Behavioral flexibility in prey selection by bacterivorous nanoflagellates

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1 NOTES Limnol. Oceanogr., 40(S), 1995, 1503-l , by the American Society of Limnology and Oceanography, Inc. Behavioral flexibility in prey selection by bacterivorous nanoflagellates Abstract-We used laboratory experiments to study the effects of changing food abundance on the selective feeding of two heterotrophic nanoflagellates on particles of similar size but differing nutritional quality (latex beads and fluorescently stained bacteria). Both Bodo saltans and Spumella sp. exhibited similar shifts in selectivity with changes in food concentration. Flagellates that were cultured under food-limiting conditions showed a modest but significant preference for beads when both particles were offered simultaneously. However, both flagellates exhibited strong discrimination against the inert beads within min after the addition of a satiating concentration of live bacteria. As bacterial abundance declined over 24 h, discrimination against the inert beads gradually relaxed. The observed pattern of concentration-dependent selectivity is in agreement with the predictions of optimal diet models. Phagotrophic protists have been identified as major consumers of bacteria and phytoplankton in many aquatic systems (Sherr and Sherr 1994). Due to their ecological importance, selective feeding by these protozoans should have considerable impact on the structure and composition of pica- and nanoplankton. Prey selectivity of suspension-feeding organisms is in various ways related to the type of feeding mechanism. Most planktonic protozoans, including nanoflagellates and ciliates, can be classified as filter feeders, direct or raptorial interception feeders, or diffusion feeders (Fenchell986). Selectivity in food uptake can involve passive selection, in which physical constraints determine particle retention or encounter rate between predator and prey, and active selection governed by the ability of the predator to distinguish between food items due to mechanical or chemical cues. Observed food preferences are often a result of both kinds of processes (DeMott 1990; Verity 199 1). For example, food selection by filter-feeding protozoans can be seen mainly as a passive, mechanical process where the morphological structure of the filtering apparatus results in size preferences (Fenchel 1986). However, chemical stimuli of the prey may change selectivity even for filter feeders due to chemosensory attraction or repulsion (see Verity 199 1). Size-selective feeding by interception-feeding nanoflagellates can be explained as a passive mechanism in which prey size and the hydrodynamics of prey capture determine encounter rate and therefore ingestion rate (Spielman 1977; Fenchel 1982). However, since direct interception feeders capture and process prey particles individually, they may be able to use chemical cues to select among particles that differ in nutritional value. The chemosensory capabilities of heterotrophic nanoflagellates and ciliates (Sibbald et al. 1987; Bennett et al. 1988; Verity 1988) are probably responsible for their observed abilities to discriminate between latex beads and bacteria (Pace and Bailiff 1987; Nygaard et al. 1988) and between live and heat-killed bacteria of the same strain (Landry et al ). Information on chemically mediated prey selection is still scarce, and it is not known whether nanoflagellates can modify their food selection behaviors as environmental conditions vary. Optimal foraging theory proposes that evolution will favor behaviors that maximize net nutritional gains. Optimal diet models predict how behaviorally flexible predators should adjust their selectivity when feeding on mixtures of low- and high-ranking prey within homogeneous patches. These models predict that discrimination against low-ranking prey should be strong when preferred prey are abundant and weak when preferred prey are scarce (see Stephens and Krebs 1986; Hughes 1993). Limnologists and oceanographers have often underestimated the sensory and behavioral capabilities of aquatic organisms. Calanoid copepods, for example, were once considered mechanical filter feeders. Recent studies have demonstrated that Calanoid copepods have considerable behavioral flexibility and exhibit the predicted pattern of concentration-dependent selection between particles that differ in nutritional value (see DeMott 1993). In this study we tested for concentration-dependent selectivity in the smallest aquatic predators, heterotrophic nanoflagellates (HNF). We used a defined laboratory system with bacterivorous flagellates as grazers and mixtures of stained bacteria and latex beads as prey particles. The experiments were designed to detect selectivity between these two qualitatively different particles and to test whether this selection changes in response to food abundance and the nutritional status of the flagellates. Our model organisms were two HNF species that were isolated from mesotrophic Schiihsee (Plan, Germany) and whose abilities to consume bacteria have been examined previously (Jiirgens 1992): Bodo saltans (Kinetoplas tidae), 3-4 x 5-8 pm, and Spurnella sp. (Chrysomonadida), 3-6 pm in diameter. Two-stage chemostats were used to obtain well-defined cultures of HNF for the selection experiments. A mixed lake water-bacterial culture was grown in a first stage with low dilution rates ( d-l) and a low carbon concentration in the mineral me-

2 1504 Notes dium (5 mg glucose liter- ). This culture served as food for grazers in the second stage. All cultures and experiments were performed at C. HNF could be kept longer at low growth rates (~0.5 d-l) in steady statelike conditions. Bacterial concentrations in the first stage were - 1 O7 cells ml-l and were reduced by grazing in the second-stage chemostats to 3-8 x lo5 cells ml-l. Despite this depletion of food resources, the HNF showed no sign of reduced activity. The food-limiting conditions were reflected in reduced HNF volumes compared to exponentially growing cells (both species pm3). We tested for selection between latex beads (0.88~pmdiam, noncarboxylated, Polyscience Inc.) and live bacteria from the first-stage chemostat, stained with the fluorochrome Rhodamine isothiocyanate (RITC) according to Landry et al. (199 1). The bacteria were not motile and the mean volume, as determined from DAPI-stained samples, was 0.22 pm3 and therefore slightly smaller than the beads (0.36 pm3). However, considering that the cell volume of DAPI-stained bacteria is generally underestimated (Suzuki et al. 1993), mean volumes of beads and bacteria in our experiments should have been very similar. Previous experiments with food-limited cultures of the same species of flagellates found that clearance and ingestion rates for both particle types were in a similar range (Jfirgens 1994). For feeding experiments, 200 ml of a suspension of HNF was taken from a second-stage chemostat and transferred to batch culture (300-ml Erlenmeyer flasks, shaking table). A series of short-term feeding trials tested the response of each HNF species to a sudden increase in bacterial abundance followed by a gradual decline. After an initial time-zero feeding trial with food-limited flagellates, additional unlabeled bacteria from the first-stage chemostat were added to a batch culture. This food addition resulted in an increase from < 0.5 x 1 O6 to - 6 x 1 O6 bacteria ml- l. Selection experiments with beads and RITC-stained bacteria (RITCB) were repeated immediately after the food addition and six or seven additional times over 24 h. By the end of the 24-h period, bacterial abundance had been reduced by grazing to approximately the initial level. This experimental design resulted in a series of similar selection experiments with flagellates from different food levels and different nutritional states. A 5-ml sample was taken from this HNF suspension for each short-term feeding trial. There were three replicate trials for each time interval. For each feeding trial, beads and RITCB were added simultaneously in about equal concentrations ( x 1 O6 particles ml- ). Grazing was stopped after 6 min with Spurnella or 10 min with Bodo by adding an equal volume of ice-cold glutaraldehyde (2% final concn). Samples for counting ingested particles and estimating abundance of bacteria were stained with DAPI (Porter and Feig 1980), filtered onto 0.2~pm black Nuclepore filters, and inspected under a Zeiss epifluorescence microscope equipped with an HBO 50-W light source. Orange fluorescence of beads and red fluorescence of RITCB could be distinguished even within the same food vacuole with the Zeiss filter set 14 (BP 5 lo-560 excitation filter, FT 580 chromatic beam splitter, and LP 590 barrier filter). Ingested particles from at least 100 flagellates were counted in each replicate. A time-zero control served as a background value. Clearance rates and selectivity were calculated from numbers of fluorescent particles ingested per flagellate, particle concentration in food suspensions, and duration of the trial. A selectivity index for latex beads, DB, was calculated according to Jacobs (1974): DB = (FB - FR)I(FB + FR). FB and FR are clearance rates for latex beads and RITCstained bacteria. Ds can range from + 1 (uptake of only beads) to - 1 (uptake of only bacteria) with a value of 0 indicating nonselective feeding. Differences in selectivity coefficients and clearance rates over time were tested with one-way ANOVAs and a posteriori comparisons of the means (least significant differences). Both HNF species exhibited significant changes in selectivity in response to temporal changes in food concentration (Figs. 1 and 2; ANOVAs for selectivity over time: Bodo, F7,16 = 57.9, P < ; Spurnella, F8,18 = 34.6, P < ). I m t la 11 y, when food bacteria were scarce and flagellates were food limited, both RITCB and latex beads were ingested at high rates. Beads yielded slightly but significantly higher clearance rates for both species (t-tests, P < 0.05). After increasing the background food level by adding chemostat bacteria, selectivity between the two prey particles changed drastically. Uptake of beads declined sharply compared to uptake of RITCB, resulting in a decline in selectivity for beads, DB, for both species from 0.2 to c-0.6. The decrease in bead uptake was especially pronounced with Bodo and the minimum Ds value was recorded at 30 min; the change was slower for Spurnella and the minimum DB was recorded at 2 h. Bacterial concentrations gradually declined in batch culture due to grazing activity of flagellates, and, simultaneously, discrimination against the beads gradually decreased. Within 16-l 8 h, the bacterial concentrations had declined to the initial time-zero levels, and flagellates again exhibited significantly higher clearance rates for beads than for RITCB. Clearance rates for both particle types declined after bacterial additions and gradually increased again as bacterial concentrations declined (Figs. 1B and 2B). Temporal changes for Spurnella were significant for both beads v-8,18 = 109.5, P < ) and RITCB (F8,18 = 14.7, P < ). Similarly, clearance rates for Bodo exhibited significant changes for beads (F7,16 = 97.5, P < ) as well as for.ritcb (F7,16 = 3.99, P = ). With both flagellate species, clearance rates changed much more for beads than for RITCB with changing food concentration (Figs. 1B and 2B). Mean clearance rates of Bodo ranged from 0.05 to 1.63 nl ind.- h-l for beads and from 0.35 to 0.90 nl ind? h-l for RITCB. Clearance rates for Spurnella ranged from 0.18 to 3.19 nl ind. - l h-l for beads and from 0.47 to 2.09 nl ind.- h-l for RITCB. At the beginning and end of the experiments when bacterial concentrations were < 1 O6 ml-l, ingestion and growth rates of HNF were presumably strongly food lim-

3 Notes y 0 E C h.c >r -4 0 t > F; > c; 0.0 a 4 C ts -2 g cz zi a b C C C b a a a b c,d d,e e c b a a - 2, I 0 A 0 -_-_ 0 I E Cu Time after food addition (h) Time after food addition (h) Fig. 1. Prey selection experiments with Bodo sultans performed before (time 0) and at several time points after food addition (unlabeled bacteria) to a batch HNF suspension. A. Bacterial concentration in the HNF batch suspension (0) and selectivity index Ds (0) in the selection experiments. Values of D > 0 indicate a preference for beads; values <O indicate a preference for RITCB. Treatments with the same letter have no significant difference in selectivity coefficient (P > 0.05). B. Clearance rates for simultaneously offered latex beads (white bars) and RITCB (shaded bars). Clearance rates and selectivity coefficients are means (+ SD) from 3 replicate trials. ited. In contrast, after addition of bacteria, flagellates were presumably satiated. These differing nutritional states were also reflected in HNF volumes, which were minimal at the beginning and end of the experiments but increased l-2 h after food addition (data not shown). We obtained precise estimates of selectivity of HNF by simultaneously offering two kinds of particles. Beads and stained bacteria were comparable in size but very different in chemical composition and nutritional value. These particles could be clearly distinguished microscopically within the same food vacuoles. In agreement with optimal diet models, discrimination against the poorquality, inert beads varied with concentration of highquality live bacteria. Discrimination was strong when live bacteria were abundant and weak or nonexistent when live bacteria were scarce. The decrease in absolute clearance rates for the highquality particles (RITCB) as well as for beads was probably mainly a result of the increased concentration of the live bacteria that produced saturating food levels of the functional response curve (Jiirgens 1992). However, decreased ingestion and clearance rates were to some extent also a consequence of increased satiation as judged from the decrease in clearance rate within the first hour after food addition, where total particle concentrations did not change very much. Functional response curves with Fig. 2. As Fig. 1, but with Spurnella sp. strongly reduced maximal ingestion rates have been reported previously for these two HNF species when they were grown under food satiation (Jiirgens 1994). Concentration-dependent selectivity can be mediated directly by the external food concentration or indirectly by the nutritional status of the predator. The classical optimal diet models first applied to vertebrates assume direct effects of food concentration whereby preingestion handling times limit maximal ingestion rates (Stephens and Krebs 1986). In contrast, an optimal diet model for suspension feeders assumes that digestive capacity places upper limits on ingestion rate (Lehman 1976). In this latter situation, feeding behavior should respond to rapid changes in food abundance with time lags. Here feeding behavior would depend on gut fullness, food vacuole formation, or biochemical correlates of hunger and satiation. A plot of selectivity vs. bacterial concentration reveals a time lag of min between addition of bacteria and maximal discrimination against beads (Fig. 3) as evidence that recent food history (satiation, food limitation) influences prey selection. When data from immediately after the food addition are excluded (Spurnella within 1 h and Bodo after 3 min), there is a strong correlation between selectivity and bacterial concentration (Pearson correlation: df = 11, r = 0.98, P < 0.001). A similar time lag between changes in food abundance and feeding selectivity for particles of contrasting food quality was also evident in experiments with Calanoid copepods (DeMott 1990, 1993). Thus, the behavior of both copepods and HNF is consistent with an optimal diet model based on digestive constraints (Lehman 1976). As predicted, selection against low-quality particles was associated with satiated feeding and reduced clearance rates on the high-quality RITCB. The mechanisms by which nanoflagellates can modulate their feeding selectivity are unknown. However, in-

4 1506 Notes Bodo salfans Spume/la sp i 7% 0.25!2.= )r > G $ P P 1 B 20 P l 6o a I I I I I I I Bacteria [1 O6 ml- ] Fig. 3. Selectivity coefficient DB (from Figs. 2 and 3) as a function of food concentration (live bacteria). Means (+ SD) from 3 replicate trials. For selectivity experiments performed shortly (within 1 h) after food addition, the starting time is given near each data point (in min). dependent of the mechanisms, our results stress that interception-feeding flagellates exhibit a degree of behavioral plasticity not previously shown for protozoans. The experimental organisms, Spurnella and Bodo, belong to different taxonomic orders (Phytomastigophora and Zoomastigophora), but their behavioral responses were similar. This finding suggests that concentration-dependent selectivity may be widespread among protozoans that use direct interception for prey capture. A few previous studies have suggested the possibility of flexible feeding behavior in phagotrophic protozoans. Nygaard et al. (1988) compared the feeding rates of a Bodo species on beads with bacterial ingestion rates calculated from population densities in a two-stage chemostat. Although the ingestion rates on bacteria were not directly measured, their results point in the same direction as ours: food-limited flagellates seemed to show less discrimination against beads. Another case for changing prey selection behavior was reported for the interception-feeding flagellate Paraphysomonas imperforata (Goldman and Dennett 1990). Within a mixture of three phytoplankton species, P. imperforata first fed preferentially on the two smallest species, but switched to the larger species when the concentration of the smaller prey was substantially reduced. Goldman and Dennett hypothesized that the flagellates sensed the relative densities of small and large prey and assumed that the switch in feeding behavior was precipitated by an increase in flagellate size. A certain plasticity has also been observed in the chemosensory behavior of marine tinntinid ciliates (Verity 1988). Prior exposure to a given food alga en- hanced its attractiveness relative to other food algae not present in the ciliates recent diet. Behavioral flexibility in food selection should be adaptive under natural spatial and temporal variation in food abundance and quality. Weaker selection under starvation increases the chance to acquire nutritive particles even when they are not bacteria (detritus and perhaps viruses and macromolecules). Under high food concentrations, more selective feeding should increase net energy uptake by excluding poor-quality particles from food vacuoles that might be occupied by high-quality particles. Food quality selection may be especially important in systems with abundant concentrations of bacteria-sized clays and other inert particles. Behavioral plasticity in prey selection has potentially important implications for the impact of protozoans on the community structure of their prey organisms and for the precision of in situ estimates of protozoan grazing. Several studies have noted that discrimination against artificial particles could lead to biased estimates of bacterial grazing rates (Pace and Bailiff 1987; Nygaard et al. 1988). The dynamic nature of selectivity between beads and bacteria found in this study shows that it is unrealistic to apply a constant correction factor for the uptake of beads in natural samples. For example, correction factors for converting bead clearance rates to RITCB clearance rates from our data range from 0.6 to 4.3 for Bodo and from 0.7 to 11.4 for Spurnella. The low values are for food-limited flagellates and the high values are for satiated flagellates. Due to a spatially and temporally patchy environment, planktonic flagellates probably experience a similarly wide range of food conditions in nature. At present we do not know the level of selectivity realized in natural protozoan communities and therefore cannot recommend appropriate tracer particles. The use of fluorescently labeled bacterioplankton (FLB) as tracers (Sherr et al. 1987) certainly diminishes the problem of discrimination but probably does not totally overcome it. For example, in studies with phagotrophic flagellates, Landry et al. (199 1) found that FLB were strongly selected against whereas Mischke (1994) found that FLB were preferred. Heat killing and staining changes surface chemistry as well as motility of bacteria, both of which can impact prey selectivity of protozoans (Verity 199 1; Gonzilez et al. 1993). Complex patterns of prey selection by raptorial-feeding protozoans will yield large uncertainties in field-grazing measurements. Therefore, tracer particles should be as similar as possible in size and quality to natural bacteria. If the selectivity pattern we found is widespread in natural flagellate communities, it implies that the strongest selectivity must be expected in more eutrophic environments where total bacterial densities (Bird and Kalff 1984) as well as the abundance of larger sized bacteria (Jiirgens 1992) are comparable to the satiating conditions of our laboratory experiments. In contrast, in oligotrophic wa- ters, where bacterial concentrations rarely exceed l-2 x 1 O6 ml-l, selection for food quality should be weak. The majority of HNF contact prey particle by particle, conferring potential for strong selectivity. However, we

5 Notes 1507 are far from understanding the sensory and behavioral constraints that underlie food selectivity in these raptorial-feeding protozoans. Moreover, we have very little information on variation in nutritional value among strains of bacteria and other picoplankton (Jiirgens and Glide 1994). In addition to discrimination between inert particles and bacteria, food-selection capabilities of HNF might include discrimination between different bacterial strains and phenotypes that differ in food value. The next step beyond quantifying bacterial grazing rates will be to test for concentration-dependent selective feeding on bacterial species or phenotypes. This dependence might be crucial in understanding bacterial population dynamics and may eventually be examined by application of newly developed methods for analysis of bacterial diversity and community structure. Max-Planck-Institute for Limnology POB 165 D Pliin, Germany References Klaus Jiirgens William R. DeMott BENNETT, S. 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