Studies on the Freshwater Ciliates of New Zealand
A General Morphology of Bursaria Truncatella Muller
[Read before the Wellington Branch, June 16, 1948; received by Editor, March 15, 1949.]
The present account of Bursuria truncatella Müller is intended as the first of a series on the ciliated protozoa of this country. This group has been neglected since late in the nineteenth century, when the studies by Kirk (1885), Maskell (1886, 1887), and Schewiakoff (1892), were published. Bursaria truncatella is a cosmopolitan species well known in Europe and North America, but not previously recorded from New Zealand. It is of large size, comparatively slow in movement, and but for the relative denseness of the cytoplasm would be an excellent ciliate for general study. The species is not well described. The denseness of the cytoplasm and general complexity of organisation have been the cause of disagreement in the accounts by various authors. Especially is this so with regard to the ingestive apparatus, which is unusually complex even for a ciliate, and in the distribution and size of the contractile vacuoles and other features; these are accordingly described at some length in this paper. It became apparent during the course of this study that among the available literature no one paper gave a short, yet complete account of this species—the only one in the genus Bursaria. Further, it was evident that some points could be added to the description. In this paper the description is extended on the basis of observations on the present material.
The large size and apparent simplicity of organisation no doubt have led many workers to study B. truncatella. That this study is not simple is evidenced by the variety of interpretations accorded various structures. Of the literature available, Kent (1880–1882) reviews the work of Ehrenberg and Stein on this species, and briefly describes the distribution of the contractile vacuoles and other readily observable structures. The adoral membranellae are interpreted as a ciliary wreath, but there is no reference to the skeletogenous rod about the ventral cleft. He gives the distribution of many of the species included in the genus Bursaria by Ehrenberg and other writers. Schuberg (1887) has published a long account of this species and covers most systematic points. The form and structure of the peristome are discussed in particular, but the contractile vanuoles and membranellae are treated briefly. A lengthy and detailed account
of the minute structure of the ectoplasm and endoplasm is given. He compares his findings with those of previous writers, Brauer and Stein especially, and revises several of their interpretations, such as Stein's concept that the mundspalte was part of a water secretory system (Wassersekretionssystem), and Brauer's statement that the organism is bilaterally symmetrical. Kahl (1927) states that his findings agree with those of Brauer except for Brauer's erroneous interpretation that the membranellae were discrete cilia. He contributes further to the knowledge of Bursaria truncatella when he describes the contractile vacuoles as being 30 to 40μ apart and estimates that 300 to 400 occur in a medium-sized specimen. Bhatia (1936) gives generic and specific descriptions. He states that “contractile vacuoles are usually absent, but sometimes many distributed all over the body.” In a short discussion of the literature dealing with Bursaria truncatella he states that, unlike Hickson, he could not find a “vertical fold” projecting into the cavity on the right side, but that there was a “distinct flap along the left edge of the peristomal field which bore fine cilia along its free edge in the prominent anterior portion only.” Wetzel's cytological investigation is not available to the writer, but it is quoted by Peschkowsky (1927). Peschkowsky deals in a comprehensive and detailed manner with the fibrillar system of the body surface and peristomal aperture and with the peristome band and the granules of the ectoplasm. The investigations, by the authors quoted above, have aided in interpretating the observations in the present work and at the same time permitted a more correct description of the morphology of Bursaria truncatella. Reference should be made to the works by Schuberg and Peschkowsky for detailed discussion of the minute structure of ectoplasm and endoplasm.
From the accounts given by the authors quoted above, Bursaria truncatella is apparently between 500 and 1500μ long, about half as long again as wide, purse or pocket-shaped and rather asymmetrical. The peristome is deep, curves towards the left and dorsally, and contains a broad zone of membranellae on its left side. There are no cilia in the peristome, but it is striated in a regular manner. There is no plate on the left of the ventral surface corresponding to the peristome plate on the right (Schuberg), or, according to Bhatia, there is a “distinct flap” on the left of the peristomal field, but none on the right. No contractile vacuoles may be present (Bhatia), or they may occur throughout the cortex (Kent). On the ventral surface is a longitudinal eleft, in the margins of which is a skeletogenous rod, the peristome band, which is continuous with a transverse band present in the anterior dorsal peristome margin (Schuberg). The surface of the body is finely striated, the striations having a slight right-handed spiral, or, as more usually illustrated, they are longitudinally disposed The body is clothed with short, fine cilia, which are situated between the striations. The macronucleus is long and flexuous. Micronuclei are numerous. There is a terminal posterior cytopyge. In this account, these contradictory statements have been examined, and further facts concerning Bursaria determined. It has been found necessary to revise the description where it is concerned with the
nature and distribution of the contractile vacuoles, the structure of the mundspalte (stomial aperture), the occurrence and form of the peristome plates (ventral flaps?) and the distribution of the cilia. Additional information has been obtained concerning the structure and number of the membranellae and the form of the micronuclei. Attention is drawn to the convergent nature of the fibrillae which are continuous with the left-hand ends of the membranellae.
Materials and Methods
Bursaria truncatella does not appear to be abundant about Wellington, having been collected but once during the two years of the present study. Kent (1880–1882) reports that it is not common in England, but that when it does occur the species is usually present in great numbers. Bhatia (1936) collected the species in the Punjab, India; Kahl (1927) describes it from Europe, and it also occurs in North America, so that though not common it is a truly cosmopolitan species. The field sample in which the Bursaria were collected in this instance was used for culturing the species. This method proved satisfactory for a limited period, the food of the natural habitat being present in abundance. Multiplication was rapid at first, but the species disappeared at the end of a few weeks. Attempts to reobtain the species have not been successful.
In order to clarify the structural relationships of the peristome, a plasticine reconstruction from a series of sections cut at seven microns and stained with Heidenhain's iron-alum haematoxylin proved invaluable. Schaudinn's fixative, used hot, gave excellent results, the animals being killed and fixed rapidly with negligible necrosis or distortion. The method detailed by Yocom (1918), (and modified by Gray, 1932), in which the protozoa are fastened to the slide by Meyer's albumin in the act of killing and fixing, was extensively utilised. When this method of fixation was followed by Ehrlich's acid haematoxylin, counter-stained with 5% eosin in alcohol, a high degree of nuclear differentiation resulted. It was found that xylol as a clearing agent was quite satisfactory in place of the recommended terpineol (Gray). A “Burton” light equipped with a hundred-watt, vertical filament projection lamp facilitated the necessary careful and continual observations. This illumination remarkably accentuates detail and is far superior to any other source tried. The figures in this paper, except for those of the reconstruction, were drawn with a camera lucida.
The body of Bursaria truncatella (Fig. 1) is markedly asymmetrical as is described by Schuberg (1887). There is a strong convexity of the right and dorsal surfaces, but the anterior one-third of the left and ventral surfaces is concave. There is quite a definite, though not a pronounced spiral tendency observable in the surface striations and in the peristome. The truncated anterior extremity has the appearance of slanting towards the right and at the same time ventrally, but this is due to unequal curvature of the convex and concave surfaces. The widest portion is immediately posterior to the mid-body region, and the body then rapidly diminishes towards the extremity
which may be bluntly pointed or rounded. This ciliate is of large size, though the New Zealand specimens appear somewhat smaller than those reported from elsewhere. Kudo (1946) gives the length as 500 to 1000°, Bhatia as “up to 1·5 millimetres long and scarcely one and one-half times as long as broad.” Specimens measured from Wellington, were from 260 to 600°. long and between 230 and 400° wide. Even at this smaller size, Bursaria is still a very large protozoon and readily visible to the naked eye as an opaque, whitish body in the culture medium.
There is not a constant length-breadth ratio in this species, but the ratio becomes greater as the adult form is reached following transverse fission. In the daughter individuals this ratio is practically unity, but in the average adult (450 to 500° long) it is approximately 1·5:1·0. This variation, together with the fact that there may be considerable distention from ingested food, requires caution to be exercised when using such measurements as descriptive features.
The cilia of Bursaria truncatella are relatively short, but the body is densely clothed (Fig. 1). Schuberg states that the basal granules of the cilia are placed around the periphery of the ectoplasmic vacuoles and that they lie between the surface striations. This irregular distribution forms a broad row, with the striations forming the boundary along the margins of the row. In preparations of the Wellington material, the basal granules were closely spaced in parallel rows and themselves formed the striations, or were so closely associated with the striations as to be indistinguishable from them (Fig. 1). On the right of the ventral cleft, the cilia extend round the margin on to the septum (described below). They accompany the septum where it passes from the base of the cleft on to the ventral face of the peristome (Fig. 1). On the left side of the cleft the cilia reach only to the edge of the peristomal excavation. Though the peristome is striated it is devoid of cilia. If the septum is regarded as being a once free edge that has become inturned, as evident in transverse sections, then the presence of cilia on it can be explained.
The striations take a right-hand spiral path across the body, particularly on the ventral surface. In the anterior half of the right side there is only a slight indication of the spiral which becomes greatly accentuated in the posterior half of the body, particularly about the base of the ventral cleft. On the left side the striations commence at the margin of the cleft and run obliquely in a direction from the anterior right extremity towards the posterior left, exhibiting only a small degree of curvature (Fig. 1). In all the specimens examined, the striations were spiralled in the above manner.
Bursaria truncatella has a complex and extensive ingestive apparatus. The peristome occupies almost the entire anterior extremity, and reaches posteriorly for more than half the length of the body. In its posterior one-third it curves towards the left and at the same time dorsally, ending in the endoplasm (Figs. 1 and 2, and Text-fig. 1). The dorsal curve begins at what Schuberg torms the peristome angle, the short region where the turn towards the left becomes apparent.
Sehuberg considers that the peristome was originally straight. If a right-hand twist has occurred in the body this would account for the spiral tendency in the peristome and surface striations. The whole of the excavation has been termed “peristomal” in the present account, though Kudo (1946) disagrees with this interpretation. To name the posterior portion of the excavation the cytopharynx, implies that a clearly differentiated cytostome is present, but this is not the case. The excavation is lined up to its apical point with a continuous zone of regularly straited, vacuolate ectoplasm, only differentiated from that of the body-surface by the absence of cilia. There would seem to be but one possible position for a cytostome, namely, just posterior to the base of the ventral cleft where the left peristomal wall recurves sharply towards the left (Figs. 1 and 2). However, there is no indication of the cytostome on the dorsal, right or ventral walls of the excavation. For these reasons the term peristome, as used by Schuberg, has been retained.
A thin sheet or flap of cytoplasm extends from the ventral righthand side of the peristome towards the mid-ventral line and almost completely closes the peristome off from the exterior. This sheet, Schuberg's “peristome plate,” turns the peristome into a pocket-like excavation (Figs. 1 to 3 and Text–fig. 1). The left-dorsal edge of the plate bears the lougitudinal ciliated ridge or septum which forms the right-hand margin to the ventral cleft. The septum is well seen only in transverse sections (Fig. 3, a to d), when it is found that its inner (dorsal) edge for about half its length contacts the dorsal wall of the peristome to the left of the membranellae, thus partly closing the peristome from the exterior. The left margin has a small medially extending plate at the anterior extremity only (Figs. 1 and 2); Kahl describes, but Schuberg denies the presence of this plate. Bhatia (1936) describes a distinct ciliated flap along the left of the peris-
Text-fig. 1—Reconstruction in plasticine of Bursaria truncatella from a series of sections cut at seven microns. The letters indicate the positions of sections shown in Fig. 3, Plate 36. Fig. la, is the complete reconstruction; lb, the same, sectioned to expose the peristome and the base of the ventral cleft.
tomal field, but this was not observed in the present material, and in fact the left of this field is entirely free of cilia. Except for the small plate at its anterior extremity, the left margin is defined only by the peristome band, discussed below, and the ends of the ciliary rows on the left of the body. The base of the cleft opens into the peristome in a right-hand spiral over the septum, thus defining a second opening for the peristome (Figs. 1 and 2, Text–fig. 1). There is an excavation in the dorsal peristomal wall, immediately posterior to this second opening, which encompasses the posterior prolongation of the septum; this excavation is the septal space of earlier writers.
Embedded in the margins and surrounding the ventral cleft is a stiffer cytoplasmic structure, the peristome band (Figs. 2 and 3). Its function, an important one, is to strengthen and support the cleft, particularly in the right-hand margin. Because of the extent of the peristome, this margin might tend to collapse inwards were such support not present. Peschkowsky (1927) gives a fully detailed account of the structure and relationships of the peristome band and the associated fibrillar system. Schuberg describes a crescent-shaped process extending posteriorly from the base of the band, and also a transverse band in the anterior margin of the peristome, but there were no indications of either of these structures in the specimens described from Wellington.
Kahl (1927) states that in contrast to all other observers he was unable to find any “stomial aperture” (mundspalte) in the peristome. It is difficult to see how this structure was missed if it were present. Schuberg discusses it and corrects Stein's interpretation of it as part of a “Wassersekretionssystem.” In Schuberg's figures, the mundspalte is shown as an endoplasmic structure. In the Wellington material, this aperture extends from the anterior margin posteriorly along the right-dorsal wall of the peristome (Figs. 1 to 3, Text–fig. 1). It maintains a constant diameter for the greater part of its length, but widens to two or three times this diameter anteriorly. Towards the deepest part of the peristome it becomes shallower, and then continues round the outer curve to the apex of the peristome (Fig. 1 and Text-fig. 1). When viewed in transverse sections there is no doubt that this groove or opening is lined with ectoplasm (Fig. 3, a to d). It is therefore not an endoplasmic structure as figured by Schuberg.
The peristomal membranellae (adoral zone) are immediately conspicuous in the living animal, but are more readily studied in permanent mounts prepared by Horvath's (1931) toluidin blue method. They are thin and very wide and somewhat crescent-shaped. In longitudinal section of a membranella, or when the basal granules are stained with toluidin blue, 2 closely parallel rows of granules are visible. Each membranella is therefore composed of 2 rows of cilia which have become fused. The membranelle zone, situated on a broad ill-defined ridge (see Fig. 3), commences anteriorly on the inner surface of the small left-hand plate, and extending posteriorly, curves on to the dorsal surface, runs an almost straight course towards the right-posterior extremity of the peristome excavation and then follows the curve of the peristome to its apex (Figs. 1 and 2, Text-fig. 1).
From the peristome angle to the apex, the membranellae become steadily narrower; anteriorly from the angle they are of an equal width, but narrow again where the zone bends towards the ventral surface. In an average-sized specimen there are approximately 90 membranellae. The left-hand ends of the basal portions of the membranellae continue into a system of well-defined fibrils. These are ectoplasmic structures converging towards a centre at the left posterior region of the ventral cleft (Figs. 1 and 2), and they are clearly visible in the living or fixed and stained specimens. Schuberg calls these fibrils, peristomal striations, and Kahl designates the region containing them as a “zone free of membranellae,” although he illustrates the presence of the fibrils in his figure. Their convergent nature is not shown by Kahl or Schuberg. It is possible that these structures belong to the neuromotor system and that the centre of convergence contains the neuromotorium. The preparations from the present material do not give a precise delineation of the system. The remainder of the peristomal excavation has a series of very fine, more or less trausverse striations (Fig. 1), which are well described by Schuberg and Peschkowsky.
There is general agreement on the structure of the endoplasm, the form of the macronucleus and the occurrence of micronuclei. Large numbers of vacuoles occur in the endoplasm. These are generally small to medium-sized, but are so numerous that usually only a thin sheet of endoplasm separates one vacuole from its neighbour (Fig. 3). When seen by dark field illumination, their surfaces reflect the light and the animal becomes an almost opaque white. Numerous, varioussized inclusions are present in the endoplasm, among which coarse, dark granules predominate and these impart a greyish-brown colour under transmitted light. It is the combined effect of vacuolation and inclusions which makes morphological observations difficult. The pronounced cyclosis in the endoplasm takes the form of irregularly occurring movements, rather than of a steady streaming. The macronucleus is visible as a ribbon-like, lighter-coloured zone in the endoplasm of the living animal. It is long, vermiform and often twisted, and has a finely granular structure when stained with Ehrlich's acid haematoxylin. In two animals an unusual condition was detected in the maeronuelens; in one specimen this took the form of a forked extremity, in the other the macronucleus was U-shaped, with a short third arm protruding from the base up between the arms of the “U.” A thin sheet of endoplasm always invests the macronucleus and isolates it from the endoplasmic vacuoles (Fig. 3). Scattered in the endoplasm are numerous mieronuclei, eleven being counted in one specimen, thirteen in another. They are vesicular, each one being formed of a small, central knot of chromatin, surrounded by a remote, clearly defined membrane.
Food vacuoles in Bursaria truncatella often reach a very large size (Fig. 3), and on occasion may attain one-third the width of the animal. They are readily distinguished from endoplasmic vacuoles by their larger size and their contents. After the vacuole completes its circuit in the endoplasm, the faeces are discharged from the posterior cytopyge. The species is a voracious feeder. In cultures, speci-
mens are often to be seen moving about slowly within a restricted area, and feeding with the anterior opening of the peristome applied to the bottom or sides of the dish. Frequently the food consists almost wholly of diatoms, but a species of the family Volvocales was taken and occasionally ciliates as well. Fragments of diatoms were found in the food vacuoles of sectioned specimens.
There has been disagreement among various writers on the distribution and sizes of the contractile vacuoles. Kent (1880–1882) states that they are found throughout the ectoplasmic cortical layer of the surface, and his illustration shows that they may be of different sizes. Schuberg (1887) draws attention to their presence in both the trophozoite and encysted condition. No encysted forms were detected during the present investigation. Kahl (1927) has estimated that 300 to 400 vacuoles occur in a medium-sized specimen (500° long); he describes them as being 30 to 40° apart, and states that each one has a distinct pore. In his figure they are drawn approximately of the one size. Bhatia (1936) in his generic description of Bursaria states that the contractile vacuoles are usually absent, or that they may occur distributed over the surface of the body. In the Wellington material, the contractile vacuoles appeared throughout the ectoplasmic cortical zone, and, unlike Kahl's material, varied considerably in size. (Fig. 1). They were first detected about the lateral and posterior margins of the body, probably because more light penetrated these regions, but careful observations revealed them at other positions throughout the surface ectoplasm. For the most part they were exceedingly small and difficult to detect, but larger vacuoles were always present. Though Kent illustrates this variation in the size of the vacuoles, later writers do not seem to recognise it.
Reproduction is by transverse fission. Its onset is first indicated by a marked darkening in colour and a corresponding increase in opacity of specimens. The comparative rigidity of the body form is lost, and a variety of shapes may be assumed; these, though not differing greatly from one another, indicate that a certain degree of plasticity has appeared. The characteristic form of Bursaria is slowly transformed during these changes and an ovoid shape is assumed. The animal becomes very sluggish in its movements during this time. While the ovoid shape is developing, a transverse furrow forms about the equator of the specimen. The margins of the ventral cleft become approximated, but do not fuse, remaining as a ciliated groove on the surface of the future anterior individual. The rest of the peristome, and the membranellae, disappears. During this period, ciliation remains uniform, the constricting furrow deepens and the macronucleus shortens and thickens prior to division. There is as yet no indication of the peristomal structures developing. In the final stages of fission, the still united daughters are dark brown, subspherical, and each now possesses a horseshoe-shaped macronucleus. Separation is effected approximately thirty minutes after the onset of constriction, the two daughters rotating in opposite directions to cause the break. When free, each of the daughters is at first almost spherical, but shortly develops an ovoid form. The peristome and its membranellae first appears as a ciliated, oblique groove on the surface
of the new individual, and this sinks inwards and enlarges. Reversal of polarity in the daughter cells, described by Lund and quoted by Kudo, was not detected in these specimens.
On the basis of previous accounts and the present study, B. truncatella is to be recognised as a markedly asymmetrical ciliate, the asymmetry being due to a right-hand torsion combined with a strong convexity on the dorsal and right sides, and an almost equally strong concavity on the anterior one-third of the ventral and left sides. It is widest posterior to the mid-body region and diminishes rapidly to the bluntly pointed or rounded extremity; the anterior extremity is broadly truncated and has the appearance of sloping towards the ventral and right sides. The cilia are relatively short and occur in rows which are identical with, or are closely associated with, the surface striations. The peristome is free of cilia and consists of a deep excavation with narrow, more or less transverse striations. In its posterior one-third it curves to the left and at the same time dorsally. On its left is a broad zone of approximately 90 membranellae; anteriorly the zone begins at the right, ventral extremity, curves on to the dorsal surface of the excavation and then proceeds to the apex of the peristome. Each membranelle is composed of 2 parallel rows of fused cilia. From the right of the ventral surface, a thin, fold-like peristomal plate extends inwards to the wid-ventral line; on the left there is a small plate at the anterior extremity only. The inner edge of the right-hand plate forms the right-hand margin to the ventral cleft; on its dorsal surface this margin bears a ciliated ridge or septum, which partly closes off the peristome from the exterior. Both the septum and its cilia continue beyond the cleft on to the ventral wall of the peristome and are encompassed by the septal space. The mundspalte or stomial aperture runs along the right dorsal wall of the excavation. It extends from the anterior extremity to the apex of the peristome, and it is an open groove lined with ectoplasm. About the margins of the ventral cleft is a skeletogenous rod, the peristome band. The contractile vacuoles are very numerous, and occur throughout the ectoplasmic cortical zone; they vary in size from minute to comparatively large. The macronucleus is finely granulated, vermiform and often twisted, and always invested by a thin sheet of endoplasm. Micronuclei are vesicular, numerous and scattered in the endoplasm. The endoplasm is vacuolated, very finely granular, but contains large numbers of irregularly shaped, more coarsely granular inclusions, some of which impart a brownish colour to the animal. A thick zone of ectoplasm maintains the body shape. It covers not only the body surface, but also completely invests the surface of the peristome; it is closely and regularly vacuolate, and when seen in section has a minutely striated appearance. The cytopyge is postero-terminal in position. The species inhabits fresh-water; in this case the pH was 7·1.
I wish sincerely to thank Professor Li. R. Richardson, Victoria University College, for his very helpful advice throughout this study, and for his assistance during the preparation of this manuscript.
Bursaria is a monospecific genus. Many workers have published accounts of B. truncatella, but these are generally incomplete, and on some points contradictory. In this account, the available literature has been examined, briefly reviewed and discussed in relation to the findings of this study. The general morphology of B. truncatella has described at some length and special attention paid to the complex ingestive apparatus. The mundspalte or stomial aperture is an open groove lined with ectoplasm, and there is a zone of approximately 90 membranellae each composed of a double row of fused cilia. The oral excavation is considered as being a large peristome which does not give rise to a cytopharynx. The contractile vacuoles vary in size and they occur throughout the ectoplasmic cortical zone. Transverse fission is described.
List of References
Bhatia, B. L., 1936. The Fauna of British India: Protozoa, Ciliophora. London.
Gray, P. 1932. A Rapid Technique for the Permanent Mounting of Minute Freshwater Organisms. J. Roy Micr. Soc., vol. 52, pp. 370–372.
Horvath, P., 1931. Sublimat-Toluidinblan für Cilienfärbung. Z. wiss. Mikr., vol. 47, pp. 463–465.
Kahl, A., 1927. Neue und ergänzende Beohachtunge heterotrichen Oiliaten. Arch. Protistenk., vol. 57, pp. 121–203.
Kent, W. S., 1880–82. A Manual of the Infusoria, vols. 1 and 2. London.
Kirk, T. W., 1885. On Some Specimens of Vorticellae Collected in the Neighbourhood of Wellington. Trans. N.Z. Inst., vol. 18, pp. 215–217.
Kudo, R. R., 1946. Protozoology. Springfield, Illinois.
Maskell, W. M., 1886. On the Fresh-water Infusoria of the Wellington District. Trans. N.Z. Inst., vol. 19, pp. 49–61.
— 1887. On the Fresh-water Infusoria of the Wellington District. Trans. N.Z. Inst., vol. 20, pp. 3–19.
Peschkowsky, L., 1927. Skelettgebilde bei Infusorien. Arch. Protistenk., vol. 57, pp. 31–57.
Schuberg, A., 1887. Uber den Bau der Bursaria truncatella; mit besonderer Berüchsichtigung der protoplasmatischen Strukturen. Morph. Jb., vol. 12, pp. 333–365.
Yocom, H. B., 1918. The Neuromotor Apparatus of Euplotes patella. Univ. Calif. Publ. Zool., vol. 18, (14), pp. 337–396.
Fig. 1—Live specimen from the ventral aspect.
Fig. 2—Ventral view of fixed and stained specimen showing the peristome and associated structures in more detail than in Fig. 1.
Fig. 3—Transverse sections (7μ) through regions corresponding to those marked in Text-fig. 1.
Abbreriations.—A.P.O., anterior peristome opening. B.C., base of ventral cleft. ECT., Ectoplasm. END., endoplasm. F., fibrlllae. M., mundspalte. MAC., maoronucleus. MEM., membranellae. P., peristome. P.B., speristome band. R.V.C., right margin of ventral cleft. S., septum. S.S., septal space.