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Food selectivity and feeding behaviour in omnivorous filter-feeding ciliates: A case study for Stylonychia
Europ. J. Protisto!. 34,446-457 (1998) December 7, 1998
European Journal of
PROTISTOLOGY
Food Selectivity and Feeding Behaviour in Omnivorous Filter-feeding Ciliates: A Case Study for Sty/onychia Gerald Pfister!" and Hartmut Arndt'« 'Institute of Limnology, Austrian Academyof Sciences, A - 5310 Mondsee, Austria 2Z oological lnstit ute, University of Cologne, Weyertal 119, D- 50923 Cologne, Germany; e-mail: [email protected]
Summary The trophic role of ciliates in microbial food webs has often been considered to be either bacterivore, herbivore or predatory. In general, single species have been assigned to only one trophic level, although early publications have already pointed to omnivorous feeding in many different groups of ciliates. However, the knowledge on the quantitative aspects of ciliate food preferences is scarce up to now. The aim of this study was to quantify food selectivity with the help of different fluorescently labeled potential prey organisms offered to the omnivorous filter-feeding ciliate Stylonychia mytilus as a model organism. Different fluorescently labeled bacteria (FLB) and algae (FLA), and inert fluorescent particles (FP) were offered in feeding experiments and evaluated with the help of epifluorescence microscopy. In addition, different live-stained fluorescent ciliates (FLCi) were offered as living prey. A vital staining method for obtaining FLCi is described. The results of this study showed a selectiveingestion of different ciliates as well as a clear selection between different algae. Bacteria and FP were ingested, but negatively selected. There was a variability in the food preference regarding different-sized cells by Stylonychia mytilus indicating ontogenetic differences in feeding habits. Young daughter cells were not able to ingest the largest prey items. Feeding rates of Stylonychia were highest during the first twenty minutes of the experiments and reached a plateau after 60 min. Capture efficiency for ciliate prey was 70% at the beginning and declined to a value of about 30% after 30 min. Such behavioural patterns may significantly overestimate feeding rates of ciliates determined by short-term experiments. Food concentration was shown to have strong effects on ciliate ingestion rates leading to a clearly detectable functional response. Various factors influencing the feeding behaviour of filter-feeding ciliates and their significance in grazing experiments are discussed. "Present address: Institute of Zoology and Limnology, University of Innsbruck, Technikerstrasse25, A-6020 Innsbruck, Austria; e-mail: [email protected] © 1998 by Gustav Fischer Verlag
Key words: food quality; grazing; live-staining; microbial web; omnivory.
Introduction Due to many investigations published during the last years, a more precise picture of the functioning of aquatic ecosystems has been achieved. The importance of the complex coupling and interactions of protozoa within microbial and macrobial food webs gained more and more attention [1, 4,10,19,41]. Ciliates form a significant component of microbial food webs in lakes [e.g. 38, 48]. Their feeding strategies are diverse and distinguishable into three categories: filter (suspension) feeders, raptorial feeders and diffusion feeders [17]. Filter-feeders are widespread within planktonic hymenostomes, heterotrichs, oligotrichs and hypotrichs [17,40]. Since food selectivity in filter-feeding ciliates is often considered to be mainly influenced by particle size of the available prey [e.g. 20, 45, 55], many filter-feeders are probably omnivores grazing on a distinct size spectrum of components from different trophic levels. However, the influence of food quality on food selection of filter-feeders is poorly investigated [e.g. 54, 65]. The presence of omnivory among several ciliate groups has been described in previous publications [d. 25, 50]. Fenchel [15] presented qualitative and semiquantitative evidence that omnivory is common among marine benthic ciliates. Omnivorous feeding seems to be an important phenomenon and widespread in nature, especially in decomposer food webs [7]. Omnivory has been considered to be an important factor influencing the resilience of planktonic communities and regulating the trophic dynamics and structure of aquatic communities [59].
Feeding behaviour in an omnivorous ciliate
In contrast to these considerations, ciliate taxa are often divided into functional groups [e.g. 8,34,43,60]. Commonly simplifications are used to classify the feeding behaviour of ciliate species into three major groups: bacterivores, herbivores and predators. They are generally considered to be primary grazers of bacteria and phytoplankton populations [e.g. 20, 39, 57]. Therefore, the role of ciliates is generally described as a link from bacteria and small phytoplankton to the metazooplankton [15, 16]. However, if numerous filter-feeding ciliates are omnivorous, their relevance to the functioning of pelagic ecosystems and microbial food webs has to be examined in an all inclusive way. The development of new methods and tracer techniques, like epifluorescence microscopy in connection with fluorescent prey items [e.g. 12,51] or radioisotope labelling [e.g. 6, 30] offer new possibilities to study the nutritional habits of ciliates in more detail. With the help of these methods a more realistic characterisation of the trophic role of single ciliate species will be possible in the future, which is a prerequisite to evaluate the role of ciliates in organic matter fluxes. In this case study the feeding behaviour of the widespread filter-feeding ciliate Stylonychia mytilus was investigated as a model organism in order to evaluate preferences for food items from different trophic levels. For this purpose a method to selectively stain different prey protists had to be developed. Six different experiments were designed to get information about (1) the extent to which a typical filter-feeding ciliate ingests ciliates, (2) the ability of filter-feeders to select for qualitatively different but similarly sized foot items, (3) the possible existence of age-specific changes in food selectivity of individual protistan cells and (4) temporal changes in the feeding activity of single specimens within minutes.
Material and Methods Experimental organisms The investigations were intended as a case study on the feeding behaviour of the well-known filter-feeding hypotrich ciliate Stylonychia mytilus. There are problems with the identification of the sibling species Stylonychia mytilus and Stylonychia lemnae. These two species are genetically different [2], the morphological difference can only be detected in the number of basal bodies of some dorsal cilia [3, 66]. Because of this difficulty in species determination, we followed the proposal of Foissner et al. [18] and treated our experimental organisms as members of the Stylonychia mytilus-complex. All the investigations were done with organisms originating from one clonal culture. One single specimen of the Stylonychia mytilus-complex was isolated from a culture of the isopod Asellus aquaticus, which was cultivated by Friederike MoBlacher at the Institute of Limnology in Mondsee, Austria.
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They prey ciliates were Euplotes moebiusi, Halteria grandinella (both isolated from a smallpond in Mondsee, Austria), and cultures of Dexiostoma campylum and Cyclidium glaucoma which were kindly provided by Miroslav Macek (Institute of Hydrobiology, Czech Academy of Sciences, Ceske Budejovice). All ciliate species were kept at 20°C and continuous dim light in batch cultures. Stylonychia was cultivated in filtered «0.1 prn) water of Lake Mondsee (Austria) and fed with Chlamydomonas reinhardti (kindly obtained from Karl Otto Rothhaupt, Limnological Institute, University of Constance, Germany) and Dexiostoma campylum. Euplotes moebiusi was kept in modified Woods Hole MBL Medium (Rothhaupt pers. comm.) and fed with Chlamydomonas reinhardti. The other ciliate taxa were cultivated in biweekly renewed wheat grain cultures. The two offered species of green algae Chlamydomonas reinhardti and Monoraphidium minutum were cultivated in a 2-litre chemostat system at 20°C and continuous dim light. Modified Woods Hole MBL Medium was used for all algae cultures. The taxonomic determination of all ciliates was achieved by live observation in addition to an improved quantitative protargol staining technique according to Skibbe [56]. Cell dimensions were measured on both living and fixed cellswith the help of an automatic image analysis system (LuciaM,Nikon). Biovolumes were calculated by approximations to simple geometric forms.
Fluorescent tracer items Four different kinds of food items were used in the feeding experiments with Stylonychia to offer a diverse composition of prey. 1) Living fluorescently labeled ciliates (FLCi): A live staining method for ciliates was developed to provide living FLCi as prey for Stylonychia. Cultures of potential prey ciliates (Euplotes moebiusi, Halteria grandin ella, Dexiostoma campylum and Cyclidium glaucoma) were stained with the fluorescent dye DAPI (4',6-Diamidino-2-phenylindoldihydrochloride, Merck) at a final concentration of 0.08% for 60 minutes. All FLCi showed a negative gravitaxis which was used to separate them from the bacteria-rich culture and staining solution. In a glass tube the ciliates moved from the staining solution on the bottom into <0.1 pm filtered lake water at the top of the tube. From this medium the FLCi were directly added to the experimental flasks at the beginning of each experiment. The low DAPI concentration in combination with the long staining time provided well-stained, living ciliates without any visible negative effect on their behaviour. FLCI's survived for at least two weeks and a significant portion of about 90% showed reproduction. 2) Untreated living green algae: The two chlorophyte taxa Chlamydomonas reinhardti and Monoraphidium minutum were offered as living food to Stylonychia without any staining or fixation treatment. Autofluorescence of chlorophyll in these two algae allowed a good determination of ingested cells in food vacuoles by epifluorescence microscopy. 3) Fluorescently labeled bacteria (FLB) and algae (FLA): The bacterial prey offered in the experiments was represented by DTAF (5-4,6-dichlorotriazin-2-yl)aminofluorescein, Sigma) stained Escherichia coli following the procedure described by Sherr et al. [52].The same staining method with slight modifications [42] was used to stain the diatom Cyclotella meneghiniana. The diatom originated from a chemostat culture (courtesy of K. O. Rothhaupt) and showed only weak
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G. Pfister and H. Arndt
autoflu orescence resulting in th e need to fluorescently label the cells. The deep fro zen FLB and FL A were thawed one hour prior to each experiment. 4) Inert fluo rescen t particles (FP): FP according to MeManus & Fuhrmann (Dyed trialdehyde resin, Radiant Co lor Col. [36]) were used in one of the experiments as a model detritus to evaluate the ingestion of very small and inert particles (minimum diameter 0.1 prn) by Stylonychia.
Food selectivity experiments All feeding experiments were run at 20 °C (± 0.5 °C, temperature-regulated water bath) in glass tubes (8 ml) under gentle continous shaking . About 20 cells of Stylonychia were transferred from the batch cultures (see above) and added to filtered lake water «0.1 prn), After one hour of acclimatisation the experiments were started by adding the cocktail of tracer organisms (see below for details). After 20 minutes the samples were fixed by adding ice-cold glutaraldehyde at a final concentration of 2% [54]. Each experiment was run in triplicate . The fixed samples were filtered on black polycarbonate filters (pore size 0.8 ]Jm,Poretics). DAPI was added at a final concentration of 0.08% to stain the Stylonychia-cells on the filter for five minu tes. Filters wer e washed three times with filtered lake water « 0.1 urn) and mou nt ed for epifluorescence microscopy between a glass slide and a cover glass. At least 15 individuals of Stylonychia mytilus were examined for each replicate wit h the help of an epifluorescence microscope (Zeiss Axiop lan), and the content of food vacuoles was registered for each specimen . Different tracer items could easily be distinguished due to their different colours and shapes.
D ifferent FLCi's wer e distin guished by th e specific shape of their well-stained nu clear apparatus. The food concentrations for all tracer items used in feeding experiments and their dimensions are shown in Table 1. Only a selected number of tracer organisms was offered in each experiment to make sure that all tracer organisms cou ld be distinguished from each other in the food vacuoles. Individual food selectivity of each Stylonychia was calculated usin g the electivity index W j according to Vanderploeg & Scavia [63], which provides the single most useful electivity index [31]. The calculation of the index was based on the determination of individual ingestion rates (I) and clearance rates (Cr.) per hour following the form ulas: 1= N/ tx60 and C ri = I /
c,
The abbreviations are: C, = concentration of prey class i [number per ml in the medium], Crj= clearance rate on single food item i [filtered volume of water per individual Stylonychia in ml per hou r], I = ingestion rate [ingested food items of prey class i per individual Stylonychia per hour], N , = number of ingested cells of prey class i per individual Stylonychia after t minutes, t = experimental time in minutes. The selectivity of Stylonychia was analysed by relating the clearance rates on a certain food item (Cr.) to the maximum filtration rate observed in each experiment (Cr ma><) following the formula: Wi = Cr;l Crmax
Table 1. Dimensions (diameter or lengthxwidth in pm, mean in brackets) and concentrations (numbers of cells per millilitre) of food items offered in the feeding experiments. Concentrations for exper iments 6a, 6b and 6c are minimum (upper row) and maximum (lower row) values. Abbreviations : Chlam = Chlamydomonas reinhardti, Cyclid = Cyclidium glaucoma, Cyclot = Cyclotella meneghiniana, Dexio = Dexiostoma campylum, Eupl. = Euplotes moebiusi, FLB = Escherichia coli (fluorescen tly labeled bacteria), FP = inert fluroescent pa rticles, Ha lt = Halteria grandinella, Monor = Monoraphidium rninutum. Prey FP FLB Cyclot Monor Chlam Cyclid Dexio Halt Eupl
Di mensions [um] 0.1-5 .6 (2.9) 1-3.5xO.6-1.l (2.9xO.8) 12- 16.5x5-7 (14.2x6.3) 7-11x3-5 (10x4) 5-9.5 (7.3) 15-22xlD-12.5 (18.5x12) 36-70x2D-30 (50x24) 20-40 (30) 5D-65x3D-34 (57x36)
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Feeding behaviour in an omnivorous ciliate
Individual electivity indices were summarized to mean values (± SD) for each experiment. A comparison of the mean electivity indices was achieved by statistical analysis (paired Student's t-test, p < 0.05) of the individually calculated electivity indices (Table 2). Linear regression analysis was performed to detected a significant correlation between cell size of Stylonychia and the volume of ingested prey.
Evaluation of the functional response Three experiments were run to evaluate a correlation between ingestion rates and food concentration. In the first set of experiments only Chlamydomonas was offered as food, in the second experiment a mixture of Chlamydomonas and Dexiostoma (each 50% of the total biovolume) were offered, and in the third experiment only Dexiostoma was provided as prey to Stylonychia. The maximum food concentration in all three experiments was 13 mrrr' per litre. Dilution was achieved using filtered lake water «0.1 urn) in eight steps to a minimum food concentration of 0.1 mm' per litre. The experiment was run for 20 min in triplicate for each food concentration. Further treatment and evaluation of ingestion rates was made in the same way as described for the food selectivity experiments.
Long term observations Feeding behaviour of individual cells of Stylonychia mytiIus was observed directly under a microscope (Reichert Neovar, magnification 100x). Observations were made at a temperature of 20°C at reduced illumination. Five millilitre of a culture medium with the living and untreated prey ciliate Dexiostoma campylum (550 cells/ml) were added to 10 mlcounting chambers. One specimen of Stylonychia was added to the counting chamber at the beginning of each observation. All contacts of the prey with the oral apparatus of the predator
449
and ingestion of prey cells by Stylonychia during a continuous time interval of 120 min were recorded. The mean capture efficiency (ingested prey/contacts of the buccal region with prey) of five different specimens of Stylonychia was monitored. The data allowed for the detection of temporal alterations in the feeding activities of Stylonychia cells. Video recording supported the observations of the feeding behaviour.
Results Food selectivity 1) Selectivity among different prey items: Stylonychia mytilus consumed a wide spectrum of food parti-
cles ranging from bacteria to algae and different sized ciliates and can clearly be classified as an omnivore. Our results show a clear selection of different prey items. The electivity indices also indicate differences in selection of different algae as well as of different ciliates (Table 2, Fig. 1). The green algae Monoraphidium minuturn and the fast moving and jumping ciliate Halteria grandinella were negatively selected in the first set of experiments (Fig. 1a). The two hymenostome ciliates were preferred (Cyclidium glaucoma, with a maximum clearance rate of 1.9x10-2 mlxh'), or non-selectively (Dexiostoma campylum) ingested. A significant (p < 0.05) positive selection of Stylonychia for Cyclidium over Monoraphidium and Halteria was recorded (Table 2). The second experiment was designed to investigate the food selection among relatively small food items. The living green algae Chlamydomonas were signifi-
Table 2. Comparison of the food selectivity of Stylonychia mytilus feeding on one food item (rows) vs the other food items (columns) present in the respective experiments. Summary of the results from experimental data shown in Fig. 1. Electivity indices compared by paired Student's t-test (p < 0.05). Abbreviations: p = significant positive selection, n = significant negative selection == no significant difference between electivity indices, - = not determined. Chlam = Chlamydomonas reinhardti, Cyclid = Cyclidium glaucoma, Cyclot = Cyclotella meneghiniana, Dexio = Dexiostoma campylum, FLB = Escherichia coli (fluorescently labeled bacteria), Halt = Halteria grandinella, IFP = large fluorescent particles (>3 um), mFP = medium fluorescent particles (1.5-3 prn), Monor = Monoraphidium minutum, sFDP = small fluorescent particles «1.5 pm), Example: Cyclotella meneghiniana (row 5) was preferred compared to fluorescent particles and Escherichia coli (columns 1-4) and was less preferred than Chlamydomonas reinhardti (colum 7). There was no statistically significant preference detected regarding Cyclotella meneghiniana vs. Cyclidium glaucoma (column 8) and Cyclotella meneghiniana vs. Dexiostoma campylum (column 9). Monoraphidium minutum (column 6) and Halteria grandinella (column 10) were not offered in this experiment. Food item
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Fig. 1. Food selectivity of Stylonychia mytilus. Mean clectivity indices (± SD) according to Vanderploeg & Scavia [63] for food items from different trophic levels. Abbreviations of species as in Tables 1 and 2. l a: Results of Experiment 1, Ib : Results of Experiment 2, l c: Results of Experiment 3.
Fig. 2. Feeding behaviour of different size classes of Stylonychia mytilus offered different potential prey algae, Cyclotella meneghiniana and Chlamydomonas reinhardti, and ciliates, Cyclidium glaucoma and Dexiostoma campylum (exp. 3). 2a: Mean electivity indices (± SD) calculated according to Vanderploeg & Scavia [63]. 2b: Mean percentage composition regarding the biovolume of the ingested food items.
cantly (p < 0.05) preferred (wi th a max imum clearance rate of 9.2x lO-3 mlx h') over all othe r foo d items offered in the experiment. FLB (E. coli) were significan tly (p < 0.05) nega tively selected against Ch lamydomonas and Cyclotella. FP of all provided size-classes were also negatively selec ted compared to Chlamydomonas. H ow ever, a slight but sign ificant (p < 0.05) higher positive select ion was found for the largest compared to the smallest FP (Table 2, Fig. 1b). In the third expe riment (Table 2, Fig. l c) th e green algae Chlamydomonas was again the preferred food item (with a maxi mum clearance rate of 1.2xl O-2 ml x h') for Stylonychia. A clear negative selection (p < 0.05) against the on ly no n-living prey in this experiment, Cyclotella meneghiniana, was recorded. 2) Size-dependent select ivity an d ingestion: In order to investigate whether there is a relat ionship between food selectivity and cell size of Sty lonychia, indi -
vidual biovolumes of the observed Stylonychia cells in two of the exper iments (exp. 3 and 4, Table 1) were calcu lated and related to the respective amount and composition of ingested prey. The experiments revealed different food preferences of different size-classes of Stylonychia (Fig. 2 and 3). In the first experiment (exp. 3, Fig. 2) an increasing selection for the large Dexiostoma by larger Sty lonychia cells was observed (Fig. 2a). The composition of the food vacuo le content of the smallest cells was abo ut 50% Ch lamydomonas and 50% Cyclidium. Stylonychia cells being larger than 2xl0 5 prrr' ingested abo ut 50% Chlamydomonas and 25% each Cyclidium and Dexiostoma. The diatom Cyclotella was hardly ingested by all size groups of Stylonychia (Fig. 2b) . Bigger cells of Stylonychia ingested more food (r 2 = 0.9981), up to about 3xl0 4 pm' per individual cell per ho ur. In the second set of experiments on ly ciliate prey was offered to Stylonychia (exp. 4, Fig. 3). An increasing food prefer-
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Fig. 3. Feeding behaviour of different size classes of Stylonychia mytilus offered the different potential prey ciliates Cyclidium glaucoma, Dexiostoma campylum and Euplotes moebiusi (exp. 4). 3a: Mean electivity indices (± SD) calculated according to Vanderploeg & Scavia [63]. 3b: Mean per centage composition regarding the biovo lume of the ingested food items .
Fig. 4. Mean feeding rates (± SD) of Stylonychia mytilus per hour calculated after different experimental times (exp. 4). 4a: Feeding rate on the green algae Chlamydomonas reinhardti . 4b: Feeding rate on the ciliate Dexiostoma campylum.
ence of larger predators for the bigger prey ciliates Dexiostoma and Euplotes was detected (Fig. 3a). Electivity indices for these two ciliates increased significantly (p < 0.05) with increasing cell volume of Stylonychia. The total amount of ingested biovolume was pos itively correlated (r2 = 0.9520) with Stylonychia size. The food vacuole in the smallest Stylonychia cells contained abo ut 50% each, Cyclidium and Dexiostoma, whereas in larger specimens the relative proportion of Dexiostoma increased to about 90% (Fig. 3b). The large Euplotes (length about 60 prn) was not ingested by cells smaller than 4.5xl0' prrr' (about 175xl00 J.1m).
lonychia per ho ur were measured for the algal prey Chlamydomonas reinhardti, minimum feeding rates of about 0.5 cells per Stylonychia per hour were calculated for the relatively large prey ciliates Halteria grandinella and Euplotes moebiusi. The feeding rates calculated for short exper imental times were generall y higher than for longer experimental times. Especia lly for larger prey, like the offere d algae and ciliates, calculate d feeding rates were much higher using experimental times shorter than abo ut 30 minutes. After this time, the calculated feeding rates declined to minimum values (exp. 3, Fig. 4). Only initial digestion of algal and ciliate prey could be observed in the experiments in the course of 75 minutes. Egestion of ciliate cells was never observed during two hours in the long term observations (exp. 5). 2) Feedin g activity and capt u re efficienc y: Longterm experiments ind icated alterations in the feeding
Feeding rates 1) Influen ce of expe rimental time: Hig hest feeding rates with a maximum ingestion of 850 cells per Sty -
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activity of Stylonychia. The ciliates showed feeding periods followed by non-feeding periods, both lasting about 20 to 30 minutes (Fig. Sa). A higher feeding activity and capture efficiency was detected at the beginning of the experiments, whereas, after ingestion of about 4 to 6 prey ciliates (Dexiostoma campylum), capture efficiency declined from about 70% to about 30% (Fig. Sb). The feeding behaviour of the individual ciliates was relatively similar. 3) Functional response: A functional response similar to Holling's type II curve [cf 7, pp. 311-314] was registered in the experiments where different concentrations of food were offered (Fig. 6). Two kinds of potential prey (Chlamydomonas reinhardti and Dexiostoma campylum) were used in this set of experiments (exp. 6a-c). Experiments dealing with the food selectivity showed that both food items were consumed by Stylonychia (see Fig. 1). Feeding rates on algal prey, ciliate prey, and a mixture of algae and ciliate prey (Fig. 6) increased with increasing food concentration following
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Fig. 6 Functional response of Sty/onychia mytilus (exp. 6). 6a: Functional response for only algal food. 6b: Functional response for only ciliate food. 6c: Functional repsonse for both algal and ciliate food. Mean values ± SD.
Feeding behaviour in an omnivorous ciliate
a hyperbolic curve. For the offered food items maximum uptake rates were reached at a concentration of about 2 to 3 mgxl', At the lowest tested food concentration (0.1 mm'xl') Stylonychia showed minimal individual ingestion rates of about 2000 prrr' prey volume per hour. This value increased to 7100 ± 1100 unr' for algal prey at the maximum food concentration of 13 mrrr'xl" (Fig. 6a). Highest ingested biovolumes (26900 ± 5600 um'xlnd.r'xh') at the highest food concentration were detected in the experiments where we offered ciliates only (Fig. 6b). About 19500 ± 4700 pm'xlnd.r'xh' were ingested in the experiments with mixed food (Fig. 6c). There was no selection regarding the two food items. The proportion of both food types in the food vacuoles did not change significantly at different food concentrations.
Discussion Food selectivity It is known that various predatory ciliates consume other ciliates [e.g. 12, 27], under certain conditions some ciliates may develop cannibalism [e.g. 26, 46]. Our present results indicate that a typical filter-feeding ciliate known as a bacterivore-herbivore can also act as
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453
an efficient predator on certain ciliates. Many ciliates in pelagic and benthic systems are filter-feeders [17] and one can conclude that omnivory is much more widespread among components of the microbial food web than currently believed. The results of the present investigation clearly indicate that even filter-feeding ciliates show preferences for certain food items depending not only on their size, but also on their "quality". A selective feeding behaviour, namely a strong preference for few components of the diverse food spectrum, was observed when many different potential prey items were offered (summarized in Fig. 7). Generally, smaller food items like small FP and bacteria were less preferred than larger ones (d. Fig. 1 and 7). On top of that even within the same size class of prey food selection of Stylonychia was different: non-mobile prey was less preferred than mobile prey. An explaination could simply be a higher chance to get caught by the filter current for mobile organisms. On the other hand, a quick and jumping movement seems to influence the food selectivity of Stylonychia in an opposite way. The oligotrich ciliate Halteriawas negatively selected in our experiments (Fig. la). We observed under the microscope that its jumping reaction allowed the prey to escape from the filter current created by Stylonychia. Jumping has been shown to reduce the vulnerability of oligotrichs to metazoan filter-feeders [e.g. 24, 61]. The possession of a
Cyclidium glaucoma (fasf moving) hymenostome ciliate (living) 10-22 urn
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'
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----. -+
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, /'
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.
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Escherichia coli bacteria (heat killed) ' 0.6-3 .5jJm " --------
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Fig. 7. General scheme of the feeding behaviour of Stylonychia mytilus regarding food items from different trophic levels (summary of expo 1-4). Arrows mark a strong (full line), medium (coated line) and low (dotted line) selection for the different food items, respectively.
454
G. Pfister and H. Arndt
hard and rigid cortex in Euplotes and its tend ency to attach to th e su bstrate (and thu s reducing th e chan ge to get caught by the filter current) are possible reasons for a weak grazing pressure performed by potential pred ators. We assume that the feeding behaviour of Stylon ychia is influ enced by a combination of all of these factors and possibly even changing w ith changing environmental conditions. There was only one exception where no selection was registered, namely in th e experiment wh ere the two different prey item s Chlamydom onas and Dexiostoma were offered. H ere no selection could be observed (Fig. 6c), po ssibly due to the stro ng affinity of Stylony chia for both of these pre y organisms. Interestingly, Stylo nychia ingested a much higher amount of food when feeding only on the large ciliate Dexiostom a, compared to the incorpor ated biovolume when feeding only on the smaller alga Chlamydomonas (Fig. 6a and 6b). We assume th at the form ation of food vacuo le memb ranes could possibly be a limiting factor in th e latter case. Th e formation of man y small vacuoles for th e small algae cells requires a larger membran e-surface than form ation of less but larger vacuol es for the ciliates. Our experiences in cultivation showed th at Sty lony chia-cultures grew well on both algae and mixed food, but significantly degenerated on an exclusive Dexiostomadiet, which could indicate an insuffici ent nutritional value of th e ciliates. Our results agree with findin gs of Lilly [33] w ho rep orted that cultures of Sty lonychia pustulata degenerated after having been fed exclusively with Tetrahymena pyrifo rm is. However, Licht enberg [32] obtained cont rary results. Her cultures of Stylonychia mytilus degenerated on only Chlamydomonas food and grew well feeding on th e ciliate Dexiostom a. In agreement with oth er investigations [d. 35], we assume that a diet containing algae better supports the growth of omnivorous ciliates than onl y ciliate food.
Food selection of juvenile and adult Sty/onychia Th e results of our experiments gave clear evidence for differ ences in the food pr eference s of different size groups of Stylonychia (Fig. 2 and 3). All experimenta l organisms of Stylony chia originated from one clone and were cultivated und er the same conditions. Our observations of clonal cultures revealed that under th e conditions of cultivation yo ung daughter cells stayed in the size group <2xl0 5 urrr' xind' for th e first three to four hours. The specimens grew up to th e size group >5x105 prrr' xind' within about 13 to 15 hours. In spite of individu al variations in growth, it was clear th at the smallest and th e largest size groups belong to just divided or ganisms ("y oung") and to or ganisms pri or to division ("old"), respectively. Thus, the observe d biovolume specific differences in feeding activity can be int er-
preted as differences comparable to ontoge netic differences known from metazoa [e.g. 62]. To our kn owledge, such" ontogenetic" differences in feeding activity of protists have not yet been describ ed. In both experiments the percentage contribution of large food items (D exiostoma and Euplotes) in the ration of juvenile Stylonych ia was significantly lower than in older and larger specimens (d . Fig. 2 and 3). Particl e size is known to be an imp ortant factor influen cing th e food selectivity of ciliates [9, 16, 49]. As our result s indicat e, pot enti al prey can simply be too large for the pr edator. The reduced predation on th e large D exiostoma and Euplotes by small Stylo ny chia may serve as examples (Fig. 2 and 3). It is known th at rigid surfa ces and appendages in Euplo tes are produced as a result of predator-induced defen ses [e.g. 22]. Compared to th e other ciliates offered in our experiments Euplotes had by far th e hardest cortex, which may have served as a certain protection from ingestion by Sty lonychia.
Capture efficiency To our knowledge th ere are no data reported on the capture efficiency and long term feedin g activity of ciliates. This interesting behaviour could have been overlooked so far due to the fact that most ciliate feeding experiment s have been carried out with small food item s, not showing remarkable changes in ciliate feedin g activity. The reason for th e decreasing captu re efficiency implied by th e observed resting phases in our experiments (Fig. 5a) could be th e time requ ired to reorganize memb rane material for th e form ation of new food vacuoles. Besides, data fro m literature indicate that digestion tim es can vary from some minutes for small particles like bacteria [53] to hours for large pr ey like ciliates [13]. We observed in th e long term feeding experiment s that all ingested ciliates we re still pre sent in Sty lonycbia's food vacuoles after 120 minutes confirming lon g digestion times for ciliate prey. Th ese lon g digestion time s could explain the need for th e observed resting phase s in our experiments. To the cont rary, Dolan & Simek [14] demonstrated recentl y for an oligotrich ciliate th at neither th e food quality nor the nutrition al stage of th e experimenta l organisms seems to have any significant influence on digestion tim es.
Methodological and general considerations Omnivorous feeding is defin ed as simultaneo usly preyin g on different trophic levels and hence ingestin g a wide range of different food item s. Although it is widespread in natural commun ities [25, 58, 59, 65], omnivory is often neglected in ecolo gical investigations. It is generally difficult to quant ify th e complexity and
Feeding behaviour in an omnivorous ciliate variability of the feedin g beh aviour of omni vor es. To get a realistic idea about complex trop hic interactions in natu ral assemblages, laborator y feeding experiments should simulate natural conditions. An essent ial need for feeding experiments is th e simultaneo us use of diverse and natural food items [23, 37, 44]. Man y attempts have been made to simulate natur al food condition s in protozoan feedin g experi ments. Fluorescent part icles [e.g. 16, 36, 49J, fluo rescently labeled bact eria [FLB , e.g. 19, 52J and algae [FLA , e.g. 47J are usual tr acers in laboratory experi ments . A disadvant age of thi s surro gate pre y is its non-living character. Hence, biochemical and behavioural interactions among protozoa and its prey are often neglected. In contrast, it is kn own that ciliates can accumulate due to chemical sub stances originating from pot ential prey [ef. 64J and morphological changes of both predators and prey indu ced by special submitting factors have often been described [e.g. 22, 27, 28, 29]. D efensive cell organelles of potenti al prey are kno wn to play an impo rtant rol e in ciliate predator-prey int eraction s [e.g. 21J. O ur experi ments based on a diverse food spectru m. In or der to keep detectable food sources as natural as possible, in addition to FP, FLB and FLA living algae (use of their autofluo rescence) and living fluroes centl y labeled ciliates (FLC i) were offered. We develop ed a stain ing protocol for ciliates, which pr ovides a techniqu e to study the selective grazi ng on different ciliate species. For FLCi no visible negative effects regarding locom otion and reproduction could be detected. This may reflect only weak effects on th e physiological state of cells caused by the staining procedure. Due to the ir different shapes of the nuclear appar atus even ingested ciliates could be taxonomically distinguished. This allowed investigations on Stylonychia's food selectivity regarding different prey ciliates. An other live-staining techniqu e for protists (flagellates) was developed by Cl even [11] using fluores cent parti cles which have been previ ously ingested by prey flagellates. However, th e experi men tal protocol is mu ch more complex and different species are difficult to distinguish. In th e future species specific genetic p robes may be used for fluore scent study of pr ey prot ists. H owever, our simp le method may be a qu ick and cheap alternative for prey species th at differ remarkably in the size or shape of thei r nuclear apparatus . We assum e that our results on the feeding beha viour of Sty lonychia reflect some general characte ristics of filter- feeding ciliates which are often neglected in grazing experiments. It is often overloo ked that filter-feeding ciliates (except for typ ical bacterivores) are probably omnivores, feeding on different trophic levels. Filt erfeeding ciliates may show prefe rences for different food part icles, which may be similar in size. It has to be considered that food selection of ciliates may change in th e
455
course of individual grow th fro m young daughter cells to cells just pri or to division. D igestion times of some ciliates for specific foo d items may vary from a few minutes to several hours. Investigation s on the capture efficiency concerni ng th e upt ake of large food particles indicated that periods of high grazing activity and periods of resting may alterate within minutes. U nfortunately, an ideal experimental setup in all of th ese respects can hardly be achieved. H owever, we hope to have pointed to some major drawb acks of cur rently applied meth od ologies in ciliate grazing stu dies. Acknowledgements: We th ank our colleagues at the Institut e of Limnology (Mondse e, Austria) for their help. Special thanks to Friederike Molilacher for cession of ciliates originating from her cultures, Walter Geiger for helpful comments on statistical analysis of our data and Karel Simek for constructive criticism on the manuscr ipt. Financial support was partially prov ided by th e Austrian FWF, project PI 03061 BIO .
References
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9 10
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European Journal of
PROTISTOLOGY
Food Selectivity and Feeding Behaviour in Omnivorous Filter-feeding Ciliates: A Case Study for Sty/onychia Gerald Pfister!" and Hartmut Arndt'« 'Institute of Limnology, Austrian Academyof Sciences, A - 5310 Mondsee, Austria 2Z oological lnstit ute, University of Cologne, Weyertal 119, D- 50923 Cologne, Germany; e-mail: [email protected]
Summary The trophic role of ciliates in microbial food webs has often been considered to be either bacterivore, herbivore or predatory. In general, single species have been assigned to only one trophic level, although early publications have already pointed to omnivorous feeding in many different groups of ciliates. However, the knowledge on the quantitative aspects of ciliate food preferences is scarce up to now. The aim of this study was to quantify food selectivity with the help of different fluorescently labeled potential prey organisms offered to the omnivorous filter-feeding ciliate Stylonychia mytilus as a model organism. Different fluorescently labeled bacteria (FLB) and algae (FLA), and inert fluorescent particles (FP) were offered in feeding experiments and evaluated with the help of epifluorescence microscopy. In addition, different live-stained fluorescent ciliates (FLCi) were offered as living prey. A vital staining method for obtaining FLCi is described. The results of this study showed a selectiveingestion of different ciliates as well as a clear selection between different algae. Bacteria and FP were ingested, but negatively selected. There was a variability in the food preference regarding different-sized cells by Stylonychia mytilus indicating ontogenetic differences in feeding habits. Young daughter cells were not able to ingest the largest prey items. Feeding rates of Stylonychia were highest during the first twenty minutes of the experiments and reached a plateau after 60 min. Capture efficiency for ciliate prey was 70% at the beginning and declined to a value of about 30% after 30 min. Such behavioural patterns may significantly overestimate feeding rates of ciliates determined by short-term experiments. Food concentration was shown to have strong effects on ciliate ingestion rates leading to a clearly detectable functional response. Various factors influencing the feeding behaviour of filter-feeding ciliates and their significance in grazing experiments are discussed. "Present address: Institute of Zoology and Limnology, University of Innsbruck, Technikerstrasse25, A-6020 Innsbruck, Austria; e-mail: [email protected] © 1998 by Gustav Fischer Verlag
Key words: food quality; grazing; live-staining; microbial web; omnivory.
Introduction Due to many investigations published during the last years, a more precise picture of the functioning of aquatic ecosystems has been achieved. The importance of the complex coupling and interactions of protozoa within microbial and macrobial food webs gained more and more attention [1, 4,10,19,41]. Ciliates form a significant component of microbial food webs in lakes [e.g. 38, 48]. Their feeding strategies are diverse and distinguishable into three categories: filter (suspension) feeders, raptorial feeders and diffusion feeders [17]. Filter-feeders are widespread within planktonic hymenostomes, heterotrichs, oligotrichs and hypotrichs [17,40]. Since food selectivity in filter-feeding ciliates is often considered to be mainly influenced by particle size of the available prey [e.g. 20, 45, 55], many filter-feeders are probably omnivores grazing on a distinct size spectrum of components from different trophic levels. However, the influence of food quality on food selection of filter-feeders is poorly investigated [e.g. 54, 65]. The presence of omnivory among several ciliate groups has been described in previous publications [d. 25, 50]. Fenchel [15] presented qualitative and semiquantitative evidence that omnivory is common among marine benthic ciliates. Omnivorous feeding seems to be an important phenomenon and widespread in nature, especially in decomposer food webs [7]. Omnivory has been considered to be an important factor influencing the resilience of planktonic communities and regulating the trophic dynamics and structure of aquatic communities [59].
Feeding behaviour in an omnivorous ciliate
In contrast to these considerations, ciliate taxa are often divided into functional groups [e.g. 8,34,43,60]. Commonly simplifications are used to classify the feeding behaviour of ciliate species into three major groups: bacterivores, herbivores and predators. They are generally considered to be primary grazers of bacteria and phytoplankton populations [e.g. 20, 39, 57]. Therefore, the role of ciliates is generally described as a link from bacteria and small phytoplankton to the metazooplankton [15, 16]. However, if numerous filter-feeding ciliates are omnivorous, their relevance to the functioning of pelagic ecosystems and microbial food webs has to be examined in an all inclusive way. The development of new methods and tracer techniques, like epifluorescence microscopy in connection with fluorescent prey items [e.g. 12,51] or radioisotope labelling [e.g. 6, 30] offer new possibilities to study the nutritional habits of ciliates in more detail. With the help of these methods a more realistic characterisation of the trophic role of single ciliate species will be possible in the future, which is a prerequisite to evaluate the role of ciliates in organic matter fluxes. In this case study the feeding behaviour of the widespread filter-feeding ciliate Stylonychia mytilus was investigated as a model organism in order to evaluate preferences for food items from different trophic levels. For this purpose a method to selectively stain different prey protists had to be developed. Six different experiments were designed to get information about (1) the extent to which a typical filter-feeding ciliate ingests ciliates, (2) the ability of filter-feeders to select for qualitatively different but similarly sized foot items, (3) the possible existence of age-specific changes in food selectivity of individual protistan cells and (4) temporal changes in the feeding activity of single specimens within minutes.
Material and Methods Experimental organisms The investigations were intended as a case study on the feeding behaviour of the well-known filter-feeding hypotrich ciliate Stylonychia mytilus. There are problems with the identification of the sibling species Stylonychia mytilus and Stylonychia lemnae. These two species are genetically different [2], the morphological difference can only be detected in the number of basal bodies of some dorsal cilia [3, 66]. Because of this difficulty in species determination, we followed the proposal of Foissner et al. [18] and treated our experimental organisms as members of the Stylonychia mytilus-complex. All the investigations were done with organisms originating from one clonal culture. One single specimen of the Stylonychia mytilus-complex was isolated from a culture of the isopod Asellus aquaticus, which was cultivated by Friederike MoBlacher at the Institute of Limnology in Mondsee, Austria.
447
They prey ciliates were Euplotes moebiusi, Halteria grandinella (both isolated from a smallpond in Mondsee, Austria), and cultures of Dexiostoma campylum and Cyclidium glaucoma which were kindly provided by Miroslav Macek (Institute of Hydrobiology, Czech Academy of Sciences, Ceske Budejovice). All ciliate species were kept at 20°C and continuous dim light in batch cultures. Stylonychia was cultivated in filtered «0.1 prn) water of Lake Mondsee (Austria) and fed with Chlamydomonas reinhardti (kindly obtained from Karl Otto Rothhaupt, Limnological Institute, University of Constance, Germany) and Dexiostoma campylum. Euplotes moebiusi was kept in modified Woods Hole MBL Medium (Rothhaupt pers. comm.) and fed with Chlamydomonas reinhardti. The other ciliate taxa were cultivated in biweekly renewed wheat grain cultures. The two offered species of green algae Chlamydomonas reinhardti and Monoraphidium minutum were cultivated in a 2-litre chemostat system at 20°C and continuous dim light. Modified Woods Hole MBL Medium was used for all algae cultures. The taxonomic determination of all ciliates was achieved by live observation in addition to an improved quantitative protargol staining technique according to Skibbe [56]. Cell dimensions were measured on both living and fixed cellswith the help of an automatic image analysis system (LuciaM,Nikon). Biovolumes were calculated by approximations to simple geometric forms.
Fluorescent tracer items Four different kinds of food items were used in the feeding experiments with Stylonychia to offer a diverse composition of prey. 1) Living fluorescently labeled ciliates (FLCi): A live staining method for ciliates was developed to provide living FLCi as prey for Stylonychia. Cultures of potential prey ciliates (Euplotes moebiusi, Halteria grandin ella, Dexiostoma campylum and Cyclidium glaucoma) were stained with the fluorescent dye DAPI (4',6-Diamidino-2-phenylindoldihydrochloride, Merck) at a final concentration of 0.08% for 60 minutes. All FLCi showed a negative gravitaxis which was used to separate them from the bacteria-rich culture and staining solution. In a glass tube the ciliates moved from the staining solution on the bottom into <0.1 pm filtered lake water at the top of the tube. From this medium the FLCi were directly added to the experimental flasks at the beginning of each experiment. The low DAPI concentration in combination with the long staining time provided well-stained, living ciliates without any visible negative effect on their behaviour. FLCI's survived for at least two weeks and a significant portion of about 90% showed reproduction. 2) Untreated living green algae: The two chlorophyte taxa Chlamydomonas reinhardti and Monoraphidium minutum were offered as living food to Stylonychia without any staining or fixation treatment. Autofluorescence of chlorophyll in these two algae allowed a good determination of ingested cells in food vacuoles by epifluorescence microscopy. 3) Fluorescently labeled bacteria (FLB) and algae (FLA): The bacterial prey offered in the experiments was represented by DTAF (5-4,6-dichlorotriazin-2-yl)aminofluorescein, Sigma) stained Escherichia coli following the procedure described by Sherr et al. [52].The same staining method with slight modifications [42] was used to stain the diatom Cyclotella meneghiniana. The diatom originated from a chemostat culture (courtesy of K. O. Rothhaupt) and showed only weak
448
G. Pfister and H. Arndt
autoflu orescence resulting in th e need to fluorescently label the cells. The deep fro zen FLB and FL A were thawed one hour prior to each experiment. 4) Inert fluo rescen t particles (FP): FP according to MeManus & Fuhrmann (Dyed trialdehyde resin, Radiant Co lor Col. [36]) were used in one of the experiments as a model detritus to evaluate the ingestion of very small and inert particles (minimum diameter 0.1 prn) by Stylonychia.
Food selectivity experiments All feeding experiments were run at 20 °C (± 0.5 °C, temperature-regulated water bath) in glass tubes (8 ml) under gentle continous shaking . About 20 cells of Stylonychia were transferred from the batch cultures (see above) and added to filtered lake water «0.1 prn), After one hour of acclimatisation the experiments were started by adding the cocktail of tracer organisms (see below for details). After 20 minutes the samples were fixed by adding ice-cold glutaraldehyde at a final concentration of 2% [54]. Each experiment was run in triplicate . The fixed samples were filtered on black polycarbonate filters (pore size 0.8 ]Jm,Poretics). DAPI was added at a final concentration of 0.08% to stain the Stylonychia-cells on the filter for five minu tes. Filters wer e washed three times with filtered lake water « 0.1 urn) and mou nt ed for epifluorescence microscopy between a glass slide and a cover glass. At least 15 individuals of Stylonychia mytilus were examined for each replicate wit h the help of an epifluorescence microscope (Zeiss Axiop lan), and the content of food vacuoles was registered for each specimen . Different tracer items could easily be distinguished due to their different colours and shapes.
D ifferent FLCi's wer e distin guished by th e specific shape of their well-stained nu clear apparatus. The food concentrations for all tracer items used in feeding experiments and their dimensions are shown in Table 1. Only a selected number of tracer organisms was offered in each experiment to make sure that all tracer organisms cou ld be distinguished from each other in the food vacuoles. Individual food selectivity of each Stylonychia was calculated usin g the electivity index W j according to Vanderploeg & Scavia [63], which provides the single most useful electivity index [31]. The calculation of the index was based on the determination of individual ingestion rates (I) and clearance rates (Cr.) per hour following the form ulas: 1= N/ tx60 and C ri = I /
c,
The abbreviations are: C, = concentration of prey class i [number per ml in the medium], Crj= clearance rate on single food item i [filtered volume of water per individual Stylonychia in ml per hou r], I = ingestion rate [ingested food items of prey class i per individual Stylonychia per hour], N , = number of ingested cells of prey class i per individual Stylonychia after t minutes, t = experimental time in minutes. The selectivity of Stylonychia was analysed by relating the clearance rates on a certain food item (Cr.) to the maximum filtration rate observed in each experiment (Cr ma><) following the formula: Wi = Cr;l Crmax
Table 1. Dimensions (diameter or lengthxwidth in pm, mean in brackets) and concentrations (numbers of cells per millilitre) of food items offered in the feeding experiments. Concentrations for exper iments 6a, 6b and 6c are minimum (upper row) and maximum (lower row) values. Abbreviations : Chlam = Chlamydomonas reinhardti, Cyclid = Cyclidium glaucoma, Cyclot = Cyclotella meneghiniana, Dexio = Dexiostoma campylum, Eupl. = Euplotes moebiusi, FLB = Escherichia coli (fluorescen tly labeled bacteria), FP = inert fluroescent pa rticles, Ha lt = Halteria grandinella, Monor = Monoraphidium rninutum. Prey FP FLB Cyclot Monor Chlam Cyclid Dexio Halt Eupl
Di mensions [um] 0.1-5 .6 (2.9) 1-3.5xO.6-1.l (2.9xO.8) 12- 16.5x5-7 (14.2x6.3) 7-11x3-5 (10x4) 5-9.5 (7.3) 15-22xlD-12.5 (18.5x12) 36-70x2D-30 (50x24) 20-40 (30) 5D-65x3D-34 (57x36)
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7 850
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Feeding behaviour in an omnivorous ciliate
Individual electivity indices were summarized to mean values (± SD) for each experiment. A comparison of the mean electivity indices was achieved by statistical analysis (paired Student's t-test, p < 0.05) of the individually calculated electivity indices (Table 2). Linear regression analysis was performed to detected a significant correlation between cell size of Stylonychia and the volume of ingested prey.
Evaluation of the functional response Three experiments were run to evaluate a correlation between ingestion rates and food concentration. In the first set of experiments only Chlamydomonas was offered as food, in the second experiment a mixture of Chlamydomonas and Dexiostoma (each 50% of the total biovolume) were offered, and in the third experiment only Dexiostoma was provided as prey to Stylonychia. The maximum food concentration in all three experiments was 13 mrrr' per litre. Dilution was achieved using filtered lake water «0.1 urn) in eight steps to a minimum food concentration of 0.1 mm' per litre. The experiment was run for 20 min in triplicate for each food concentration. Further treatment and evaluation of ingestion rates was made in the same way as described for the food selectivity experiments.
Long term observations Feeding behaviour of individual cells of Stylonychia mytiIus was observed directly under a microscope (Reichert Neovar, magnification 100x). Observations were made at a temperature of 20°C at reduced illumination. Five millilitre of a culture medium with the living and untreated prey ciliate Dexiostoma campylum (550 cells/ml) were added to 10 mlcounting chambers. One specimen of Stylonychia was added to the counting chamber at the beginning of each observation. All contacts of the prey with the oral apparatus of the predator
449
and ingestion of prey cells by Stylonychia during a continuous time interval of 120 min were recorded. The mean capture efficiency (ingested prey/contacts of the buccal region with prey) of five different specimens of Stylonychia was monitored. The data allowed for the detection of temporal alterations in the feeding activities of Stylonychia cells. Video recording supported the observations of the feeding behaviour.
Results Food selectivity 1) Selectivity among different prey items: Stylonychia mytilus consumed a wide spectrum of food parti-
cles ranging from bacteria to algae and different sized ciliates and can clearly be classified as an omnivore. Our results show a clear selection of different prey items. The electivity indices also indicate differences in selection of different algae as well as of different ciliates (Table 2, Fig. 1). The green algae Monoraphidium minuturn and the fast moving and jumping ciliate Halteria grandinella were negatively selected in the first set of experiments (Fig. 1a). The two hymenostome ciliates were preferred (Cyclidium glaucoma, with a maximum clearance rate of 1.9x10-2 mlxh'), or non-selectively (Dexiostoma campylum) ingested. A significant (p < 0.05) positive selection of Stylonychia for Cyclidium over Monoraphidium and Halteria was recorded (Table 2). The second experiment was designed to investigate the food selection among relatively small food items. The living green algae Chlamydomonas were signifi-
Table 2. Comparison of the food selectivity of Stylonychia mytilus feeding on one food item (rows) vs the other food items (columns) present in the respective experiments. Summary of the results from experimental data shown in Fig. 1. Electivity indices compared by paired Student's t-test (p < 0.05). Abbreviations: p = significant positive selection, n = significant negative selection == no significant difference between electivity indices, - = not determined. Chlam = Chlamydomonas reinhardti, Cyclid = Cyclidium glaucoma, Cyclot = Cyclotella meneghiniana, Dexio = Dexiostoma campylum, FLB = Escherichia coli (fluorescently labeled bacteria), Halt = Halteria grandinella, IFP = large fluorescent particles (>3 um), mFP = medium fluorescent particles (1.5-3 prn), Monor = Monoraphidium minutum, sFDP = small fluorescent particles «1.5 pm), Example: Cyclotella meneghiniana (row 5) was preferred compared to fluorescent particles and Escherichia coli (columns 1-4) and was less preferred than Chlamydomonas reinhardti (colum 7). There was no statistically significant preference detected regarding Cyclotella meneghiniana vs. Cyclidium glaucoma (column 8) and Cyclotella meneghiniana vs. Dexiostoma campylum (column 9). Monoraphidium minutum (column 6) and Halteria grandinella (column 10) were not offered in this experiment. Food item
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Fig. 1. Food selectivity of Stylonychia mytilus. Mean clectivity indices (± SD) according to Vanderploeg & Scavia [63] for food items from different trophic levels. Abbreviations of species as in Tables 1 and 2. l a: Results of Experiment 1, Ib : Results of Experiment 2, l c: Results of Experiment 3.
Fig. 2. Feeding behaviour of different size classes of Stylonychia mytilus offered different potential prey algae, Cyclotella meneghiniana and Chlamydomonas reinhardti, and ciliates, Cyclidium glaucoma and Dexiostoma campylum (exp. 3). 2a: Mean electivity indices (± SD) calculated according to Vanderploeg & Scavia [63]. 2b: Mean percentage composition regarding the biovolume of the ingested food items.
cantly (p < 0.05) preferred (wi th a max imum clearance rate of 9.2x lO-3 mlx h') over all othe r foo d items offered in the experiment. FLB (E. coli) were significan tly (p < 0.05) nega tively selected against Ch lamydomonas and Cyclotella. FP of all provided size-classes were also negatively selec ted compared to Chlamydomonas. H ow ever, a slight but sign ificant (p < 0.05) higher positive select ion was found for the largest compared to the smallest FP (Table 2, Fig. 1b). In the third expe riment (Table 2, Fig. l c) th e green algae Chlamydomonas was again the preferred food item (with a maxi mum clearance rate of 1.2xl O-2 ml x h') for Stylonychia. A clear negative selection (p < 0.05) against the on ly no n-living prey in this experiment, Cyclotella meneghiniana, was recorded. 2) Size-dependent select ivity an d ingestion: In order to investigate whether there is a relat ionship between food selectivity and cell size of Sty lonychia, indi -
vidual biovolumes of the observed Stylonychia cells in two of the exper iments (exp. 3 and 4, Table 1) were calcu lated and related to the respective amount and composition of ingested prey. The experiments revealed different food preferences of different size-classes of Stylonychia (Fig. 2 and 3). In the first experiment (exp. 3, Fig. 2) an increasing selection for the large Dexiostoma by larger Sty lonychia cells was observed (Fig. 2a). The composition of the food vacuo le content of the smallest cells was abo ut 50% Ch lamydomonas and 50% Cyclidium. Stylonychia cells being larger than 2xl0 5 prrr' ingested abo ut 50% Chlamydomonas and 25% each Cyclidium and Dexiostoma. The diatom Cyclotella was hardly ingested by all size groups of Stylonychia (Fig. 2b) . Bigger cells of Stylonychia ingested more food (r 2 = 0.9981), up to about 3xl0 4 pm' per individual cell per ho ur. In the second set of experiments on ly ciliate prey was offered to Stylonychia (exp. 4, Fig. 3). An increasing food prefer-
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Fig. 4. Mean feeding rates (± SD) of Stylonychia mytilus per hour calculated after different experimental times (exp. 4). 4a: Feeding rate on the green algae Chlamydomonas reinhardti . 4b: Feeding rate on the ciliate Dexiostoma campylum.
ence of larger predators for the bigger prey ciliates Dexiostoma and Euplotes was detected (Fig. 3a). Electivity indices for these two ciliates increased significantly (p < 0.05) with increasing cell volume of Stylonychia. The total amount of ingested biovolume was pos itively correlated (r2 = 0.9520) with Stylonychia size. The food vacuole in the smallest Stylonychia cells contained abo ut 50% each, Cyclidium and Dexiostoma, whereas in larger specimens the relative proportion of Dexiostoma increased to about 90% (Fig. 3b). The large Euplotes (length about 60 prn) was not ingested by cells smaller than 4.5xl0' prrr' (about 175xl00 J.1m).
lonychia per ho ur were measured for the algal prey Chlamydomonas reinhardti, minimum feeding rates of about 0.5 cells per Stylonychia per hour were calculated for the relatively large prey ciliates Halteria grandinella and Euplotes moebiusi. The feeding rates calculated for short exper imental times were generall y higher than for longer experimental times. Especia lly for larger prey, like the offere d algae and ciliates, calculate d feeding rates were much higher using experimental times shorter than abo ut 30 minutes. After this time, the calculated feeding rates declined to minimum values (exp. 3, Fig. 4). Only initial digestion of algal and ciliate prey could be observed in the experiments in the course of 75 minutes. Egestion of ciliate cells was never observed during two hours in the long term observations (exp. 5). 2) Feedin g activity and capt u re efficienc y: Longterm experiments ind icated alterations in the feeding
Feeding rates 1) Influen ce of expe rimental time: Hig hest feeding rates with a maximum ingestion of 850 cells per Sty -
452
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activity of Stylonychia. The ciliates showed feeding periods followed by non-feeding periods, both lasting about 20 to 30 minutes (Fig. Sa). A higher feeding activity and capture efficiency was detected at the beginning of the experiments, whereas, after ingestion of about 4 to 6 prey ciliates (Dexiostoma campylum), capture efficiency declined from about 70% to about 30% (Fig. Sb). The feeding behaviour of the individual ciliates was relatively similar. 3) Functional response: A functional response similar to Holling's type II curve [cf 7, pp. 311-314] was registered in the experiments where different concentrations of food were offered (Fig. 6). Two kinds of potential prey (Chlamydomonas reinhardti and Dexiostoma campylum) were used in this set of experiments (exp. 6a-c). Experiments dealing with the food selectivity showed that both food items were consumed by Stylonychia (see Fig. 1). Feeding rates on algal prey, ciliate prey, and a mixture of algae and ciliate prey (Fig. 6) increased with increasing food concentration following
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Feeding behaviour in an omnivorous ciliate
a hyperbolic curve. For the offered food items maximum uptake rates were reached at a concentration of about 2 to 3 mgxl', At the lowest tested food concentration (0.1 mm'xl') Stylonychia showed minimal individual ingestion rates of about 2000 prrr' prey volume per hour. This value increased to 7100 ± 1100 unr' for algal prey at the maximum food concentration of 13 mrrr'xl" (Fig. 6a). Highest ingested biovolumes (26900 ± 5600 um'xlnd.r'xh') at the highest food concentration were detected in the experiments where we offered ciliates only (Fig. 6b). About 19500 ± 4700 pm'xlnd.r'xh' were ingested in the experiments with mixed food (Fig. 6c). There was no selection regarding the two food items. The proportion of both food types in the food vacuoles did not change significantly at different food concentrations.
Discussion Food selectivity It is known that various predatory ciliates consume other ciliates [e.g. 12, 27], under certain conditions some ciliates may develop cannibalism [e.g. 26, 46]. Our present results indicate that a typical filter-feeding ciliate known as a bacterivore-herbivore can also act as
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453
an efficient predator on certain ciliates. Many ciliates in pelagic and benthic systems are filter-feeders [17] and one can conclude that omnivory is much more widespread among components of the microbial food web than currently believed. The results of the present investigation clearly indicate that even filter-feeding ciliates show preferences for certain food items depending not only on their size, but also on their "quality". A selective feeding behaviour, namely a strong preference for few components of the diverse food spectrum, was observed when many different potential prey items were offered (summarized in Fig. 7). Generally, smaller food items like small FP and bacteria were less preferred than larger ones (d. Fig. 1 and 7). On top of that even within the same size class of prey food selection of Stylonychia was different: non-mobile prey was less preferred than mobile prey. An explaination could simply be a higher chance to get caught by the filter current for mobile organisms. On the other hand, a quick and jumping movement seems to influence the food selectivity of Stylonychia in an opposite way. The oligotrich ciliate Halteriawas negatively selected in our experiments (Fig. la). We observed under the microscope that its jumping reaction allowed the prey to escape from the filter current created by Stylonychia. Jumping has been shown to reduce the vulnerability of oligotrichs to metazoan filter-feeders [e.g. 24, 61]. The possession of a
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;, ... ,
.
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-----.--,-:.'-': '::''
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Escherichia coli bacteria (heat killed) ' 0.6-3 .5jJm " --------
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Fig. 7. General scheme of the feeding behaviour of Stylonychia mytilus regarding food items from different trophic levels (summary of expo 1-4). Arrows mark a strong (full line), medium (coated line) and low (dotted line) selection for the different food items, respectively.
454
G. Pfister and H. Arndt
hard and rigid cortex in Euplotes and its tend ency to attach to th e su bstrate (and thu s reducing th e chan ge to get caught by the filter current) are possible reasons for a weak grazing pressure performed by potential pred ators. We assume that the feeding behaviour of Stylon ychia is influ enced by a combination of all of these factors and possibly even changing w ith changing environmental conditions. There was only one exception where no selection was registered, namely in th e experiment wh ere the two different prey item s Chlamydom onas and Dexiostoma were offered. H ere no selection could be observed (Fig. 6c), po ssibly due to the stro ng affinity of Stylony chia for both of these pre y organisms. Interestingly, Stylo nychia ingested a much higher amount of food when feeding only on the large ciliate Dexiostom a, compared to the incorpor ated biovolume when feeding only on the smaller alga Chlamydomonas (Fig. 6a and 6b). We assume th at the form ation of food vacuo le memb ranes could possibly be a limiting factor in th e latter case. Th e formation of man y small vacuoles for th e small algae cells requires a larger membran e-surface than form ation of less but larger vacuol es for the ciliates. Our experiences in cultivation showed th at Sty lony chia-cultures grew well on both algae and mixed food, but significantly degenerated on an exclusive Dexiostomadiet, which could indicate an insuffici ent nutritional value of th e ciliates. Our results agree with findin gs of Lilly [33] w ho rep orted that cultures of Sty lonychia pustulata degenerated after having been fed exclusively with Tetrahymena pyrifo rm is. However, Licht enberg [32] obtained cont rary results. Her cultures of Stylonychia mytilus degenerated on only Chlamydomonas food and grew well feeding on th e ciliate Dexiostom a. In agreement with oth er investigations [d. 35], we assume that a diet containing algae better supports the growth of omnivorous ciliates than onl y ciliate food.
Food selection of juvenile and adult Sty/onychia Th e results of our experiments gave clear evidence for differ ences in the food pr eference s of different size groups of Stylonychia (Fig. 2 and 3). All experimenta l organisms of Stylony chia originated from one clone and were cultivated und er the same conditions. Our observations of clonal cultures revealed that under th e conditions of cultivation yo ung daughter cells stayed in the size group <2xl0 5 urrr' xind' for th e first three to four hours. The specimens grew up to th e size group >5x105 prrr' xind' within about 13 to 15 hours. In spite of individu al variations in growth, it was clear th at the smallest and th e largest size groups belong to just divided or ganisms ("y oung") and to or ganisms pri or to division ("old"), respectively. Thus, the observe d biovolume specific differences in feeding activity can be int er-
preted as differences comparable to ontoge netic differences known from metazoa [e.g. 62]. To our kn owledge, such" ontogenetic" differences in feeding activity of protists have not yet been describ ed. In both experiments the percentage contribution of large food items (D exiostoma and Euplotes) in the ration of juvenile Stylonych ia was significantly lower than in older and larger specimens (d . Fig. 2 and 3). Particl e size is known to be an imp ortant factor influen cing th e food selectivity of ciliates [9, 16, 49]. As our result s indicat e, pot enti al prey can simply be too large for the pr edator. The reduced predation on th e large D exiostoma and Euplotes by small Stylo ny chia may serve as examples (Fig. 2 and 3). It is known th at rigid surfa ces and appendages in Euplo tes are produced as a result of predator-induced defen ses [e.g. 22]. Compared to th e other ciliates offered in our experiments Euplotes had by far th e hardest cortex, which may have served as a certain protection from ingestion by Sty lonychia.
Capture efficiency To our knowledge th ere are no data reported on the capture efficiency and long term feedin g activity of ciliates. This interesting behaviour could have been overlooked so far due to the fact that most ciliate feeding experiment s have been carried out with small food item s, not showing remarkable changes in ciliate feedin g activity. The reason for th e decreasing captu re efficiency implied by th e observed resting phases in our experiments (Fig. 5a) could be th e time requ ired to reorganize memb rane material for th e form ation of new food vacuoles. Besides, data fro m literature indicate that digestion tim es can vary from some minutes for small particles like bacteria [53] to hours for large pr ey like ciliates [13]. We observed in th e long term feeding experiment s that all ingested ciliates we re still pre sent in Sty lonycbia's food vacuoles after 120 minutes confirming lon g digestion times for ciliate prey. Th ese lon g digestion time s could explain the need for th e observed resting phase s in our experiments. To the cont rary, Dolan & Simek [14] demonstrated recentl y for an oligotrich ciliate th at neither th e food quality nor the nutrition al stage of th e experimenta l organisms seems to have any significant influence on digestion tim es.
Methodological and general considerations Omnivorous feeding is defin ed as simultaneo usly preyin g on different trophic levels and hence ingestin g a wide range of different food item s. Although it is widespread in natural commun ities [25, 58, 59, 65], omnivory is often neglected in ecolo gical investigations. It is generally difficult to quant ify th e complexity and
Feeding behaviour in an omnivorous ciliate variability of the feedin g beh aviour of omni vor es. To get a realistic idea about complex trop hic interactions in natu ral assemblages, laborator y feeding experiments should simulate natural conditions. An essent ial need for feeding experiments is th e simultaneo us use of diverse and natural food items [23, 37, 44]. Man y attempts have been made to simulate natur al food condition s in protozoan feedin g experi ments. Fluorescent part icles [e.g. 16, 36, 49J, fluo rescently labeled bact eria [FLB , e.g. 19, 52J and algae [FLA , e.g. 47J are usual tr acers in laboratory experi ments . A disadvant age of thi s surro gate pre y is its non-living character. Hence, biochemical and behavioural interactions among protozoa and its prey are often neglected. In contrast, it is kn own that ciliates can accumulate due to chemical sub stances originating from pot ential prey [ef. 64J and morphological changes of both predators and prey indu ced by special submitting factors have often been described [e.g. 22, 27, 28, 29]. D efensive cell organelles of potenti al prey are kno wn to play an impo rtant rol e in ciliate predator-prey int eraction s [e.g. 21J. O ur experi ments based on a diverse food spectru m. In or der to keep detectable food sources as natural as possible, in addition to FP, FLB and FLA living algae (use of their autofluo rescence) and living fluroes centl y labeled ciliates (FLC i) were offered. We develop ed a stain ing protocol for ciliates, which pr ovides a techniqu e to study the selective grazi ng on different ciliate species. For FLCi no visible negative effects regarding locom otion and reproduction could be detected. This may reflect only weak effects on th e physiological state of cells caused by the staining procedure. Due to the ir different shapes of the nuclear appar atus even ingested ciliates could be taxonomically distinguished. This allowed investigations on Stylonychia's food selectivity regarding different prey ciliates. An other live-staining techniqu e for protists (flagellates) was developed by Cl even [11] using fluores cent parti cles which have been previ ously ingested by prey flagellates. However, th e experi men tal protocol is mu ch more complex and different species are difficult to distinguish. In th e future species specific genetic p robes may be used for fluore scent study of pr ey prot ists. H owever, our simp le method may be a qu ick and cheap alternative for prey species th at differ remarkably in the size or shape of thei r nuclear apparatus . We assum e that our results on the feeding beha viour of Sty lonychia reflect some general characte ristics of filter- feeding ciliates which are often neglected in grazing experiments. It is often overloo ked that filter-feeding ciliates (except for typ ical bacterivores) are probably omnivores, feeding on different trophic levels. Filt erfeeding ciliates may show prefe rences for different food part icles, which may be similar in size. It has to be considered that food selection of ciliates may change in th e
455
course of individual grow th fro m young daughter cells to cells just pri or to division. D igestion times of some ciliates for specific foo d items may vary from a few minutes to several hours. Investigation s on the capture efficiency concerni ng th e upt ake of large food particles indicated that periods of high grazing activity and periods of resting may alterate within minutes. U nfortunately, an ideal experimental setup in all of th ese respects can hardly be achieved. H owever, we hope to have pointed to some major drawb acks of cur rently applied meth od ologies in ciliate grazing stu dies. Acknowledgements: We th ank our colleagues at the Institut e of Limnology (Mondse e, Austria) for their help. Special thanks to Friederike Molilacher for cession of ciliates originating from her cultures, Walter Geiger for helpful comments on statistical analysis of our data and Karel Simek for constructive criticism on the manuscr ipt. Financial support was partially prov ided by th e Austrian FWF, project PI 03061 BIO .
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