An Occurrence in Wellington Harbour of Cyclotrichium meunieri Powers, a Ciliate causing Red Water, with Some Additions to Its Morphology

Transactions and Proceedings of the Royal Society of New Zealand, Volume 78, 1950, Page 86

An Occurrence in Wellington Harbour of Cyclotrichium meunieri Powers, a Ciliate causing Red Water, with Some Additions to Its Morphology By Brian M. Bary and R. G. Stuckey Department of Zoology, Victoria University College, Wellington, N.Z. [Received by Editor, July 12, 1948; issued separately, February, 1950.] Introduction In Wellington Harbour lanes of deep brownish-maroon coloured water, extending over an area estimated to be three miles long and half a mile wide, were observed during a spell of fine warm weather in April, 1948, and again in August, 1948. An examination of the organism causing the discoloration showed it to be Cyclotrichium meunieri Powers, originally described from the Gulf of Maine, U.S.A. (Powers, 1932). His description was from fixed material, as he was unable to slow down living specimens sufficiently for study. By using 0·3% hydrogen peroxide we have been able to overcome this difficulty. This makes possible the description of structural details which are visible only in living material. An account of the behaviour and habits of the species is included. Material and Methods Cyclotrichium meunieri disintegrates rapidly under even the most gentle centrifuging. It was concentrated therefore by making use of its habit of settling in regions of diffuse light. If left in sunlight in a deep container it collects in the bottom three inches of water. By placing this concentrate in an opaque dish in sunlight and covering it except for a small aperture, the organisms collect on the side away from the light and may then be pipetted into fixative. The material was fixed in hot Schaudinn's fluid, embedded and cut at 3μ. The sections were stained in various ways; iron haematoxylin counterstained with orange G, and Mallory's triple stain were used to demonstrate the general structure; picro-nigrosin was used to bring out details of the pellicle, cilia and basal granules, and Horvath's toluidin blue to determine the distribution of the cilia and cirri. Feulgen's technique was best suited for demonstrating the details of the nuclear complex, especially in whole mounts, where nuclear structure is masked by the chromatophores. Hydrogen peroxide, diluted to 0·3% with sea water, was the only satisfactory narcotic among the various ones tried. This produced a narcosis lasting several minutes before death occurred. Attempts to culture the species were not successful.

Observations The most outstanding characteristic of live specimens of Cyclotrichium is their rapid movements, which Powers likened to the zig-zag darting of Halteria. Although the movements are very similar, Cmeunieri moves much more rapidly, and specimens are able to traverse 30 to 50 times their own length in less than a second. When in motion the smaller half of the organism is directed anteriorly and the larger half, which contains the “cytostome” (Powers), is posterior. When the organism momentarily becomes stationary, the smaller “anterior” portion becomes uppermost; as the longitudinal axis reaches the vertical, the specimen again darts away. Partially narcotised specimens rotate about the longitudinal axis in either a clockwise or an anticlockwise direction, but in untreated animals rotation could not be observed. It was possible to keep untreated specimens alive and under observation for at most 6 minutes. The temperature of the harbour water was 16·1°C. when collections were made, and if this temperature is exceeded by more than a degree or so in the laboratory, death and immediate disintegration occurs in all specimens. When an individual disintegrates, the cilia and cirri break up first, and then the equatorial band from which these structures arise, constricts. This constriction forces the anterior and posterior portions of the body apart, whereupon they rapidly disintegrate, but retain their original brownish-maroon shade for half a minute or so. Two to 4 opaque, colourless spheres, presumably nuclear structures, are extruded from the “cytostome.” On further breakdown the colour of the chromatophores becomes yellow-green. Almost immediately on the death of large numbers of this ciliate, the brownish-maroon shade of the water changes to a rusty-pink which begins to disappear after about 24 hours, being replaced by a persistent, yellow-green colour. When individuals are under the influence of hydrogen peroxide they become more or less atypical as death approaches. The corona of cilia about the smaller portion of the body becomes reversed, with reversal beginning at the tips of the cilia and progressing towards their origin. At the same time all the cirri are directed posteriorly. Frequently the cilia develop a slow rowing action as the movements of the organism become more sluggish. Cyclotrichium meunieri tends to congregate in diffuse light. This behaviour was evident in the harbour, and was shown to occur under experimental conditions. On bright sunny days when collections were made, few specimens were present in the first 9 inches of water, but below this extended downwards in concentration for 3 to 4 feet. The most convincing demonstration of this sensitivity to light was obtained experimentally. A 4-foot length of three-quarter inch glass tubing was filled with water containing specimens, placed horizontally, and a 6-inch length subjected to a light intensity of 300 foot-candles. At the end of 1 hour, though all specimens were dead, on either side of the intensely illuminated section there was a zone of aggregated specimens, in one case 4 inches wide, the other 10 inches wide. The light intensity in these zones ranged between 50 and 2 foot-candles. There were few specimens in the remainder of the tube and these were not aggregated into zones.

Though active multiplication must have occurred in the harbour, not one division stage was observed among either living or dead specimens. It was not possible therefore to make any observations of this process for comparison with those made by Powers, in which he describes seeing 6 chromosomes in each of the 2 daughter nuclei at late telophase in binary fission. The shape of living specimens is remarkably constant though the size varies considerably, the dimensions ranging between 22 and 47μ long, 19 and 41μ wide, at the widest part. The body consists of 2 semi-spherical to elliptical portions separated by a wide ciliary zone. The anterior half of the body is slightly dome-like and narrower than the truncated posterior portion. Except in the ciliary zone, the body is covered by greenish-maroon, concavo-convex platelets, the “chromatophores” (Powers). These lie beneath the pellicle (Pl. 13, figs. 2 and 3), completely masking internal structures and giving a vacuolated appearance to the body. Fauré-Fremiet (1924) discusses the cytostome of known Cyclotrichia and concludes that it is present in some species, not in others, and when present it lies at the anterior extremity. Powers describes a funnel-shaped, pellicular-lined depression as the cytostome. He implies that it is anteriorly placed. In our studies of the living organism we find that this structure is invariably posterior in position in terms of the moving animal. There is a shallow depression, approximately half the width of the body, from which leads a narrower tube ending blindly in the endoplasm. The depression is lined by both chromatophores and pellicle, the tubular portion only by pellicle. Pl. 13, Fig. 1, indicates the extent of the depression in the living animal, while in Pl. 13, Fig. 2, although the depression is contracted, the tubular portion is present. No evidence that this is a cytostome can be advanced. The fact that it is situated postero-terminally—which would be, to our knowledge, the first record of a cytostome so placed, and also from the fact that no ingested food material either in vacuoles or other form could be demonstrated, leads us to consider that the structure is not a functional cytostome. No satisfactory alternative function, however, has been ascribed to the structure. The ciliary zone (Pl. 13, Figs. 1–4) occupies between one-fifth and one-quarter the length of the animal and encircles either the mid-body region or is slightly posterior to this. The zone is free of chromatophores, is colourless and translucent, and from it arise the cilia and cirri. In the majority of specimens the diameter of the ciliary zone is greater than for adjacent regions of the body (Fig. 1), but in some specimens it is flat (Figs. 2 and 3), and is then contained within the full body contour. In slowly-moving narcotised specimens, the cilia are seen to arise from longitudinal rows of granules in the zone (“striations,” see Powers), and then to extend anteriorly forming a transparent “corona.” The length of the cilia could not be determined definitely, but they are about 20μ long in an average-sized animal (35μ long). The cirri (Figs. 1 and 2) arise from the most posterior of the granules of the longitudinal rows and extend outwards in 3 planes, one anteriorly at approximately 45 degrees to the longitudinal axis, one at 90 degrees and the third at 45 degrees posteriorly. The length of the cirri is in the vicinity of 25μ The typical springing

Fig. 1—Cyclotrichium meunieri Powers; a composite drawing of the live animal, showing the anterior corona of cilia, the cirri and the posterior depression. Fig. 2—Composite figure of whole mounts. Fig. 3—Tangential longitudinal section. Fig. 4—A portion of a transverse section through the ciliary zone. Fig. 5—A series illustrating the nuclear complex and showing the normal complement (a) and extra-nuclear vesicles (b to c). Degenerating stages of the macronucleus, Dl to Dv. Abbreviations: C., cirri; CB., ciliary bunches or compound cilia; CH., chromatophores; CS., cytoplasmic sheet or strand; CR., ciliary row, or longitudinal row of granules (striation); CY., “cytostome”; Dl–v. degenerating stages of the macronucleus; G., basal granules; MAC., macronucleus: MIC., micronucleus; MG., metaplasmic granules; N., nodule on ciliary rootlet; P., pellicle; PL., plastin body; PY., pyrenoid bodies; R., ciliary rootlet; V., vacuoles about the pyrenoid bodies.

movement of these organisms appears to result from a single, vigorous, co-ordinated beat by these cirri. A study of whole mounts and sections (see Figs. 2–5) largely confirms Powers' descriptions of the morphology of C. meunieri. The dimensions of fixed local material are slightly greater than for Powers' specimens: the length lies between 21 and 51μ, the width between 19 and 46μ, while the cilia are from 8 to 11μ and the cirri from 12 to 15μ long. The shorter length of cilia and cirri of fixed material is due to these structures assuming a close zig-zag pattern at death. In the Wellington material the structure of the ciliary zone is reasonably in accord with Powers' findings, though there are some differences. There is no increase in the size of the basal granules (see Fig. 2) towards the narrower (anterior) portion of the body as Powers describes. Usually 10 granules occur to a row, although counts as low as 6 were recorded. From 43 to 106 longitudinal rows of granules may be present, with 70 to 90 in the majority of specimens. There is therefore a greater range and higher average number of rows than in Powers' material, in which he recorded 52 to 60 rows. The very numerous, fine cilia and the large associated basal granules in fixed material, and the uneven movements of the living organism, lead Powers to deduce that compound cilia, possibly cirri or membranelles are present, and he illustrates this. He does not mention the possibility that both groups of cilia (compound cilia) and cirri may be present. We find that numerous, very fine cilia arise in groups from the large basal granules (Figs. 3 and 4), penetrate the pellicle through a single pore and then usually spread fanwise. These, we consider, are discrete, but compound cilia, not cirri or membranelles. The basal granules would of necessity be compound, through no indication, other than their large size, can be obtained. Each cirrus (Figs. 1 and 2) arises from the most posterior granule of each row. These granules appear, in both sections and whole mounts, to be squarish in outline and transversely elongated in contrast to the spherical or slightly longitudinally elongated granules of the ciliary groups. The cirri were evidently not detected by Powers, but in local material they lie close to the surface of the posterior portion of the body (Fig. 2). They are compact and solid structures at their point of origin, but they break into bundles of discrete cilia within a short distance of the origin. A ring of transversely elongate, minute granules (Fig. 2), much narrower than those of the cirri, is sometimes seen immediately anterior to the granules of the cirri. The significance of these granules could not be determined. Ciliary rootlets (neuromotor fibrils) extend into the surface of the endoplasm from the innermost portions of the basal granules. They can be seen in both transverse and longitudinal sections. Prolonged searching failed to disclose any longitudinal or transverse fibres. Some individual rootlet fibres, however, show small nodules at a regular depth below the basal granules (Fig. 4), and these may be the junctions of longitudinal and transverse fibres. The macro- and micronucleus of local individuals (Fig. 5a) are typically as Powers describes them, including the “extra-nuclear vesicles” which he reported. The macronucleus is slightly irregular in outline, sub-spherical to spherical, and contains a plastin core

(Powers). The micronucleus is more or less oval, stains densely with the Feulgen reaction and is usually compact, although there is often a tendency towards vesiculation. There is never more than a single micronucleus in a non-dividing stage. “Extra-nuclear vesicles” (Figs. 5, b to e) may be present in addition to macronucleus and micronucleus. These vesicles stain but lightly with the Feulgen reaction. Of 51 specimens checked, 23 contained no extra-nuclear vesicles; 18, 1; 6, 2; and 4, 3 of these bodies. Figure 5 illustrates a series showing the nuclear complex; it seems clear from these, and from further similar stages observed, that the extra-nuclear vesicles are degenerative phases of the macronucleus. This is borne out by clumping and knotting of the chromatin in some (Fig. 5, d′, d″, d‴), which is a pycnotic condition, and similar to the degenerating phases of chromatin bodies during endomixis (Woodruff and Erdmann, 1914). Other stages suggest karyorrhexis in which chromatin masses are prominent at first just within the nuclear membrane. and later are distributed irregularly in the cytoplasm. Both pyenosis and karyorrhexis are common pathological conditions during necrosis. No membrane is present in the more advanced stages of degeneration and a marked irregularity of shape occurs, while the plastin bodies disappear. If the bodies were formative phases of the macronucleus, irregularities in chromatin distribution would not be evident, and a membrane would be present; both of these conditions are realised, for example, in the macronuclear anlage in endomixis. The endoplasm contains many granules of irregular shape and size (Figs. 3 and 4), and these Powers calls the “metaplasmic” granules. Large pyrenoid bodies (Figs. 2 to 4), each one contained in a vacuole, are associated with the chromatophores. The pyrenoids in local specimens are generally sub-spherical to spherical, and are of more regular shape than those illustrated by Powers. The vacuole in many cases has what appears to be either a sheet or a strand of cytoplasm stretching across it and subdividing it (Figs. 2 and 3). This is best seen in fragmented specimens stained with Mallory's Triple. Discussion The literature consulted during this study has shown that discoloration of sea water by swarms of ciliates has been several times recorded in the past. Darwin, in the Voyage of the Beagle, reported sailing through large areas of red-water along the coast of Peru. He describes organisms having the characteristic movements and appearance of Cyclotrichium meunieri. Hart (1934) gives a short account and figure of a species which we identify as probably Cyclotrichium meunieri which he found blooming along the east coast of Cape Peninsula, South Africa. In his account, he designates the organism “Mesodinium or some closely allied genus”; later. (1943). he names Mesodinium rubrum Lohmann 1908 as the causative organism. Paulsen in 1934 stated that a phenomenon, similar to that in Hart's (1934) account, and caused “by apparently the same organism” was reported from Iceland in 1909. Clemens (1935) has recorded M. rubrum occurring in sufficient numbers to cause red-water along the British Columbia coast.

From an analysis of the above literature, it appears to us that two similar, but distinct, ciliates are being confused—the “Mesodinium rubrum” recorded by Hart, and the “Mesodinium rubrum” of Lohmann. Lohmann's (1908) paper is unobtainable, but his planktonic Halteria rubra is included by Hamburger and Buddenbrock (1911), where it is named Mesodinium rubrum, Lohmann's figures being reproduced. This organism has deep red or colourless chromatophores, a large spherical “trunk section” and an anterior, spherically-shaped oral cone which, as illustrated, is very small, but which is stated to increase in size with an increase in number of chromatophores. The cirri are said to be similar to those of Mesodinium pulex and tentacles are not described about the oral aperture. Colourless individuals are recorded which are 10 to 18μ long, the coloured specimens being 20 to 50μ long. The description and figure are not at all precise and there appears to be no more than a superficial resemblance to Cyclotrichium meunieri in the size, the possession of reddish chromatophores and spherical macronucleus. The cirri and cilia arise anteriorly from a ring, thus delimiting the proportionately small oral cone. One figure is of a specimen with an enlarged anterior portion which is shown as a truncated cone. Chromatophores, described as plate-like, are illustrated as numerous, small, more or less oval bodies in the posterior, globular portion of the body. To us it appears that Cyclotichium meunieri is a distinct species, and that Hart wrongly identified his specimens as Mesodinium rubrum. This being so, the two ciliates, Mesodinium rubrum and Cyclotrichium meunieri, both with chromatophores, and both adapted to a pelagic existence, have been recorded as blooming in coastal waters. To date, C. meunicri only has been found for certain from New Zealand, but recently a single specimen of a species remarkably similar to, and provisionally identified as, M. rubrum was obtained from a litre sample taken at four metres from Wellington Harbour. Summary Cyclotrichium meunieri Powers occurred in extensive blooms in Wellington Harbour during April and August, 1948. The cause of the blooming of this ciliate has not been investigated. Observations on living material satisfactorily slowed with 0·3% hydrogen peroxide, show that Powers' interpretation of the antero-posterior axis is incorrect: the “cytostome” is posterior in position, and because of this and the absence of food vacuoles, it is considered not to be a cytostome. This species is shown to possess both compound cilia and a posterior row of cirri, the latter not described by Powers, who interpreted the former as possibly membranelles or cirri. Ciliary rootlets are described as part at least of the neuromotor apparatus. Analysis of the nuclear complex shows that the “extra-nuclear vesicles” are necrotic macronuclei undergoing typical pycnosis and karyorrhexis. C. meunieri seeks an optimum light intensity and has well-marked phototropic responses. No stages in fission or indications of other forms of reproduction were observed.

Acknowlegments We wish to thank Professor L. R. Richardson, Victoria University College, for his many helpful suggestions during the course of this investigation, and for his ready assistance with the preparation of the manuscript. References Clemens, W. A., 1935. Red Water-bloom in British Columbia Waters. Nature, vol. 135, p. 473. Darwin, Charles, 1831. Journal of Researches into the Geology and Natural History of the Various Countries visited during the Voyage of the H.M.S. Beaglc round the World. J. M. Dent & Sons, London. Fauré-Fremiet, E., 1924. Contribution à la connaissance des Infusoires plank-toniques. Bull. biol., suppl. vi, pp. 1–171. Hamburger, Cl. and Buddenbrock, W. von, 1911. Nordische Ciliata mit Ausschluss der Tintinnoidea. (In Nordisches Plankton Lfg. 15). Kiel u. Leipzig (Lipsius and Tischer), 1911, no. xiii, 1–152. Hart, T. J., 1934. Red “Water-bloom” in South African Seas. Nature, vol. 134 (3386), pp. 459–460. — 1943. Darwin and “Water-bloom.” Nature, vol. 152 (3866), pp. 661–662. Lohmann, H., 1908. Untersuchungen zur Feststellung des vollständigen Gehaltes des Meeres an Plankton. Wiss. Meeresunters., Kiel (N.F.), 10, Abt. Kiel, 1908 (129–370), Taf. ix–xvii. Paulsen, Ove., 1934. Red “Water-bloom” in Iceland Seas. Nature, vol. 134 (3399), p. 974. Powers, P. B. A., 1932. Cyclotrichium meunieri sp.nov. (Protozoa, Ciliata); cause of Red Water in the Gulf of Maine. Biol. Bull. Woods Hole, vol. 63, (1), pp. 74–80. Woodruff, L. L., and Erdmann, R., 1914. A Normal Periodic Re-organisation Process without Cell Fusion in Paramecium. J. Exp. Zool., vol. 17, pp. 425–517.