The Effect of Environmental Factors on the Life History of the Ciliate, Vorticella microstoma* During these studies the author held a Research Scholarship from Victoria University College.

Transactions and Proceedings of the Royal Society of New Zealand, Volume 82, 1954-55, Page 705

The Effect of Environmental Factors on the Life History of the Ciliate, Vorticella microstoma* During these studies the author held a Research Scholarship from Victoria University College. By J. D. Stout, Department of Zoology, Victoria University College [Read before Wellington Branch March 16, 1954; received by Editor, March 17. 1954.] Summary In the absence of oxygen, and in response to suddenly reduced oxygen tensions, the trophic vorticellid forms a telotroch which will not settle unless oxygen is supplied. The telotroch will survive two days in the complete absence of oxygen. A telotroch is also formed in response to high carbon dioxide tensions, which the telotroch survives much better than the trophic vorticellid. Desiccation of the culture also causes the trophic vorticellid to form a telotroch. The factors affecting encystment and excystment are discussed in relation to the present experiments. Introduction The life history of Vorticella microstoma is shown diagrammatically in Text-fig: 1. There is both a sexual and a non-sexual cycle, and the nonsexual cycle normally includes a division and a cystogenic cycle. In the non-sexual cycle the stalked trophic ciliate, with its peristome everted, feeds and increases in size Division takes place by longitudinal fission, which is slightly asymmetric so that one daughter cell remains attached to the parent stalk. The other forms a posterior ciliary wreath and with its peristome retracted becomes a free-swimming non-trophic telotroch. The polarity of the telotroch is the exact reverse of the stalked individual, the ‘posterior ciliary wreath’ of the settled form now being foremost in locomotion. The telotroch rotates in a counter-clockwise direction. Ultimately it settles, secretes a stalk, resorbs the posterior ciliary wreath, everts the peristome and begins feeding as a normal trophic individual. On the exhaustion of food, and sometimes under the influence of other environmental factors, the peristome is retracted and the ciliate secretes a cyst membrane (Brand, 1923). When stimulated to excyst, the ciliate escapes from the cyst membrane as a telotroch and then reverts to the normal trophic cycle (Stout, 1954). Under certain circumstances conjugation takes place (Finley, 1936, 1939) Preconjugation division leads to the formation of a macrogamete and a microgamete, the latter very much smaller but otherwise morphologically identical with the telotroch. The free-swimming microgamete becomes fused with a macrogamete and conjugation takes place, the microgamete being completely absorbed. The microgamete can conjugate with its sister macrogamete but not with an ordinary trophic vorticellid which the macrogamete otherwise resembles. Unlike the telotroch it dies if it fails to conjugate. It cannot metamorphose into a normal tropaic individual, although the macrogamete can. After conjugation the vorticellid reverts to the non-sexual cycle.

Although the normal vorticellid has two stages, one sessile and trophic and the other non-trophic and free-swimming, occasionally the stalked individual becomes detached from its base. When this happens the individual will swim freely in the medium propelled by the beat of its peristomial cilia which normally cause a current of food particles to be swept towards the vestibule. One change not mentioned in the above account is the habit of direct telotroch formation by the trophic vorticellid. This is a characteristic response to a number of environmental factors of which low oxygen tension and high carbon dioxide tension are two demonstrated in the present paper. Previously it has been held (Brand, 1923) that the vorticellid encysts in response to “adverse” conditions but this is rarely the case, except in response to starvation. The most common response is the formation of a free-swimming telotroch which is able to survive such conditions as lack of oxygen and high carbon dioxide tension for a comparatively long time. Text-fig 1.—Diagram of the life history of Vorticella microstoma. The sexual cycle has been described by Finley (1939) and cystment and division by Brand (1923) and Stout (1954). The present studies are concerned with excystment and the direct metamorphosis of the trophic to the telotroch form (shown by the broken line) in response to exceptional environmental conditions. It is apparent that this metamorphosis interrupts all three normal cycles, namely the sexual cycle, encystment and division. Material Vorticella microstoma was obtained from an anaerobic meat digestion plant (Stout, 1954). Cultures were prepared directly in Syracuse watch glasses from the fluid by the addition of loops of bacteria. Telotrochs were also washed free of other protozoa and permitted to grow in bacterial suspensions. The systematics of Vorticella microstoma Ehrb. is dealt with by Noland and Finley (1931) and its ecology illustrated by Lackey (1938). Essentially it is a saprobic species, first described as V. hians by O. F. Muller (1773), and common in polluted water. Excystment and encystment in relation to bacteria are described in a former paper (Stout, 1954). Methods A modification of Kitching's (1939) apparatus was used for the experiments. A small chamber with inlet and outlet tubes was placed on the stage of the microscope. A hanging drop preparation of the ciliates was sealed to the chamber

and the atmosphere of the chamber then replaced with either purified nitrogen, carbon dioxide, oxygen, an oxygen-carbon dioxide mixture, or unpurified nitrogen from which the oxygen had not been wholly removed. Before passing through the chamber the purified nitrogen was washed in dilute sulphuric acid, to remove traces of ammonia, alkaline pyrogallol, to remove traces of oxygen, and distilled water. On escaping from the chamber this nitrogen was passed through distilled water and alkaline pyrogallol. The other gases were passed through distilled water before and after passing through the chamber. The gases used were from commercially prepared cylinders. Rubber leads were used to connect the apparatus. Effect Of Anoxia And Lowered Oxygen Tensions The reactions of Vorticella microstoma to suddenly reduced oxygen tensions or complete oxygen lack are quite definite. The trophic ciliate forms a posterior ciliary wreath, detaches itself from its stalk and becomes a free-swimming non-trophic telotroch. The telotroch will not settle unless a trace of oxygen is present. The cysts are stimulated by suddenly decreased oxygen tensions and many will excyst. The following are typical experiments. A hanging drop containing five trophic vorticellids and one dividing vorticellid was placed in the chamber and the air replaced with purified nitrogen. Within half an hour the first telotroch had formed and a second was being formed. Within four hours all seven ciliates had formed telotrochs. After twenty-four hours three telotroehs were still free-swimming while four were stationary and attempting to settle. After thiry hours all seven were attempting to settle and had ceased swimming freely in the hanging drop. After forty-eight hours only four viable telotrochs remained. These were still attempting to settle but no stalk or peristome was formed and there was no pulsation of the contractile vacuole. The drop was taken from the chamber after fifty hours and returned to air. Within an hour and a half one of the ciliates had formed a stalk and a peristome and was feeding. Some difficulty was experienced in preserving the chamber free of oxygen If the flow of purified nitrogen was slowed or stopped, oxygen tended to diffuse back into the chamber. Consequently by leaving the drop in the chamber and turning the nitrogen flow on and off, the reactions of the ciliates to the presence or absence of oxygen could be observed. A hanging drop of ten trophic vorticellids was placed in the chamber and the nitrogen flow turned on for forty minutes. The ciliates stopped feeding and one telotroch was formed. The flow of nitrogen was stopped, permitting oxygen to diffuse back into the chamber, and all recommenced feeding. The nitrogen flow was then turned on again and after an hour all the ciliates had formed telotrochs. The flow was stopped and the ciliates settled and recommenced feeding a id dividing as the oxygen diffused back into the chamber. After sixty-six hours there were about two hundred trophic ciliates in the hanging drop. The nitrogen flow was turned on once more and after three hours there were about one hundred and twenty telotrochs and after six and a half hours about one hundred and fifty. After twenty-four hours there were still about one hundred viable telotrochs and no trophic individuals. The nitrogen flow was stopped, and within two and a half hours all the telotrochs had settled and were normal trophic forms.

Hanging drops of starved V. microstoma containing many cysts and a few trophic individuals were placed in the chamber. The drops also had cysts and a few trophic forms of Oxytricha pellionella. Unpurified nitrogen, still retaining a trace of oxygen, was passed through the chamber. The reactions of the ciliates are summarised in Table 1. Although the vorticellids react at first by forming telotrochs, they subsequently become adapted to the reduced oxygen tension and resettle. Table 1.—Reaction of cysts of Vorticella and Oxytricha to suddenly reduced oxygen tensions. The effect of an atmosphere of unpurified nitrogen containing a trace of oxygen. Time 0 30 mins. 45 mins. 18 hrs. 30 mins. Vorticella 9 trophic crliates many cysts telotroch formation 1 trophic 8 telotrochs 100 trophic 2 telotiochs 140 excysting 30 cysts Oxvtriha 0 trophic many cysts 45 trophic 11 excysting Time 0 30 mins. 1 hr. 5 hrs 30 mins. Vorticella 6 trophic many cysts 1 telotroch 30 telotrochs excystment 50 telotrochs 200 trophic Oxytricha 4 trophic many cysts 20 trophic The vorticellids showed the same reactions to the removal of oxygen if pure carbon dioxide was used to replace the air in the chamber. The following experiment shows the alternate effects caused by nitrogen with a trace of oxygen, pure carbon dioxide, and pure oxygen. Although the trophic ciliates initially form telotrochs, they ultimately settle again in a nitrogen atmosphere containing a trace of oxygen. They do not settle in an atmosphere of carbon dioxide. A fresh drop of about twenty-eight trophic vorticellids was placed in the chamber and unpurified nitrogen passed through. Within twenty minutes posterior ciliary wreaths were being formed and within forty-five minutes there were about eighteen telotrochs. After an hour one had settled and the rest were still telotrochs. After two and a quarter hours, six had settled. Feeding and division took place. After twenty-one hours there were about fifty-five trophic and dividing ciliates and about eight telotrochs. The unpurified nitrogen flow was turned off and pure carbon dioxide turned on for quarter of an hour. Two hours later there were about sixty telotrochs and three forming. Pure oxygen was then passed through the chamber and, within an hour, sixty-six vorticellids were settled, one was settling and one was about to settle. In one experiment using pure carbon dioxide, a thousand telotrochs, formed after passing pure carbon dioxide through the chamber, survived twenty hours in this atmosphere. After this time the chamber was flushed through with unpurified nitrogen and within an hour all but ten of the telotrochs had settled. Effect Of Carbon Dioxide In the previous experiments the effect of carbon dioxide is somewhat masked by the effect of the removal of oxygen which causes telotroch formation. The telotroch proved very resistant to an almost pure carbon dioxide atmosphere, surviving many hours, as in the last experiment. The trophic ciliate however succumbed

very rapidly It first becomes narcotized and later cytolysis takes place. This occurred in the following experiments where an oxygen-carbon dioxide mixture was employed. A hanging drop containing six Oxytricha pellionella, twenty-six trophic vorticellids and many cysts was placed in the chamber and pure oxygen turned on for an hour. There was no change in either the encysted or trophic forms with the exception of a single vorticellid at the margin of the drop which formed a telotroch is the edge of the drop dried. The oxygen-carbon-dioxide mixture (about 1:1) vas then passed through for twenty minutes. There was no excystment or telotroch formation but the ciliates were narcotized and the gases were therefore turned off. Forty minutes later there were six telotrochs, the other ciliates were still narcotized and after an hour and twenty-five minutes there were no telotrochs all the ciliates being either settled and feeding, narcotized, or dead. Even when pure oxygen was passed through some of the ciliates failed to recover from the initial treatment of carbon dioxide. Pure carbon dioxide was then passed through the chamber for some minutes. After twenty minutes there were eleven telotrochs and after thirty minutes there were twenty telotrochs and no trophic ciliate. A trace of oxygen was let into the chamber but the ciliates were left in a predominately carbon dioxide atmosphere for seventeen hours when there were still thirty-five to forty telotrochs. The experiment was then stopped. Further experiments subjecting trophic vorticellids to a mixed oxygen-carbon dioxid atmosphere for half an hour generally showed no telotroch formation but narcosis and death of the trophic ciliates. However in one experiment under such conditions about twelve trophic ciliates were placed in the chamber and the oxygen-carbon dioxide mixture turned on. After three hours these ciliates had formed telotrochs. It would appear that provided the carbon dioxide concentration does not narcotize the trophic ciliate it will react by forming a telotroch. This reaction takes place in the presence of oxygen so that the response appears to be due to the raised carbon dioxide tension and not to the removal of oxygen. Moreover the telotroch does not settle in a high carbon dioxide atmosphere even when oxygen is present. Discussion The detachment of sessile ciliates from the substrate under “adverse” conditions is comparatively well known. Stentor so reacts to strong light and settles again in less intense illumination (Jennings, 1915). Naturally such reactions are of considerable ecological importance and Brand (1946) in particular quotes the case of Vorticella nebulifera which he describes as forming a telotroch when subjected to low oxygen tensions. To some extent the ciliates are capable of adaptation and as Jennings (1915. p. 67) remarked, ‘acclimatization to non-optimal conditions is an ever present factor in the behaviour of organisms.’ In the present experiments telotrochs which had been formed following a sudden drop of oxygen tension subsequently settled provided there was a trace of oxygen present However in the complete absence of oxygen the telotroch failed to settle although it survived over forty-eight hours, a long period for a non-trophic free-swimming ciliate It is not. unreasonable to assume that in this case the lack of oxygen definitely interfered with the metabolism of the ciliate and inhibited its metamorphosis to the trophic form. This is comparable to the inhibition of the ‘activity’ system by various drugs (Fisher and Stern, 1942; McElroy, 1948) and the inhibition of excystment and division in ciliate facultative anaerobes

by lack of oxygen (Thomas, 1942; Watson, 1944; Brown, 1939). For this reason the claims of Kolkwitz and Marsson (1909) and Nikitinsky and Mudrezowa-Wyss (1930) that V. microstoma survives the absence of oxygen must be qualified. V. microstoma survives only as a cyst or as a telotroch, it cannot feed or divide under such conditions. The presence of trophic vorticellids is an indication of the presence of oxygen. Of the factors causing encystment of V. microstoma absence of food is generally accepted as essential. Enriques (1907) regarded chemical influences as also affecting the encystment of V. microstoma and putrefaction and desiccation have been suggested as possible factors (Brand, 1923). Brand himself inferred from his experiments that starvation and lack of oxygen were the principal causes of encystment and that desiccation also would cause encystment. Brand did not find that either the pH or the accumulation of Paramecium metabolites affected encystment. The present experiments have shown that lack of oxygen causes telotroch formation and not encystment and this is also true of desiccation. Brand (1946) himself recorded that V. nebulifera formed a telotroch when subjected to low oxygen tensions. Starvation therefore appears to be the principal cause of encystment. However, in a previous paper (Stout, 1954) encystment is recorded in dense bacterial suspensions and cysts were recovered from an anaerobic fluid which had ample bacterial food. In these cases the cysts were larger than those formed in well aerated cultures. It was suggested that encystment in these cases was due to the intense bacterial activity associated with a low redox potential. Hayes (1938) recorded encystment of Dileptus anser under similar conditions. It appears therefore that although starvation is normally the principal cause of encystment, under exceptional circumstances the vorticellids may encyst in the presence of bacteria, although in this case the cysts are larger than those formed under starvation conditions. Brand (1923) found that excystment was affected by a number of factors including bacteria, oxygen and pH. Moreover he found that cysts differed in their response, some excysting under one set of conditions and others under different conditions. An alkaline pH he thought was inhibitory and neutralization, in removing this inhibitory factor, permitted excystment to take place. Aeration and neutralization were sufficient by themselves to cause excystment of over 30% of the cysts. The addition of bacteria caused the excystment of about 70%. The three factors accounted for the excystment of almost all the cysts and Brand therefore believed that they were the most significant. Cysts formed in the anaerobic fluid and in dense bacterial suspensions have been found to excyst following aeration (Stout, 1954) which implies that 30% of Brand's cysts may have been formed in response to such anaerobic or reducing conditions. In the present experiments cysts subjected to very low oxygen tensions were also found to excyst. This was also the case with cysts of Oxytricha pellionella and has been found to be the case with Colpoda steinii. No satisfactory explanation of this phenomenon is apparent. Normally, however, bacteria are required for excystment, neither yeast nor hay extract being effective (Stout, 1954). In this respect V. microstoma resembles Didinium (Beers, 1946) for which bacteria or bacterial metabolites are a necessary excystment factor. However in the case of Didinium, which is a carnivore, the bacteria cannot be thought of as a nutritional factor and although Vorticella is bacteriophagous it does not necessarily imply that the effect of the bacteria is nutritional.

Perhaps the most remarkable result of the present experiments is the resistance of the telotroch to very high carbon dioxide tensions and the response of the trophic ciliate to increased carbon dioxide tensions. It is apparent that the resistance of the telotroch combined with the readiness with which the telotroch is formed is a considerable asset in the ecology of the species. It is hoped that this aspect will be discussed more generally in a later paper. Acknowledgments The author is indebted to Prof. L. R. Richardson for helpful criticism during the present studies which form part of a thesis submitted to the University of New Zealand in partial fulfilment of the requirements for the degree of Doctor of Philosophy. The cylinder gas was supplied by N.Z. Industrial Gases Ltd., who kindly loaned the necessary control valves for the experiments. References Beers, C. D., 1946 Excystment in Didinium nasutum, with special reference to the role of bacteria. J Exp. Zool., 103: 201–232. Brand, T. Von, 1923. Encystierung ber Vorticella microstoma und hypotrichen Infusorien. Arch. Protistenk., 47: 59–100. — 1946. Anaerobiosis in Invertebrates. No. 4 of the Brodynamica monographs edited by B. J. Luyet, Normandy, Mo., 328 pp. Brown, M. G., 1939. The blocking of excystment reactions of Colpoda duodenaria by absence of oxygen. Biol. Bull., 77: 382–390. Enriques, P., 1907. La conjugazione e il differenziamento sessuale negli infusori. Arch. Protistenk., 9: 195–296. Finley, H. E., 1936. A method of inducing conjugation within Vorticella cultures. Trans. Amer. Micr. Soc., 55: 323–326. — 1939. Sexual differentiation in Vorticella microstoma. J. Exp. Zool., 81: 209–229. Fisher, K. C., and Stern, J. R., 1942. The separation of an ‘activity’ metabolism from the total respiration of yeast by the effects of ethyl carbamate. J. Cell. Comp. Physiol., 19: 109–122. Hayes, M. L., 1938. Cytological studies on Dileptus anser. Trans. Amer. Micr. Soc., 57: 11–25. Jennings, H. S., 1915. Behaviour of the Lower Organisms Columbia University Press, New York, 366 pp. Kitching, J. A., 1939. The effects of lack of oxygen and of low oxygen tensions on Paramecium. Biol. Bull., 77: 339–353. Kolkwitz, R., and Marsson, M., 1909. Oekologie der tierischen Saprobren. Int. Rev. Hydrobiol., 2: 126. Lackey, J. B., 1938. A study of some ecologic factors affecting protozoa. Ecol. Monogr., 8: 501–527. McElroy, W. D., 1947. The mechanism of inhibition of cellular activity by narcotics. Quart. Rev. Biol., 22: 25–58. Muller, O. F., 1773. Vermium terrestr. et fluviatil., S. animal infusor., etc. historia. Hafnia et Lipsiae. Nikitinsky, J. and Mudrezowa-Wyss, F. K., 1930. Uber die Wirkung der Kohlensaure, des Schwefelwasserstoffs, des Methans und der Abwesenheit des Sauerstoffs auf Wasser-organismen. Central. Bact., Abt. 2, 81: 167–198. Noland, L. E., and Finley, H. E., 1931. Studies on the taxonomy of the genus Vorticella. Trans. Amer. Micr. Soc., 50: 81–123. Stout, J. D., 1954. Some observations on the Ciliate Fauna of an Experimental Meat Digestion Plant. Trans. Roy. Soc. N.Z., 82: 199–211. Thomas, J. O., 1942. The Anaerobic Carbohydrate Metabolism of Tetrahymena geleii. Dissertation, Stanford University. Watson, J. M., 1944. Studies on the morphology and bionomics of a little known holotrichous ciliate—Balantiophorus minutus. II. The effect of environmental factors. J. Roy. Micr. Soc., 64: 31–67.