The Effect of Electrostatic Charge of Food Particles on Capture Efficiency by Oxyrrhis marina Dujardin (Dinoflagellate)

Protist, Vol. 150, 375-382, December 1999 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protist

Protist

ORIGINAL PAPER

The Effect of Electrostatic Charge of Food Particles on Capture Efficiency by Oxyrrhis marina Dujardin (Dinoflagellate) Astrid Hammera,1, Cordula Gruttnerb , and Rhena Schumanna a b

University of Rostock, Department of Biology, Experimental Ecology, Freiligrathstr. 7-8, 18055 Rostock, Germany microcaps GmbH, Friedrich-Barnewitz-Str. 4, 18119 Rostock, Germany

Submitted July 20,1999; Accepted October 14,1999 Monitoring Editor: Michael Melkonian

Laboratory experiments were carried out to investigate the effect of food quality, measured as surface charge of the particles, on capture efficiency and ingestion rate by the heterotrophic dinoflagellate Oxyrrhis marina. Fluorescent particles in two size classes of around 1 and 4 IJm and of 7 different qualities were offered to the flagellate: carbohydrate and albumin particles, the algae Synechocysfis spec. and Chlorella spec., carboxylated microspheres, silicate particles and bacteria. Rates of particle uptake showed significant differences depending on particle size and quality, and ranged from oto 4 particles cell-1 h-1 • Ingestion rates were up to 4 times higher for 4 IJm particles than for 1 IJm particles, which indicates strong size-selective feeding. Our main result is that the surface charge or zeta potential, of artificial particles, i.e. carboxylated microspheres (~-107 mY) and silicate particles, strongly differ from more natural and natural food (~-17 mY). For both size classes Oxyrrhis had ingestion rates up to 4 times higher for particles with less negative charge, such as albumin particles or algae. Thus, the zeta potential of the model food should be considered in experimental design. Particles with a zeta potential similar to that of natural food, e.g. albumin, seem to be the preferred model food.

Introduction Recent research has confirmed that heterotrophic protozoa play an important part as major consumers of pico- and nanoplankton (e.g. Archer et al. 1996; Goldman et al. 1989; Pernthaler et al. 1996) and therefore in the control of carbon flux through the microbial food web in natural waters. Thus, it is necessary to obtain a comprehensive understanding of their physiology, and in particular those aspects that relate to their ingestion of food, such as their feeding 1 Corresponding author; fax 49-381-498-2011 e-mail [email protected]

selectivity. Preference for certain prey items may be explained by discrimination on grounds of size, chemical composition, or a combination of both. Previous studies (Baldwin and Newell 1991; Gonzalez et al. 1990; Hansen et al. 1994; Holen and Boraas 1991; Simek and Chrzanowski 1992; Tobiesen 1990) demonstrated that size is of major importance as a selective criterion. Chemical discrimination of food by protozoa (Bennett et al. 1988; Buskey 1997; Landry et al. 1991; Verity 1991) or metazoan suspension feeders (Gallager et al. 1991) has scarcely been investigated, as it is presumed to be too subtle and difficult to quantify (Flynn et al. 1996). One way 1434-4610/99/150/04-375 $ 12.00/0

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of obtaining a chemical classification of food is to record its surface charge. The cell surface can be understood as a multivalent ion containing negatively and positively charged groups, which ionise as a function of pH, temperature and ionic strength of the suspending medium. The net surface charge or zeta potential can be measured by particle microelectrophoresis. (Benderliev et al. 1988; Honda et al. 1998; Smith et al. 1998). The role of cell surface charges has been considered to be important when discussing interactions in mycoparasitism, i.e. between microorganisms and host surfaces (Smith et al. 1998) or in separation of microorganisms (E. coli) by chitosan-conjugated magnetic particles (Honda et al. 1998). In the explanation of capture efficiency and particle selectivity by proto- (Sanders 1988) or metazoan (Gallager et al. 1991; Gerritsen and Porter 1982) the zeta potential of particle surfaces has scarcely been used as a relevant parameter. At any rate, these studies observed only filtrating suspension feeders, a ciliate, the zooplankter Daphnia and mollusc larvae. There exist no data about raptorial feeders. To study the influence of surface charge of food particles on capture efficiency and selective grazing in flagellates, the widespread species Oxyrrhis marina, an omnivorous phagotrophic dinoflagellate, was used as a model organism. Size selective feeding of Oxyrrhis has been widely studied. So this flagellate is described as a raptorial feeder (Goldman et al. 1989) and therefore, is specialised to some extent on larger particles (Flynn et al. 1996; Hansen et al. 1996; Schumann et al. 1994; Tobiesen 1990). However, there is no unanimity over chemical discrimination of food in Oxyrrhis (Flynn and Fielder 1989; Tarran 1992). Flynn and Fielder (1989) suggested that the uptake of free amino acids by Oxyrrhis is associated with chemosensing. In contrast following Tarran (1992), chemosensitivity seems to be non-existent in this flagellate. This would correspond to Flynn et al. (1996) and H5hfeld and Melkonian (1998). The authors reported for this organism that cells come into contact with their prey by accident and that predation depends mainly on the probability of collision. Our hypothesis is that great differences between the surface charge of organism and food enhance the probability of collision and contact duration between them and consequently the uptake rate of food by the organism. In this study, the ingestion rates of a wide range of particle classes were estimated and compared with regard to their surface charge measured as zeta potential. To distinguish effects of surface properties from size selection, we applied particles in two size classes of around 1 and 4 IJm.

Results Table 1 summarises all food particles used and their properties. Fluorescent particles of two size classes (about 1 and 4 IJm) and of 7 different surface properties were offered to Oxyrrhis marina. On the one hand, we used larger particles (4 IJm) as models for algal food: starch particles, albumin particles, the cyanobacterium Synechocystis spec.; the chlorophyte Chlorella spec. and carboxylated spheres, and small particles, simulating bacteria as food: starch particles, albumin particles, silicate particles, carboxylated spheres and the bacterium Pseudomonas f1uorescens, DTAF stained. Silicate particles were not ingested. In all other experiments, Oxyrrhis marina showed linear uptake of particles to a maximum value, after which particles per cell remained constant due to an equilibrium between ingestion, digestion or egestion. Slopes of ingestion were significantly non-zero in all cases, except for that with silicate particles (Table 1). The time elapsed between the addition of particles and levelling off the uptake curve ranged from 40 to 120 min, depending on particle type. Figure 1 shows the pattern of particle ingestion in the protists during an experiment. Rates of particle uptake for Oxyrrhis ranged from o to 4 particles cell-1 h-1 (Table 1). Calculated clearance rates are presented in the same table. Ingestion rates were up to 4 times higher for 4 IJm particles than for 1 IJm particles, which indicates strong size-selective feeding. The correlation between particles/individual and time was given by r2 > 0.95 for

5

,,

,.,.'- - -b--.--;-~-=-=-'::'-1:-:-:_;-;;_:-:_=--_..-1

120

160

200

240

280

mm

Figure 1. Ingestion of Syneccocystis spec. (pee 6803, initial concentration 106 ml-1) by Oxyrrhis marina, mean ± 95% confidence interval, dashed line: linear regression.

Food Particle Capture by Oxyrrhis

377

Table 1a. 1 IJm particles: mean of equivalent spherical diameter (ESD), ingestion rate (lR) and significance of its slope (r2 and p) and calculated clearance rate (CR). particle class

origin

silicate particles carboxylated microspheres bacteria albumin particles

microcaps Polyscience BA321 microcaps

ESD (IJm)

1.00 0.90 1.00 0.90

IR (part ind h-1) 0.00 0.06 1.34 1.00

SO

0.00 0.05 0.30 0.19

IR-slope r2

p

0.87 0.93

<0,05 <0,05

CR (nl ind h-1)

0.06 1.34 1.00

Table 1b. 4 IJm particles: mean of equivalent spherical diameter (ESD), ingestion rate (lR) and significance of its slope (r2 and p) and calculated clearance rate (CR). particle class

origin

carboxylated microspheres starch particles Synechocystis Chlorella albumin particles

Polyscience microcaps PCC 6803 CCMP255 microcaps

ESD (IJm)

4.00 4.20 2.35 3.70 3.60

all 4 IJm particles (p < 0.01), as compared to only 0.87 for FLB (p < 0.05) and 0.93 for 1 IJm albumin particles (p < 0.05), Le., small particles. Comparing 5 particle types of 4 IJm diameter revealed significant differences in absolute rates between slopes of ingestion rate (p < 0.001, KruskalWallis-test, graph pad prism 2, Table 2). Ingestion rates ranged between 1.7 particles cell- 1 h- 1 for carboxylated microspheres and 4.0 particles cell-1 h-1 for starch particles. Highly significant differences between rates of ingestion for starch particles and

IR (part ind h-1) 1.68 4.02 2.88 2.82 3.10

SO

0.13 0.39 0.18 0.29 0.23

IR-slope r2

p

0.96 0.95 0.99 0.97 0.98

<0,0001 <0,0001 0.004 0.002 0.002

CR (nl ind h-1)

1.68 4.02 2.88 2.82 3.10

carboxylated microspheres particles were observed (Table 1b). We did not detect any significant differences in absolute rates between slopes of ingestion rate of Synechocystis, Chlorella, and 4 IJm albumin particles (p > 0.05): slopes and intercepts are equal (Table 2). Mean ingestion rates ranged between 2.8 and 3.1 particles cell-1 h-1 • Because Oxyrrhis is rather specialised on larger food particles (Flynn et al. 1996; Hansen et al. 1996; Schumann et al. 1994; Tobiesen 1990), particles around 1IJm in diameter were ingested at low rates

Table 2. Differences between ingestion rates (Mann-Whitney-test) of particles by Oxyrrhis cms1

FLB

Albumin1 IJm

cms4IJm

FLB

ns Albumin 1 IJm

ns ns cms4IJm

Synechocystis

Chlorella

Albumin 4 IJm

Starch 4 IJm

Synechocystis

ns

ns

ns

Chlorella

ns Albumin 4 IJm

ns ns Starch 4 IJm

Silicate 1 IJm ns cms1IJm

FLB: fluorescently labelled bacteria, cms: carboxylated microspheres, ***p < 0.001, **p < 0.01, *p < 0.05 ingestion rates highly significant to significant different; ns ingestion rates not significant different.

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Hammer et al.

Table 3. Zeta potential for each particle class at pH 7.8

particle class

zeta potential (mV)

1 IJrn particles

silicate particles carboxylated microspheres albumin particles FLB

-70.3 -60.8 -20.0 -18.4

4 IJrn particles

carboxylated microspheres Chiarella Synechocystis albumin particles starch particles

-107.0 -41.4

-36.9 -25.0 -17.3

of between 0 and 1.3 particles cell-1 h-1 • Differences of ingestion rates between 4 particle types were highly significant. Silicate particles were not ingested, 1 IJm carboxylated microspheres at rates near to zero. Ingestion rates of FLB and 1 IJm albumin particles were equal (Table 1band 2). The zeta potentials of the particles used were in the order of -107.0 for 4 IJm carboxylated microspheres and -17.3 mV for starch particles (Table 3). Measurements indicated that particularly the zeta potential of artificial particles, i.e. carboxylated microspheres and silicate particles, strongly differ from more natural and natural food. The surface charges of Chlorella and Synechocystis as well as FLB and

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1 IJm albumin particles showed only weak differences. Starch particles and 4 IJm albumin particles were less negative as natural food of 4 IJm. Surface charges, measured as zeta potential at different pH, provide not only information about contact probabilities, but also about the chemical composition of particle surfaces. Under weak basic pH 7.8 of our culture medium, surfaces of carboxylated microspheres are negatively charged (107 mV) due to dissociation (deprotonisation) of the carboxyl group. The surface of silicate particles is characterised by silica groups, which are thus negatively charged under basic pH. Natural food such as Chlorella, Synechocystis or Pseudomonas are different in the composition of surface groups, such as phosphatidic, sulphate, carboxyl and amino groups. Their net charge at pH 7.8 is even less negative than silicate particles and carboxylated microspheres. Starch and protein particles possess hydroxyl and amino groups and are nearly neutrally charged under basic pH, where dissociation (protonisation) does not occur. The amount of particles ingested per flagellate per hour (slope of ingestion rate) versus zeta potential was plotted for each particle used (Fig. 2 and 4). The two size classes were considered separately. Both slopes were significantly non-zero. The correlation coefficient for the linear regression was 0.90 for 4 IJm particles (p < 0.01) (Fig. 2) and 0.96 for 1 IJm particles (p < 0.05) (Fig. 3). From both figures, it is evident that Oxyrrhis has a higher ingestion rate for particles with less negative charge.

OIl

0.0 -80

.S

... -70

O+----,---,-----;,-----,----j -60

-50

-40

-30

-20

-10

zeta potential (mV) Figure 2. Relationship between zeta potential of 1 IJm particles and ingestion rate of these particles by Oxyrrhis marina, r2 = 0.96, cms: carboxylated microspheres,

dashed line: 95% confidence interval, pH 7.8. • silicate, 0 ems, • albumin, 6. FLB.

-125

-100

-75

-50

-25

o

zeta potential (mY)

Figure 3. Relationship between zeta potential of 4 IJm particles and ingestion rate of these particles by Oxyrrhis marina, r2 = 0.86, cms: carboxylated microspheres, dashed line: 95% confidence interval, pH 7.8. 0 cms, T Chlorella, D Synechocystis, • albumin, 6. starch.

Food Particle Capture by Oxyrrhis

Discussion As emerges from this and other studies (Goldman et al. 1989; Hansen et al. 1996; Schumann et al. 1994), Oxyrrhis marina is highly versatile, being capable of grazing on a wide assortment of particles. However, this organism shows a preference for food with an ESD 4~m, which is consistent with Oxyrrhis specialised as a raptorial feeder (Goldman et al. 1989). According to Fenchel (1986), below a size ratio of the radii of predator to prey of about 10:1 or lower raptorial feeding would be expected. Our results of the uptake experiments indicated size-selective feeding of Oxyrrhis marina. The flagellate cleared 4 ~m particles at a faster rate then 1 ~m particles. Estimates of clearance rates for 4 ~m particles were in the same range as reported in previous studies on Oxyrrhis, obtained under similar high initial food concentrations of at least 106 particles ml-1, e.g., with the clearance rates of 90-100 nl ind-1 d-1 reported by Goldman et al. (1989) with /sochrysis ga/bana and Dunaliella tertio/ecta as prey; or of 55 nl reported by Davidson et al. (1995) with /sochrysis ga/bana as food. The 70-100 nl we calculated for Oxyrrhis for natural and semi-natural food particles fits to the middle of this range. Waterhouse and Welschmeyer (1995) estimated much higher clearance rates for this flagellate under lower particle concentrations, e.g. 6 x 103 particles ml-1. Clearance rates determined by using 1 ~m natural (FLB) and semi-natural (albumin) particles were much lower, that is in the range of 25-30 nl cell-1 d-1. Sherr et al. (1988) and Gonzalez et al. (1990) reported similar rates, 10-30 and 25 nl cell-1 d-1, respectively, for mixed species assemblages of flagellates grazing on FLB as made from cultures of natural bacterial assemblages. For each of the two size classes, we investigated several particles each different in quality, and we observed significantly different uptake rates. Hence, prey size cannot be the only explanation for faster clearance of natural and quasi natural food as compared to carboxylated microspheres. Recent studies which employed fluorescent microspheres to measure the ingestion rates of protozoa (McManus and Fuhrman 1986; Pace and Bailiff 1987; Sherr et al. 1987; Sieracki et al. 1987) provided evidence that some protists, particularly raptorial feeders, discriminate against microspheres. Often chemosensory ability is suggested as an explanation for this phenomenon. However, Oxyrrhis seems to be unable to detect food from a distance (Tarran 1992). Particle uptake does not seem to be associated with chemosensing. Hohfeld and Melkonian (1998) reported that Oxyrrhis marina comes into contact with

379

its prey by accident. Thus, predation could depend mainly on the probability of collision (Flynn et al. 1996). This probability should be higher, if the differences in surface charge are higher. Examples from mycology, microbiology and ecology show the importance of considering the zeta potential as a factor for cell to cell recognition. Smith et al. (1998) reported that the adsorption of conidia to their mycoparasite Coniothyrium minitans depends on the difference between the zeta potential of the two components. The zeta potential of conidia decreased with increasing culture age, so the electrostatic force binding the cells decreased too. Honda et al. (1998) tested the harvesting of various microorganisms by positively charged chitosan-magnetic particles. Differences in adsorbed amounts were considered to be mainly due to the different zeta potential of the microorganisms tested (bacteria). The recovery was most effective with the strongest negatively charged bacteria E. coli. Gerritsen and Porter (1982) reported that the retention of small particles by the filter feeding zooplankter Daphnia was increased when the particle surface charges were neutralised. Sanders (1988) found that surface properties of microspheres seemed to affect grazing of the suspension feeding ciliate Cyclidium. He concluded that simple mechanical filtration did not explain differences in clearance rates by Cyclidium on proteintreated versus carboxylated microspheres. The clearance rate on protein-treated microspheres were approximately 2 times greater than on the same sized carboxylated microspheres. The electrophoretic mobilities were significantly different, -29 (~m s-1)/(voltage cm-1) for carboxylated and -17 for protein-treated microspheres. The capture efficiency of Oxyrrhis for less negative particles such as starch particles or natural food is much higher than for more negative particles, e.g. carboxylated microspheres for both size classes. From this result, one may conclude that the surface potential of Oxyrrhis is highly negative. Unfortunately, Oxyrrhis can only be maintained in a bacteria containing medium and even washing procedures using filtration do not reduce bacterial numbers to values below those of the flagellates. Therefore, it is difficult to measure the surface charge of Oxyrrhis without disturbing the particles. According to Smith et al. (1998), most microbial cell surfaces should be electrically charged and usually negatively so. A better attachment of particles to organism-surfaces will be obtained with lesser negatively to positively charged particles, like the albumin particles used here. This is particularly important for raptorial feeders like Oxyrrhis, which captures larger particles and depends on contact probability and duration. Some

380

A. Hammer et al.

tentative calculations suggest the following. By using the derivative of Lagaly (MOiler 1996) we obtained a maximum repulsion force of 1-1 .5 x 10-13 N between Oxyrrhis marina and particles in a 1 M solution (ASW medium). The distance between the flagellate and high negatively charged carboxylated microspheres must not exceed 0.5-2 ~m for an attraction to arise. Contrary to this, lesser negative particles are attracted by the flagellate from a distance of 6-1 0 ~m and possibly more. If all protozoa ingest carboxylated microspheres more slowly than they ingest natural particles of the same size, as we have shown here with Oxyrrhis marina, then grazing experiments involving the use of carboxylated microspheres should result in underestimated values. Otherwise overestimation up to 50% would occur with starch particles as model food only. Albumin particles were ingested at rates similar to those of natural food. They seem to be perfect model food particles, at least for determining ingestion rates. Further work with various organisms, covering various types of food capture strategies and particles of appropriate size, will be needed to enlighten the effects of surface charge of food on the ingestion rate. To distinguish between effects of charge and biochemical composition, particles of similar charge but with different chemical nature or one particular particle type charged to different potentials should be constructed and applied.

Methods Cultures of Oxyrrhis marina Dujardin (strain CCAP 1133/5) were cultivated on autoclaved ASW medium with a base of 33 g 1-1 sea salt (Sigma) and one boiled wheat grain per 25 ml as proposed by CCAP. The flagellate had an average length of 15 ~m and a calculated biovolume of 950 ~m3. Cultures were grown in static flasks at 15°C in complete darkness without aeration. Oxyrrhis in mid-exponential growth phase was used for all experiments. We prepared fluorescently labelled bacteria (FLB) as described by Sherr et al. (1987) by DTAF (5-(4,6dichlorotriazinyl) aminofluorescein) staining. Pseudomonas f1uorescens, strain BA 321 isolated by MINKWITZ from the Baltic sea, was grown overnight in nutrient broth and harvested by centrifugation before FLB preparation. Synechocystis spec. (PCC 6803) and Chlorella spec. (CCMP 255) were grown on BG11 medium. Carboxylated microspheres particles, made of polystyrene, were purchased from Polysciences, Inc.. Starch and protein microspheres, a new class of organic model food parti-

c1es, have been produced by modification of the emulsion-precipitation method (Mosbach and Schroder 1979) and labelled covalently with DTAF. These and yellow silicate particles (SICASTAR®) were provided from microcaps-GmbH, Rostock, Germany. All particles were diluted or harvested by centrifugation and resuspended in ASW-medium. Prior to use in feeding experiments, we sonicated the microspheres for 4 s with a Bandelin Sonopuls HD 60. Microscopic examination confirmed that clumping of particles was rare. Equivalent spherical diameter of particles were measured with an ImageAnalysis-System (CUEZ (Galai)) and coulter counter® Multisizer 2. Particles and flagellates were enumerated after filtration onto irgalan-blackstained 0.2-~m-pore-size polycarbonate filters (Isopore) with an epifluorescence microscope (Olympus BH-2 RFA). The following filter sets were used: for the algae BP 490, for FLB and microcaps particles BP 545, for DAPI counts BP 400 (UV), broad band excitation. The experimental design followed those described by Sanders (1988) and Gonzalez et al. (1990). The grazing experiments were performed under the same conditions as for culturing. To ensure, that the presence of bacteria did not affect the ingestion in the experiments, Oxyrrhis marina was separated from the culture medium by inverse filtration. Copious amounts of autoclaved ASW-medium were used to wash the flagellates. Resuspension in ASWmedium was carried out, resulting in flagellate density of approximately 1000 ml-1. 50 ml aliquots of Oxyrrhis culture were transferred to flasks and then placed in the dark at 15°C for 1 h to allow the protozoa to recover from handling shock. Flagellate suspensions were inoculated with particles so that the initial concentration of the particles was 106 ml-1. A time course started after the addition of particles. At t = 0 min, 5 ml subsamples were immediately fixed with ice-cold glutaraldehyde (same salinity as ASW, 1% final concentration) for enumeration of bacteria, particles, flagellates and as a control. A sequence of samples was taken at 10-20 min intervals (for 80-100 min), immediately narcotised with carbonated water (10% final concentration) and fixed as mentioned above to avoid both preservation-induced egestion of spheres by flagellates and lysis of them. Within 3 days, fixed samples were stained with 4' ,6'-diamidino-2-phenylindole (DAPI) (Sherr et al. 1987) for approximately 5 min, filtered onto 0.2~m-pore-size irgalan-black-stained Isopore filters and examined by epifluorescent microscopy. A minimum of 100 flagellates were inspected for each time point subsample to determine the average number of particles per cell. Slopes of particle in-

Food Particle Capture by Oxyrrhis

crease per cell were determined via regression analysis for each experiment. Mann- and Whitney-tests (graph pad prism 2) were used for comparing average values of slopes. Clearance rates were calculated by dividing the ingestion rate by the concentration of particles per nl. To obtain a zeta-potential-pH-profil, zeta potentials of particles at a range from pH 3 to 9 were determined with a Zeta-Autotitrator (Malvern Instr.), which is composed of a titrator, titration vessel, pump, zetamaster and computer. Particle suspensions (10 Ill) were injected into the titration vessel filled with 100 ml deionised water. By supplying alkaline/acid solutions from the titrator to the titration vessel, the adjustment of the pre-set pH-values between 9 and 3 occurs. Samples were taken from a zetamaster. The zeta potential of the suspended particles was determined from their velocity (electrophoretic mobility) in an applied electrical field. Special attention was paid to the zeta potential at pH 7.8, the pH of Oxyrrhis medium ASW.

Acknowledgements We thank the two anonymous reviewers for their helpful comments on the manuscript and Michael Kuhn for linguistic improvements. This research was supported by a doctoral fellowship from the Deutsche Forschungsgemeinschaft to A.H.

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