Feeding by Euplotes Mutabilis (Ciliophora, Hypotrichida)*

ZOBODAT - www.zobodat.at Zoologisch-Botanische Datenbank/Zoological-Botanical Database Digitale Literatur/Digital Literature Zeitschrift/Journal: Denisia Jahr/Year: 2008 Band/Volume: 0023 Autor(en)/Author(s): Wilks Sandra A., Sleigh Michael Artikel/Article: Feeding by Euplotes mutabilis (Ciliophora, Hypotrichida) 383-388 ©Biologiezentrum Linz/Austria, download unter www.biologiezentrum.at Feeding by Euplotes mutabilis (Ciliophora, Hypotrichida)* S a n d r a A. W I LK S & M i c h a e l A. S LEI GH Abstract: Experiments on the capture and ingestion of microspheres and algal cells of different sizes presented at different concentrations, alone or in combination, showed that feeding activity and ingestion rates were dependent on the concentration and size of food particles; larger particles were preferred to smaller ones and algal cells to microspheres of similar sizes, at the same concentration. The membranelles of Euplotes create water currents carrying suspended particles, filter particles from the current and direct them to the cytostome. Specialised frontal membranelles may play some part in recognition of prey, but the principal selection site is probably the membrane lining the forming food vacuole at the cytostome. Key words: Algal prey, ciliate food selection, lectin binding, membranelles, microspheres. Introduction lated marine species, identified as Euplotes mutabilis, which was maintained on the bacterium Vibrio na- Features of the ciliature of ciliates have long been triegens, and had been used in a study on the fate of in- used to characterise differences between them at all gested food materials by ZUBKOV & SLEIGH (1995). This taxonomic levels from sub-phylum down to species. food bacterium is much smaller than the tracer particles Ciliary patterns reflect the adaptations of the organism used to observe flow patterns, and also smaller than the concerned to the particular mode of life, principally lo- spacing between membranelles, which led us to take an comotion, including relevant aspects of behaviour, and interest in the feeding mechanism of this ciliate. feeding. In principle, a comparison between the ciliary patterns of two ciliates should allow an understanding The results of two of our studies related to the feed- of a major part of how they are fitted for their particu- ing of Euplotes mutabilis have already been reported. In lar ecological niche. Our knowledge of the differences the first the relationship between the rate of ingestion between the patterns of ciliature of ciliates is far in ex- of inert microspheres and the concentration and size of cess of our understanding of how cilia, and especially presented particles was investigated by WILKS & SLEIGH distinctive patterns of ciliature, are used in the life of (1998). It was found that microspheres of all five diam- these ciliates. One bold approach to studying the inte- eters from 0.57 to 10 µm were captured and ingested at grated use of cilia in ciliate locomotive behaviour was rates that depended upon particle size and concentra- exploited by RICCI (e.g. 1990) in constructing tion, but that particles with diameters of 1.90 and ‘ethograms’ of moving ciliates, and ways of using cilia in 3.06 µm were taken in largest numbers at a particular collecting food have been studied by various workers. concentration. However, the volume of ‘food’ ingested per cell per hour increased with microsphere diameter If a dense suspension of particles is pipetted around at any particular concentration, and a greater volume of a surface-dwelling motile ciliate, such as Euplotes or 10 µm microspheres was ingested at a concentration of Stylonychia, then flow patterns are set up like those de- 104 particles ml-1 than of any smaller particle at a con- scribed by MACHEMER (1966). These are created by the centration of 106 ml-1. It was interesting that in these membranelles of the adoral zone (AZM). Some of the experiments the proportions of cells that ingested par- particles are intercepted by the membranelles and are ticles decreased sharply as concentration was reduced then seen to progress towards the cytostome at the rear from near 100 % at 106 ml-1, to 75 % at 10 5 ml-1, 25 % end of the buccal area and may be taken into food vac- at 104 ml-1 and about 10 % at 103 ml-1. uoles. We observed this feeding activity in a locally-iso- In the other study the patterns of lectin binding ILKS * The authors dedicate this article to Prof. Dr. Wilhelm FOISSNER, sites in E. mutabilis were determined by W & on the occasion of his 60th birthday. Denisia 23 (2008): SLEIGH (2004). Living cells, including those of prey or- 383-388 ©Biologiezentrum Linz/Austria, download unter www.biologiezentrum.at taraldehyde preserves ingested algal cells better. Each experiment was repeated three times and the number of ingested cells in at least 100 ciliate cells was counted in each replicate experiment and presented as a mean val- ue with standard error, making these experiments exactly comparable with those on microspheres completed a few weeks previously. Four types of photosynthetic cells were used as prey organisms. The algae Chlorella sp. (CCAP 211), nearly spherical and about 4.5 µm in diameter, Isochrysis galbana (Plymouth Culture Collection “I”), nearly spherical and about 3 µm in diameter, and Rhodomonas sp. (CCAP 995/2), whose elongate cells have an equivalent spherical diameter of about 9 µm, were all grown in 100 ml Nunclon culture flasks containing 40 ml of Guil- lard’s f/2 medium (see GUILLARD 1975). The cyanobacterium Synechococcus sp. (strain WH7803 from Warwick University), with a diameter of about 2 µm, was grown Fig. 1: The percentage of Euplotes observed to feed on microspheres or algae in artificial seawater medium with added trace metals, when these were presented at different concentrations from 102 to 106 ml-1. For this figure the data for microspheres or algae of all sizes used were boric acid and EDTA. All were cultured at a room tem- combined together at each concentration. perature of 20 ± 5 °C in natural daylight. Although the cyanobacterium Synechococcus is not strictly an alga, it ganisms, expose carbohydrates of the glycocalyx on is a comparable photosynthetic cell, and will be referred their surface which may be a useful property for the to as an alga for brevity. recognition of potential prey by a ciliate, using other carbohydrates on their own surface as binding sites. Car- We had expected that the autofluorescence of bohydrate recognition sites on the surface of a ciliate ca- chlorophyll in these cells would be sufficient for them to pable of binding to prey may be located by the use of be easily distinguished for counting within the food vac- lectins that specifically bind to those carbohydrates. uoles of Euplotes, but this was not the case for three of Several fluorescent lectins were found to bind princi- these types, particularly Synechococcus. It was therefore pally to the cytostome area and to food vacuoles; con- necessary to use fluorescent labeling of the prey, which canavalin A showed the strongest and most persistent has been common practice in such feeding studies (e.g. labelling of this type. However, one lectin, wheat germ SHERR et al. 1987). It was decided to stain algae with agglutinin (WGA), also rapidly labelled a group of 8–9 DTAF (5(4,6-dichlorotriazinyl)aminofluorescein, ob- frontal membranelles of all E. mutabilis cells. Both tained from Sigma), following the method used by frontal membranelles and the cytostome lining could be RUBLEE & GALLEGOS (1989). This involved gentle cen- involved in positive or negative food selection. trifugation to concentrate the cells, which were re-suspended in 0.02 M TRIS-NaCl buffer (2.42 g l-1 TRIS, The present report is concerned with further parts of 8.5 g l-1 NaCl) containing 0.2 mg ml-1 DTAF (Sigma) the same study attempting to relate these results to the and incubated for 2–3 hours in a water-bath at 54– real life of the ciliates by seeking answers to two princi- 60 °C, before being washed three times in the TRIS pal questions. 1. Will real prey be treated in the same buffer and re-suspended using a vortex mixer. Stained way as inert microspheres? 2. How will the cell behave cells can be stored in this medium for months if kept when presented simultaneously with prey of different frozen. Before use in experiments the cells were thor- sizes, as in nature? The findings of experiments are then oughly washed three times in artificial seawater (ASW) combined into a discussion on the feeding of this ciliate. and then re-suspended in ASW. Isochrysis galbana was too fragile to survive the heating involved in labelling Materials and methods by this method, but fortunately had sufficient autofluorescence to be used live in experiments. The ciliates, their preparative handling and the pro- cedures for performing experiments were exactly as de- The interplay between relative sizes and relative scribed for experiments on the uptake of microspheres concentrations of even two different particles may be by WILKS & SLEIGH (1998), except that ‘algal’ cells were investigated by varying the parameters in many combi- provided as potential prey, and 0.5 % (v/v) glutaralde- nations. Two of the simpler patterns were tested experi- hyde was used as a fixative in place of TCA, since glu- mentally. In the first one type of particle was presented 384 ©Biologiezentrum Linz/Austria, download unter www.biologiezentrum.at Table 1: Comparisons of the numbers of algae and the volume of algal material ingested at typical ambient concentrations for algae of various sizes, compared with numbers and volumes of microspheres of the nearest equivalent sizes ingested at the same concentrations.

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