VOLUME 36, No. 4 DECEMBER 1959 THE EFFECT OF ANAEROBIC CONDITIONS ON TWO HETEROTRICH CILIATE PROTOZOA FROM PAPYRUS SWAMPS BY L. C. BEADLE AND J. R. NILSSON* Department of Zoology, Makerere University College Kampala, Uganda (Rtceived 3 April 1959) That a number of aquatic invertebrate animals can live for long periods in the complete absence of oxygen, and that many are actually called upon to do so in their natural habitat, is a reasonable conclusion to draw from the extensive work on this subject which has been reviewed by von Brand (1946). Since most animal tissues can derive energy from an anaerobic glycolysis, the major problem of adaption to prolonged lack of oxygen would appear to be the removal of (or acclimatization to) the products, mainly fatty acids, of an anaerobic metabolism. Many of the previous experiments were, however, open to criticism in view of the uncertainty that com- pletely anaerobic conditions were attained. It is difficult also to establish beyond doubt that an animal has no access to traces of oxygen in nature, and the methods used for oxygen estimation were not likely to be sensitive to low concentrations in water containing reducing substances. The experiments described here were carried out in the hopes of getting more precise information on the effects of anaerobic conditions on the survival of species of ciliate protozoa from papyrus swamps, using an improved method for oxygen estimation and at the same time measuring the oxidation-reduction potential in the water as an additional confirmation of anaerobic conditions when chemical analysis failed to detect oxygen. The water in the interior of papyrus swamps is characteristically deficient in oxygen and is often completely anaerobic to within an inch of the surface (Beadle, 1932; Carter, 1954). But around the margins and in natural and cut clearings the water in pools may contain detectable or even abundant oxygen. A preliminary survey by one of us (J. R. N.) has shown, as might be expected, that certain protozoa are commonly found in the interior of the swamp and others are restricted to the better aerated water outside dense papyrus. The two species chosen for these experiments clearly differ in this respect and are both easily cultured in the laboratory. MATERIALS Both of the ciliates were collected from a papyrus swamp ten miles from Kampala on the Mubende road. (1) Bursaria sp. (fam. Bursariidae). From examination of cultures now established in the Carlsberg Laboratory it is clear that this is a new species not yet described • Present address Carlsberg Laboratory, Copenhagen. 37 Exp. Biol. 36, 4 584 L. C. BEADLE AND J. R. NILSSON and is not B. truncatella as originally thought. It was found mainly in open unshaded pools and not in the interior of the swamps, and thus would appear to be sensitive to low concentrations of oxygen. It fluctuates greatly in numbers, and on one occasion in August 1955 there was a distinct whitish layer near the surface caused by the swarming of this ciliate. (2) Blepharisma undulans (fam. Spirostomidae). This was common in water densely shaded by papyrus in which dissolved oxygen is often extremely low. It is peculiar in the possession of the pink photodynamic pigment 'Zoopurpurin' (ArchikovBkij, 1905), which disappears in the light and reappears in the dark. It is chemically related to the blue ' stentorin' found in Stentor coeruleus, both belonging to the meso-naphthodianthone group of compounds (Emerson, 1930A; Mcller, 1958). Pigmented specimens are killed by strong light, but not in the absence of oxygen (Giese, 1946). Giese & Zeuthen (1949) found that strong light greatly increased the oxygen uptake of pigmented specimens before killing them, but only a slight increase was induced in specimens previously bleached in the light. In view of this apparent photo-oxidative action of the pigment two of the following experiments were designed to compare the resistance of pigmented with that of unpigmented forms to a low concentration of oxygen. Both ciliates were cultured on agar plates 8-10 mm. deep in which sterile wheat grains were embedded and which were flooded with Pringsheim's salt solution* to a depth of 3-4 cm. The plates were inoculated with the ciliates, together with some unsterilized swamp water from which grew the organisms serving as food. The Bursaria cultures were kept on a shelf in dull light. The culture medium for Blepharisma was prepared and inoculated in the same way, but the pH was adjusted to 8-o which was found by Giese (1953) to be the optimum. The cultures were kept in the dark. When colourless specimens were needed cultures were kept for several days on the open bench, though not in the direct light. The colourless Blepharisma in the lighted cultures were rather smaller and more slender than the pigmented forms in the darkened cultures, the latter containing some of the giant forms noted by Giese (1946). Subcultures were made every 2 or 3 weeks. METHODS For each experiment 4 or 5 1. of swamp water containing a small amount of fine organic debris and inoculated with ciliates were stirred vigorously in an aspirator jar while being run into five or more pairs of 200 ml. glass-stoppered reagent bottles. This was done to ensure that each bottle contained the same number of ciliates and amount of suspended organic matter as well as the same initial concentration of oxygen. The bottles were filled to the top, stoppered without including air and kept under water. In experiments with pigmented Blepharisma the bottles were kept in the dark; with Bursaria and colourless Blepharisma they were exposed to dull light during the day. • K,HPOi, ooa g.; NaCl, 002 g.; Ca(NO,). 4H,O, oao g.; Mg SO4. 7H,O, o-oa g.; Fe SO4. 7H,O, 0-002 g.; water, 1 1. Anaerobic conditions on two heterotrich ciliate protozoa 585 Once daily the condition of the ciliates was noted after examination through a hand-lens, and a pair of bottles was opened, one for estimation of oxygen, the other for measurement of pH and oxidation-reduction potential. Dissolved oxygen was estimated by an adaption of the double-iodine modification of the Winkler method involving a preliminary clarification with aluminium hydroxide (Beadle, 1958). With waters of oxidation-reduction potential too low to be consistent with the presence of dissolved oxygen this method may give small negative values for oxygen content. For oxidation-reduction potential a bright platinum electrode was lowered to near the bottom of the second sample of the pair which was connected to a saturated calomel reference electrode through a KCI-agar bridge. The pH was then measured by substituting a glass electrode for the platinum electrode. A Cambridge pH meter was used for both measurements. The pH ranged from 6* 1 to 7-9, and a correction was applied to the measured oxidation-reduction potential to adjust the figure to pH 7-0 (£7), and the potential of the reference electrode was corrected for tempera- ture. Reasonably steady potentials were obtained after 5 min. and the readings were taken after 10 min. The final measurements in each experiment were made on the day on which the ciliates were found to have died. The temperature in the laboratory during all the experiments was 22-25 ° C. In the course of any one experiment it would have ranged over less than 2° C. With the object of destroying all organisms other than those introduced with the inoculation from the cultures, all the experiments were repeated using swamp water which had previously been boiled for about 10 min. and then aerated. In this way it was hoped both to retard the processes leading to anaerobic conditions and per- haps to alter the nature of the chemical reactions involved. If, in spite of this, the survival of the ciliates bore the same relation to oxygen concentration as in the experiments with unboiled water, the conclusion that oxygen is the primary factor determining survival would be more justified, though by no means certain. Each experiment involving several pairs of bottles and lasting several days was repeated three to nine times. The three curves reproduced in each figure are the two most divergent together with one intermediate curve from each set of experiments. RESULTS Bursaria sp. (Figs. 1 A, B) Without exception the ciliates were always dead on the day on which the oxygen concentration approached zero. This happened also in the experiments using boiled water, in which the oxidation-reduction potential did not drop as much as in two at least of the experiments using unboiled water. In both sets however anaerobic conditions were reached in 3 days. Blepharisma undulans. Pigmented form (Fig. 2 A, B) The experiments showed clearly and without exception that this species will withstand 1-3 days of anaerobiosis. This survival period does not seem to be influenced either by the length of time taken for the oxygen to be exhausted or by 37-2 586 L. C. BEADLE AND J. R. NILSSON the course or level of the oxidation-reduction potential curve. Considerable changes in these were produced by previous boiling of the water. Blepharisma undulans. Colourless form (Fig. 3 A, B) The absence of the pigment made no significant difference to the survival in oxygen-free water. Previous boiling of the water did not retard the onset of an- aerobic conditions and the results of the two sets of experiments were remarkably similar. Fig. 1. Bursaria vp. A. Unboiled, aerated swamp water. Three of five experiments. B. Previously boiled and aerated swamp water. Three experiments in all. pH, start to finish: a, 7-3-6-9; b, 7-3-6-5; c, 6-3-6-2; d, 7-9-6-6; e, 7-9-6-7;/, 7'5-6-7- DISCUSSION Bursaria sp.