Limnol. OctBanogr..

35(l),
0 1990, by the American

1990, 16-23 Society of Limnology

and Oceanography,

Inc.

Differences in particle size-dependent feeding efficiencies of closely related rotifer species

Karl 0. Rothhaupt
Max-Planck-Institute of Limnology, Department of Physiological Ecology, P.O. Box 165, D-2320 Plan, FRG Abstract Size-selective feeding of four Brachionus strains was studied with three experimental setups: selection between polystyrene spheres of different sizes in short-term ( 10 min) feeding experiments, selection between pairs of dual-labeled algal taxa in short-term feeding experiments, and selection among three algal taxa in long-term (24 h) feeding experiments. Food size preferences were related to body sizes between strains but not within one strain (Brachionus calyciflorus). Brachionus angularis preferred food items <5-~m equivalent spherical diameter (ESD), Brachionus rubens strain F fed most efficiently on particles -5km ESD, and B. caZycijlorus on particles of - lo-pm ESD. Brachionus rubens strain B ingested particles from 3.5 to 12-pm ESD equally well. Apart from particle size effects, feeding was unselective. Polystyrene spheres of the appropriate sizes were ingested readily.

Exploitative competition for food is believed to be a major structuring element of zooplankton communities. Consequently, there has been much recent interest in the interspecific role of food Limitation (e.g. Lampert 1985). Herbivorous zooplankton differ in their feeding strategies and modes of food selection (e.g. DeMott 1986; Bogdan and Gilbert 1987), raising the possibility of differential resource use. Within genera, food-size preferences are often related to body sizes. Gilbert and Bogdan (1984) gave evidence for such a pattern from in situ studies with the rotifers Polyarthra and Keratella. Food-size partitioning by differently sized congeners is believed to be an important phenomenon among herbivorous zooplankton (e.g. Hutchinson 1965; Gilbert and Bogdan 1984), and its potential for allowing steady state coexistence was shown in laboratory experiments with the rotifers Brachionus rubens and Brachionus calyciJlorus (Rothhaupt 1988). Rotifers of the genus Brachionus have a cosmo:politan distribution, short generation times, and are easy to culture. They are thus -LPresent address: Institute of Lake Research and Fisheries, Untere SeestraBe 8 1, D-7994 Langenargen, FRG. Acknowledgments Much of this work was stimulated by W. R. DeMott when he was a Max-Planck research fellow in P16n. W. Lampert, J. Grover, and Z. M. Gliwicz commented on the manuscript.

an ideal model system for testing theoretical concepts that predict the outcome of direct or indirect interactions from ecophysiological traits. Rotifer feeding modes have been related to features in the corona morphology (Edmondson 1965). Brachionus spp. are regarded as generalist suspension feeders that are able to collect and process numerous small cells simultaneously (Gilbert and Bogdan 1984). Due to this mode of feeding, one could expect them to ingest particles equally well over most of their food size range. The aim of this paper is to compare the food size preferences of four differently sized strains of the genus Brachionus. Three different experimental protocols were involved: selection between polystyrene spheres of different sizes, long-term feeding experiments with algae: comparable in size to the spheres, and short-term, dual-label experiments involving seven algal species from 1- to 18-pm ESD (equivalent spherical diameter). The study centered around two basic questions: are food size preferences related to body size between and within species, and how do feeding efficiencies change over the range of ingestible particle sizes? .Materials and method:; Experimental animals-Brachionus angularis (adult lorica length 100-l 40 pm; all length measurements are my unpublished data) was obtained frolm N. Walz and was 16

Brachionus

size selectivity

17

Table 1. Food algae used in selectivity experiments. ESD is equivalent spherical diameter (pm); culture number is from the culture collection at the University of GWingen.
ESD Shape Culture No.

Synechococcus elongatus Chlorella minutissima Monoraphidium minutum Chlamydomonas reinhardii Cyclotella meneghiniana Chlamydomonas sphaeroides Micractinium pusillum

2 3.5 6 8.5 12 18

Oblong Spherical kcuate Spherical Cylindrical Spherical Aggregates

1.80 243- 1 11-32 10200la 58.72 13.81

originally isolated from a lake near Munich (Walz 1983). Brachionus rubens strain F (120-l 80 pm) came from a culture kept at the University of Frankfurt and was isolated from a pond near Frankfurt (Halbach et al. 1983). Brachionus rubens strain B (ZOO-260 pm) was isolated from the Grol3er Binnensee near Plijn. Brachionus calyctflorus (220285 pm) was hatched from resting eggs that were obtained from M. Schliiter. Animals were maintained in the laboratory in 300ml Erlenmeyer flasks with modified Chu- 12 medium (Miiller 1972). They were fed Monoraphidium (about 2.5 mg C liter-) every 2 d, and medium was renewed every 2 weeks. All culturing and experimentation was done at 20_+ 1C. Food algae (Table 1) -All chlorophytes and the diatom Cyclotella were grown in modified Chu-12 medium. The blue-green Synechococcus was cultured in the medium of OFlaherty and Phinney ( 19 70). Reagentgrade chemicals (Merck) were used throughout. Algae were cultured in continuous light in chemostats (residence time, - 1.3 d) or in frequently diluted batch cultures. Light extinction (800 nm) of algal cultures was measured to estimate carbon content with previously established calibration curves. Carbon content was determined by combustion and measurement of infrared absorption (Krambeck et al. 198 1). The ESD of algae was determined with a Coulter counter TA II (140~pm aperture). ESD was taken as the mode of the size distribution. Selection between polystyrene spheres Experiments with spheres followed the methods described by DeMott (1986). Experiments were run in two parallel jars with 20 ml of medium each. Rotifers were fed 1 : 1 mixtures of spheres of either Z- and 6-pm diameter or 6- and 12qm diameter

(5 X lo3 ml- of each size). Monoraphium cells were usually present in a concentration 10 times higher than the beads ( lo5 cells ml-l). About 50 animals that had been prefed for at least 1 h at the appropriate cell concentrations without spheres were pipetted into the jars. Feeding time was restricted to 10 min, which is below gut passage times. Then the animals were collected on 5&m sieves, narcotized in carbonated water (30 s), and fixed in a Petri dish with a few drops of formaldehyde (40%). About 20 animals from each jar were transferred to microscope slides, and tissues were cleared with tissue solubilizer (Soluene-350, Packard). The number of spheres in the gut was counted under a compound microscope. In one experiment with B. calyciflorus, the length of the lorica was also measured with an ocular micrometer to the nearest 1.5 pm. Long- term feeding experiments-Tubes holding 25 ml of food suspension received 125 animals each (four replicates for B. rubens, three for B. calycijlorus). Three tubes without animals served as controls. Food suspensions were Chlamydomonas sphaeroides (0.4 mg C liter-), Monoraphidium minutum (0.2 mg C liter-l), and Chlorella minutissima (0.1 mg C liter-l). The tubes were kept for 24 h in the dark on a rotating wheel at about 1 rpm. After incubation the animals were counted and the algae fixed with Lugols solution. After settling for at least 24 h in cylindrical chambers (25-mm diam, 25-ml vol), the algae were counted under an inverted microscope (Utermohl 1958). At least 800 cells of each species were counted for the small algae; two diameters of the counting chamber for Chlarnydomonas. Calculation of clearance rates followed standard procedures (Peters 1984). Dual- label experiments - Two food algae,

Brachionus size selectivity

19

; T g 3 w 5 w Jz

6420 . 12lo-

Brachionus angularis

Brachionus Strain F + H-l Brachionus calycif lorus

ru bens

86:O

"0

0.5

1.5

2.5

DIAMETER Fig. 2. Clearance rates for different sizes of polystyrene spheres, calculated from the selectivity experiments in Fig. 1 (mean rS1 SD).

experiments with B. rubens strain F, selectivities between 6- and 12-pm spheres remained unchanged without algae and in the presence of different densities of Monoraphidium (Table 2). In another experiment, 6-pm beads were used as a tracer to estimate clearance and ingestion rates at different concentrations of Monoraphidium. Algal cell densities were 10 times higher than
Table 2. Brachionus rubens strain F. Selectivity between 6- and 12-brn polystyrene spheres in media without algae and in different concentrations Monoraphidium. (n = No. of animals).
Monoraphidium (mg C liter-l) (cells ml-l) Spheres ml (per si7e) No. of ingested spheres
n 6m

0.5

1.5

2.5

FOOD CONCENTRATION (mg C liter) Fig. 3. Clearance and ingestion rates of Brachionus rubens on Monoraphidium, determined with 6-pm spheres as tracers. Lines are rates determined in radiotracer experiments (Rothhaupt 1990). Short dashes-Monoraphidium (3.5~pm ESD); long dashesChlamydomonas reinhardii (6-pm ESD).

12crm

D,

0.1 0.5 2.5

2x lo4 105 5x105

104 10 5x103 5~10~

22 32 33 48 27 33 38 36 61 39

337 59 58 81 279 339 355 39 82 63

4 3 2 4 8 16 15 1 1 1

0.976 0.903 0.933 0.906 0.944

0.910 0.959 0.950 0.976 0.969

the concentration of spheres. Clearance and ingestion rates were comparable to results with radiolabeled algae (Fig. 3). Long- term feeding experiments - In these experiments the algae were chosen to be similar in size to the beads. Monoraphidium was chosen for the medium size because its distinct shape facilitates microscopic identification. Brachionus rubens strain F and B. calyciflorus had similar particle size-dependent clearance rates on the algae as they had in the experiments involving plastic spheres (Fig. 4). Again, B. calycijlorus ingested the biggest food item most efficiently, whereas B. rubens strain F had highest clear-

20
-iL: 7 cl 6 12 10 8, -I Brachionus - StrainF

Rothhaupt
rubens

Brachionus angularis

Brachionus Strain F
8 5 w 6 2 12 10 8 4 2 6i 0 Cm. Mm.

ru bens

Brachionus Strain B

ru bens

Fig. 4. Clearance rates from long-term feeding experiments with three algae offered simultaneously (mean k I SD). C.m.-Chlorella minutissima (2-pm ESD); M.m.- Monoraphidium minutum (3.5~pm ESD); C.S.Chlamydomonas sphaeroides (12~pm ESD).

ante rates on the medium-sized alga (cf. Fig. 2). ES0 (yrn) Dual-label experimentsAmong the sevFig. 5. Relative clearance efficiencies (mean + 1 en food algae (Table I), the most rapidly SD) for seven algae (cf. Table 1) of different sizes ingested proved to be Monoraphidium (ESD (ESD). The most rapidly ingested food item for each strain is marked with a black dot (efficiency = 1). = 3.5 blrn) for B. angularis, Chlamydomonas reinhardii (ESD = 6 grn) for both B. rubens strains, and Cyclotella meneghiniana (ESD among rotifer size classes (Table 3) but the = 8.5 pm) for B. calyciflorus. All other algae largest animals had significantly higher were offered in pairwise comparison with ingestion rates than the smaller size classes. them. The results show (Fig. 5) that B. anDiscussion gularis most efficiently ingested algae <5The experiments show that in the genus pm ESD. Brachionus rubens strain F had highest efficiencies on particles - 5-pm ESD Brachionus ingestion is largely particle sizeand B. calyciflorus on particles - IO-pm dependent. All strains examined, with the ESD. Brachionus rubens strain B had the exception of B. rubens, strain B, have a relwidest efficiency spectrum and ingested al- atively narrow spectrum of efficiently ingae from 5 5- to L: lo-pm ESD equally well. gestible particle sizes. Apart from particle For this strain, there was no significant dif- size effects, feeding appears to be unselective. Larger species prefer larger food items. ference in clearance rates among MonoraSuch patterns may be important in allowing phidium, Cyclotella, and both Chlamydomonas species (ANOVA: IT = 1.06; 3,11 dc the coexistence of congeneric herbivorous P = 0.406). Small particles were ingested zooplankton (Hutchinson 1965; Gilbert and with relatively low efficiency by all rotifer Bodan 1984). Laboratory experiments have shown that strains. the outcome of competition between B. ruEficts of body size within a species-In an experiment with B. calycz$orus, the an- bens strain F and B. clzlyctjlorus can be preimals were offered 6- and 12-pm spheres. dicted from the food offered and that both When counting the ingested beads, lorica species can coexist on a mixed diet (Rothlength was measured for each animal. There haupt 1988). Brachionus rubens strain F was no significant difference in selectivities more efficiently ingests the small M. minu-

Brachionus size selectivity

21

Table 3. Selectivilies between 6- and 12-pm beads and ingestion rates of four size classes of Brachionus cu/~@Zoru~(lorica length). Ingestion rates are expressed as the sum of the relative volumes of ingested spheres (6-pm spheres: volume = 1; 12-pm spheres: volume = 8). Number of animals (n) is not identical for selectivities and ingestion rates, because no D, can be calculated for animals that did not ingest any beads. Only the biggest size class has a significantly higher ingestion rate (Tukeys multiple range test. P -=z0.05).
4
Length (pm) n Estimate SE n Ingestion rate Estimate SE

141-168 171-19s 20 l-228 23 l-285

24 58 27 17 I;= 1.471;

-0.222 -0.349 -0.111 -0.376 3, 122 df

0.133 0.060 0.115 0.130

P = 0.226

25 62.6 74 60.2 33 60.8 17 139.0 F = 7.738; 3, 145 df

11.9 6.6 12.3 18.9 P < 0.001

turn (ESD = 3.5 pm) and is the superior competitor, reaching a given population growth rate at lower food concentrations when this alga is fed. The same is true for B. calyciflorus with C. sphaeroides (ESD = 12 pm) as food. Using Tilmans (1982) graphical method, I predicted coexistence or extinction of one or both species from the supplied concentrations of both foods. The predictions were tested in semicontinuous laboratory experiments at two dilution rates (equivalent to experimentally imposed mortality rates) with six different food preparations. There was a high degree of correspondence between prediction and experiment: the outcome of exploitative competition was correctly predicted by the model in 11 of 12 cases. In contrast to the pattern between species, selectivities do not change significantly with body size within B. calyciflorus. This pattern may be due to eutelic body growth in rotifers. There are no more cell divisions after hatching from the egg (Ruttner-Kolisko 1972) and it may be that the cilia and cirri, constituting the feeding apparatus on the corona, do not grow isometrically with body size. Very small particles generally are ingested with low efficiency. The relative clearance efficiency for the smallest food alga tested (Synechococcus, ESD = 1 pm) is highest for B. angularis ( Wsyn = 0.494&O. 119) and lowest for B. calyciflorus ( WSYn = 0.043 kO.0 12). Synechococcus is large relative to field bacteria. It thus may be concluded that Brachionus species are not significant grazers on unattached bacteria or phototrophic picoplankton. Low ingestion

rates on bacteria-sized particles have been reported for B. rubens (Pilarska 1977) and for B. calyciflorus (Starkweather et al. 1979; Seaman et al. 1986). Brachionus ingests inert particles if they are of the appropriate size (DeMott 1986). There are differences in selectivity, however, between polystyrene spheres and algae of similar sizes: Brachionus angularis did not ingest any 12-pm beads, but algae of this size were still ingested, although with low efficiency (WChla12= 0.197+0.137 for C. sphaeroides); B. rubens strain F selected more strongly against 12-pm spheres (012 = -0.938kO.03) than against C. sphaeroides in dual-label experiments (&hla 12= -0.225-1-0.136) and in long-term feeding experiments (&hl a 12= -0.65620.03); B. rubens strain B selected against 12-ym sphercs, whereas algae from 3.5 to 12-pm ESD were ingested equally well in radiotracer experiments. In all these cases,particles are near the upper food size limit, and the differences may be due to different size-frequency distributions of algae and spheres. The modes of the size distributions of algae and spheres are identical, but algal cell sizes have broader variances, extending into smaller and more ingestible size classes(Fig. 6). Another explanation may be the different texture of particles. It is possible that hard spheres cannot be ingested if they are bigger than the mouth opening, but that softer and more flexible algae of the same size can still be eaten. Hard, artificial particles like plastic or glass spheres seem to be unsuitable to estimate the maximal size of ingestible natural food (Nadin-Hurley and Duncan 1976).

22
80 60

Rothhaupt

60

12

12

CHANNEL

No.

Fig. 6, Comparison of the size distributions of Chlamydomonas reinhardii (left) and of Chlamydomonas sphaeroides (right) with polystyrene spheres of similar sizes. Measurements were done with a Coulter counter. Algae--@; spheres-O.

a bioassay tool for ecotoxicological tests in aquatic environments. Ecotoxicol. Environ. Safety 7: 484513. HUTCHINSON, G. E. 1965. The ecological theater and the evolutionary play. Yale. .fAcOEIs, J. 1974. Quantitative measurement of food selection. A modification of the forage ratio and Ivlevs electivity index. Oecologia 14: 4 13-4 17. KRAMBECK, H.J., W. LAMPERT, AND H. BREDE. 1981. Messung geringer Mengen von partikularem Kohlenstoff in natiirlichen GewHssem.GIT Fachz. Lab. 25: 1009-1012. LAMPERT, W. [ED.]. 1985. Food limitation and the structure of zooplankton communities. Ergeb. Limnol. 21. ~ AND B. E. TAYLOR. 1985. Zooplankton grazing in a eutrophic lake: Implications of diel vertical migration. Ecology 66: 68-82. MILLER, H. 1972. Wachstum und Phosphatbedarf von Nitzschia actinastroides (Lemm.) v. Goor in statischer und homokontinuierlicher Kultur unter Phosphatlimitierung. Arch. Hydrobiol. Suppl. 38, p. 399-484. NADIN-HURLEY, C. M., AND A. DUNCAN. 1976. A comparison of daphnid gut particles with the sesReferences ton present in two Tharnes reservoirs throughout BOGDAN, K. G., AND J. J. GILBERT. 1987. Quantithe season. Freshwater !Biol. 6: 109-123. tative comparison of food niches in some fresh- O'FLAHER-IY, L.M., AND H.K. PHINNEY. 1970. Rewater zooplankton. A multi-tracer-cell approach. quirements for the maintenance and growth of I 0ec:ologia 72: 33 l-340. Aphanizomenon j7os-aquae in culture. J. Phycol. DEMO-IT, W. R. 1986. The role of taste in food se6: 95-97. lection by freshwater zooplankton. Oecologia 69: PETERS,R. H. 1984. Methods for the study of feeding, 334-340. grazing, and assimilation by zooplankton, p. 336-. 1988. Discrimination between algae and ar412. In J. A. Downing and F. H. Rigler [eds.], A manual on methods for the assessment of secondtific:ial particles by freshwater and marine copeary productivity in fresh waters, 2nd ed. IBP pods. Limnol. Oceanogr. 33: 397-408. Handbook 17. Blackwell. EDMONI)SON, W. T. 1965. Reproductive rate of planktonic rotifers as related to food and temperRLARSKA, J. 1977. Eco-physiological studies on Brachionus rubens Ehrbg. (Rotatoria). 1. Food seature in nature. Ecol. Monogr. 31: 6 l-l 11. lectivity and feeding rate. Pol. Arch. Hydrobiol. GILBERT, J. J., AND K. G. BOGDAN. 1984. Rotifer 24: 3 19-328. grazing: In situ studies on selectivity and rates, p. ROIHHAUPT, K. 0. 1988. E4echanistic resource com97-l 33. Zn Trophic interactions within aquatic petition theory applied to laboratory experiments ecosystems. AAAS Select. Symp. 85. Westview. with zooplankton. NatLlre 333: 660-662. HALBACH, U.,M. SIEBERT, M. WE:STERMAYER,AND C.

In conclusion, the experiments reported here confirm the view that apart from particle size, Brachionus spp. have a generalist, poorly discriminating feeding mode. Similar to other rotifer genera (Gilbert and Bogdan 1984; Bogdan and Gilbert 1987), particle size-dependent ingestion efficiencies may differ significantly between species or strains. Ingestion efficiency is not constant over the range of ingestible particle sizes within one species. Particle size is thus an important feature of food quality. Whenever functional or numerical responses of Brachionus species are determined and compared, the results may depend crucially on the size of the food items used. This fact is important in determining the trophic role of Brachionus species in nature and aquaculture.

WISSEL. 1983. POpUkitiOII eCOlOgy of rotifers as

Brachionus size selectivity

-. 1990. Changes of the functional responses of the rotifers Brachionus rubens and Brachionus caIyciJlorus with particle sizes. Limnol. Oceanogr. gewasser 26: 99-234.
SEAMAN, M. T., M. GOPHEN, B. Z. CAVARI, AND B. f&OULAY. 1986. Brachionus calyciflorus Pallas as agent for the removal ofE. coli in sewageponds. TILMAN, D.

23

35: 24-32. RUTTNER-KOLISKO, A. 1972. Rotatoria. Die Binnen-

Hydrobiologia 135: 55-60.

STARKWEATHER, P.L.,J.J. GILBERT,AND T.M. FROST. 1979. Bacterial feeding by the rotifer Brachionus calyciflorus: Clearance and ingestion rates, behavior and population dynamics. Oecologia 44: 2630.

1982. Resource competition and community structure. Princeton. UTERM~HL, H. 1958. Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mitt. Int. Ver. Theor. Angew. Limnol. 9. 38 p. VANDERPLOEG, H. A., AND D. SCAVIA. 1979. Calculation and use of selectivi ty coefficients of feeding: Zooplankton grazing. Ecol. Model. 7: 135-149. WALZ, N. 1983. Continuous culture of the pelagic rotifers Keratella cochlearis and Brachionus angularis. Arch. Hydrobiol. 98: 70-92.

Submitted: 8 February 1989 Accepted: 7 June 1989 Revised: 3 October 1989