The Growth of Mixed Populations of Chilomonas paramecium and Tetrahymena patula

Experimental studies of the relations between predator and prey have not been as numerous, nor have they been made on as many different species, as have studies on competition between organisms. Gause (1934, 1935a, 1935b) showed that their interaction did not invariably lead to periodic oscillations of population density, as predicted in the mathematical theories of Lotka (1925) and Volterra (1926), but that the occurrence of oscillation depended on certain characters of the species under investigation, and on environmental factors. He observed (Gause, 19356) oscillations with Paramecium bursaria and Saccharomyces pombe, provided that yeast was added to the medium from time to time, and that the population of the predator was decreased by taking samples. In other experiments (Gause, 1935a), where Bursaria truncatella was predator and Paramecium bursaria prey, periodic fluctuations did not appear, however, and Bursaria died out before the paramecia were destroyed. Gause does not mention that the relationship’ was more complex here than in his previous experiments on Paramecium bursaria and Saccharomyces pom.be-, but evidently the food chain of Bursaria-Paramecium-Saccharomyces was more complex than the simple relation of predator to prey. According to Sandon (1932), Bursaria truncatella feeds on ciliates as well as taking other food. In Gause’s experiments Bursaria may have preyed both on Paramecium and on yeast. According to Lund’s observations (1914a, 19146), Bursaria truncatella is selective in its feeding, since it accepted fragments of hard-boiled yolk stained with dyes insoluble in water, but refused fragments of yolk stained with dyes soluble in water ; but Schaeffer (1917), was unable to agree that Bursaria had any food-selecting mechanism whatsoever ; the occasional refusal of food was only part of a general reaction to more or less injurious stimuli. If Bursaria is not selective it is probable that it was indeed feeding on both paramecia and yeast in Gause’s experiments.

Gause (1934) reported that Didinium nasutum, a voracious predator, destroyed Paramecium caudatum and then died out; fluctuations in the densities of their populations did not normally appear. Only when their interaction was modified by the presence of a ‘refuge’ (a sediment of yeast) or when, as in other experiments, he introduced both species into the culture at regular intervals of time, did fluctuations occur.

Brown (1940) observed the growth of mixed populations of Leucophrys patula preying on Glaucoma pyriformis. He was interested in the logarithmic phase of the growth of Leucophrys and he prepared both theoretical and observed growth curves for that phase, but followed the growth of the cultures for 4 days only, so that his experiments give very limited information about predatorprey relations. In his equations for rates of growth, Brown took the values for Leucophrys from the graph representing its growth in single populations, but he did not define the conditions under which it was grown in single populations. At that time L. patula (Tetrahymena patula according to Corliss, 1952) had not yet been established in bacteria-free cultures. It is doubtful, therefore, whether he was justified in taking values for its growth from graphs based on figures derived from growth under quite different conditions. Nor is it clear why Brown ascribed the end of the exponential phase to the high concentration of predator in the one instance, and to the exhaustion of food in the other. The density of Leucophrys was as high in the second instance as in the first, or even higher, as is shown by the curves. He does not state from how many cultures or samples data were obtained.

The study made by Dewey and Kidder (1940) of the growth of Perispira ovum with Euglena gracilis gives more details and covers a longer period than Brown’s data. It is interesting to note that Perispira continued multiplying for some time after the extinction of its prey, and at the expense of the size of individuals.

Lilly’s study (1942) of the nutrition of Stylonychia pustulata and Pleuro-tricha lanceolata is also important as a contribution to knowledge of the growth of mixed populations. It reveals how small details gathered from observation of the species may be of value in explaining the growth of the population. At the same time, it implies how many variables are necessarily neglected, if the growth of mixed populations—for example, the interaction between predator and prey—is approached purely mathematically. Lilly confirmed that the length of the lag phase for these ciliates depended on the age of the inoculum. He also found that the size of the organisms is important in relation to the duration of the lag phase, which lasted longer if the inoculum consisted of small organisms, because these are not able to ingest large food organisms until they have increased in size. Lilly compared the phases of growth of both predators, and reported that they were characteristic for each species. Differences between the growth of their populations also appeared when the food was exhausted: Pleurotricha continued to grow, whereas Stylonychia did not.

According to this review of previous work, the growth of mixed populations of two protozoan species which are related as predator to prey, has previously been studied either in small communities composed of three species at least, or on two species in bacteriafree cultures but without control experiments. It seemed desirable, therefore, to examine the predatorprey relationship in a community of two species with control cultures of the single species.

In preliminary experiments, two species of Protozoa were sought, able to live separately in pure axenic culture in the same organic medium, but which, when brought together, interact as predator and prey. Chilomonas paramecium serves as prey to many ciliates, but since most of these are carnivorous forms that cannot live without food organisms, even in an organic medium, choice was restricted to those few ciliates which have been obtained in bacteria-free cultures. Among the flagellates, Peranema trichophorum feeds upon Chilomonas (Chen, 1950), but it cannot live with Chilomonas in the same organic medium. An attempt was first made to use Tetrahymena vorax (strain V₂) as predator. Mixed cultures were inoculated with macrostomatous forms only, because these are known to be carnivorous. They fed on Chilomonas, but after 3 to 4 days all macrostomatous forms had disappeared, and the population was composed of microstomatous forms only, which are not predaceous (Kidder, Lilly, and Claaff, 1940). This species, therefore, was abandoned. Subsequently, Tetrahymena patula (Müller) Corliss was found to be suitable for the present experiments; it takes Chilomonas as food (fig. 1, A) and can also live as a saprophyte. Subcultures were obtained from the Culture Collection of Algae and Protozoa, Botany School, Cambridge.

Maupas (1888) wrote on Tetrahymena patula under the name Leucophrys patula, giving a description of the. species and many observations on its life cycle. Fauré-Fremiet (1948) extended Maupas’s description, retaining the name Leucophrys. Recently, however, Corliss (1952, 1953) gave a full account of the history, systematics, and morphology of this species as Tetrahymena patula. T. patula was isolated before 1942 by Fauré-Fremiet (Corliss, 1952).

It was in principle desirable to perform experiments with the same medium as had been used in the experiments on T. pyriformis (Mucibabic, 1957) but this was not possible because in the medium of o-i% proteose peptone and o-i% sodium acetate, T. patula did not continue to multiply after one or two initial divisions. The medium became satisfactory, however, when the concentration of proteose peptone was increased from o-1% to 1%.

Cultures in 1% proteose peptone were inoculated with 10 Chilomonas and 10 Tetrahymenapatula, and control cultures were inoculated with 10 organisms of each species separately. All cultures were maintained at 22-5° C. The number of organisms was counted every day as in previous studies (1956); but since the density of population of Chilomonas differed greatly from that of Tetrahymena patula from the third day onwards, the size of the two populations could not be determined in the same way. Five samples from each culture were counted in order to determine the size of the Chilomonas population; while the entire population of Tetrahymenapatula in each culture was counted throughout the period of the experiment. As the culture had to be diluted before the population of Chilomonas could be counted, Tetrahymena patula was counted before dilution. The counting-procedure was as follows.

One drop of culture was placed on a slide. Since during the counting of T. patula it was not possible to pick up T. patula alone, the organisms collected in the capillary micro-pipette during counting were not discarded, but were delivered into a test-tube containing 5 to 10 ml of fresh medium. The next drop was placed on the same spot on the slide, and so on, till the whole culture had been examined. When the last drop had been taken, 0-3 ml of the medium was pipetted into the culture test-tube. This was also examined, in order to pick up any organisms that might have remained on the wall of the test-tube. The individuals of Chilomonas remaining in the drop on the slide were washed from the slide into the large test-tube in which the whole culture had been diluted. For washing, 5 ml of the fresh medium was used. Finally, the necessary quantity of medium was added to complete the required dilution. From this diluted culture 5 samples were then counted, in order to determine the size of the Chilomonas population.

Population growth of Ch. paramecium and Tetrahymena patula in terms of total number of organisms is shown in fig. 2 and table 1 (p. 256). The table shows mean size of the population, standard deviation, and number of observations for both species in single and mixed cultures. Table 1 and fig. 2 reveal that the growth rate of the Chilomonas population is very similar in single and mixed cultures. A very pronounced lag-phase appeared in both single and mixed cultures in this medium. It lasted for 2 days. The maximum numbers of Chilomonas in single and mixed cultures do not differ significantly.

At the beginning, the growth of the population of Tetrahymena patula is very similar in single and mixed cultures. Differences appear from the 3rd day onwards and later become greater. From the 6th day, the population of Tetrahymena in single cultures begins to degenerate: the organisms become very opaque, flattened, and abnormal, and their number decreases rapidly. In mixed cultures, however, tetrahymenae were multiplying until the 32nd day. On the 6th day they began to take Chilomonas more noticeably than before, and organisms with ingested individuals of Chilomonas were frequent (fig. i, A). The following day the population of Tetrahymena patula was nearly twice as great. This outbreak of division of Tetrahymena after increased predatory activity on the 6th day was observed in both series of experiments. The population of Tetrahymena increased from then onwards with small fluctuation till the end of experiment. Since the single culture of Tetrahymena had died out by the 18th day, counting of mixed cultures was not continued after the 20th day, but occasional observations were made from time to time. On the 32nd day the entire population of Chilomonas and of Tetrahymena was counted in one culture; the population of Chilomonas was then very small (only 474 organisms), while that of Tetrahymena included over a thousand individuals. After two months the Tetrahymena population was still dense, although no Chilomonas were to be found, in the originally mixed culture.

Data in table 1 for the Chilomonas population in single and mixed cultures were statistically compared, as well as data for the Tetrahymena population. On the 4th day only the population of Chilomonas is significantly larger in single than in mixed cultures. On the 7th day, however, the population of Chilomonas is larger in mixed than in single cultures to a degree that is almost statistically significant.

The difference between the size of Tetrahymena populations in single and mixed cultures is significant from the 6th day of population growth, the population being larger in mixed than in single cultures.

Table 2 summarizes data for population growth of Chilomonas in single and mixed cultures in terms of biomass (total volume of organisms). Total volumes of organisms were calculated from the data in tables 1 (above) and 5 (p. 259) (for method see Mucibabid, 1957). The biomass of Chilomonas is initially smaller in mixed than in single cultures. Later, for a short time, it surpasses the amount in single cultures; but as soon as it reaches the maximum stationary phase, the Chilomonas population in mixed cultures begins to decrease in total volume.

Tetrahymena patula is known to be a polymorphic species (fig. 1, B). Forms with a large peristome+mouth (macrostomatous), with a small mouth (microstomatous), and with a small slit instead of an open mouth, have previously been observed in bacteria-free cultures, as well as in cultures with bacteria. Transitional forms between the microstomatous and macrostomatous forms have also been described. In the present work, during the first series of experiments, it was noticed that the composition of the Tetrahymena population changes during population growth. For this reason, in the second series of experiments the number of each form of Tetrahymena present was recorded. This prolongs and increases the difficulty of counting, because one has to wait until the organisms are in such a position that the mouth can be seen. Occasionally it was not possible to determine by observation in life to which type an organism belonged. Transitional forms between microstomatous and macrostomatous forms were counted as macrostomatous forms.

The percentage of each form of organism from the whole population was calculated, in order to make the results comparable. These are shown in table 3 (p. 258). The table reveals that slit-mouthed forms never appear in single populations, while microstomatous forms are more frequent during the logarithmic phase of population growth than at other times, both in single and in mixed cultures. Their number in mixed populations increases on the 7th day, at the time when the rate of population growth suddenly increases.

The proportion of macrostomatous forms increases during population growth. Their percentage is greatest when the single cultures are in the stationary phase. At the same time their percentage is also maximal in mixed populations ; later their number decreases when the slit-mouthed forms appear in greater number. These macrostomatous forms are known to be cannibalistic (fig. i, c). They prey on the slit-mouthed forms as well as on Chilomonas, so that the percentage of both macrostomatous and slit-mouthed forms oscillates. In cultures a month and two months old, slit-mouthed forms are the most numerous; the percentage of macrostomatous forms is smaller than before, but they are large and healthy; microstomatous forms are poorly represented.

This division of forms of Tetrahymena patula into three groups is only approximately correct; it does not reflect the variety of shapes in which macrostomatous forms may appear in the same culture. Besides the forms that are rounded at the posterior end, as mentioned by previous authors, there are also forms with a re-entrant posterior end (fig. 1, D), forms with longitudinal folds (fig. i, E), and forms with an obtusely pointed end. The last were mentioned by Maupas (1888). In cultures 8 days old or more, macrostomatous forms were observed in a striking and characteristic attitude, as if looking for food: the body tilted with the mouth downwards, apparently examining the substratum, while the organism slowly advanced.

On the whole, there are no statistically significant differences between the percentages of microstomatous or macrostomatous forms present in single and mixed populations. The most important difference is the occurrence of slit-mouthed forms in mixed populations only.

Tables 4 and 5 contain data on changes in size and shape of Chilomonas paramecium and Tetrahymena patula during the growth of their single and mixed populations. These data were obtained in the same way as in previous experiments on the growth of mixed populations of Chilomonas paramecium and Tetrahymena pyriformis (1957). The volume of individual organisms of T. patula, however, cannot be calculated from the data obtained from the photographs, because of the irregular shape of the animal, and for this reason data for the volume of T. patula do not appear in table 5.

Length, width, and volume of individual organisms of Chilomonas, both in single and mixed cultures, decrease during population growth. The size of individual organisms increases only when the numbers of the Chilomonas population in mixed cultures begin to decrease.

The ratio of length to width of Chilomonas shows a decrease after inoculation, in single and mixed cultures, during the lag phase. This means that the organisms become more plump. In other phases of population growth, changes in shape of Chilomonas are not great. In mixed cultures only is there a pronounced decrease in the ratio at the end of the stationary phase, when the population of Chilomonas starts decreasing.

The length of Tetrahymena patula decreases, both in single and mixed cultures, after inoculation. Later, the length increases slightly in single cultures, though the organisms never regain the length of freshly inoculated organisms. In mixed cultures, however, an increase in length is very marked on the day following the increased ingestion of Chilomonas. A further increase in length was recorded when the organisms become cannibalistic; at the beginning of population growth they are not cannibalistic. The width of organisms also increases in mixed cultures at the same time.

The ratio of length to width of Tetrahymena patula decreases after inoculation, and the organisms are plump at the beginning of the logarithmic phase. Later, however, they become slender, even more slender than freshly inoculated organisms. Data for microstomatous forms are scarce, but such as they are, they are shown in table 5 (p. 259) under the data for macrostomatous forms. The lowest row (on the 14th day) comprises data for the slit-mouthed forms.

The variation in shape of Chilommas is greater in mixed than in single cultures, as can be seen from the coefficients of variation of the ratio length : width (coefficient of variation = relative standard deviation, that is, the standard deviation expressed as a percentage of the mean). The variation in size and shape of Tetrahymena patula is very great in mixed cultures. At the beginning of growth, variation is smaller than later.

Using the i-test (Fisher, 1950), the data from tables 4 and 5 have been statistically compared, in order to determine the significance of difference of means between single and mixed cultures. Significant differences in length between Chilomonas from single and mixed cultures exist from the beginning of the logarithmic phase of growth till the end of observations. Differences in width of organisms from single and mixed populations, as well as differences in the ratio of length to width, are usually not significant.

The comparison of results for Tetrahymena patula shows that differences in width of organisms from single and mixed cultures are always significant or nearly significant. Differences in length of Tetrahymena are significant from the 6th day, but differences in the ratio of length to width are less significant than those for length or width.

The mean maximal and minimal values for length, width, and ratio of length to width, for members of the single populations were compared. This comparison was also made for members of the mixed populations. Values of t for Chilomonas for the difference between mean maximal value at 2 days and minimal value at 10 days, from single populations, were: 3-167 for length; 5-681 for width; and 1-756 for the ratio of length to width. Corresponding values for Chilomonas from mixed cultures were: for length (2 and 10 days old), 13-022; for width (2 and 8 days old), 7-547; for the ratio length : width (L : W) (6 and 14 days old), 2-737. These values show that the differences are highly significant, with the exception of the difference in the ratio of length to width in organisms in single population which, however, is nearly significant ; that is to say, the organisms in single culture change significantly in size during population growth, while those in mixed culture change significantly both in size and in shape. The corresponding values of t for Tetrahymena patula in single population were: for length (2 and 6 days old), 3-40; for width (2 and 6 days old), 5-70; for the ratio L : W (4 and 6 days old), 3-506. In mixed populations the values were: for length (4 and 14 days old), 5-276; for width (6 and 14 days old), 5-568; for the ratio L : W (2 and 8 days old), 5-416. These are all highly significant; so that both size and shape of Tetrahymena patula change significantly during population growth, both in single and in mixed cultures.

According to the mathematical theories of Lotka (1925) and Volterra (1926), the interaction between predator and prey is characterized by cyclical variation in numbers of both species. In the present experiments, however, such periodic oscillations in population numbers have not appeared. After reaching the stationary phase, the population of the prey decreased continuously until it was exterminated, while the population of the predator increased steadily. At the beginning of population growth, the size of the Chilomonas population in single and mixed cultures does not differ significantly; for a short time, however, the population of Chilomonas is greater in mixed than in single cultures. This is, surprisingly enough, at the time when Tetrahymena ingests Chilomonas more readily than it did before. This must mean that the multiplication rate of Chilomonas in mixed cultures is greater at that time than it is in single cultures.

It has been shown that the composition of the population of Tetrahymena patula is affected by the presence or absence of Chilomonas; it also changes with the age of population. Slit-mouthed forms do not appear in single cultures, while in mixed cultures their percentage increases in old cultures. Corliss (1953) shows slit-mouthed forms as a transitory stage between microstomatous and macrostomatous forms ; but in the present experiments, slit-mouthed forms did not appear in single cultures, though both macrostomatous and microstomatous forms were present. It was observed that the slit-mouthed forms swim quickly, and Maupas (1888) also recorded a greater rate of swimming in slit-mouthed forms. He found them in cultures with a small quantity of food. In the present experiments also, the slit-mouthed forms do not appear from the beginning, but only from the seventh day. In one culture only, a single slit-mouthed form was noticed on the 4th day.

Maupas ascribed to them the role of the distribution of species. Fauré-Fremiet (1948) observed slit-mouthed forms in cultures of Tetrahymena (‘Leucophrys’) patula, previously fed with Colpidium. They appeared only in starving cultures. He observed a progressive reduction in the size of the mouth of microstomatous forms; indeed, in some forms the mouth became vestigial, and such organisms were unable to ingest bacteria. He mentions that these forms became the prey of the macrostomatous forms; this was also observed in the present experiments. Fauré-Fremiet and Mugard (1949) have described macrostomatous and microstomatous forms of Espejoia mucicola. The microstomatous forms are migratory and were observed to transform themselves to macrostomatous forms as soon as they entered the mucilage covering the thallus of Batrachospermum. In Espejoia the occurrence of microstomatous and macrostomatous forms does not depend on the age of population, as observed in the present experiments on Tetrahymena patula, but on the presence or absence of the appropriate mucilage.

It has been observed that T. patula does not prey continuously on Chilomonas. After a day of voracious feeding, they seem to ‘rest’, since on the following day Tetrahymena could not be seen to take Chilomonas. Volterra and Lotka’s equations for the interaction of organisms are based on the supposition that the rate of feeding is constant, and that the finding of prey by predator is a matter of chance. In old cultures, with ‘clouds’ of degenerating Chilomonas, Tetrahymena patula was observed to gather in these clouds. It is questionable whether either this aggregation or the discontinuity in feeding is the result of chance.

I would like to express my gratitude to the Heads of the Botany School and of the Department of Zoology, Cambridge University, for research facilities, and to rhany assistants who have spared no trouble in helping me. Sincere thanks are due to Professor E. G. Pringsheim, Dr. G. Salt, Professor S. Stankovic, and Professor G. E. Briggs for stimulating discussions and for useful suggestions in relation to my work. In particular I am indebted to Dr. L. E. R. Picken for constant help and encouragement.