Structure and Coiling of the Stalk in the Peritrich Ciliates Vorticella and Carchesium

J. Cell Sci. io, 95-122 (1972) qr Printed in Great Britain STRUCTURE AND COILING OF THE STALK IN THE PERITRICH CILIATES VORTICELLA AND CARCHESIUM W. B. AMOS Tlte Department of Zoology, Downing Street, Cambridge, England SUMMARY The stalk of Carcliesium and Vorticella coils by the action of a contractile organelle. The organelle lies within a thread of cytoplasm which is encased in a complex extracellular tube. Study with the light microscope and the electron microscope suggests that the structure of the tube and the course of the organelle determine the form of the coiling. The contractile organelle contains a system of interconnected membranous tubules and the cytoplasm around it also contains membranous saccules. Both tubules and saccules extend along the length of the stalk. INTRODUCTION Vorticella and Carchesium have stalks which are capable of coiling helically. The coiling is brought about by the action of a contractile organelle which differs markedly from muscle in its physiological properties. This paper is concerned with the structure of the stalks and seeks to explain some aspects of the coiling in structural terms. Experiments on the activation of the stalks after glycerination are described elsewhere (Amos, 1971, and in preparation). Faure-Fremiet (1905) examined the stalks of both genera with the light microscope and found them to be similar. They consisted of a cylindrical sheath containing the contractile thread, or spasmoneme, which pursued a helical course throughout the length of the sheath. Longitudinal fibres lay on the inner surface of the sheath on the opposite side to the spasmoneme. Faure-Fremiet regarded these fibres, the bdtonnets, as stiffeners which, because of their position, ensured that the sheath would bend into a helix, rather than merely collapse, when the spasmoneme contracted. Randall & Hopkins (1962) confirmed his description with the electron microscope in the case of Vorticella, but presented a different account of Carchesium. In Carchesium, they reported an axial spasmoneme separated equally on all sides from the sheath by an annulus containing many tubular fibres. This structure, which seems ill suited to helical coiling, is visible in electron micrographs of a related peritrich, Zoothamnium, obtained by Faure-Fremiet, Favard & Carasso (1963). Since the Carchesium studied here agrees with Faur^-Fremiet's description of the genus, it seems likely that the organisms examined with the electron microscope by Randall and Hopkins were not Carchesium but Zoothamnium. The present account of the Carchesium stalk is therefore believed to be the first extensive one, though the spasmoneme was described by Carasso & Favard (1966). 96 W. B. Amos Many studies have been made of the coiling of Vorticella and it has recently been analysed with a high speed camera by Jones, Jahn & Fonseca (1970), so observations here are chiefly confined to the type of deformation involved, and its relation to the structure of the stalk. MATERIAL AND METHODS Carchesium was collected from a pond near Kenn in north Somerset. The species (see Fig. 3) resembled Carchesium polypinum L., as shown by Stein (1854), but the published descriptions of this species are inadequate for identification. All the organisms used came from the same clone. They were cultured in a soil extract prepared by autoclaving 80 g of loam in 1 1. of glass-distilled water, filtering and autoclaving once more. One small drop of fresh milk was added to 25 ml of soil extract in a Petri dish before inoculation. Subcultures were made weekly. Vorticella convallaria L. was obtained from the Culture Collection of Algae and Protozoa in Cambridge. It was cultured in a salt solution nutrified with the Glaxo preparation ' Complan'. The salt solution contained 17 mM NaCl, 1-2 mM NaHCO3, 0054 mM KC1 and 0-054 mM CaCl2. It was sterilized and poured over a sterile solid layer of 3 % agar, made up in the same solution, in a Petri dish. One small drop of an autoclaved 2 % suspension of Complan in water was added and the culture was inoculated by inserting a glass slip from a previous culture, to which the organisms had attached themselves. Subcultures were made monthly. Light microscopy was carried out with a Zeiss Standard WL microscope equipped for phase contrast and Nomarski's differential interference contrast. Photographs were taken with electronic flash. The methods used for electron microscopy were largely conventional. The organisms were fixed between o and 30 C in a solution containing 25 % glutaraldehyde and 27 mM sodium cacodylate buffer adjusted to pH 7-4 at 2 °C by means of o-i N HC1. After exposure to 2 changes of this medium for a total period of 2 h at 0-3 °C the specimens were washed for 2 h in 4 changes of a similar medium, but with 6 % sucrose instead of glutaraldehyde. They were then postfixed in 1 % osmium tetroxide with 6 % sucrose and 50 mM veronal acetate buffer. The buffer was made up to give a pH of 74 at 2 °C. The fixed material was washed for 1 h in the same medium but without osmium tetroxide and with only 4 % sucrose. In 2 subsequent washes, each 15 min long, the sucrose concentration was reduced to 2 % and then to zero. No experiment was made to test the value of the graded reduction in sucrose concentration, but it is believed that it might have lessened the osmotic stress on the fixed material. Dehydration in ethanol, treatment with propylene oxide and embedding in Araldite were carried out according to Luft's method (1961). Sections were cut with glass and diamond knives, and grey sections were picked up on grids with carbon-coated Celloidin films. They were stained with a saturated solution of uranyl acetate in 50 % ethanol for 30-50 min and also with the lead citrate solution of Reynolds (1963) for 3—5 min. The electron microscope was a Philips EM 200 operated at an accelerating voltage of 60 kV. OBSERVATIONS Principal features Faure-Fremiet (1905) has described the formation of the stalk in Vorticella, which begins with the attachment of the body to a submerged surface. A tube of extracellular material is secreted which apparently elongates by the addition of new material at one end. At the same time, an outpushing of the cytoplasm of the body enters the tube. In Carchesium, but not in Vorticella, the body subsequently divides many times and the stalk becomes bifurcated after each division, so that a colony of many indivi- duals or zooids is formed. The spasmoneme of each zooid is restricted to its own branch, and there is no cytoplasmic continuity between zooids. Structure and coiling of peritrich stalk 97 The size and structure of the stalk vary with its position in the Carchesium colony. In the species examined here, the stalk of a newly attached zooid is 7-10/tm in diameter, but as it grows in length it increases in diameter to 10-18 /im, SO that the final form is a slender cone tapered towards the base. The main trunk of the colony loses Plasma membrane Fibrillar matrix Cytoplasm Spasmoneme Mitochondrion Sheath Batonnet Fig. 1. Diagram of the main structures in the stalk of Carchesium. The number of batonnets is reduced for the sake of clarity. its ability to coil after thirty or so zooids have been produced. It swells somewhat and becomes filled with a refractive granular material. The chief features of a contractile region are shown in Fig. 1. Inside the sheath runs the cytoplasmic strand in a left- handed helix, with a pitch of about 140 /tm in the main trunk and 60-100 /im in the branches. The spasmoneme, which is embedded in the strand, has the form of a ribbon running helically, with the outer surface flattened against the inside of the 98 W. B. Amos sheath. The other surface of the spasmoneme, facing inwards, is covered with a layer of cytoplasm containing many spherical mitochondria. The cytoplasmic strand has a circular cross-section in which the spasmoneme lies eccentrically, the cytoplasmic layer being 4 /tm deep on the inner surface of the spasmoneme but so thin on the outer surface that it is barely detectable with the light microscope. The transverse section of the spasmoneme is elliptical when the stalk is extended, with the minor axis lying radially. In the spasmoneme of a branch of a colony the major axis is about 4 /MH and the minor 1-5 /an. The minor axis increases when the stalk coils, so that the cross- section of the spasmoneme becomes almost circular when the organelle is fully contracted (Fig. 5). The batonnets lie longitudinally on the inner surface of the sheath. They are straight and parallel to one another but staggered in such a way that a helical palisade is formed (see Figs. 1, 6) which runs around the stalk on the opposite side to the spasmoneme. The scopula, the junction between stalk and zooid, is a highly organized region, in which the cytoplasmic strand becomes continuous with the cytoplasm of the body of the zooid (Fig. 7). The spasmoneme is continuous with longitudinal myonemes in the body. Protruding from the base of the body of the zooid are rod-shaped structures, the scopula organelles, which occur quite generally in this position in peritrichs. Their fine structure is somewhat similar to that of the basal bodies of cilia (Randall & Hopkins, 1962; Faure-Fremiet, Favard & Carasso, 1962). The base of the main stalk, which is normally attached to the substrate, is sealed by a transverse disk to which the cytoplasmic strand is attached by about 15 elongated processes which splay out from a swelling at its tip.

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