Hydrobiologia 491: 159–166, 2003. E. van Donk, M. Boersma & P. Spaak (eds), Recent Developments in Fundamental and Applied Research. 159 © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

An approach to measure grazing on living heterotrophic nanoflagellates

Kirsten Christoffersen1 & Juan M. Gonzalez´ 2 1Freshwater Biological Laboratory, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød, Denmark E-mail: [email protected] 2IRNAS-CSIC, P.O.Box 1052, 41080 Sevilla, Spain E-mail: [email protected]

Received 21 August 2001; in revised form 4 June 2002; accepted 22 August 2002

Key words: ciliate, grazing, fluorescence, heterotrophic nanoflagellates, live-labelling, FITC, macromolecules

Abstract The complicated routes by which organic material is channelled up to higher trophic levels via bacteria and proto- zoans is a major issue in aquatic . Because of the fragile nature of it is not straightforward to perform experimental studies of prey–predator interactions. Here we present an approach for the assessment of ciliate grazing on living heterotrophic nanoflagellates. Stationary phase cultures of a heterotrophic nanoflagellate (Cafeteria sp.) were live-stained by allowing them to take up fluorescently labelled macromolecules. Controls revealed that this label persisted for several hours. Fluorescently labelled living flagellates (FLLF) were added into enriched natural assemblages of marine oligotrich and uptake of FLLF was monitored over time. Oligotrich ciliates did not incorporate fluorescent-labelled macromolecules but a linear FLLF uptake over time was observed for 20–30 min at 20 ◦C. Ingestion rates were 21–46 FLLF h−1 at a concentration of about 2×104 FLLF ml−1, which corresponded to clearance rates of 0.7–0.8 µl ciliate−1 h−1. These results are in the same order of reported ciliate grazing on of similar size. This method represents a direct approach to measure ciliate grazing specifically on living heterotrophic nanoflagellates.

Introduction al., 1987; Sherr 1988) but it has been shown that marine bacteria are too small to be efficiently grazed Bacteria and phagotrophic protists play an important by most ciliates and larger organisms (Fenchel, 1980; role in aquatic food-web structure, nutrient cycling Sherr, 1988; Bernard & Rassoulzadegan, 1990). How- pathways, and biogeochemical dynamics (e.g. Fuhr- ever, both ciliates and flagellates have been reported man and Azam, 1982; Azam et al., 1983). About one- as important phytoplankton grazers as they habitually half of the phytoplankton production is channelled ingest a wide range of phytoplankton species (Bernard through bacteria that represent an important fraction & Rassoulzadegan, 1990; Sherr et al., 1991; Verity, of total biomass. In addition, bacterial communities 1991a,b; Li et al., 1996). Microzooplankton inverteb- show a relatively constant standing stock, which has rates, i.e. , have also been reported as import- led researchers to consider bacteria as either a sink or ant phytoplankton grazers (e.g. Hansen et al., 1994). a link of organic biomass in pelagic . Phytoplankton cells are generally easy to enumerate Phagotrophic protists, mainly heterotrophic nan- because of the autofluorescence of their pigments (e.g. oflagellates, are the dominant bacterivores in most Li et al., 1996). These results showed that phytoplank- aquatic ecosystems (e.g. Sherr et al., 1987, Gonzàlez ton biomass is actually channelled through different et al., 1990). Ciliates, mostly nano-ciliates (<20 µm trophic levels. in diameter), have been reported as important bac- Although heterotrophic nanoflagellates are thought terial grazers only in particular ecosystems (Sherr et to the major bacterial consumers (but see Jürgens et 160 al., 1996), relative few studies on their fate in pelagic naturally occurring nanoflagellates take up macro- food webs are available. Most of the studies have been molecules, such as dissolved proteins and polysac- directed to estimate grazing on phytoplankton cells, charides; but ‘naked’ dinoflagellates and oligotrich which were traditionally considered as the most im- ciliates do not. Marchant & Scott (1993) provided fur- portant carbon pool in aquatic ecosystems. There is ther evidence and Christoffersen et al. (1996) found increasing evidence, however, that <1 µm-diameter- that very different species of nanoflagellates were cells might be much more important in aquatic ecosys- able to concentrate relatively low molecular weight tems as previously expected (e.g. Azam et al., 1983). polysaccharides. Recently, it has been shown that Heterotrophic nanoflagellates represent the most prob- natural assemblages of heterotrophic nanoflagellates, able link between bacterial biomass and higher trophic and specially choanoflagellates, were able to take levels, and as such the fate of heterotrophic nanoflagel- up viruses and virus-size particles of 50 nm in dia- late populations are likely to affect food-web structure meter (González & Suttle, 1993). The clear advantage and nutrient cycling. of using such labelled macromolecules to label het- Experimental studies of protozoans feeding on erotrophic nanoflagellates as prey is that the ciliates other protozoans are not trivial basically because such grazing on them are often able to ingest fluorescent organisms are very delicate. Most methods require particles in the size range (i.e. bacteria) lengthily incubations that are likely to affect the pro- while these ciliates are unable to incorporate fluor- cess studied. Tedious counting hours at a microscope escent macromolecules (Sherr, 1988; Tranvik et al., are necessary (Putt, 1991; Landry, 1994). In addi- 1993). tion, most heterotrophic flagellates are fast-swimmers, Although the above results are important for the which also makes using non-motile stained cells not , the focus for this study is the a proper method in ciliate grazing experiments. Be- possibility of using those techniques for labelling het- cause phagotrophic ciliates are able of prey discrim- erotrophic nanoflagellates to be used in uptake experi- ination (Stoecker, 1988; Verity, 1991b; Li et al., ments. The aim of this study is to present and evaluate 1996), a label that might hide or damage possible a direct approach based on this ability of nanoflagel- receptors at the exterior of nanoflagellate cells could lates to incorporate fluorescent dyes, which allows the render measurements of grazing difficult. Possible al- assessment of ciliate grazing on living-heterotrophic ternatives are use of radioisotopes (Putt, 1991), but nanoflagellates. the fate of radioisotopes in water samples cannot be controlled. Size-fractionation also shows important Materials and methods inconveniences because heterotrophic nanoflagellates and ciliates are damaged during the required filtra- A culture of the heterotrophic nanoflagellate Cafeteria tions. The dilution technique (Landry, 1994), does sp. (4–5 µm in diameter) was previously isolated from not permit the exact identification of the actual fate an Oregon coast sample and maintained in 0.2-µm- of heterotrophic nanoflagellates and requires very long filtered seawater with one boiled wheat grain per 100 incubations (>24 h) especially in oligotrophic wa- ml seawater. ters. Many other methods, taking the advantages of For the uptake experiments, heterotrophic nan- using labelled mini cells (e.g. Vazquez-Dominquez, oflagellates were grown in 0.2-µm-filtered seawater 1999), labelled micro-spheres and labelled bacteria supplemented with yeast extract (0.2 g l−1)at15◦C (e.g. Cleven, 1996; Dolan & Simek, 1997), immuno- in the dark (González et al., 1993). After 1 week, fla- labelling of ingested prey (Christoffersen et al., 1997) gellate number was about 105–106 cells ml−1 while or various vital stain protocols (e.g. Li et al., 1990; bacterial number was lowered to about 106 cells ml−1. Smalley et al., 1999) have emerged during the recent At this stage, labelled macromolecules or particles years. were added to the flagellate culture and incubated at Several studies have shown that heterotrophic 15 ◦C for 2 h from 30 min to several hours. nanoflagellates can take up dissolved organic mat- The ability of different fluorescent-labelled mac- ter (DOM) with a molecular weight of 55–2000 Kd romolecules to label the food vacuoles of nanoflagel- (Sherr, 1988; Tranvik et al., 1993; Marchant & Scott, lates were assayed: FITC-dextran (molecular weight 1993). It is possible to visualize labelled food vacuoles 2000 Kdaltons; Sigma Co.), FITC-Concanavalin A in heterotrophic nanoflagellates by using epifluores- (molecular weight 55 Kdaltons; Sigma Co.), FITC- cence microscopy. Tranvik et al. (1993) showed that ferritin, FITC-Albumin (66 Kdaltons; Sigma Co.), and 161

Cascade Blue-Concanavalin A (55 Kdaltons; Poly- Concanavalin A (10 mg l−1; see ‘Results’) as the sciences). Stock solutions (1 mg ml−1)ofFITC- fluorescent compound because it gave the best la- conjugates were prepared by dissolving the FITC- belling of the flagellate food vacuoles. After incuba- conjugate in phosphate buffer saline solution (pH 7.4), tion of the flagellate culture in the presence of FITC- whichcontainedin1l:8gNaCl,0.2gKCl,0.2g Concanavalin A, flagellates were inspected to check KH2PO4, and 0.15 g Na2HPO4. Concentration of for an adequate labelling of their food vacuoles. Im- labelled macromolecules was usually 10 mg l−1 al- mediately, FLLF were inoculated into duplicate ciliate though concentrations from 5 to 30 mg l−1 were enrichments and 0.2-µm-filtered controls. tested. cultures were diluted about 1:100 which reduced the We also assayed fluorescent virus-sized particles number of flagellates to about 103–104 ml−1.This (50 nm-diameter) and fluorescently labelled bacteria represented about 10–20% of total number of flagel- (FLB) in order to create fluorescently labelled food lates in the ciliate enrichment. The number of bacteria vacuoles of heterotrophic nanoflagellates through pha- from flagellate cultures did not represent a signific- gocytosis. Virus-sized particles were used at a con- ant number after addition to the ciliate enrichments centration of 107–109 fluorescent particles ml−1,and (about 0.1% of total bacterial number). Incubations particle suspensions were prepared as described by were performed at 15 ◦C in dim light without shaking. González & Suttle (1993). Fluorescently labelled bac- Two grazing experiments were performed in which teria were prepared as described by Sherr et al. (1987) subsamples of 10 ml were collected from the duplic- by using ‘Y’, a marine bacterial isolate from Oregon ate ciliate cultures periodically at time zero to 30 min waters (González et al., 1993). Final concentrations of (Exp. I), and at time zero to 180 min (Exp. II). The FLB were about 25–40% of total bacterial number in subsamples were immediately preserved by the Lugol- the flagellate cultures. Finally we tested several fluor- Formaldehyde decolouration technique as described escent dyes for their ability to live staining nanoflagel- by Sherr et al. (1987). Two types of controls were per- lates. The dyes were: DTAF (5-([4,6-dichlorotriazin- formed: (1) 0.2 µm-filtered ciliate enrichments were 2-yl]amino)-fluorescein, FITC (fluorescein isothiocy- used as controls for checking label in flagellates and anate), and Rhodamine 123. The dyes were tested (2) ciliate enrichments plus FITC-Concanavalin A at at final concentrations that apparently did not dam- a final concentration of 0.1 mg l−1, which was the age nanoflagellates, since they continued swimming final concentration of FITC-Concanavalin A in the and grazing on bacteria during the staining period. In experimental samples with FLLF. The latter type of these cases, flagellate cultures were prepared as above controls was performed to check for possible uptake of and incubated for 2–3 h in the presence of virus-sized free FITC-Concanavalin A by ciliates and flagellates particles, FLB or a fluorescent dye. present in the ciliate enrichments. Subsamples from Ciliates were obtained from seawater collected at the duplicate controls were treated as above. Coos Bay, Oregon, U.S.A. (43◦ 21 N, 124◦ 20 W) Preserved subsamples from grazing and control at high tide (temperature 15 ◦C; salinity 31‰). Wa- experiments were stained with DAPI (4,6-diamidino- ter samples were concentrated by passing through 2-phenylindoldihydrochloride) according to Porter & 10 µm-pore-size meshes (no vacuum was applied). Feig (1980) as modified by Sherr et al. (1987). Counts Concentrates were supplemented with phytoplankton were performed with a Zeiss Universal epifluores- cultures (1 ml from each species l−1) of different cence microscopy equipped with standard blue and taxa: a coccoid cyanobacterium (Synechococcus sp.) UV filter sets. A total of 20–40 fields (depending of the a chrysophyte (Pelagococcus subviridis)twopras- density of flagellates) were inspected at 1250× magni- inophytes (Pycnococcus provasolii and Micromonas fication and all flagellates (i.e. labelled and unlabelled) pusilla) and a (). as well as the number of vacuoles per flagellate were Phytoplankton cultures were kindly provided by Dr. counted. Ciliates in DAPI-stained preparations were Lynda Shapiro. Phytoplankton cultures were grown enumerated by inspecting the entire filters at 400× in f/2 medium under natural light at room temperat- magnification; FLLF and taken up by ciliates were ure. Several oligotrich ciliate species of 20–80 µm enumerated by switching to 1250× magnification and in diameter/length were observed in the cultures but blue excitation light. a Strombidium-type always dominated. FLLF per ciliate was estimated in two ways; either For ciliate grazing experiment on fluorescently (a) by dividing the average number of flagellate- labelled living flagellates (FLLF), we selected FITC- flagellate food vacuoles ciliate−1 by the number of 162

flagellate-flagellate food vacuoles flagellate−1 or (b) by direct counting of FLLF in ciliates (see ‘Res- ults’). The linear portion of the uptake curve (FLLF ciliate−1 over time) was used to estimate uptake rates (or ingestion rates) (FLLF ciliate−1 min−1) by lin- ear regression analyses. Clearance rates (µl ciliate−1 h−1) were calculated by dividing uptake (or ingestion) rates by the number of FLLF µl−1. Absolute inges- tion rates (flagellates l−1 h−1) of flagellates by ciliates were estimated by multiplying clearance rates by total number of flagellates µl−1 and dividing by the num- ber of ciliates l−1. Statistical analyses were performed according to Sokal & Rohlf (1981). Figure 1. An example of the total density of Cafeteria sp. and the density of labelled flagellates after exposure for Concanavalin A − (10 mg l 1) for 2 h and subsequently diluted with filtered seawa- ter (= time zero). Each value is the mean of 20 grids counted in the microscope and the bars are standard error of the mean. The Results and discussion average labelling efficiency in this experiment was 63% (SD = ± 9%, n = 5). Living heterotrophic nanoflagellates were indirectly stained as most dyes tested (DTAF, FITC, Rhodam- ine 123) only stained bacteria. Stained bacteria could ciliate assemblages since bacterial number was at a be seen in living nanoflagellate food vacuoles. How- minimum and flagellate number at a maximum. ever, stained bacteria represent a source of error when FITC-Concanavalin A provided also the best res- ciliates are grazing on flagellates since those bacteria ults as a labelling technique for uptake experiments were always present in the experimental ciliate as- of heterotrophic nanoflagellates by ciliates. Other semblages and small ciliates (<30-µm-diameter) were FITC-labelled macromolecules assayed in this study able to ingest bacteria. The use of virus-sized fluor- provided significant fluorescent background and lower escent particles (50 nm-diameter) allowed us to label fluorescence intensity of the flagellate food vacuoles. flagellate food vacuoles, but high concentrations of FITC-Concanavalin A produced bright, well-defined particles (>108 ml−1) were necessary for an accept- flagellate food vacuole that was easily visualized by able labelling, thus causing many clusters of fluores- a blue filter set. Such flagellate food vacuoles per- cent particles (González & Suttle, 1993), which were sisted for up to 180 min and thus long enough to subsequently ingested by ciliates during the uptake measure uptake of heterotrophic flagellates by ciliates experiments. (Fig. 2, Exp. I). Using natural microbial assemblages The use of fluorescently flagellate macromolecules constituted by a multiple species of as a labelling technique for living heterotrophic nan- and heterotrophic nanoflagellates, we obtained hetero- oflagellates showed satisfactory results when FITC- trophic nanoflagellates stained as well as laboratory labelled-concanavalin A was used (see Fig. 1 for an cultures (Sherr, 1988; Tranvik et al., 1993). example). FITC-labelled Concanavalin A was taken The use of stationary phase flagellates presented up by Cafeteria cells stationary phase culture within one additional advantage, besides a maximum uptake the 2 h incubation period and did not label natural of FITC-compounds: the flagellates did not divide dur- bacteria. The labelling efficiency for the case shown ing the incubation. This is an important prerequisite in Figure 1 was 63% (SD = ± 9%) but the effi- for an accurate estimation of grazing rates (McManus ciency was higher in the feeding experiments (Exp. & Okubo, 1991). Steady-state conditions in the flagel- I: 72±10%, Exp. II: 85±11%). Previously published late culture provided the best opportunity for using the results showed that a maximum label with fluores- nanoflagellates as markers for the grazing by ciliates. cent macromolecules was obtained in a late stationary Since number of nanoflagellates in the flagellate cul- phase of the heterotrophic nanoflagellate growth, that ture was maximum (105–106 ml−1), a small volume of is, after 8–10 days incubation of a culture of Cafeteria culture could be added in order to obtain an adequate sp. (González et al., 1993; Christoffersen et al., 1996). final number of flagellate flagellates in the experi- This also provided the best conditions as inoculum for mental assemblages. This also reduced the concen- 163

− − − − Table 1. Ingestion rates (IR, cell ciliate 1 h 1) and clearance rates (CR, µl ciliate 1 h 1)of different types or species of ciliates feeding on live-stained flagellate prey using various dying techniques. Abbreviations are: Env = environmet, M = marine, F = freshwater, HNF = heterotrophic nanoflagellates, FLB = fluorescently labeled bacteria (heat-killed), FITC = fluorescein isothiocyanate, CMFDA = 5-chloromethyl-fluorescein diacetate, DAPI =  4 ,6-diamidino-2-phenylindoldihydrochloride, ND = no data. IR and CR are given as min and max values as reported by the authors

Ciliate (type/species) Env Labelling Prey IR CR Reference

Strombilidium M FITC HNFa 21–46 0.7–0.8 This study Tintinnids (5 species) M CMFDA Algaeb 0.2–15 ND Kamiyama et al. (2001) Favella (2 species) M Autofluor. Algaec 0.5–45 0.4–17.4 Kamiyama & Arima (2001) Tintinnids (5 species) M CMFDA Algaed 0.2– 2.4 ND Kamiyama (2000) Laboea strobila MCMFDAAlgaed 1.8 ND Kamiyama (2000) Euplotes vannus F DAPI/FITC Flagellatese 2–53 ND Premke & Arndt (2000) Strobilidium/Halteria F FLB Flagellatesf 1–15 3.8– 8.7 Cleven (1996) Codonella/Tintinnidium F FLB Flagellatesf 3–39 1.3–6.3 Cleven (1996)

a ; b Heterocapsa circularisquama; c Pavlova lutheri, Chattonella verruculosa, Heterosigna akashiwo, Heterocapsa circularisquama, Prorocentrum dentatum, P. triestinum, H. triquetra, Gymnodinium mikimotoi, Alexandrium catenella, Gyrodinium intriatum; d Heterocapsa circularisquama; e Cafeteria roenbergensis, , B. sorokini, Spumella sp., sp., Oxyrrhis marina; f Spumella sp.

tration of flagellate macromolecules to a low enough level to avoid the labelling of heterotrophic nanofla- gellates and ciliates during the grazing experiments. Numbers of FLLF in the ciliate assemblages were about 10–20% of the total number of heterotrophic nanoflagellates. The number of flagellate food vacuoles in cili- ates increased over time until a plateau was reached (Fig. 2). The control of ciliate assemblages plus FITC- Concanavalin A showed that ciliates were not able to accumulate FITC-Concanavalin A in their food vacuoles at the final concentration of labelled mac- romolecules in the uptake experiments with ciliate assemblages. The heterotrophic nanoflagellates were inoculated into the ciliate assemblages without addi- tional treatment, that is without washing or another extra manipulation. Any manipulation at this point would certainly damage the flagellates and reduce the retention time of their labelled food vacuoles. FLLF in the food vacuoles of ciliates were easily detected and enumerated. Flagellate food vacuoles in- side ciliates were clearly assigned as belonging to one or several flagellates, because food vacuoles in flagel- lates showed a typical distribution in the basal side of the flagellate body (Fenchel, 1991). In addition, fla- gellate food vacuoles of flagellates had a diameter of Figure 2. Labelled food vacuoles of FLLF (Cafeteria sp.) and cili- about 1 µm and flagellates had a diameter of about ates with time from experiment I (a) and II (b). Bars represent the 4 µm on average. The time course of ciliate feed- means from 14 to 20 individual ciliates. Error bars are SE of the mean. The average (± SD) labelling efficiency in these experiments ing on Cafeteria followed the same pattern in both was 86% ± 11% (n = 4) and 72% ±10% (n = 5), respectively. experiments with a saturation level after 20–30 min (Fig. 3). We compared the uptake of flagellates by 164

live-stained preys either by using their autofluorescens (Kamiyana & Arima, 2001) or by using a vital stain (Kamiyana, 2000; Kamiyana et al., 2001). Our experiments both with natural assemblages and cultures have shown that the use of fluorescent bacterial-size particles lead to very high label of ciliate food vacuoles by direct uptake of these bacterial-sized particles, which seriously interfered with estimates of grazing on heterotrophic nanoflagellates. Ciliate as- semblages in our study were dominated by oligotrich ciliates (mostly Strombidium)of<30 µm in diameter but contained also ciliates of up to approx. 80 µm Figure 3. The calculated ciliate ingestion rates of Cafeteria sp. with in diameter. Larger ciliates have been reported to from zero to 30 min from experiment I (upper part) and II (lower scarcely ingest bacteria (Fenchel, 1980; Bernard & part). Bars represent between 14 and 20 individual ciliates. Error bars are SE of the mean. The regression equations for 0–30 min are: Rassoulzadegan, 1990; Verity, 1991b) and with these y(I) = 0.55x + 0.42 (r2 = 0.999), y(II) = 0.30x + 0.68 (r2 = 0.966). organisms live labelling methods might be used satis- factorily (Cleven, 1996; Lessard et al., 1996; Li et al., 1996; Smalley et al., 1999; Kaniyama, 2000). ciliates estimated from (i) the counting of flagellate The uptake of nanoflagellates by ciliates was linear food vacuoles and estimation of ingested flagellates over time until a plateau was reached after 20–30 min and (ii) the direct estimation of ingested flagellates incubation and remained stable (i.e. not significantly per ciliate by microscopic visualization. No signific- different) for about 180 min (Fig. 2, upper and lower ant differences (ANOVA) were recorded between both panel). Based on linear regression analyses of the first procedures. 30 min of the uptake curves, ingestion rates of 33 and − − Ciliates (<30 µm-diameter) have been shown to 18 FLLF ciliate 1 h 1 were obtained. This is equiv- − − ingest nanoplankton cells as well as bacteria. These alent to clearance rates of 0.69 and 0.84 µl 1 h 1, ciliates are often inefficient bacterial grazers as repor- respectively. The slopes of the regression lines were ted by several authors (Bernard & Rassoulzadegan, significantly different (p>0.05) which probably can 1990; Verity, 1991b; Lessard et al., 1996) although be attributed to the fact that we used natural ciliate as- smaller ciliates may ingest bacterioplankton at high semblages. The results allowed the calculation of ab- − rates (Sherr et al., 1987). The ease of measuring preda- solute ingestion rates of 21 and 46 flagellates ciliate 1 − tion on nanophytoplanktondue to the autofluorescence h 1 by knowing the total nanoflagellate abundance of their pigments (Bernard & Rassoulzadegan, 1990; and the number of labelled nanoflagellates. The clear- Verity, 1991b; Li et al., 1996) is reflected in the higher ance rates obtained by using FLLFs are within the abundance of available data on ciliate grazing on relative broad capacity of ciliates to filter feed (e.g. phytoplankton than on heterotrophic cells. Apart from Jürgens et al., 1996) and also fit to the results obtained the fact that they lack autofluorescence, heterotrophic by comparable methods using live staining (Table 1). nanoflagellates are fragile and difficult to handle dur- Verity (1991a,b), using monospecific cultures of ing labelling protocols. Nevertheless, a number of ciliates and selected prey cultures, showed that some protocols to label flagellates to be used as prey for species of tintinnids and oligotrich ciliates ingested ciliates are available (Table 1). Most of these proto- and grew on aplastidic nanoplankton; however, the cols use fluorescent microspheres or flagellate bacteria highest ciliate growth rates were observed on phyto- to mark flagellates and then use these flagellates to plankton cells. It is also known that cultured ciliates measure grazing rates by ciliates (Dolan & Coats, hardly grow on monospecific prey assemblages. In ad- 1991; Cleven, 1996; Smalley et al., 1999). However, dition, it has been shown that ciliates are not simple, Premke & Arndt (2000) used live stained nanoflagel- mechanical feeders (Stoecker, 1988; Verity, 1991b; lates (Cafeteria, Bodo, Spumella) and a dinoflagellate Li et al., 1996), and they are able to discriminate (Oxyrrhis) as food for a filter-feeding freshwater cili- prey in base of different prey characteristics besides ate (Euplotes), and concluded that the method was prey volume. Whether phagotrophic ciliates are graz- very useful to study interactions between protists. Not ing mostly on phytoplankton cells or similarly on only protists but also phytoplankton can be used as both phytoplankton and heterotrophic flagellates will 165 depend on the relative importance of bacteria, het- González, J. M., E. B. Sherr & B. F. Sherr, 1993. Differential erotrophic flagellates and phytoplankton biomass, as feeding by marine flagellates on growing versus starving, and well as the growth efficiency of ciliates on these po- on motile versus nonmotile, bacterial prey. Mar. Ecol. Prog. Ser. 102: 257–267. tential prey populations. A direct demonstration of the González J. M. & C. A. Suttle, 1993. Grazing by marine nanoflagel- specific ingestion of heterotrophic nanoflagellates by lates on viruses and viral-sized particles: ingestion and digestion. ciliates as shown in the present study might help to Mar. Ecol. Prog. Ser. 94: 1–10. Hansen, B., P. K. Bjørnsen & P. J. Hansen, 1994. The size ratio approach a better understanding of the importance of between planktonic predators and their prey. Limnol. Oceanogr. food web interactions within the microbial food web. 39: 395–403. Jürgens, K., S. A. Wickham, K. O. Rothhaupt & B. Santer, 1996. Feeding rates of macro- and microzooplankton on heterotrophic Acknowledgements nanoflagellates. Limnol. Oceanogr. 41: 1833–1839. Kamiyama, T., 2000. Application of a vital staining method to meas- ure feeding rates of field ciliate assemblages on a harmful alga. Professors E. and B. Sherr kindly hosted and suppor- Mar. Ecol. Progr. Ser. 197: 299–303. ted the experimental work. Two anonymous referees Kamiyama, T. & S. Arima, 2001. Feeding characteristics of two provided valuable comments on an earlier version tintinnid ciliate species on phytoplankton including harmful spe- cies: effects of prey size on ingestion rates and selectivity. J. exp. of the manuscript. The Faculty of Science, Univer- mar. Biol. Ecol. 257: 281–296. sity of Copenhagen, Denmark, financed KC and JMP Kamiyama, T., H. Takayama, Y. Nishii & T. Uchida, 2001. Graz- acknowledge support from the Spanish Ministry of ing impact of the field ciliates assemblage on a bloom of Science and Technology (Ramon y Cajal program). the toxic dinoflagellate Heterocapsa circularisquama. Plankton Biol. Ecol. 48: 10–18. 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