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MICROBIAL ECOLOGY

Microb Ecol (2001) 42:168176 DOI: 10.1007/s002480000116 2001 Springer-Verlag New York Inc.

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates
J. Boenigk,1 C. Matz,2 K. Jurgens,2 H. Arndt1
1

Department of General Ecology and Limnology, Zoological Institute, University of Cologne, D-50923 Cologne, Germany 2 Department of Physiological Ecology, Max Planck Institute of Limnology, D-24306 Plon, Germany Received: 30 August 2000; Accepted: 28 November 2000; Online Publication: 8 May 2001

B S T R A C T

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of grampositive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. Selective digestion could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.

Present address (JB): Institute for Limnology, Austrian Academy of Sciences, Mondseestr.5, 5370 Mondsee, Austria Correspondence to: J. Boenigk; E-mail: jens.boenigk@uni-koeln.de

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Introduction
The concept that simple food chains can explain the carbon flow through aquatic communities has changed, and more complicated food webs are now assumed to be a suitable approximation [1, 28]. The significance of bacterial production and of bacteriaprotozoa interactions for carbon flow through aquatic ecosystems is widely accepted. Heterotrophic nanoflagellates especially are believed to be important consumers of bacteria and to be mainly responsible for carbon transfer to higher trophic levels [10]. Food selection by heterotrophic nanoflagellates is suggested as being one important factor in explaining the size distribution of natural bacteria [5, 13, 16]. However, size selection seems to be a passive mechanism that depends on contact probabilities and morphological limitations of the feeding structures [2, 11]. In contrast to these indirect predator-induced shifts in the bacterial community, optimal foraging theory predicts a generally active selection for food of a higher nutritional value [3, 34]. Selection by heterotrophic nanoflagellates for bacterial characteristics other than size has been described (e.g., surface hydrophobicity [26] or mobility [15]). Active selection by the flagellate as well as contact probabilities may both be responsible for shifts in the bacterial population and are generally difficult to distinguish. However, it is generally assumed that selection occurs during capture and ingestion. The occurrence of prey particles in the food vacuole is assumed to be an indication for ingestion as well as for digestion of these particles and therefore is used as a basis for the calculation of these parameters [15, 33]. Thus, the number of ingested particles is generally used for the calculation of ingestion and clearance rates, which are the basis for the estimation of carbon flow through the microbial food web. In contrast to the general assumptions of these calculations, Boenigk et al. (submitted) could show that indigestible particles are ingested at similar rates but are excreted 23 min after ingestion. However, the importance of this mechanism for prey selection of protists within natural picoplankton has not yet been studied. If the digestion rates of different bacteria vary, the effect of flagellate grazing would be species-specific. It would not be possible to predict the grazing impact of flagellates on the bacterial community as a whole. Therefore, a new interpretation of grazing experiments may be necessary when species-specific digestion can be shown to be an important component of food selection. Here we examined the ingestion and digestion process of six different food particles

for three species of heterotrophic interception feeding flagellates in different physiological states.

Methods
Organisms and Culture Conditions
For observation we chose three interception-feeding flagellate species that attach to the substratum. Cafeteria roenbergensis Fenchel and Patterson 1988 [12] was isolated from the Baltic Sea at a salinity of 10 near Kloster (Hiddensee, Germany) by A.P. Mylnikov. The strain of Spumella sp. was isolated by K. Jurgens from Lake Schohsee, Germany, and the Ochromonas species (strain M) was isolated by D. Springmann from Lake Constance, Germany. The flagellates were grown on Pseudomonas putida MM1 in 100 ml Erlenmeyer flasks at 17C and under permanent light. For the experiments concerning adaptation to specific food particles some flagellates were grown on Bacillus subtilis for 8 days before the experiments started. Exponentially growing cultures of Pseudomonas putida MM1 and Bacillus subtilis, respectively, were repeatedly inoculated with flagellate culture to render the cultures monoxenic. The bacterial species in the cultures are referred to as culturing diet. Flagellates in the exponential growth phase were cultured at a permanent food concentration of >5 106 bacteria ml1. For the experiments with starved flagellates, the culture medium was exchanged for new bacteria-free medium and the flagellates were kept at a food concentration of below 105 bacteria ml1 for 6 to 8 hr before the experiments were started. Pseudomonas putida MM1 was isolated from barley rhizosphere [4], Bacillus subtilis was from the DSMZ, Braunschweig (CatNo. DSM10, type strain), Pseudomonas sp. CM10 was isolated from Lake Schohsee by C. Matz, Synechococcus sp. (strain BO 8809) was isolated by A. Ernst from Lake Constance, and Synechococcus elongatus was from the culture collection of the Max Planck Institute for Limnology in Plon, Germany [6]. Basic medium for the culti vation as well as for the experiments was an artificial algae mineral medium (WC medium [17]). All bacterial species were transferred to WC medium supplemented with 100 mg glucose per liter. Following inoculation the bacteria were allowed to grow for about 48 to 72 hr before the experiment started to reach stationary phase. Bacterial cell volume (Table 1) was calculated from image analysis after DAPI-staining [31] according to Posch et al. [32]. Swimming speed of bacteria (Table 1) was measured using a calibrated video screen.

Live Video Microscopy


The methodology is described in more detail elsewhere [2, Boenigk et al., submitted]. Briefly, flagellates were transferred to petri dishes with a diameter of 55 mm with a cover glass at the bottom. The cover glass allowed the use of a 100 oil immersion objective to obtain high optical resolution. The flagellates were allowed to attach to the bottom of the observation chamber and to adapt to the experimental conditions for 30 min after the transfer. Immediately before the observation was started the medium was repeatedly ex-

170 Table 1. Cell volume [m3] and speed [m s1] of food particlesa Cell volume (m3) Pseudomonas putida MM1 Pseudomonas sp. CM10 Bacillus subtilis Synechococcus sp. BO 8809 Synechococcus elongatus Latex beads 0.61 0.41 0.42 0.12 0.60 0.23 0.26 0.09 0.32 0.14 0.22 0.02 Speed (m s1) 26.5 (1.4 0.2) 42.5 (1.6 0.3) 13.7 (1.2 0.3) 7.8 (0.9 0.2) 8.8 (1.0 0.2) 7.3 (0.9 0.3)

J. Boenigk et al. the initial 5 min of the experiment as well as from the following 5 min. For each flagellate species the influence of food quality and satiation status on ingestion rate was tested using analysis of covariance (ANCOVA). We assumed the contact rate to converge asymptotically at a limit as described by a functional response type 2 [29]. Because of this assumption, the reciprocal values of the contact rates were used as covariates, as we did not want to investigate the particle-specific contact probabilities but the particlespecific digestion. By this procedure the ingestion rates were recalculated for a constant contact probability. Afterwards the six different combinations of flagellates and food conditions (Spumella, Ochromonas, and Cafeteria; starved and satiated) were tested separately to obtain more insight into species-specific differences. Ingestion rates and contact rates were log-transformed to obtain a normal distribution of the data. For each flagellate species the influence of food quality and satiation status on vacuole passage time of excreted particles (latex beads and both species of Synechococcus were excreted by Spumella and Ochromonas) was tested using analysis of variance (2-way ANOVA). Differences between the ingestion rates of the different prey particles were tested using Tukeys post-hoc test. Minimum vacuole passage times could be measured for digested particles only. Passage times were log-transformed to obtain a normal distribution of the data. For the experiments with Pseudomonas putida and Bacillus subtilis the influence of culturing diet, satiation status, and food bacteria on ingestion rate was tested using analysis of covariance (ANCOVA). Contact rates were used as covariates to eliminate the influence of different food concentrations. In addition, the influence of the culturing diet and food bacteria was tested separately for starved cells as well as for growing cells of each flagellate species using ANCOVA. For the calculation of ingestion rates of starved flagellates, only data from the initial 5 min of an experiment were taken in this test.

a Cell volume (mean SD) was calculated using an automated image analysis according to Posch et al. [32]. Speed (mean) was measured from a calibrated video screen and includes active swimming as well as a passive drift of the particles. Thus, swimming speed represents the mobility of the particles under the experimental conditions. In parentheses: logtransformed mean and SD.

changed for bacteria-free medium. This procedure allowed us to lower the food bacterial concentration within 3 min below 105 bacteria ml1. After the washing procedure the petri dish was inspected with an inverted microscope and one attached flagellate cell was selected for the detailed observation of the particle uptake. Then the particle type to be inspected was added at a final concentration of 12 107 ml1. Pseudomonas putida, Bacillus subtilis, Pseudomonas sp. CM10, Synechococcus sp. (strain BO 8809), Synechococcus elongatus, and latex beads were tested as food particles. In addition, as artificial particles latex beads were tested (Flouresbrite Plain YG, 0.75 m, Polysciences, Inc., USA). All experiments were carried out at 18 20C with flagellates grown on Pseudomonas putida. In addition, the experiments with Bacillus subtilis and Pseudomonas putida were also carried out with flagellates grown on Bacillus subtilis. Fifteen individual flagellate cells were observed for each combination (food particle and satiation status of the flagellate) for a time period of 10 min. All encountered, ingested, and excreted particles were noted. Observations of the feeding process and size measurements were carried out with a Zeiss Axiovert S100 equipped with a Plan Neofluar 100/1.3 oil objective according to Boenigk et al. [2]. Briefly, the microscope was connected with a zoom adapter to a MC1009/S video camera (AVT Horn, Aalen, Germany). The output of the video camera was fed to on S-VHS recorder (AG 7355, Panasonic), recording at 25 frames s1.

Results
The range of ingestion rates across all particle types was 5.548.1 particles flag.1 hr1 for Spumella, 2.7175.7 for Ochromonas, and 1.066.1 for Cafeteria, respectively (Tab. 2). All factors (flagellate species, satiation status, and quality of the food particle) were found to have a significant (p < 0.001) influence on the ingestion rate. In addition, all combinations of factors were significant (p = 0.003 for the combination flagellate species and satiation status and p < 0.001 for all other combinations). However, the specific characteristics of the food particles as measured in the experiments, i.e., size and motility, could not explain the variability of the ingestion rates. To gain more insight into food particlespecific differences each species was tested separately.

Data Analysis
Statistical analyses were carried out using the SPSS software (Version 8.0.0, SPSS Inc.). Ingestion rates were measured for each individual cell of the experimental sets. As ingestion rates were not constant for starved flagellates, ingestion rates were calculated from

Feeding Strategies of Flagellates Table 2. Observed ingestion ratesa Latex beads Spumella Satiated Starved (initial 5 min) Starved (from 5 min on) Ochromonas Satiated Starved (initial 5 min) Starved (from 5 min on) Cafeteria Satiated Starved (initial 5 min) Starved (from 5 min on)
a

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Synechococcus elongatus 16.4 (1.2 0.3) 30.4 (1.5 0.2) 11.7 (1.1 0.3) 133.6 (2.1 0.1) 175.7 (2.2 0.3) 115.9 (2.1 0.2) 3.1 (0.6 0.3) 13.6 (1.1 0.4) 2.3 (0.5 0.3)

Synechococcus sp. BO 8809 37.1 (1.5 0.1) 48.1 (1.6 0.5) 30.3 (1.5 0.3) 39.1 (1.6 0.2) 83.8 (1.9 0.2) 23.9 (1.4 0.4) 10.5 (1.0 0.1) 6.5 (0.9 0.6) 6.1 (0.9 0.2)

Bacillus subtilis 5.5 (0.8 0.6) 34.6 (1.6 0.2) 17.2 (1.3 0.3) 42.9 (1.6 0.2) 40.0 (1.6 0.2) 2.7 (0.6 0.8) 3.6 (0.7 0.2) 12.8 (1.1 0.5) 1.0 (0.3 0.3)

Pseudomonas sp. CM10 16.6 (1.2 0.1) 20.4 (1.3 0.2) 7.1 (0.9 0.3) 50.3 (1.7 0.2) 67.1 (1.8 0.2) 30.9 (1.5 0.4) 7.1 (0.9 0.3) 2.0 (0.5 0.6) 2.1 (0.5 0.5)

Pseudomonas putida MM1 18.2 (1.3 0.3) 45.6 (1.7 0.2) 15.6 (1.2 0.1) 44.9 (1.7 0.3) 86.1 (1.9 0.2) 7.7 (0.9 0.8) 11.2 (1.1 0.3) 66.1 (1.8 0.2) 18.2 (1.3 0.5)

21.2 (1.3 0.1) 22.0 (1.4 0.5) 15.0 (1.2 0.2) 17.6 (1.2 0.2) 120.3 (2.1 0.2) 58.4 (1.8 0.3) 10.9 (1.1 0.4)

Ingestion rates (I [particles individuum1 hr1]; n = 15) are corrected for contact probabilities (because of size and swimming speed of the particles). In parentheses: log-transformed mean and SD.

Satiation and Food Vacuole Content Ingestion rates depended strongly on the satiation of the flagellates and on the actual content of the food vacuoles. Ingestion rates of satiated flagellates, i.e., flagellates cultured at high food concentration, remained constant during the whole observation time because of more or less constant filling of the food vacuoles. In contrast, for starved flagellates the ingestion rate differed depending on the actual filling status of the food vacuoles. During the initial phase of the experiment (first 5 min) when no or only few particles were ingested, the ingestion rates were significantly higher compared with those of satiated flagellates (p = 0.001 for Spumella and Ochromonas, p = 0.002 for Cafeteria). After this initial phase, ingestion rates of starved flagellates were significantly lower (p < 0.001 for Ochromonas, p = 0.001 for Cafeteria) or similar (p = 0.993 for Spumella) compared with ingestion rates of satiated flagellates (Fig. 1). Food Quality and Preculturing Diet In addition to the satiation status of the flagellates, the nature of the food particles also had a significant influence on the ingestion rate (p < 0.001 for all species, satiated as well

as starved, except starved Spumella (p = 0.24)). However, the particle characteristics size and motility were not correlated with the ingestion rates. The p-values of the pairwise comparisons between the different food particles are shown in Table 3. The culturing diet, i.e., the bacteria fed during preculture before the experimentseither Pseudomonas putida or Bacillus subtilishad a significant influence on the ingestion rate for Spumella (p = 0.006) and for Cafeteria (p < 0.001; see Table 4). In addition, the combination of experimental diet and culturing diet, i.e., the bacteria fed during the experiment, was found to be significant (p = 0.033 for Spumella; p < 0.001 for Cafeteria). We tested satiated and starved flagellates separately to obtain detailed information on the influence of the culturing diet. For starved flagellates the combination of experimental diet and culturing diet had a significant influence on the ingestion rate (p = 0.03 for Spumella; p < 0.001 for Cafeteria): Feeding rates of both flagellates were higher when they were precultured on the same food organism as used in the ingestion experiment (Table 4). For Ochromonas the same trend was observed but it was not significant (p = 0.151). For satiated Spumella the ingestion rates feeding on Pseu-

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cies were observed for latex beads and Synechococcus. These were not excreted by Cafeteria (vacuole passage time >20 min) but by Spumella and Ochromonas (Fig. 2) after only short vacuole passage time (Table 5). The fast excretion of the Synechococcus species corresponds with the fact that the flagellates were not able to grow when fed on these species as the only food (Matz, unpublished data). Vacuole passage time of the excreted particles also varied with the particle type and food satiation of the flagellates. Vacuole passage time was significantly longer for starved flagellates compared with that of satiated flagellates (p = 0.002 for Spumella; p = 0.015 for Ochromonas). For satiated Spumella and Ochromonas, vacuole passage time of beads was significantly shorter than those of the two Synechococcus species (p = 0.001). For Ochromonas, significant differences between the two Synechococcus species were also apparent.

Discussion
Methodical Considerations In general, ingestion and digestion rates are calculated either from changes in abundance of predator and prey [e.g., 18] or directly from food vacuole content in short-term uptake experiments [e.g., 33]. These methods are limited by a temporal resolution of at least several minutes and by the assumption of a uniform feeding behavior during the investigation, which can hardly be proved. Video microscopy allows a temporal resolution in the range of milliseconds and therefore offers the opportunity to analyze the fate of individual prey particles [2]. Under the microscope the flagellates are exposed to high light intensities that are, however, comparable to those of surface waters during a sunny day. The heating by the microscope lamp may be more problematic but is reduced by using a large water volume in the observation chamber allowing vertical and horizontal compensating water currents. Tests with different light intensities showed no irregularity in the flagellates behavior except very high light intensities due to full light and open aperture (unpublished data).

Fig. 1. Ingestion rates for satiated flagellates (during the first 15 min), for starved flagellates during the first 5 min of the experiment, and for starved flagellates from 5 to 10 min. Mean and standard deviation are calculated from log-transformed data.

domonas putida and Bacillus subtilis, respectively, were significantly lower when precultured on Pseudomonas (p = 0.001) compared with the corresponding ingestion rates for Spumella precultured on Bacillus. For Ochromonas the same trend was founds, but it was not significant (p = 0.150). In contrast, the ingestion rate of satiated Cafeteria was significantly higher when precultured on Pseudomonas (p = 0.03). Thus, the effect of the preculturing diet on the ingestion rates is dependent on the satiation of the flagellates.

Digestion Process The fate of ingested particles was different depending on particle characteristics. All flagellates digested Bacillus and both Pseudomonas species and the vacuole passage time, defined as the time from ingestion of particle to its excretion, was >20 min. Strong differences between the flagellate spe-

Ingestion Rates Ingestion rates were in the range of reported rates for all three species [e.g., 2, 13, 19, 23]. However, significant differences in the ingestion rate were found between the particles. Lower ingestion rates for larger food particles on the one hand have been reported and were mainly explained by

Feeding Strategies of Flagellates Table 3. Pairwise comparision of the ingestion rates of Spumella, Ochromonas, and Cafeteria feeding on different food particlesa Pseudomonas putida Spumella Pseudomonas putida Pseudomonas sp. CM10 Bacillus subtilis Synechococcus sp. BO 8809 Synechococcus elongatus Latex beads Ochromonas Pseudomonas Pseudomonas sp. CM10 Bacillus subtilis Synechococcus sp. BO 8809 Synechococcus elongatus Latex beads Cafeteria Pseudomonas Pseudomonas sp. CM10 Bacillus subtilis Synechococcus sp. BO 8809 Synechococcus elongatus Latex beads
a

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Pseudomonas sp. CM10 .016* .017* .006** .699 .386 .645 .113 .094 .000*** .000*** .000*** .021* .333 .005* .151

Bacillus subtilis .044* .836 .000*** .001** .002** .000*** .019* .942 .000*** .004** .000*** .002** .001** .581 .021*

Synechococcus sp. BO 8809 .218 .427 .443 .004** .038* .929 .540 .000*** .000 .004** .000*** .010* .519 .000*** .021*

Synechococcus elongatus .021* .988 .794 .314 .732 .000*** .000*** .000*** .000*** .000*** .000*** .000*** .266 .048* .440

Latex Beads .001** .389 .240 .065 .353 .055 .645 .000*** .040* .087

.478 .000*** .011* .690 .996 .062 .721 .780 .000*** .016* .476 .004** .810 .001** .026*

Significant differences between the ingestion rates (corrected for contact probability) for different food particles, p-values of (ANCOVA) for starved flagellates (initial 5 min) (right up) and growing flagellates (left down).

the feeding capacity of the flagellates [e.g. 19]; higher ingestion rate, on the other hand, have been reported and were explained by a higher contact probability [2, 9]. In our investigation the influence of species-specific contact probabilities was eliminated. Thus, the combination of the flagellate vacuole capacity and the cell volume of the different food particles may be one main reason for different ingestion rates. In addition, mobility of the prey organisms may

increase their chance to escape from feeding after being captured and thus decrease the ingestion rate of flagellates feeding on these particles. Size and motiliy, respectively, seem to have an influence not only on contact probability but also on escape probability, but neither size nor motility could explain the observed variability in our experiments. Other factors such as surface characteristics of the prey may also alter the ingestion rate [26], and therefore these factors should be

Table 4. Ingestion rates for Spumella, Ochromonas, and Cafeteria feeding on Pseudomonas putida (MM1) and Bacillus subtilis (DSM10), respectivelya Spumella Culture MM1 MM1 DSM10 DSM10
a

Ochromonas Starved Satiated 63 (1.7 0.2) 47 (1.6 0.2) 118 (2.0 0.2) 47 (1.6 0.2) Starved 91 (1.9 0.2) 45 (1.6 0.2) 94 (1.9 0.3) 56 (1.7 0.2) Satiated

Cafeteria Starved 70 (1.8 0.2) 18 (1.1 0.5) 22 (1.2 0.4) 21 (1.3 0.2)

Food MM1 DSM10 MM1 DSM10

Satiated 21 (1.3 0.3) 12 (0.8 0.6) 36 (1.5 0.2) 20 (1.3 0.2)

51 (1.7 0.2) 37 (1.6 0.2) 46 (1.6 0.2) 53 (1.7 0.1)

15 (1.1 0.3) 4 (0.7 0.2) 5 (0.8 0.1) 3 (0.5 0.4)

Flagellates were precultured on either MM1 or DSM10 for one week before the experiment started. Mean and SD of the log-transformed data are given in parentheses (n = 15). Ingestion rates were not corrected for contact probabilities. The data for starved flagellates were calculated from the time interval 05 min.

174 Table 5. Vacuole passage timesa Spumella Satiated Synechococcus sp. 8809 Synechococcus elongatus Latex beads
a

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Ochromonas Satiated 6.6 (0.9 0.2) 3.3 (0.6 0.2) 1.6 (0.4 0.2) Starved 5.0 (0.7 0.2) 3.2 (0.6 0.1) 4.0 (0.7 0.2)

Starved 3.3 (0.6 0.3) 4.0 (0.6 0.3) 2.7 (0.5 0.2)

3.0 (0.5 0.3) 3.2 (0.6 0.3) 1.3 (0.3 0.2)

Vacuole passage time (min) for Spumella and Ochromonas feeding on the Synechococcus species and on beads (n = 15). Vacuole passage time for Bacillus and both Pseudomonas species and for all particles in Cafeteria was >20 min (not included in the table). In parentheses: log-transformed mean and SD.

Fig. 2. Percentage of the ingested particles that were excreted within the first 10 min of the experiment (particles ingested within 10 min = 100%) for satiated and starved flagellates.

investigated for a variety of food particles. Even though motility and size have been described as altering contact probability [15, 25], particle selection in advance to ingestion seems to be not very common at moderate food concentrations [2]. In addition to bacterial characteristics, ingestion rates depend on factors related to the nutritional and physiological conditions of the flagellates. Starved flagellates generally fed at higher rates compared to satiated flagellates. As starved flagellates contain no or only few food vacuoles when the experiment is started, they may ingest more particles in the initial phase of the experiment compared with satiated flagellates. After this initial feeding phase the food vacuoles are filled with ingested particles and the flagellates ability to ingest further particles is significantly lower. This agrees with the finding that starved

flagellates feed at lower rates compared with well-fed flagellates [8]. Higher ingestion rates could be observed for starved flagellates when precultured on the same bacterial strain as that offered as food. Starved flagellates should have a similar vacuole capacity independent of the culturing diet; thus, the ingestion rate should not depend on the vacuole capacity but on food capture and prey handling. Food capture may be more efficient when the flagellates are used to the food particles. In contrast, satiated flagellates fed at significantly different rates depending on the preculture conditions. Flagellates precultured on good digestible prey should have higher feeding capacities and show higher feeding rates. For Spumella and Ochromonas the digestion of Bacillus is slow compared with the digestion of Pseudomonas, whereas it is the other way round for Cafeteria. Even though the culturing diet had a significant effect on the ingestion rate, overall influence seemed to be weak compared with factors such as size of the bacteria, biochemical composition of the bacterial surface, and digestibility of the bacterial strain. This is supported by the finding that culturing the flagellates on the Synechococcus species increased individual variability concerning vacuole passage times but, in general, flagellates did not become able to digest the cyanobacteria (unpublished data). Although the influence of the culturing diet was only weak, the grazing impact of natural HNF communities on bacteria may also depend on the temporal variability in the bacterial community, inaddition to food concentration and size distribution of bacteria. Not only the effect of rapid changes in the species composition of bacterioplankton (e.g.,

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larger cells, biochemical composition) but also the temporal component of such a change itself may serve as a protection mechanism. This might be one reason for sometimes relatively high planktonic bacterial concentrations occurring despite substantial protozoan grazing pressure [22].

Digestion Process One focus of this investigation was the particle-specific selective digestion as described by Boenigk et al. (submitted). In contrast to the only weak selection during food uptake, digestion depended strongly on the type of ingested food particle and on the flagellate species. The two basic digestion strategies described by Boenigk et al. (submitted) could be verified: Cafeteria showed no differences in the handling of different food particles. All particles, independent of their digestibility, were stored for at least 20 min in the food vacuoles. This agrees with findings of Dolan and Simek [7], who reported a digestion rate of about 1% of the food vacuole content per minute for Bodo saltans species feeding on Synecchococcus. In contrast, Spumella and Ochromonas rapidly excreted unsuitable food particles. For these flagellates the fate of ingested particles seems to depend mainly on their biochemical characteristics. The fast excretion of beads and of cyanobacteria compared with the other bacteria may indicate that prey selection takes place inside the food vacuoles. As a consequence the flow of materials out of the food vacuole may be of importance. Such an explanation seems to be more reasonable than the assumption that extracellular receptors can explain the mechanisms of food selectivity. Thus, for natural picoparticles a continuum of particle-specific digestion times (from rapid excretion as observed for beads to complete digestion as observed for several bacteria) must be assumed. Differential digestion as described for metazooplankton [29, 30] has also been reported for protozoans [14], but selection is generally assumed to take place during food capture and processing of the prey organism [e.g., 35]. An ingested prey organism should be digested and therefore the predator should profit from the ingested bacterium, whereas the bacterium should be destroyed and therefore the number of bacteria should be reduced. Thus, the population dynamics of the prey become strongly coupled to the predator dynamics. In contrast, selective digestion opens several possibilities. Bacteria may be completely digested or, in contrast, excreted as an intact, viable bacterium not affected by the vacuole passage. In both these cases the assumptions of a strong coupling between population dynamics of predator

and prey are still the same as in the classical model. However, bacteria may be partially digested and excreted, not digested but killed by the physiological conditions of the food vacuole, or survive the food vacuole passage. Surviving food vacuole passage does not mean that the flagellate has not profited from these particles. The flagellate may obtain some profit from metabolic products of the ingested bacteria without strong consequences for the bacteria as must be assumed, for example, for the uptake of endosymbionts. Even though differential digestion is well known from higher animals, e.g., for zooplankton feeding on algae [e.g., 27, 29], and in principle also from protozoans [14], the excretion of particles within only 23 min after the ingestion has not yet been described for protozoans. The prey organisms not only may be unaffected by vacuole passage but may receive some profit. Gut passage, for example, is known as a dispersal mechanism for seeds of many higher plants [e.g., 21, 24] and as selective advance for algae fed by zooplankton [e.g., 30]. The exact mechanisms and the fate of nondigested bacteria must be evaluated to estimate the consequences for carbon flow.

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