sustainable than bacterial flora control.
Acknowledgements The authors would like to express their gratitude to Dr. Isao Koike, Ocean Research Institute of the University of Tokyo, for providing the use of a mass spectrometer. The authors are grateful for a scholarship from the National Science Council, Republic of China, and the support of Dr. I-Chiu Liao, director of the Taiwan Fisheries Research Institute (T.F.R.I.).
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