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Can cytochalasin B be used as an inhibitor of feeding in grazing experiments on ciliates?
European Journal of
Europ.j. Protisto!' 30, 309-315 (1994) August 29, 1994
PROTISTOLOGY
Can Cytochalasin B be used as an Inhibitor of Feeding in Grazing Experiments on Ciliates? Raymond J. G. Leakey1, Sandra A. Wilks 1,2 and Alistair W. A. Murray1 1British Antarctic Survey, High Cross, Madingley Road, Cambridge, UK 2Department of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex, UK
SUMMARY The effects of cytochalasin B and dimethyl sulphoxide (DMSO) on ciliate ingestion and growth were investigated in laboratory cultures of the freshwater ciliate Tetrahymena pyriformis and the marine ciliate Uronema marinum, in order to assess whether cytochalasin B could be used to inhibit grazing selectively in natural ciliate communities. The ciliates were exposed to a final concentration of 0-10 f-lg ml- t cytochalasin B dissolved in 0.4 % DMSO, and to 0-0.4 % DMSO only, for between 1 and 48 hours, then fed carmine particles as tracers of particle ingestion. DMSO had no effect on particle ingestion. Cytochalasin B reduced particle ingestion in U. marinum and T. pyriformis at respective concentrations of ~0.1 f-lg ml- 1 and ~ 1.0 f-lg ml-\ achieving a maximum effect after 4-8 hours. The effect of cytochalasin B was greater on U. marinum, with a minimum of only 4 % of the population feeding after 4 hours exposure to 10 f-lg ml-\ as compared to a minimum of 48 % recorded for T. pyriformis after 8 hours exposure to the same concentration. Cytochalasin Band DMSO at these concentrations had no effect on ciliate growth rates over 48 hours suggesting that the drug has potential as a means of reducing ciliate grazing in field samples.
Introduction Planktonic ciliates (phylum Ciliophora) are an ecologically important component of the marine plankton. Along with other protozoans, they are considered to play a significant role in trophic flux and nutrient cycling, especially as major consumers of algal production [2,9]. Accurate measurements of ciliate grazing are therefore necessary to fully elucidate the importance of these protozoans in marine ecosystems. Established methods for measuring protozooplankton grazing often require that the water medium be manipulated in order to fractionate or serially dilute the natural community (eg. [3, 16]), or disperse evenly particulate tracers of ingestion (eg. [1]). While such methods are applicable to planktonic studies, they cannot be used to study ciliate grazing in physically structured marine environments where ciliates are quantitatively important, such as Antarctic sea ice [13]. Selective eukaryote metabolic inhibitors, which diffuse through aqueous media, offer a means of inhibiting protozoan grazing on prokaryotes without excessive manipulation of samples [18,33], and need to be effective for the duration of experiments lasting several hours. Estimates of protozoan bacterivory are © 1994 by Gustav Fischet Verlag, Stuttgart
therefore possible, although the validity of such estimates is limited by the lack of target specificity commonly exhibited by eukaryote inhibitors [36,38]. To date no chemical has been tested which selectively inhibits protozoan grazing without affecting other physiological functions within eukaryotic cells. Consequently, selective chemical inhibitors have not been used to estimate protozoan herbivory. The fungal metabolite cytochalasin B affects the ion balance, membrane transport system, motility, and morphology of a wide range of cell types [5, 7, 35, 39]. The drug binds to the end of growing actin filaments reducing the rate of monomer addition, and it is thought that its effect on cell motility and morphology derive from its disruptive action on microfilament structure and function [10,28]. Cytochalasin B exhibits a variety of morphological effects on several freshwater ciliates [8, 11, 14,23,27,34,37,41]. In particularthe drug induces reversible inhibition of food vacuole formation in the freshwater bacterivorous ciliates Tetrahymena pyriformis and Paramecium caudatum in a dose dependent manner, with cell growth continuing at concentrations that inhibit vacuole formation [11,27,37]. For example, at a concentration of 7.5 f-lg ml- 1 cytochalasin B inhibits food vacuole formation but not growth in T. pyriformis 0932-4739/94/0030-0309$3.50/0
310 . R.
J. G. Leakey, S. A. Wilks and A. W. A. Murray
[27]. Therefore it may be possible to use cytochalasin B selectively to reduce ciliate grazing in field samples at concentrations which do not interfere with other physiological processes in ciliates or their prey. In studies of cytochalasin B action dimethyl sulphoxide (DMSO) has often been used as a carrier solvent. As with cytochalasin B, DMSO exhibits a variety of dose-dependent morphological effects on freshwater ciliates, including reduced cell division and reduced food vacuole formation [12,20,21,24,26,30, 34). It is therefore necessary to distinguish between the effects of cytochalasin Band DMSO. In this study we have used carmine particles as tracers of particle ingestion to investigate the effects of cytochalasin Band DMSO on grazing by two free-living bacterivorous ciliates; the freshwater species Tetrahymena pyriformis and the marine Uronema marinum. The effects of cytochalasin Band DMSO on ciliate population growth were also investigated in order to assess whether cytochalasin B could be used to reduce grazing in natural ciliate communities at concentrations which do not affect ciliate growth.
Material and Methods
Culturing Procedures Ciliate cultures were obtained from the Culture Collection of Algae and Protozoa (Ambleside, UK) and grown in autoclaved proteose peptone (2 % w/v) and yeast extract (0.25 % w/v) medium at 14 °C (± 1 0c). Tetrahymena pyriformis (Ehrenberg) (CCAP no. 1630/1W) was grown axenically and Uronema marinum (Dujardin) (CCAP no. 1686/2) grown non-axenically. To prevent salinity changes all culture media and experimental reagents were prepared using deionised water for T. pyriformis or filtered seawater for U. marinum. All glass and polycarbonate culture and experimental vessels were acid-washed (10 % HCI) for 24 hours, rinsed with deionised water and finally autoclaved. Experimental reagents were obtained from Sigma Chemical Co. (Dorset, UK).
Effects of Cytochalasin Band DMSO on Ciliate Grazing Activity To determine the effects of cytochalasin Band DMSO on the grazing activity of ciliates, food vacuole counts were undertaken on Tetrahymena pyriformis feeding axenically on carmine particles and yeast, and Uronema marinum feeding on carmine particles, yeast and bacteria, in the presence and absence of cytochalasin B or DMSO. Four replicate cytochalasin B experiments and three replicate DMSO experiments were undertaken for each species, with each replicate derived from a separate sub-culture population. For each replicate experiment, four 15 ml samples were taken from one sub-culture and subjected to a final concentration of either 0, 0.1, 1.0 or 10.0 Ilg ml- I cytochalasin B (taken from a 200 Ilg ml- I stock solution dissolved in 0.4 % DMSO), or 0, 0.004, 0.04 or 0.4 % tv/v) DMSO. The samples were then incubated at 14°C for either 1, 2, 4, 8, 24 or 48 hours giving a total of 24 separate concentration/time treatments. At the end of each treatment, carmine particles were added (from a 2 mg ml- I stock suspension) to a 2 ml sub-sample from each replicate treatment to give a final particle concentration of 100 Ilg ml- I.
The sub-samples were then gently inverted to mix the contents Each sub-sample was then and incubated for 10 minutes at 14 fixed with a 2 % tv/v) final concentration of glutaraldehyde and a 200 III aliquot then drawn gently through a 0.2 Ilm pore size polycarbonate Nuclepore filter, with a Whatman 0.2 Ilm pore size cellulose nitrate backing filter. The filters were mounted carefully on glass slides, using Leitz immersion oil and examined at X 625 magnification under fluorescent illumination (Leitz Laborlux S microscope, SOW Osram HBO mercury burner, Ploemopak illuminator with filter block 12, and NPL Fluotar 50/1.00 oil objective lens). Individual ciliate cells were identified by their bright green autofluorescence, the intensity of which fades rapidly. Within each ciliate, food vacuoles containing carmine particles appeared deep red, in contrast to those without carmine which were yellow. A total of 50 cells were selected randomly from the entire area of the filter, and the numb,er of vacuoles containing carmine particles within each cell determined. From these data the mean number of carmine-containing vacuoles per cell and the percentage of cells feeding were calculated.
0c.
Effects of Cytochalasin Band DMSO on Ciliate Population Growth To determine the effects of cytochalasin Band DMSO on ciliate growth rates, population abundance was determined at 1 hand 48 h from each replicate treatment from all cytochalasin Band DMSO experiments. Cell counts were undertaken on the 2 ml sub-samples used for cell vacuole counts. After gentle mixing, the samples were diluted tenfold, and a 1 ml aliquot removed and placed in a Sedgewick-Rafter counting chamber. For each replicate, at least 50 cells were counted using a Leitz Laborlux S microscope at x 125 magnification under bright field illumination. Population growth rates for each replicate treatment were calculated from changes in numbers over time, assuming exponential growth, using the following equation [40J: r = (lit) 10g.,(N/No)
where r N t
intrinsic rate of increase (day-I) cell numbers time
Data Analysis Data from the five experiments were analyzed using GENSTAT 5.2 [29J to determine significant differences between the cytochalasin B or DMSO treatments. Ciliate growth rate data were analyzed by analysis of variance. Where the data were either counts of vacuoles with or without carmine, or values for the percentage of cells feeding, they followed a binomial distribution. These latter data were therefore analyzed using a generalised linear model (GLM) with a binomial error distribution and a logit link function [6, 17J. The logit transformation is given by
loge [1
~ p]
where p is the probability of a vacuole containing carmine. This transformation leads to an additive scale on which effects can be estimated. Tables of fitted proportions for each combination of time and concentration were constructed using the coefficients from the fitted model together with their standard errors as calculated under the assumed binomial error. Figures presented in this paper
Effect of Cytochalasin B on Ciliates . 311 are based on these tables. The GLMs fitted the data well. In all cases the residual deviance was approximately equal to its degrees of freedom as would be expected for a X2 random variable. This indicates that the binomial error assumption was tenable for these data.
Results Mean abundance of Tetrahymena pyriformis and Uronema marinum increased during all experiments. Mean a
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growth rates ranged from 0.06 to 0.41 dar1 (Table 1) with no significant effect of DMSO or cytochalasin B. After ten minutes exposure to carmine particles, 88-98 % of Tetrahymena pyriformis cells and 93-99 % of Uronema marinum cells had ingested carmine in the absence of cytochalasin B or DMSO (Figs. 1 and 3). Carmine containing vacuoles (CCV) were clearly observed in both ciliate taxa, with approximately 2 CCV cell- 1 recorded in these untreated cells throughout the duration of each experiment (Figs. 2 and 4). Exposure of both ciliate taxa to ~0.4 % DMSO for between 1 and 48 hours prior to incubation with carmine particles had no significant
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Fig. 1. Mean percentage (with standard error) of (a) Tetrahymena pyriformis and (b) Uronema marinum cells with carmine containing vacuoles after 10 minutes exposure to carmine particles, when treated with 0-1O!tg ml- 1 cytochalasin B for up to 48 hours. Key to Figs. 1 and 2: ( - - ) = 0.0 !tg ml- l cytochalasin B. (-------) = 0.1 !tg ml- l cytochalasin B. (----) = 1.0!tg ml- l cytochalasin B. (---) = 10.0!tg ml- l cytochalasin B.
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Fig. 2. Mean number (with standard error) of carmine containing vacuoles observed in (a) Tetrahymena pyriformis and (b) Uronema marinum cells after 10 minutes grazing, when treated with 0-10 !tg ml- l cytochalasin B for up to 48 hours. Concentrations as in Fig. 1.
Table 1. Mean growth rate r, with standard error (s, e. m) and degrees of freedom (df), of Tetrahymena pyriformis and Uronema marinum populations treated with different concentrations of cytochalasin B or DMSO for 48 hours at 14 °C Mean Growth Rate r (day-I)
s.e. m (df)
Cytochalasin B (!tg ml- l ) T. pyriformis U. marinum
0,0 0.1409 0.1943
0.1 0.1401 0.1689
1.0 0.1701 0.2141
10.0 0.1428 0.1870
0.0184 (9) 0.0124 (9)
DMSO (%) T. pyriformis U. marinum
0.000 0.0897 0.4019
0.004 0.0615 0.4094
0,04 0.0735 0.3784
0.4 0.0657 0.4081
0.0112 (6) 0.0168 (6)
312 . R.
J. G. Leakey, S. A. Wilks and A. W. A. Murray
effect on ingestion; the mean percentage of cells feeding and number of CCV cell- 1 ranging from 92-99 % and 1.9-2.2 cell-l, respectively (Figs. 3 and 4). By contrast, exposure to cytochalasin B reduced ingestion of carmine particles in both T. pyriformis and U. marinum, with the percentage of cells feeding and number of CCV cell- 1 generally tending to decline with increasing cytochalasin B concentration, and with increasing duration of exposure to cytochalasin B (Figs. 1 and 2). At higher concentrations, the effect of cytochalasin B was greater on Uronema marinum than on Tetrahymena pyriformis; although the effect of exposure time was generally similar for both taxa (Figs. 1 and 2). For example, treatment with 10 lAg ml- 1 cytochalasin B resulted in the greatest inhibition of particle uptake, with only 48 % of the T. pyriformis population and 4 % of the U. marinum population feeding after 4-8 hours exposure to the drug. Treatment with 1 lAg ml- 1 cytochalasin B had slightly less effect on ingestion, with a minimum of 53 % of the T. pyriformis population and 15 % of the U. marinum population feeding. At both 1 and 10 lAg ml- 1 particle uptake decreased with increasing exposure time up to 4 hours in U. marinum and 8 hours in T. pyriformis, with most of the decrease observed in U. marinum occurring
within the first hour's exposure to cytochalasin B. Thereafter, further exposure to cytochalasin B led to a slight decrease in inhibition. Treatment with 0.1 lAg ml- 1 cytochalasin B had no effect on carmine particle ingestion in Tetrahymena pyriformis with an average of 87-94 % of the population continuing to feed and 1.6-2.0 CCV observed cell- 1 (Figs. 1 and 2). By contrast, ingestion by Uronema marinum was inhibited at this concentration but to a lesser extent than at higher concentrations. Here, the effect of cytochalasin B increased with time, with a minimum of 45 % of the population still feeding after 48 hours.
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Fig. 4. Mean number (with standard error) of carmine containing vacuoles observed in (a) Tetrahymena pyriformis and (b) Uronema marinum cells after 10 minutes grazing, when treated with 0-0.4 % DMSO for up to 48 hours. Concentrations as in Fig. 3.
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Fig. 3. Mean percentage (with standard error) of (a) Tetrahymena pyriformis and (b) Uronema marinum cells with carmine containing vacuoles after 10 minutes exposure to carmine particles, when treated with 0-0.4 % DMSO for up to 48 hours. Key to Figs. 3 and 4: ( - - ) = 0.0 % DMSO. (-------) = 0.004 % DMSO. (----) = 0.04 % DMSO. (- - -) = 0.4 % DMSO.
Cytochalasin Band DMSO at the concentrations used had no significant effect on Tetrahymena pyriformis and Uronema marinum population growth, even after 48 hours exposure to the drugs. Previous studies have reported little effect of cytochalasin B on T. pyriformis, or DMSO on T. pyriformis, Paramecium aurelia, P. caudatum and P. tetraurelia growth at concentrations similar to those used in our study [12,20,21,26,27,30,34]. There is evidence that T. pyriformis acquires nutrients by pinocy-
Effect of Cytochalasin B on Ciliates . 313 tosis in order to maintain growth when particle ingestion has been inhibited by cytochalasin B [23,25], and it is possible that U. marinum also uses this nutrient uptake mechanism to maintain growth when treated with cytochalasin B. In natural environments organic nutrient concentrations may not be sufficient to maintain ciliate growth by pinocytosis; however, the continued growth of ciliate populations in culture media demonstrates a lack of pronounced effects of cytochalasin B or DMSO at the concentrations used on other physiological functions. In control samples Tetrahymena pyriformis and Uronema marinum ingested carmine particles readily during ten minutes exposure to carmine suspension, and food vacuoles containing carmine could be clearly distinguished from those without. Carmine particles have a size corresponding to that of bacteria and are therefore suitable bacterial substitutes for bacterivorous ciliates selecting on size criteria alone [19]. The percentage of cells feeding on carmine in control samples varied between 88-98 % and 93-99 % in T. pyriformis and U. marinum, respectively. Food vacuoles are not formed by T. pyriformis cells undergoing division, and in an exponentially multiplying population up to 10 % of cells may be dividing [4,25]. The ciliates in cultures used in this study all continued to multiply throughout the 48 hours of each experiment. It is therefore likely that the 1 % to 12 % of cells that did not ingest carmine were undergoing division. The capacity of individual ciliate cells to form food vacuoles varies. Previous studies have observed mean values of 3.2 and 4.2 food vacuoles cell- 1 for T. pyriformis cells exposed to carmine particles for ten minutes at 28°C [4, 19, 22]. In this study both T. pyriformis and U. marinum formed approximately two food vacuoles cell- 1 at 14°C. Food vacuole formation in T. pyriformis increases with increasing temperature with little or no vacuole formation at < 10°C and maximum vacuole formation at 28 °C [19]. Our mean values of 1. 7-2.2 CCV cell- 1 observed for T. pyriformis and U. marinum therefore appear reasonable. DMSO failed to inhibit the uptake of carmine particles by Tetrahymena pyriformis and Uronema marinum at concentrations ~0.4 %. DMSO exhibits a number oftoxic effects on ciliate cells at higher concentrations including reduced cell division and reduced food vacuole formation [12, 20, 21, 24, 26, 30, 34]. However, concentrations similar to those used in this study are reported to have little or no effect on food vacuole formation in T. pyriformis and Paramecium caudatum [12, 23]. DMSO at 0.4 % v/v final concentration would therefore appear to be a suitable carrying compound for cytochalasin B for studies involving U. marinum as well as T. pyriformis. Exposure to 1 and 1 ~g ml- 1 cytochalasin B inhibited ingestion of carmine particles by both Tetrahymena pyriformis and Uronema marinum, with greatest inhibition recorded after 4 to 8 hours exposure to a concentration of 10 ~g ml- 1. Inhibition of particle ingestion by cytochalasin B has been previously reported for T. pyriformis [15,23,27,31,32], Paramecium caudatum [11,37], and Pseudomicrothorax dubius [14], with a concentration of 7.5 ~g ml- 1 reducing food vacuole formation in T.
°
pyriformis to 71 % of that of control cells after one hour exposure to the drug [27]. In this study a similar reduction in food vacuole formation to 63 % of that of the control was recorded for T. pyriformis after exposure to 10 ~g ml- 1 cytochalasin B for one hour. Uronema marinum appears to be more sensitive to cytochalasin B than Tetrahymena pyriformis. This may be due to morphological differences, such as its smaller size, to the osmolarity of its environment, or to the different culture methods. It is unlikely that the presence of bacteria within the U. marinum culture medium would have influenced cytochalasin B toxicity by metabolising DMSO because any reduction in DMSO concentration would have tended to decrease the drug's solubility and ability to permeate the cell membranes [12]. However, inhibition of ingestion may have been underestimated in T. pyriformis cells due to the accumulation of carmine particles in an open vacuole at the cell cytostome. Open vacuoles have been observed in T. pyriformis cells treated with cytochalasin B and resemble normal food vacuoles when observed by light microscopy [23]. In this study most cytochalasin B-treated T. pyriformis cells were observed with a single CCV located in the cytostome region, and these may have been open food vacuoles. It has been suggested that cytochalasin B interferes with the 'sealing off' of food vacuoles in T. pyriformis by disrupting a specialized region of contractile cytoplasm surrounding the cytostome and composed of a network of microfilaments [23]. This is consistent with more recent studies which show that cytochalasin B binds to the end of actin filaments [10, 28]. Cytochalasin B presumably inhibits ingestion in U. marinum in a similar manner, although few single CCV were observed in the cytostome regions of these ciliates. Thus, the exact mechanism by which cytochalasin B affects ciliate ingestion has yet to be confirmed. In order to function to full effect as a selective inhibitor of ciliate grazing in field samples, cytochalasin B would have to achieve rapid and complete inhibition of ingestion in all ciliates without affecting other physiological functions of ciliates or their prey. Cytochalasin B clearly reduces particle ingestion in Tetrahymena pyriformis and Uronema marinum at concentrations which do not appear to affect population growth. However, the drug did not inhibit ingestion completely, and maximum reduction of ingestion was attained only after 4 to 8 hours, precluding its use in situations where more effective techniques for estimating grazing rates are available. Ingestion was, however, reduced substantially by cytochalasin B treatment, with only 4 % of U. marinum cells feeding after 4 hours exposure to the drug. The use of cytochalasin B in experiments lasting up to 24 hours could therefore generate limited data on ciliate grazing in physically structured environments, such as sea ice, where established techniques cannot be used. Further studies are required to determine whether cytochalasin B reduces ingestion in natural populations of ciliates at concentrations which do not affect the ciliate growth, and to assess whether such concentrations influence other protozoan grazers and their algal prey.
314 . R. J. G. Leakey, S. A. Wilks and A. W. A. Murray
Acknowledgements We are grateful to M. A. Sleigh, J. Priddle, A. Clarke, and two anonymous reviewers for critically reading the manuscript. The research was undertaken as part of a sandwich student placement awarded to S. A. Wilks at the British Antarctic Survey.
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19 Nilsson J. R. (1972): Further studies on vacuole formation in Tetrahymena pyriformis GL. Compt. Rend. Trav. Lab. Carlsberg, 39, 83-110. 20 Nilsson J. R. (1974): Effects of DMSO on vacuole formation, contractile vacuole function, and division in Tetrahymena pyriformis GL. J. Cell Sci., 16, 39-47. 21 Nilsson J. R. (1976a): Reversible inhibition of RNA synthesis in Tetrahymena pyriformis GL by dimethyl sulphoxide: an electron microscope autoradiographic study. Trans. Amer. Microsc. Soc., 95, 403-414. 22 Nilsson J. R. (1976b): Physiological and structural studies on Tetrahymena pyriformis GL. Compt. Rend. Trav. Lab. Carlsberg, 40, 215-355. 23 Nilsson J. R. (1977a): Fine structure and RNA synthesis of Tetrahymena during cytochalasin B inhibition of phagocytosis. J. Cell Sci., 27, 115-126. 24 Nilsson J. R. (1977b): Effects of dimethyl sulphoxide on Tetrahymena pyriformis GL. Fine structural changes and their reversibility. J. Protozool., 24, 275-283. 25 Nilsson J. R. (1979): Phagotrophy in Tetrahymena. In: Levandowsky M. and Hunter S. H. (eds.): Biochemistry and physiology of protozoa. 2. ed., vol. 2, pp.339-379. Academic Press, New York. 26 Nilsson J. R. (1980): Effects of dimethyl sulphoxide on ATP content and protein synthesis in Tetrahymena. Protoplasma,
103, 189-200. 27 NilssonJ. R., Ricketts T. R. and Zeuthen E. (1973): Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis GL. Exptl. Cell Res., 79, 456-459. 28 Ohmori H., Toyama S. and Toyama S. (1992): Direct proof that the primary site of action of cytochalasin on cell motility processes is actin. J. Cell BioI., 116, 933-941. 29 Payne R. W., Lane P. W., Ainsley A. E., Bicknell K. E., Digby P. G. N., Harding S. A., Leech P. K., Simpson H. R., Todd A. D., Verrier P. J., White R. P., Gower J. c., Tunnicliffe-Wilson G. and Patterson L. J. (1987): GENSTAT 5 Reference Manual. 1. ed. Clarendon Press, Oxford. 30 Reisner A. H. and Bucholtz C. (1977): The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in Paramecium. J. Cell Physiol., 90, 169-178. 31 Ricketts T. R. and Rappitt A. F. (1975): A radioisotopic and morphological study of the uptake of materials into food vacuoles by Tetrahymena pyriformis GL-9. Protoplasma, 86, 321-337. 32 Rothstein T. L. and Blum J. J. (1974): Lysosomal physiology in Tetrahymena. III. Pharmacological studies on acid hydrolase release and the ingestion and egestion of dimethylbenzanthracene particles. J. Cell BioI., 62, 844-859. 33 Sherr B. F., Sherr E. B., Andrew T. L., Fallon R. D. and Newell S. Y. (1986): Trophic interactions between heterotrophic protozoa and bacterioplankton in estuarine water analyzed with selective metabolic inhibitors. Mar. Ecol. Progr. Ser., 32, 169-179. 34 Sibley J. T., Paul M. D. and Hanson E. D. (1977): Subcellular effects of cytochalasin Band dimethylsulphoxide on Paramecium aurelia. J. Protozool., 24, 595-604. 35 Tanenbaum S. W. (1978): Cytochalasins, biochemical and cell biological aspects. North Holland Publishing Company, Amsterdam. 36 Taylor G. T. and Pace M. L. (1987): Validity of eucaryote inhibitors for assessing production and grazing mortality of marine bacterioplankton. Appl. Envir. Microbiol., 53, 119-128. 37 Tolloczko B. (1977): Endocytosis in Paramecium. III. Effect of cytochalasin B and colchicine. Acta Protozool., 16, 185-193.
Effect of Cytochalasin B on Ciliates· 315 38 Tremaine S. C. and Mills A. L. (1987): Inadequacy of the eucaryote inhibitor cycloheximide in studies of protozoan grazing on bacteria at the freshwater-sediment interface. App!. Envir. Microbio!., 53, 1969-1972. 39 Tsuchiya W., Okada Y., Yano J., Inouye A., Sasaki S. and Doida Y. (1981): Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells. ExptI. Cell Res., 133, 83-92.
40 Verity P. G. (1986): Growth rates of natural tintinnid populations in Narragansett Bay. Mar. Eco!. Progr. Ser., 29, 117-126. 41 Wasik A. and Sikora J. (1980): Effects of cytochalasin Band colchicine on cytoplasmic streaming in Paramecium bursaria. Acta ProtozooI., 19, 103-110.
Key words: Cytochalasin B - Dimethyl sulphoxide - Grazing - Tetrahymena - Uronema Raymond
J. G. Leakey, British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 OET, UK
Europ.j. Protisto!' 30, 309-315 (1994) August 29, 1994
PROTISTOLOGY
Can Cytochalasin B be used as an Inhibitor of Feeding in Grazing Experiments on Ciliates? Raymond J. G. Leakey1, Sandra A. Wilks 1,2 and Alistair W. A. Murray1 1British Antarctic Survey, High Cross, Madingley Road, Cambridge, UK 2Department of Biology and Biochemistry, Brunei University, Uxbridge, Middlesex, UK
SUMMARY The effects of cytochalasin B and dimethyl sulphoxide (DMSO) on ciliate ingestion and growth were investigated in laboratory cultures of the freshwater ciliate Tetrahymena pyriformis and the marine ciliate Uronema marinum, in order to assess whether cytochalasin B could be used to inhibit grazing selectively in natural ciliate communities. The ciliates were exposed to a final concentration of 0-10 f-lg ml- t cytochalasin B dissolved in 0.4 % DMSO, and to 0-0.4 % DMSO only, for between 1 and 48 hours, then fed carmine particles as tracers of particle ingestion. DMSO had no effect on particle ingestion. Cytochalasin B reduced particle ingestion in U. marinum and T. pyriformis at respective concentrations of ~0.1 f-lg ml- 1 and ~ 1.0 f-lg ml-\ achieving a maximum effect after 4-8 hours. The effect of cytochalasin B was greater on U. marinum, with a minimum of only 4 % of the population feeding after 4 hours exposure to 10 f-lg ml-\ as compared to a minimum of 48 % recorded for T. pyriformis after 8 hours exposure to the same concentration. Cytochalasin Band DMSO at these concentrations had no effect on ciliate growth rates over 48 hours suggesting that the drug has potential as a means of reducing ciliate grazing in field samples.
Introduction Planktonic ciliates (phylum Ciliophora) are an ecologically important component of the marine plankton. Along with other protozoans, they are considered to play a significant role in trophic flux and nutrient cycling, especially as major consumers of algal production [2,9]. Accurate measurements of ciliate grazing are therefore necessary to fully elucidate the importance of these protozoans in marine ecosystems. Established methods for measuring protozooplankton grazing often require that the water medium be manipulated in order to fractionate or serially dilute the natural community (eg. [3, 16]), or disperse evenly particulate tracers of ingestion (eg. [1]). While such methods are applicable to planktonic studies, they cannot be used to study ciliate grazing in physically structured marine environments where ciliates are quantitatively important, such as Antarctic sea ice [13]. Selective eukaryote metabolic inhibitors, which diffuse through aqueous media, offer a means of inhibiting protozoan grazing on prokaryotes without excessive manipulation of samples [18,33], and need to be effective for the duration of experiments lasting several hours. Estimates of protozoan bacterivory are © 1994 by Gustav Fischet Verlag, Stuttgart
therefore possible, although the validity of such estimates is limited by the lack of target specificity commonly exhibited by eukaryote inhibitors [36,38]. To date no chemical has been tested which selectively inhibits protozoan grazing without affecting other physiological functions within eukaryotic cells. Consequently, selective chemical inhibitors have not been used to estimate protozoan herbivory. The fungal metabolite cytochalasin B affects the ion balance, membrane transport system, motility, and morphology of a wide range of cell types [5, 7, 35, 39]. The drug binds to the end of growing actin filaments reducing the rate of monomer addition, and it is thought that its effect on cell motility and morphology derive from its disruptive action on microfilament structure and function [10,28]. Cytochalasin B exhibits a variety of morphological effects on several freshwater ciliates [8, 11, 14,23,27,34,37,41]. In particularthe drug induces reversible inhibition of food vacuole formation in the freshwater bacterivorous ciliates Tetrahymena pyriformis and Paramecium caudatum in a dose dependent manner, with cell growth continuing at concentrations that inhibit vacuole formation [11,27,37]. For example, at a concentration of 7.5 f-lg ml- 1 cytochalasin B inhibits food vacuole formation but not growth in T. pyriformis 0932-4739/94/0030-0309$3.50/0
310 . R.
J. G. Leakey, S. A. Wilks and A. W. A. Murray
[27]. Therefore it may be possible to use cytochalasin B selectively to reduce ciliate grazing in field samples at concentrations which do not interfere with other physiological processes in ciliates or their prey. In studies of cytochalasin B action dimethyl sulphoxide (DMSO) has often been used as a carrier solvent. As with cytochalasin B, DMSO exhibits a variety of dose-dependent morphological effects on freshwater ciliates, including reduced cell division and reduced food vacuole formation [12,20,21,24,26,30, 34). It is therefore necessary to distinguish between the effects of cytochalasin Band DMSO. In this study we have used carmine particles as tracers of particle ingestion to investigate the effects of cytochalasin Band DMSO on grazing by two free-living bacterivorous ciliates; the freshwater species Tetrahymena pyriformis and the marine Uronema marinum. The effects of cytochalasin Band DMSO on ciliate population growth were also investigated in order to assess whether cytochalasin B could be used to reduce grazing in natural ciliate communities at concentrations which do not affect ciliate growth.
Material and Methods
Culturing Procedures Ciliate cultures were obtained from the Culture Collection of Algae and Protozoa (Ambleside, UK) and grown in autoclaved proteose peptone (2 % w/v) and yeast extract (0.25 % w/v) medium at 14 °C (± 1 0c). Tetrahymena pyriformis (Ehrenberg) (CCAP no. 1630/1W) was grown axenically and Uronema marinum (Dujardin) (CCAP no. 1686/2) grown non-axenically. To prevent salinity changes all culture media and experimental reagents were prepared using deionised water for T. pyriformis or filtered seawater for U. marinum. All glass and polycarbonate culture and experimental vessels were acid-washed (10 % HCI) for 24 hours, rinsed with deionised water and finally autoclaved. Experimental reagents were obtained from Sigma Chemical Co. (Dorset, UK).
Effects of Cytochalasin Band DMSO on Ciliate Grazing Activity To determine the effects of cytochalasin Band DMSO on the grazing activity of ciliates, food vacuole counts were undertaken on Tetrahymena pyriformis feeding axenically on carmine particles and yeast, and Uronema marinum feeding on carmine particles, yeast and bacteria, in the presence and absence of cytochalasin B or DMSO. Four replicate cytochalasin B experiments and three replicate DMSO experiments were undertaken for each species, with each replicate derived from a separate sub-culture population. For each replicate experiment, four 15 ml samples were taken from one sub-culture and subjected to a final concentration of either 0, 0.1, 1.0 or 10.0 Ilg ml- I cytochalasin B (taken from a 200 Ilg ml- I stock solution dissolved in 0.4 % DMSO), or 0, 0.004, 0.04 or 0.4 % tv/v) DMSO. The samples were then incubated at 14°C for either 1, 2, 4, 8, 24 or 48 hours giving a total of 24 separate concentration/time treatments. At the end of each treatment, carmine particles were added (from a 2 mg ml- I stock suspension) to a 2 ml sub-sample from each replicate treatment to give a final particle concentration of 100 Ilg ml- I.
The sub-samples were then gently inverted to mix the contents Each sub-sample was then and incubated for 10 minutes at 14 fixed with a 2 % tv/v) final concentration of glutaraldehyde and a 200 III aliquot then drawn gently through a 0.2 Ilm pore size polycarbonate Nuclepore filter, with a Whatman 0.2 Ilm pore size cellulose nitrate backing filter. The filters were mounted carefully on glass slides, using Leitz immersion oil and examined at X 625 magnification under fluorescent illumination (Leitz Laborlux S microscope, SOW Osram HBO mercury burner, Ploemopak illuminator with filter block 12, and NPL Fluotar 50/1.00 oil objective lens). Individual ciliate cells were identified by their bright green autofluorescence, the intensity of which fades rapidly. Within each ciliate, food vacuoles containing carmine particles appeared deep red, in contrast to those without carmine which were yellow. A total of 50 cells were selected randomly from the entire area of the filter, and the numb,er of vacuoles containing carmine particles within each cell determined. From these data the mean number of carmine-containing vacuoles per cell and the percentage of cells feeding were calculated.
0c.
Effects of Cytochalasin Band DMSO on Ciliate Population Growth To determine the effects of cytochalasin Band DMSO on ciliate growth rates, population abundance was determined at 1 hand 48 h from each replicate treatment from all cytochalasin Band DMSO experiments. Cell counts were undertaken on the 2 ml sub-samples used for cell vacuole counts. After gentle mixing, the samples were diluted tenfold, and a 1 ml aliquot removed and placed in a Sedgewick-Rafter counting chamber. For each replicate, at least 50 cells were counted using a Leitz Laborlux S microscope at x 125 magnification under bright field illumination. Population growth rates for each replicate treatment were calculated from changes in numbers over time, assuming exponential growth, using the following equation [40J: r = (lit) 10g.,(N/No)
where r N t
intrinsic rate of increase (day-I) cell numbers time
Data Analysis Data from the five experiments were analyzed using GENSTAT 5.2 [29J to determine significant differences between the cytochalasin B or DMSO treatments. Ciliate growth rate data were analyzed by analysis of variance. Where the data were either counts of vacuoles with or without carmine, or values for the percentage of cells feeding, they followed a binomial distribution. These latter data were therefore analyzed using a generalised linear model (GLM) with a binomial error distribution and a logit link function [6, 17J. The logit transformation is given by
loge [1
~ p]
where p is the probability of a vacuole containing carmine. This transformation leads to an additive scale on which effects can be estimated. Tables of fitted proportions for each combination of time and concentration were constructed using the coefficients from the fitted model together with their standard errors as calculated under the assumed binomial error. Figures presented in this paper
Effect of Cytochalasin B on Ciliates . 311 are based on these tables. The GLMs fitted the data well. In all cases the residual deviance was approximately equal to its degrees of freedom as would be expected for a X2 random variable. This indicates that the binomial error assumption was tenable for these data.
Results Mean abundance of Tetrahymena pyriformis and Uronema marinum increased during all experiments. Mean a
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growth rates ranged from 0.06 to 0.41 dar1 (Table 1) with no significant effect of DMSO or cytochalasin B. After ten minutes exposure to carmine particles, 88-98 % of Tetrahymena pyriformis cells and 93-99 % of Uronema marinum cells had ingested carmine in the absence of cytochalasin B or DMSO (Figs. 1 and 3). Carmine containing vacuoles (CCV) were clearly observed in both ciliate taxa, with approximately 2 CCV cell- 1 recorded in these untreated cells throughout the duration of each experiment (Figs. 2 and 4). Exposure of both ciliate taxa to ~0.4 % DMSO for between 1 and 48 hours prior to incubation with carmine particles had no significant
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Fig. 1. Mean percentage (with standard error) of (a) Tetrahymena pyriformis and (b) Uronema marinum cells with carmine containing vacuoles after 10 minutes exposure to carmine particles, when treated with 0-1O!tg ml- 1 cytochalasin B for up to 48 hours. Key to Figs. 1 and 2: ( - - ) = 0.0 !tg ml- l cytochalasin B. (-------) = 0.1 !tg ml- l cytochalasin B. (----) = 1.0!tg ml- l cytochalasin B. (---) = 10.0!tg ml- l cytochalasin B.
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Table 1. Mean growth rate r, with standard error (s, e. m) and degrees of freedom (df), of Tetrahymena pyriformis and Uronema marinum populations treated with different concentrations of cytochalasin B or DMSO for 48 hours at 14 °C Mean Growth Rate r (day-I)
s.e. m (df)
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10.0 0.1428 0.1870
0.0184 (9) 0.0124 (9)
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0.004 0.0615 0.4094
0,04 0.0735 0.3784
0.4 0.0657 0.4081
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312 . R.
J. G. Leakey, S. A. Wilks and A. W. A. Murray
effect on ingestion; the mean percentage of cells feeding and number of CCV cell- 1 ranging from 92-99 % and 1.9-2.2 cell-l, respectively (Figs. 3 and 4). By contrast, exposure to cytochalasin B reduced ingestion of carmine particles in both T. pyriformis and U. marinum, with the percentage of cells feeding and number of CCV cell- 1 generally tending to decline with increasing cytochalasin B concentration, and with increasing duration of exposure to cytochalasin B (Figs. 1 and 2). At higher concentrations, the effect of cytochalasin B was greater on Uronema marinum than on Tetrahymena pyriformis; although the effect of exposure time was generally similar for both taxa (Figs. 1 and 2). For example, treatment with 10 lAg ml- 1 cytochalasin B resulted in the greatest inhibition of particle uptake, with only 48 % of the T. pyriformis population and 4 % of the U. marinum population feeding after 4-8 hours exposure to the drug. Treatment with 1 lAg ml- 1 cytochalasin B had slightly less effect on ingestion, with a minimum of 53 % of the T. pyriformis population and 15 % of the U. marinum population feeding. At both 1 and 10 lAg ml- 1 particle uptake decreased with increasing exposure time up to 4 hours in U. marinum and 8 hours in T. pyriformis, with most of the decrease observed in U. marinum occurring
within the first hour's exposure to cytochalasin B. Thereafter, further exposure to cytochalasin B led to a slight decrease in inhibition. Treatment with 0.1 lAg ml- 1 cytochalasin B had no effect on carmine particle ingestion in Tetrahymena pyriformis with an average of 87-94 % of the population continuing to feed and 1.6-2.0 CCV observed cell- 1 (Figs. 1 and 2). By contrast, ingestion by Uronema marinum was inhibited at this concentration but to a lesser extent than at higher concentrations. Here, the effect of cytochalasin B increased with time, with a minimum of 45 % of the population still feeding after 48 hours.
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Cytochalasin Band DMSO at the concentrations used had no significant effect on Tetrahymena pyriformis and Uronema marinum population growth, even after 48 hours exposure to the drugs. Previous studies have reported little effect of cytochalasin B on T. pyriformis, or DMSO on T. pyriformis, Paramecium aurelia, P. caudatum and P. tetraurelia growth at concentrations similar to those used in our study [12,20,21,26,27,30,34]. There is evidence that T. pyriformis acquires nutrients by pinocy-
Effect of Cytochalasin B on Ciliates . 313 tosis in order to maintain growth when particle ingestion has been inhibited by cytochalasin B [23,25], and it is possible that U. marinum also uses this nutrient uptake mechanism to maintain growth when treated with cytochalasin B. In natural environments organic nutrient concentrations may not be sufficient to maintain ciliate growth by pinocytosis; however, the continued growth of ciliate populations in culture media demonstrates a lack of pronounced effects of cytochalasin B or DMSO at the concentrations used on other physiological functions. In control samples Tetrahymena pyriformis and Uronema marinum ingested carmine particles readily during ten minutes exposure to carmine suspension, and food vacuoles containing carmine could be clearly distinguished from those without. Carmine particles have a size corresponding to that of bacteria and are therefore suitable bacterial substitutes for bacterivorous ciliates selecting on size criteria alone [19]. The percentage of cells feeding on carmine in control samples varied between 88-98 % and 93-99 % in T. pyriformis and U. marinum, respectively. Food vacuoles are not formed by T. pyriformis cells undergoing division, and in an exponentially multiplying population up to 10 % of cells may be dividing [4,25]. The ciliates in cultures used in this study all continued to multiply throughout the 48 hours of each experiment. It is therefore likely that the 1 % to 12 % of cells that did not ingest carmine were undergoing division. The capacity of individual ciliate cells to form food vacuoles varies. Previous studies have observed mean values of 3.2 and 4.2 food vacuoles cell- 1 for T. pyriformis cells exposed to carmine particles for ten minutes at 28°C [4, 19, 22]. In this study both T. pyriformis and U. marinum formed approximately two food vacuoles cell- 1 at 14°C. Food vacuole formation in T. pyriformis increases with increasing temperature with little or no vacuole formation at < 10°C and maximum vacuole formation at 28 °C [19]. Our mean values of 1. 7-2.2 CCV cell- 1 observed for T. pyriformis and U. marinum therefore appear reasonable. DMSO failed to inhibit the uptake of carmine particles by Tetrahymena pyriformis and Uronema marinum at concentrations ~0.4 %. DMSO exhibits a number oftoxic effects on ciliate cells at higher concentrations including reduced cell division and reduced food vacuole formation [12, 20, 21, 24, 26, 30, 34]. However, concentrations similar to those used in this study are reported to have little or no effect on food vacuole formation in T. pyriformis and Paramecium caudatum [12, 23]. DMSO at 0.4 % v/v final concentration would therefore appear to be a suitable carrying compound for cytochalasin B for studies involving U. marinum as well as T. pyriformis. Exposure to 1 and 1 ~g ml- 1 cytochalasin B inhibited ingestion of carmine particles by both Tetrahymena pyriformis and Uronema marinum, with greatest inhibition recorded after 4 to 8 hours exposure to a concentration of 10 ~g ml- 1. Inhibition of particle ingestion by cytochalasin B has been previously reported for T. pyriformis [15,23,27,31,32], Paramecium caudatum [11,37], and Pseudomicrothorax dubius [14], with a concentration of 7.5 ~g ml- 1 reducing food vacuole formation in T.
°
pyriformis to 71 % of that of control cells after one hour exposure to the drug [27]. In this study a similar reduction in food vacuole formation to 63 % of that of the control was recorded for T. pyriformis after exposure to 10 ~g ml- 1 cytochalasin B for one hour. Uronema marinum appears to be more sensitive to cytochalasin B than Tetrahymena pyriformis. This may be due to morphological differences, such as its smaller size, to the osmolarity of its environment, or to the different culture methods. It is unlikely that the presence of bacteria within the U. marinum culture medium would have influenced cytochalasin B toxicity by metabolising DMSO because any reduction in DMSO concentration would have tended to decrease the drug's solubility and ability to permeate the cell membranes [12]. However, inhibition of ingestion may have been underestimated in T. pyriformis cells due to the accumulation of carmine particles in an open vacuole at the cell cytostome. Open vacuoles have been observed in T. pyriformis cells treated with cytochalasin B and resemble normal food vacuoles when observed by light microscopy [23]. In this study most cytochalasin B-treated T. pyriformis cells were observed with a single CCV located in the cytostome region, and these may have been open food vacuoles. It has been suggested that cytochalasin B interferes with the 'sealing off' of food vacuoles in T. pyriformis by disrupting a specialized region of contractile cytoplasm surrounding the cytostome and composed of a network of microfilaments [23]. This is consistent with more recent studies which show that cytochalasin B binds to the end of actin filaments [10, 28]. Cytochalasin B presumably inhibits ingestion in U. marinum in a similar manner, although few single CCV were observed in the cytostome regions of these ciliates. Thus, the exact mechanism by which cytochalasin B affects ciliate ingestion has yet to be confirmed. In order to function to full effect as a selective inhibitor of ciliate grazing in field samples, cytochalasin B would have to achieve rapid and complete inhibition of ingestion in all ciliates without affecting other physiological functions of ciliates or their prey. Cytochalasin B clearly reduces particle ingestion in Tetrahymena pyriformis and Uronema marinum at concentrations which do not appear to affect population growth. However, the drug did not inhibit ingestion completely, and maximum reduction of ingestion was attained only after 4 to 8 hours, precluding its use in situations where more effective techniques for estimating grazing rates are available. Ingestion was, however, reduced substantially by cytochalasin B treatment, with only 4 % of U. marinum cells feeding after 4 hours exposure to the drug. The use of cytochalasin B in experiments lasting up to 24 hours could therefore generate limited data on ciliate grazing in physically structured environments, such as sea ice, where established techniques cannot be used. Further studies are required to determine whether cytochalasin B reduces ingestion in natural populations of ciliates at concentrations which do not affect the ciliate growth, and to assess whether such concentrations influence other protozoan grazers and their algal prey.
314 . R. J. G. Leakey, S. A. Wilks and A. W. A. Murray
Acknowledgements We are grateful to M. A. Sleigh, J. Priddle, A. Clarke, and two anonymous reviewers for critically reading the manuscript. The research was undertaken as part of a sandwich student placement awarded to S. A. Wilks at the British Antarctic Survey.
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Key words: Cytochalasin B - Dimethyl sulphoxide - Grazing - Tetrahymena - Uronema Raymond
J. G. Leakey, British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 OET, UK