FEEDING BY CYCLIDIUM SP. (CILIOPHORA, SCUTICOCILIATIDA) ON PARTICLES OF DIFFERENT SIZES AND SURFACE PROPERTIES. Robert W Sanders

BULLETIN OF MARINE SCIENCE, 43(3): 446-457,1988 FEEDING BY CYCLIDIUM SP. (CILIOPHORA, SCUTICOCILIATIDA) ON PARTICLES OF DIFFERENT SIZES AND SURFACE PROPERTIES Robert W Sanders ABSTRACT The effect of size, surface properties, and concentration of food particles on feeding by the scuticociliate Cyclidium sp. as investigated. A range of particle sizes at concentrations bracketing natural picoplankton densities (1-10 4 to S IO B ml-l) as offered to the ciliates. Uptake of micro spheres and of single cells of the cyanobacterium Microcystis aeruginosa as directly related to feeding time for up to 15 minutes. Clearance rates on 0.6 Mmmicrospheres ere alays higher than for 0.93 Mmmicrospheres hen compared for similar concentrations or volumes offered. Maximum clearance on 0.93 11m spheres as at an intermediate sphere concentration. This atypical functional response as due to a change in simming behavior hich reduced ingestion at lo concentrations of 0.93 Mm spheres. When Cyclidium as offered either carboxylated or protein-coated 0.6 Mm microspheres, there as no significant difference in clearance rates at any particle concentration, Hoever, clearance rates on 0.93 11m protein-coated spheres ere significantly greater than on the same size carboxylated spheres at concentrations < IO B 'ml- ', The electrophoretic mobilities (e.m., an indicator of surface charge) of protein-coated and carboxylated spheres ere significantly different. Clearance rates on carboxylated and amide-modified spheres (0.93 and 0.94 Mm,respectively) ere similar hether they ere offered alone or together at concentrations beteen - 4. 10' and 8 IO' ml- '. Carboxylated and amide-modified spheres had similar e.m. These data suggest that interactions beteen food particle size, concentration, and surface properties alter behavior and food capture by Cyclidium in a complex manner. Use of fluorescent microspheres of the same size, but ith different coatings, has the potential for elucidating feeding behavior by protozoa in the laboratory and ultimately in situ, here the prevalence of selective feeding by protozoa has direct bearing on regulation of picoplankton community structure. Different groups of ciliates are specialized for grazing on attached microbiota, or raptorial feeding on relatively large particles, or as suspension feeders on particles hich are small compared to their on size. Many raptorial feeders can select beteen prey types and may even specialize on one prey species (Fenchel, 1968; Tucker, 1968; Luckinbill, 1973; Sleigh, 1973). Hoever, there is disagreement about hether suspension feeding ciliates select particles on the basis of properties other than size and shape. According to Fenchel (1980a; 1986) "filter feeders," including Cyclidium, can discriminate only according to mechanical properties of the food particles by sieving them beteen cilia. Ingestion of inert particles (Cox, 1967; Ricketts, 1971; Spittler, 1973; Fenchel, 1980a; Bersheim, 1984; Sanders and Porter, 1986) also suggests that many ciliates are not selective feeders or that capture is not based on nutritional value. Yet, there is evidence that some suspension feeding ciliates chose beteen food particles of similar size. The planktonic marine ciliate Balanion sp. selected dinoflagellates hich supported its fastest groth over other microalgae (Stoecker et a1., 1986), and the tintinnid ciliate Favella preferentially ingested dinoflagellates over other alga of similar size (Stoecker et a1., 1981). Bahr (1954) found Paramecium initially ingested both carmine and dead bacteria hen they ere mixed together. Later the carmine and then the bacteria ere rejected and empty food vacuoles ere formed. Fenchel (1980a; 1986) argues that effects like these are fortuitous and a result of mechanical properties of the oral apparatus. 446

SANDERS: EFFECTS OF FOOD SIZE & SURFACE PROPERTIES ON CILIATE FEEDING 447 A sieving model has also been used to describe filter feeding by metazoans (Boyd, 1976; Brendelberger et ai., 1986). Hoever, some metazoan suspension feeders appear to capture particles by some mechanism or mechanisms other than sieving. The adhesion of particles to the tube feet of a suspension feeding brittle star is strongly dependent on the surface charge of the particles (LaBarbera, 1978). The zooplankter Daphnia magna also retained particles hich ere smaller than the mesh size of their feeding appendages, and retention of the smallest particles by Daphnia as increased hen the particle surface charges ere neutralized (Gerritsen and Porter, 1982). The present study evaluates the utility of microspheres for testing hether properties of food particles exert an influence on ciliate feeding parameters hich can not be explained solely by mechanical filtration. MATERIALS AND METHODS The scuticociliate Cyclidium sp. as isolated from Lake Oglethorpe, Georgia and maintained on suspensions of the bacterium Klebsiella sp. in aged autoclaved tap ater. Cyclidium in log phase groth as used for all experiments. Ciliates had an average length of 18 f.tm and a calculated biovolume of 940 f.tm 3 To assure that the presence of bacteria did not affect microsphere ingestion in the experiments, ciliates ere separated from the culture medium by gravity filtration over a 3-f.tm Nuclepore filter. Copious amounts of filter-sterilized, aged tap ater, ith a ph matched to the culture medium, ere used to gently rinse the ciliates free of bacteria. This method gave better separation of ciliates from bacteria than centrifugation. After resuspension in aged 0.22-f.tm Nuclepore filtered tapater, final bacterial densities in the experimental chambers, determined from epifluorescent counts (Hobbie et a!., 1977), ere usually less than 10 4 ml-'. It as important to make experimental suspensions ith the same ater hen comparing ingestion of different particle types, since the electrophoretic mobility and surface properties change ith ph and ater chemistry (Gerritsen and Bradley, 1987). Ciliates ere acclimated to experimental chambers (3 replicate 50 ml beakers or glass scintillation vials) at 25 C for 20 to 40 min prior to addition of microspheres. Ciliate density in the experiment using 1.17- f.tm microspheres as approximately 2,000 ml-'. Densities in all other experiments ere <400 ml-'. There as no apparent effect of croding on ingestion by Cyclidium. Fluorescent micro spheres of several sizes and surface properties ere fed to Cyclidium (Table I). In most experiments only one type of particle as offered per replicate. These treatments included: 0.6-f.tm, 0.93-f.tm and 1.17-f.tmdiameter carboxylated (cx) spheres; 0.94-f.tm amide-modified (fx) spheres; 0.6-!Lm and 0.93-!Lm protein-treated (pr) microspheres; and the cyanobacterium Microcystis aeruginosa ("". 1.0!Lm). Cilitates ere given a mixture of 0.88-f.tm cx and 0.94-!Lm fx microspheres in one time course experiment. Carboxylated (0.93 /.tm) and amide-modified (0.94 f.tm) spheres ere also offered in a mixture to the ciliates. Ingested spheres from mixed suspensions ere differentiated by their fluorescence (red or green) ith different epifluorescent filter sets. The 0.88- and 1.17-!Lmmicrospheres ere purchased from Polysciences, Inc. All other spheres ere Covaspheres. For some treatments, 0.6- and 0.93-!Lm carboxylated spheres ere protein-coated by incubation for 24 h ith bovine serum and the coupling agent EDAC (Eastman Kodak). Prior to use in feeding experiments, all microspheres ere sonicated for 30 s ith a Branson cell disrupter. Microscopic examination confirmed that clumping after this treatment as rare and essentially all spheres remained singlets for the duration of the experiments. Microcystis as gron on Woods Hole medium (Nichols, 1973) and passed through a I-!LmNuclepore filter to yield a suspension of single cells. Details of the experimental setup of Cyclidium fed Microcystis ere reported elsehere (Gerritsen et a!., 1987). Appropriate dilutions of Microcystis or sonicated microspheres ere added ith gentle mixing to ciliate suspensions. Subsamples at t = 0 ere immediately fixed ith ice-cold, buffered gluteraldehyde (2% final concentration) for enumeration of bacteria and spheres. Additional samples ere taken at 3 to 6 min intervals in all time course experiments. When comparing particles ith different surface properties, feeding as stopped by fixing hole samples after 3 to 5 min depending on microsphere concentration. Fixed samples ere stained ith primulin for approximately 8 h, filtered onto 2-f.tm Nuclepore filters and examined by epifluorescent microscopy (Sanders and Porter, 1986). Background coincidence of non-ingested microspheres ith Cyclidium as minimal and as accounted for by subtraction of time zero subsamples. Ingested fluorescent microspheres could be individually distinguished in all treatments except hen microsphere feeding suspensions had concentrations greater than 10"'ml- I Ingestion as then estimated from food vacuole diameter by assuming hexagonal close packing of spheres (Fenchel, 1980a). At least 50 ciliates ith ingested spheres ere examined for each treatment.

448 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988 Table I. Particle types and concentrations offered to Cyclidium sp, (In the case of mixed suspensions, the concentration for each microsphere type is listed) Particle size and type Time course experiments 1.17-!lm carboxylated (cx) 0.94-!lm amide-modified (fx),\jicrocystis aeruginosa Mixture of 0.94-!lm fx and 0.88-!lm ex Selectivity experiments 0.94-!lm amide-modified 0.93-!lm carboxylated Mixture of O.94-!lm fx and 0.93-!lm ex Size/surface chemistry experiments 0.6-!lm carboxylated 0.6-!lm protein coated 0.93-!lm carboxylated 0.93-!lm protein coated Concentration (los'ml- l ) 5.5 8.2 1.1 0.12 3.8 0.47 3.6 5.0 2.5 5.7 4.8 48 700 1,300 5.6 590 2,300 4,900 2.7 15 100 1,000 1.4 39 180 1,300 At lo microsphere concentrations, as many as 300 ciliates ere examined before 50 ith ingested spheres ere encountered. Ciliates ithout spheres ere included hen average ingestion and clearance rates ere calculated. Electrophoretic mobility of spheres ere measured ith a model 80 Zeta Meter equipped ith a cylindrical electrophoresis cell using standard microelectrical techniques (Gerritsen and Bradley, 1987). Relative motility of Cyclidium in the presence of different microsphere concentrations as examined under a dissecting microscope. For each microsphere concentration, 15 ciliates ere observed for approximately 3 min each and the proportion of time spent simming as determined. At lo concentrations of 0.93-!lm spheres, variability as greater and simming times of 25 ciliates ere observed. RESULTS To accurately estimate clearance on a given particle type several criteria must be met (Fenchel, 1986). The ater in hich the experiments are run should be free of non-test particles and be similar in temperature and chemistry to the ciliates' culture media. Otherise clearance may be underestimated or the ciliates may refuse to feed. Furthermore, uptake of particles should be linear. These conditions ere carefully monitored in the experiments reported here. Ciliates ere resuspended in aged, O.22-J.lm filtered tap ater (ph = 7.5) hich as also used for their culture. Acridine orange direct counts indicated that concentrations

SANDERS: EFFECTS OF FOOD SIZE & SURFACE PROPERTIES ON CILIATE FEEDING 449 A 0.6 J.U1l A..J 2.0..J 0 1.6 c:c 1.2 Do 0.8 lii:: 0.4 ~ ::) 0.0 0 5 10 15.c 80 'E 60 (.) ~ ~..J 0 40 ~ t ~ 20 i5 0 10 5 10 6 10 7 10 8 10 9 B..J..J 0 c:c Do lii:: < Ii: ::) 3.2 2.4 1.6 0.8 0.0 0 5 10 FEEDING TIME ( MINUTES) B.c 0.93 J.U1l 40 'E 30 0 20 ~ c:c i ~ 10 I J I!~..J III (.) 0 15 10 5 10 6 10 7 10 8 10 9 MICROSPHERE CONCENTRATION (# I ml) Figure I. (Left) Time course of particle uptake by Cyc/idium sp. fed fluorescent carboxylated (cx) and amide-modified (fx) microspheres, and Microcystis aeruginosa. A. 1.17-p.m cx (5.5 lo' ml- I )- solid diamond; Microcystis (1.1 1O' ml- 1 )-open diamond; Microcystis (1.2 104 ml-l)-open square. B. 0.94-p.m fx (8.2 IO' ml- 1 )-solid square; 0.94-p.m fx (3.8 1O' ml- 1 )-solid diamond; O.88-p.m cx (4.7.10 4 ml- 1 )-open square. The O.94-p.m fx at 3.8 1O' ml- 1 and the O.88-p.m cx ere offered together. Error bars are 95% confidence intervals calculated from ingestion rates of at least 50 individual ciliates. Figure 2. (Right) Clearance rates (nanoliters h- 1 ) of Cyclidium sp. on microspheres. A. O.6-p.mspheres. Carboxylated-solid squares; protein treated-open squares. B. 0.93-p.m spheres. Carboxylated-solid diamonds; protein treated - open circles. Error bars are as in Figure 1. of contaminating bacteria ere alays negligible relative to microsphere concentrations. Ingestion by Cyclidium as linear over 15 min for all tested particles, including both 0.88- and 0.94-spheres hen the to ere offered together (Fig. 1). At similar microsphere concentrations, ingestion rates ere greater for 0.6 ~m than for O.93-~m spheres, hich is indicative of size-selective feeding. This trend as evident hether concentrations ere compared as numbers ml- 1 or as volume'ml-i (Table 2). The volume of ingested 0.93 ~m spheres as approximately equal to that of ingested 0.6-~m spheres (233- versus 276-~m3) only hen the larger spheres occupied >6 times the space in the feeding suspension (>4'10 7 ~m3 ml-i). Phagocytosis as not limiting the volume ingested at this level, since 637-~m3 of 0.6-~m spheres ere ingested. This further suggests size-selective feeding. Vacuole diameters ere noted only for 0.6-~m suspensions> 5.10 7 'ml- I, since individual particles could be distinguished in all other treatments. Particle type did not affect volume, but concentration of spheres did. The average vacuole volumes for Cyclidium in suspensions of 0.6 ~m spheres ere 7.6-, 7.2-, 19.3-,

450 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988 Table 2. Ingestion rates (±95% confidence intervals) of Cyclidium fed 0.6-lLm and 0.93-lLm microspheres in number and volume ingested per hour Particle concentration Ingestion rate Particle type #'ml-i.um3 ml-l # h-'.um3 h-1 0.6 carboxylated 4.8 x 10' 5.4 X 10" 23 (3.7) 3 (0.4) 4.8 x 10 6 5.4 X 10' 173 (15.0) 20 (1.7) 7.0 x 10 7 7.8 X 10 6 2,441 (359.0) 276 (40,6) 1.3 x 10 8 1.4 X 10 7 5,631 (922.0) 637 (104.3) 0.6 protein-treated 5.6 x 10' 6.3 X 10 4 25 (4.0) 3 (0.4) 5.9 x 10 7 6.5 X 10 6 2,168 (249.0) 245 (28.2) 2.3 x 10 8 2.6 X 10 7 8,358 (898.2) 945 (101.6) 4.9 x 10 8 5.5 X 10 7 8,067 (1,084.0) 912 (122.6) 0.93 carboxylated 2.7 x 10' 1.1 X 10' 2 (0.5) I (0.4) 1.5 x 10 6 6.7 X 10' 25 (6.0) II (4.9) 1.0 x 10 7 4.4 X 10 6 155 (25.1) 68 (25.1) 1.0 x 10 8 4.4 X 10 7 537 (133.8) 233 (111.2) 0.93 protein-treated 3.2 x 10' 1.4 X 10' 4 (1.0) 2 (0.8) 3.9 x 10 6 1.7 X 10 6 124 (13.9) 54 (11.6) 1.8 x 10 7 7.6 X 10 6 372 (28.1) 162 (23.4) 1.3 x 10 8 5.7 X 10 7 568 (52.0) 247 (43.2) 22.9-, and 21.2-J,Lm 3 for particle concentrations of 5.9,107, 7 107, 1.3'108,2.3' 10 8, and 4.9,10 8 spheres'ml- I, respectively. Vacuole volumes of Cyclidium in microsphere suspensions :::::7.10 7 'ml- I ere significantly less than those in suspensions ~1.3 108 ml-1 (I-test, P < 0.001). There as no significant difference beteen ingestion and clearance of carboxylated (cx) and protein-treated (pr) 0.6-/-tm microspheres. Mean clearance rate ranged beteen 17 and 49 nl ciliate-1. h- I and as highest at lo microsphere density (Fig. 2). Clearance rates of 0.93 /-tm cx and 0.93 J,Lm pr micro spheres ere significantly different at concentrations belo 10 8 'ml-1 (Table 3, Fig. 2). No difference in clearance rates of carboxylated (cx, 0.93 J,Lm) and amide-modified (fx, 0.94 J,Lm) micro spheres by Cyclidium as noted hen they ere offered together at densities of 2.5' 10 5 and 5.7' 10 5 ml- 1, respectively (x 2 = 1.000, P > 0.25) or separately at similar concentrations. Clearance rates on both 0.93-J,Lm cx Table 3. Clearance rates on carboxylated (cx) and protein-treated (pr) 0.93-lLm microspheres. Cyclidium from the same culture ere offered each microsphere type and concentration in separate chambers. Clearance rates on cx and pr spheres are significantly different for similar concentrations belo 10 8 'ml- ' Clearance rates (nl' h- 1 ) Microsphere concentration (#'ml-') 0.93 ex 0.93 pt Mann-Whitney U-test 2.7 X 10' 8.0 3.2 X 10' 13.6 P < 0.02 1.5 x 10 6 16.3 3.9 x 10 6 32.5 P < 0.001 1.0 x 10 7 15.5 1.8 x 10 7 21.2 P < 0.03 1.0 x 10 8 5.2 1.3 x 10 8 4.3 P> 0.2

SANDERS: EFFECTS OF FOOD SIZE & SURFACE PROPERTIES ON CILIATE FEEDING 451 Table 4. Electrophoretic mobility (e.m.) of carboxylated (cx), protein-treated (pr), and amide-modified (fx) particles (the e.m. as measured in aged tap ater) Particle type ph Electrophoretic mobility!ilm's~'/(vollage em-')l 0.60 cx 0.60 pr 0.93 cx 0.93 pr 0.94 fx 7.82 7.82 7.33 7.33 7.79-0.27-0.16-0.29-0.17-0.27 and 0.94-,um fx micro spheres ere < 12 nl' h- I, hich agrees ith those on carboxylated microspheres in the previous set of experiments (Table 3). Measurement of electrophoretic mobility indicated that protein-treated and carboxylated spheres did have different surface properties (Table 4). The surface charges of the carboxylated and amide-modified particles ere similar. Particles ith the same coating had similar electrophoretic mobility regardless of size (Table 4). For 0.6-,um spheres, clearance rates ere relatively constant over the ide range of particle concentration offered and then decreased at very high particle densities. Hoever, for the 0.93-,um particles, rates dropped off at both the high and lo extremes (Fig. 2). A decrease in clearance as expected at high food concentrations due to limitations of phagocytosis (Fenchel, 1986). The lo clearance of both carboxylated and protein-treated 0.93-,um spheres at concentrations of < 5.10 5. ml- 1 as unexpected, since clearance rates are generally maximized at lo particle concentrations. The unusual functional response on the 0.93-,um microspheres suggested some modification of feeding behavior. When observed in different microsphere suspensions, Cyclidium spent> 96% of the time stationary and presumably feeding in all treatments except for the 0.93-,um at 3 10 5 ml-1 Ciliates in that treatment ere simming an average of 10% of the time (Fig. 3). DISCUSSION Particle size as of major importance for food capture by Cyclidium sp., as has been observed for other ciliates (revieed by Fenchel, 1986). Cyclidium sp. ingested 0.6-,um particles at high rates relative to all the larger micro spheres tested. When 1.97-,um micro spheres ere offered to Cyclidium sp., they ere ingested inefficiently and by very fe of the ciliates. The upper size limit of ingestible particles appeared to be beteen 1.5 and 2,urn, although factors such as shape and flexibility can alter this (Fenchel, 1986). Cyclidium ingested rod-shaped bacteria ith a maximum dimension greater than 2.5,urn and a idth :::::0.8,urn (personal observation). Fenchel (1980b) found that clearance of a related species, Cyclidium glaucoma, also as reduced nearly to zero hen 2-,um latex beads ere offered. A minimum ingestible size as not determined for Cyclidium sp., but has been demonstrated for other ciliates (Fenchel, 1980a; Jonsson, 1986). Size-selective feeding has generally been explained by sieving models in hich cilia or meshes filter particles from solution (Boyd, 1976; Fenchel, 1980a; 1986; J0rgenson, 1983). Yet, simple mechanical filtration did not explain differences in clearance rates by Cyclidium on protein-treated (pr) versus carboxylated (cx) microspheres. Surface properties of food affected capture rates in some metazoan

452 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988 > a: <C z o ~ ti ::E t= u. o z o t=a: o Q. oa: Q. 1.0 0.9 0.8 0.7 0.6 It) CD It) CD 0 0 0 0,...,...,...,... >< >< >< >< M CD M CD 0.93 J..lm 0.60 J..lm MICROSPHERE SIZE Figure 3. Proportion of time spent stationary in ater relative to time simming in suspensions of different sizes and concentrations of carboxylated microspheres. Numbers ithin columns are microsphere concentrations in number' ml- 1 Error bars are 95% confidence intervals for 15-25 individuals observed for 3 min each. suspension feeders (LaBarbera, 1978; Gerritsen and Porter, 1982). Surface properties ofthe microspheres appeared to affect Cyclidium feeding, but discrimination beteen pr and cx spheres as only observed for the 0.93-~m beads. There as no difference in clearance of amide-modified (0.94 ~m) and carboxylated (0.93 ~m) spheres. The cx and pr microsphere types had different electrophoretic mobility (e.m.), and the e.m. as independent of size (Table 4). This indicated that the surface charges of the cx and pr spheres ere different. The amide-modified (0.94 ~m) and carboxylated (0.93 ~m) spheres had similar electrophoretic mobility. These data suggest that an interaction of size and surface properties affected feeding on the larger, less efficiently captured spheres. The clearance rate of Cyclidium fed Microcystis as as high as 69 nl h- 1 (Gerritsen et at, 1987), hich is greater than that on any microsphere tested. Microcystis ere approximately spherical and had diameters beteen 0.8- and l.l-~m. The grazing rates on the cyanobacter ere higher than expected from the data here hich shos more efficient collection of smaller particles. One difference in the experimental setups hich may have affected grazing rates as the acclimation period. Cyclidium fed Microcystis ere kept in particle-free ater for up to 3 h, hile ciliates fed beads ere in particle-free ater for < 1 h. Higher clearance on

SANDERS: EFFECTS OF FOOD SIZE & SURFACE PROPERTIES ON CILIATE FEEDING 453 Microcystis may have been due to elevated feeding rates by "starved" ciliates, although little is knon about the effect of starvation on protozoan ingestion. Alternatively, the surface chemistry of the Microcystis may have affected ingestion. The electrophoretic mobility of Microcystis is -1.71 at a ph of 7 in aged tap ater (Gerritsen and Bradley, 1987). A greater total volume of 0.6-~m spheres could be ingested compared to 0.94 ~m (Table 2). This suggested a more efficient capture of smaller particles, hich is consistent ith Cyclidium specializing as a bacterivore. Fenchel (1986) reported a similar phenomenon for Glaucoma scintillans feeding at high particle densities (1'10 7 to 18 10 7 ~m3.ml- I ), and related it to food vacuole formation in that species. G. scintillans had food vacuoles hich ere 4-5 times larger hen 0.36- ~m spheres ere ingested compared to hen l.l-~m spheres ere consumed (Fenchel, 1986). The result of this as that the relative clearance rates on the to sizes of particles ere a function offood vacuole formation rather than of filtration. Particle concentration affected vacuole formation in Cyclidium sp. Average vacuole volume for Cyclidium in the present ork as approximately If3less hen 0.6-~m spheres ere offered at ~7'106 ~m3'ml-1 compared to suspensions of 1'10 7 to 6.10 7 ~m3.ml- I Vacuole size as not significantly different beteen cx and pr treatments for the 0.6-~m spheres. Vacuole sizes ere not determined for the 0.93- or 0.94-~m spheres, but it is possible that the differences in clearance of the larger cx and pr spheres may have been due to differential vacuole formation. The unexpected decrease in clearance at lo concentrations ofo.93-~m spheres as due to behavioral changes. Cyclidium sp. remained stationary in the ater column hen feeding, or alternatively, occasionally attached to a substrate. Floating as interspersed ith short periods of rapid simming. In the presence of 0.93-~m microspheres at lo density, Cyclidium spent more time simming than stationary relative to other particle types and concentrations (Fig. 3). The proportion of time spent simming as also more variable in the lo density 0.93- ~m treatment. The reason for the erratic feeding behavior in this treatment, but not at higher concentrations as not clear. The simming behavior of Cyclidium could be interpreted as threshold feeding, i.e., searching for better feeding patches hen acceptable "food" particles are sparse. In the spatially heterogeneous food environment of the plankton, this behavior could increase encounters ith food patches. Landry and Hassett (1985) found this explanation to be consistent ith behavior of the copepod Calanus pacificus. Conversely, the crustacean Daphnia magna did not alter simming patterns in response to algal food patches (Porter et ai., 1982). Other ciliates are knon to change feeding behavior depending on food sources. Curds and Vandyke (1966) observed empty food vacuole formation in Paramecium caudatum and Histriculus vorax hen these ciliates ere fed unfavorable bacteria. In Paramecium this as the result of a reversal of ciliary beat prior to the closure of the vacuole. These experiments demonstrate the utility of fluorescent micro spheres for examining selective feeding based on surface properties. Size and shape are removed as complicating factors hen similar-sized spheres are the only "food source." The particles in the present experiments ere chemically different. Hoever, other treatments, such as coatings derived from a favorable food source or a toxic one (e.g., Chromobacterium violaceum), may have more ecological significance than bovine serum or amide treatments. Use of fluorescent beads has the advantage that individual particles can be counted ithin food vacuoles. This allos concentrations of bacteria-sized spheres hich are ecologically real (i.e., 106to 10 7

454 BULLETIN OF MARINE SCIENCE, VOL 43, NO, 3, 1988 ml- I ) to be offered in short experiments. Single microspheres can be counted and high concentrations or long feeding times can be avoided. Furthermore, to (or more) particle types can be distinguished ithin food vacuoles if spheres ith different fluorescence are used. Fluorescent micro spheres have been used to estimate grazing by bacterivores in situ (B0rsheim, 1984; Bird and Kalff, 1986; Sherr and Sherr, 1987; Pace and Bailiff, 1987; R. W. Sanders, K. G. Porter, S. J. Bennett and A. E. DeBiase, unpub. data). Selective ingestion could alter conclusions dran from these experiments. Sherr and Sherr (1987) found that some marine spirotrichous ciliates preferred fluorescently labeled bacteria (FLBs) to microspheres, hile scuticociliates and peritrichs did not. Pace and Bailiff (1987) found no difference in ingestion ofo.57-tlm protein-coated microspheres and bacteria by a marine Cyclidium sp. in continuous culture. Using FLBs in a freshater system, I found different ciliate species preferentially ingested FLBs or microspheres or shoed no preference (Sanders et ai., in prep.). This selectivity may have been due, in part, to the different sizes and shapes of the FLBs and microspheres. Although both had similar volumes, the FLBs ere distinctly rod-shaped and up to 1 tlm or more in length (Sherr et ai., 1987, figs 2 and 7). There is unlikely to be a perfect tracer particle for all in situ experiments. This ould be true even if size and shape ere the only factors determining feeding selectivity, since natural food particles differ in these parameters. For Cyclidium sp. only particles ofa relatively large, non-optimal size shoed an effect of surface properties on clearance. If this observation is true for ciliates in general, then selectivity based on surface properties ould affect estimates of ciliate bacterivory hen the tracer particles ere greater in size than those found in the environment. ACKNOWLEDGMENTS I thank J. Gerritsen, K. G. Porter and an anonymous revieer for comments hich improved this manuscript. This research as supported by NSF Grant BSR-8407928 to K. G. Porter. The Zooplankton Behavior Symposium in hich this paper as presented as supported by Skidaay Institute of Oceanography. Contribution no. 36 of the Lake Oglethorpe Limnological Association. LITERATURE CITED Bahr, H. 1954. Untersuchungen tiber die Rolle der Ciliaten als Bakterienvernichter im Rahmen der biologischen Reinigung des Abassers. Zeitschrift fur Hygiene und Infektionskrankheiten 139: 160-181 (cited in Curds, C. R., 1977). Bird, D. and J. Kalil 1986. Bacterial grazing by planktonic lake algae. Science 231: 493-495. B0rsheim, K. Y. 1984. Oearance rates of bacteria-sized particles by freshater ciliates, measured ith monodisperse fluorescent latex beads. Oecologia 63: 286-288. Boyd, C. 1976. Selection of particle sizes by filter-feeding copepods: a plea for reason. Limno\. Oceanogr. 21: 175-180. Brendelberger, H., M. Herbeck, H. Lang and W. Lampert. 1986. Daphnia's filters are not solid alls. Arch. Hydrobio\. 107: 197-202. Cox, F. E. G. 1967. A quantitative method for measuring the uptake of carbon particles by Tetrahymenapyriformis. Trans. Amer. Microsc. Soc. 86: 261-267. Curds, C. R. 1977. Microbial interactions involving protozoa. Pages 69-105 in F. A. Skinner and J. M. Shean, eds. Aquatic microbiology. Academic Press, Ne York. -- and J. M. Vandyke. 1966. The feeding habits and groth rates of some fresh-ater ciliates found in activated-sludge plants. J. App\. Eco\. 3: 127-137. Fenchel, T. 1968. The ecology of the marine microbenthos. II. The food of marine benthic ciliates. Ophelia 5: 73-121. --. I980a. Suspension feeding in ciliated protozoa: functional response and particle size selection. Microb. Eco\. 6: 1-11.

SANDERS: EFFECTSOFFOODSIZE& SURFACEPROPERTIES ONCILIATEFEEDING 455 1980b. Suspension feeding in ciliated protozoa: structure and function of feeding organelles. Arch. Protistenkd. 123: 239-260. ---. 1986. Protozoan filter feeding. Prog. Protistology I: 65-113. Gerritsen, J. and S. W. Bradley. 1987. Electrophoretic mobility of natural particles and cultured organisms in fresh aters. Limnol. Oceanogr. 32: 1049-1058. --- and K. G. Porter. 1982. The role of surface chemistry in filter feeding by zooplankton. Science 216: 1225-1227. ---, R. W. Sanders, S. W. Bradley and K. G. Porter. 1987. Individual feeding variability of protozoan and crustacean zooplankton analyzed ith flo cytometry. Limnol. Oceanogr. 32: 691-699. Hobbie, J. E., R. J. Daley and S. Jasper. 1977. Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33: 1225-1228. Jonsson, P. R. 1986. Particle size selection, feeding rates and groth dynamics of marine planktonic oligotrichous ciliates (Ciliophora: Oligotrichina). Mar. Ecol. Prog. Ser. 33: 265-277. J0rgensen, C. B. 1983. Fluid mechanical aspects of suspension feeding. Mar. Ecol. Prog. Ser. 11: 89-103. Landry, M. R. and R. P. Hassett. 1985. Time scales in behavioral, biochemical, and energetic adaptations to food-limiting conditions by a marine copepod. Arch. Hydrobiol. Beih. 21: 209-221. LaBarbera, M. 1978. Particle capture by a Pacific brittle star: experimental test of the aerosol suspension feeding model. Science 201: 1147-1149. Luckinbill, L. S. 1973. Coexistence in laboratory populations of Paramecium aurelia and its predator Didinium nasutum. Ecology 54: 1320-1327. Nichols, W. H. 1973. I: Groth media-freshater. Pages 7-24 in J. R. Stein, ed. Phycological methods. Cam bridge Press, London. Pace, M. L. and M. D. Bailiff. 1987. Evaluation ofa fluorescent microsphere technique for measuring grazing rates of phagotrophic microorganisms. Mar. Ecol. Prog. Ser. 40: 185-193. Porter, K. G., J. Gerritsen and J. D. Orcutt, Jr. 1982. The effect offood concentration on simming patterns, feeding behavior, ingestion, assimilation, and respiration by Daphnia. Limnol. Oceanogr. 27: 935-949. Ricketts, T. R. 1971. Endocytosis in Tetrahymena pyriformis. The selectivity of uptake of particles and the adaptive increase in cellular acid phosphatase activity. Exp. Cell Res. 66: 49-58. Sanders, R. W. and K. G. Porter. 1986. Use of metabolic inhibitors to estimate protozooplankton grazing and bacterial production in a monomictic eutrophic lake ith an anaerobic hypolimnion. Appl. Environ. Microbiol. 52: 101-107. Sherr, B. F., E. B. Sherr and R. D. Fallon. 1987. Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl. Environ. Microbiol. 53: 958-965. Sherr, E. B. and B. F. Sherr. 1987. High rates of consumption of bacteria by pelagic ciliates. Nature 325: 710--711. Sleigh, M. 1973. The biology of protozoa. Arnold Publishers, Ltd., London. 315 pp. Spittler, P. 1973. Feeding experiments ith tintinnids. Oikos, suppl. 15: 128-132. Stoecker, D. K., R. R. L. Guillard and R. M. Kavee. 1981. Selective predation by Favella ehrenbergii (Tintinnia) on and among dinoflagellates. BioI. Bull. 160: 136-145. ---, T. L. Cucci, E. M. Hulburt and C. M. Yentsch. 1986. Selective feeding by Balanion sp. (Ciliata: Balanionidae) on phytoplankton that best support its groth. J. Exp. Mar. BioI. Ecol. 95: 113-130. Tucker, J. B. 1968. Fine structure and function of the pharyngeal basket in the ciliate Nassula. J. Cell Sci. 3: 493-515. DATEACCEPTED: April 11, 1988. ADDRESS:Department of Zoology, University of Georgia, Athens, Georgia 30602. ApPENDIX: DISCUSSION AFTER SANDERS J. Atema: You suggested that beads can also be used successfully in in situ studies. Do e kno enough about the actual mechanisms of ciliate feeding to assume that captures of beads realistically reflect captures of small food particles? I onder hether e are sacrificing biological significance for statistical significance by using beads?

456 BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, ]988 R, Sanders: In our field ork reported elsehere, e favored using micro spheres over bacteria primarily because of the relative ease of enumerating the ingested spheres. Preliminary experiments shoed that several ciliates ingested microspheres at rates similar to those on fluorescently labeled bacteria. The laboratory experiments reported here, hich tested the effect of surface properties on ingestion, required only that the ciliates ingest the microspheres. It as not necessary that the rates be the same as on bacteria. Hoever, from the ork of the Sherrs, M. Pace, and myself, it ould appear that beads are a good model of bacterivory for scuticociliates such as Cyclidium. M. Landry: Chemosensory mechanisms have been demonstrated to be very important in the feeding of marine Protozoa, for example Peter Verity's ork (this volume) shos that even a slightly senescent food culture elicits an obvious response. I tend to accept this vie since e have conducted recent experiments shoing that marine flagellates and ciliates select living over heat-killed bacteria by a factor of four hen presented ith both prey simultaneously. On the other hand, it seems to be OK to use inert beads in freshater feeding studies in the lab and in lakes. Are there real differences beteen conditions in marine and freshater systems or beteen bacterial and algal consumers that make these vies compatible? Beads ere used in studies of copepod feeding some time ago, but not recently since the animals generally select for living cells. R. Sanders: Areas of high food concentration appear to attract some protozoa. Hoever, preferential attraction to a high concentration of a particular food does not necessarily mean that there ill be preferential ingestion. There may be real differences for marine versus freshater systems in the utility of the approach hich I used here. Surface chemistry may not be an important factor in marine systems here there are abundant free ions. There may also be differences beteen feeding on algae and bacteria. An individual bacterium contains less carbon than most algal cells. Therefore, a grazer may be able to invest more handling time per algal particle than for the much smaller bacteria and obtain enough nutrients. This could allo for more selectivity by the algavore. K. Porter: We have tried several methods of determining bacterivory in the lake here e ork. Of all the methods, the fluorescent microspheres appeared to ork best in our system. That does not necessarily mean that it is the best method in all systems. R. Sanders: When e used micro spheres in unaltered lakeater from Lake Oglethorpe, grazing appeared to approximately balance bacterial production. Community grazing rates calculated from the use of 0.6 ILm micro spheres and fluorescently labeled bacteria ere equivalent. Some species ingested more micro spheres than bacteria and some ingested more bacteria. There may not be a perfect tracer particle, but I feel that our estimates ofbacterivory are not far from reality. P. Verity: A number of studies have demonstrated that the use of inert microspheres in copepod feeding experiments produces data hich are difficult to interpret. Scientists orking ith protozoan zooplankton are fortunate to have such data available in designing and interpreting their on experiments. Hoever, numerous differences exist in the feeding behaviors of protozoan and metazoan zooplankton. Those of us ho ork ith protozooplankton feel that our studies ould suffer if e carried metazoan biases ith us into the laboratory.

SANDERS: EFFECTS OF FOOD SIZE & SURFACE PROPERTIES ON CILIATE FEEDING 457 M. Gliicz: Does your data suggestthat they should be ingesting inorganic particles in lakes? R. Sanders: I have not observed ingestion of clay particles, hich are sometimes abundant in southeastern U.S. lakes during spring runoff. I am not sure about other inorganic particles. D. Stearns: In your 15 min grazing experiments comparing different plankton groups, did you take into consideration periodicities in feeding intensity by different groups? For example, cladocerans sho a diel periodicity in feeding. R. Sanders: We did not address that factor, although e have planned for diurnal studies in the future. Little is knon about feeding periodicity in protists. Bird found that Dinobryon had the same clearance rate during the day and night. I am not aare of any other studies ith protists hich address this question.