THE FINE STRUCTURE OF CRICOSPHAERA CARTER AE

J. Cell Sci. 4, 561-567 (1969) Printed in Great Britain

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THE FINE STRUCTURE OF CRICOSPHAERA CARTER AE I. EXTERNAL

MORPHOLOGY

R. N. PIENAAR Department of Plant Biology, University of Natal, Durban, South Africa

SUMMARY The external morphology of Cricosphaera carterat is demonstrated using shadow, replicating and thin section techniques. The covering of the cell was found to be composed of three forms of scales which differ from one another in size and ornamentation. The largest of the three scales serves as the organic matrix on which the calcium carbonate is deposited to form the characteristic coccolith. The structure of these scales is discussed. INTRODUCTION

During a recent study of coccolith formation in Cricosphaera carterae Braarud & Fagerland several interesting features regarding the external covering of the motile coccolith-bearing stage were observed. It was felt that these observations were worth recording due to the fact that at present the systematics of these algae is based on hard morphology although results of recent work indicate that the classification will be revised. MATERIALS AND METHODS

C. carterae was obtained from the Culture Collection of Algae, University of Indiana, U.S.A. The cells were cultured in an enriched sea water (medium i,Provasoli, 1958) and subjected to a cycle of 16 h of light alternating with 8 h of darkness. Gold-palladium shadowed preparations were prepared by micropipetting a drop of culture medium containing cells on to a Formvar-coated grid. The cells werefixedin osmium tetroxide vapour and shadowed with gold palladium. The technique used for the preparation of replicas was identical to that described by Pienaar (1966). Thin sections were prepared byfixingthe cells in 6% phosphate buffered glutaraldehyde in 5 % sucrose at a pH of 7-15, post-fixed in 2% osmium tetroxide and finally embedded in Araldite. Sections were cut using a Huxley ultra microtome and stained with lead citrate (Reynolds, 1963). Observations were made using a Philips EM 200. OBSERVATIONS

General

The motile coccolith-bearing stage of C. carterae when studied with the aid of the optical microscope was found to be covered by a well-developed layer of coccoliths

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(Fig. 1). A detailed study revealed the external covering of the cell to be more complex. It was found to be covered by three types of scales; large elliptical scales (which serve as the organic matrix on which the crystals of calcium carbonate are deposited to form the coccoliths), circular unmineralized scales, and finally small elliptical to rectangular unmineralized scales. Large elliptical scales

The general shape of these scales is elliptical with the longitudinal polar regions slightly pointed (Fig. 4). The size varied from 0-90x0-67 to 1-90x1-01/1. The shadowed preparations revealed the proximal surface to be ornamented with ridges radiating from a central region (Figs. 5, 6) whereas the distal surface appeared to be devoid of any patterning. A study of sectioned material (Fig. 7) supported this conclusion. Along the periphery of the distal surface crystals of calcium carbonate are deposited in a manner specific for this stage of the life history. The elliptical scales are therefore the organic matrix on which the calcium carbonate is deposited. Once deposition has taken place the scales can be referred to as coccoliths. The only work on the structure of the coccolith of C. carterae was done by Braarud, Gaarder, Markali & Nordli (1952). These workers used transmission micrographs which do not reveal much detail of the structure of the coccolith except to provide some indication of its general shape and the arrangement of the crystals. Sectioned material revealed additional information. A horizontal section parallel to the longitudinal axis of the coccolith revealed that the coccolith was composed of large ' H '-shaped elements which alternated with rectangular elements except at the longitudinal polar regions. Here only the rectangular elements were found to be present (Fig. 11). The term element is being used because it is not known whether each element (in C. carterae) is a crystal of calcium carbonate. The ' H '-shaped elements when seen in a longitudinal section perpendicular to the transverse axis of the coccolith (Figs. 9, 14) were found to be composed of similar regions to those found in the CoccoUthus huxleyi by Watabe (1967) viz. an upper and a lower region which are joined together by a central cylinder. Occasionally the central cylinder was found to possess rectangular outgrowths (Figs. 9, 14). In a section cut through the longitudinal polar region and perpendicular to the transverse axis the elements appeared to be rectangular to triangular in shape and the three regions could not be distinguished (Fig. 7). Figures 12 and 13 represent tangential sections through mature coccoliths. The rectangular elements which are interspersed with the ' H' shaped elements appear to fill in the elliptical space between the main elements as depicted in the original drawing of the coccolith of C. carterae (Braarud et al. 1952, Fig. 4, p. 132). From the electron micrographs (Figs. 5, 6) it is evident that the final size of the mature coccolith is larger than that of the organic matrix scale due to the development of the calcium carbonate elements. The size of the mature coccolith was found to vary from 2-20 x 1-41 to i-oo x 0-70 /t with the average size being 1-43 x 0-96 /t.

External morphology of Cricosphaera

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Circular scales

These scales are subcircular to circular in outline and range in size from 0-89 to o-8o fi in diameter with the average diameter being 0-82 /.t. The scales were observed in sectioned material as well as shadowed preparations. On comparing the results a consistent difference between the two techniques was always obtained. The sectioned material revealed the scale to be composed of radiating ridges which in every case appeared to proceed beyond the margin of the scale (Figs. 15, 17). Shadowed scales reveal an additional form of ornamentation. Using this technique the scales were found to be ornamented with radiating ridges and were surrounded by a raised rim. Besides the rim a system of almost concentric ridges is found to traverse the radiating system (Figs. 3, 8). The picture obtained from the shadowed material bears some resemblence to the distal view of the scales that cover the motile stage of Coccolithus pelagicus (Crystallolithus hyalinus) described by Manton & Leedale (1963). The puzzling feature is that out of all the sectioned and shadowed scales studied no deviation from the ornamentation characteristic of that particular method of preparation is found. The author is of the opinion that the shadowed preparations represent the true structure of the scales, but it is not possible to indicate with certainty which is the distal or proximal surface or even whether, in the case of C. carterae, there is a difference in ornamentation. The replicated coccolith seen in proximal view (Fig. 6) clearly shows the organic matrix scale and two circular scales. If these scales are in situ it would indicate that the proximal surface exhibits the described ornamentation. Because all the shadowed preparations had this structure it could be possible that the same ornamentation is to be found on both surfaces. In the case of the sectioned material it is probable that the concentric ridges were destroyed during preparation.

Small elliptical scales

These scales vary from elliptical to rectangular in shape. They are found to exhibit the same patterning as that described for the circular scales when seen in sectioned and shadowed preparations (Figs. 2, 8, 17) and the same explanation is postulated. The size of these scales varies from 0-50x0-36/6 to 0-34x0-23/* with the average size being 0-42 x 0-28 /t.

Relationships between the scales

Of the three types described the circular scales are the most abundant followed by the coccoliths and finally the small elliptical scales. The larger elliptical scales usually have calcium carbonate deposited on the periphery of the distal surface but occasionally unmineralized elliptical scales were encountered (Fig. 4). Figure 16 illustrates the positions in which the various scales are found on the exterior of the cell. The exact position of the smaller elliptical scales is not known. Manton & Leedale (1963) suggested that these smaller scales may occur at the region of flagella insertion in the case

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of the motile stage of Coccolithus pelagicus but no evidence of this was found in C. carterae. All three types of scales are formed within the cell; unlike the motile stage of Coccolithus pelagicus (Manton & Leedale, 1963), calcium carbonate deposition takes place intracellularly (Pienaar, in preparation). The scales and coccoliths are found to the exterior of the cell and are embedded in a mucilaginous-like covering which is more easily observed in optical-microscope preparations (Fig. 10). This work was supported by a grant from the University of Natal Research Fund.

REFERENCES

T., GAARDER, K. R., MARKALI, J. & NORDLI, E. (1952). Coccolithophorids studies in the electron microscope. Observations on Coccolitlius huxleyi and Syracosphaera carterae. Nytt Mag. Bot. 1, 129—134. MANTON, I. & LEEDALE, G. F. (1963). Observations in the microanatomy of Crystallolithus hyalinus Gaarder & Markali. Arch. Mikrobiol. 47, 115-136. PIENAAR, R. N. (1966). Microfossils from the Cretaceous System of Zululand studied with the aid of the electron microscope. 5. Afr. J. Set. 62, 147-157. PROVASOLI, L. (1958). A catalogue of laboratory strains of free-living and parasitic Protozoa. J. Protozool. 5, 1-38. REYNOLDS, S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 20, 313-332. WATABE, N. (1967). Crystallographic analysis of the coccolith of Coccolithus huxleyi. Calc. Tiss.

BRAARUD,

Res. 1, 114-121.

(Received 26 June 1968) Fig. 1. Optical micrograph of Cricosphaera carterae. Note the well developed layer of coccoliths surrounding the cells, x 1400. Fig. 2. Gold-palladium shadowed preparation of small elliptical scales. Reversed print, x 40800.

Fig. 3. General view of shadowed circular scales all exhibiting the same ornamentation. Reversed print, x 27 500. Fig. 4. Large elliptical scale which was found disassociated from the calcium carbonate elements. Direct print, x 27500. Fig. 5. Carbon-platinum shadowed replica of the proximal surface of a mature coccolith. The organic matrix scale is still in situ. Reversed print, x 23 800. Fig. 6. Carbon-platinum shadowed replica of the proximal surface of a mature coccolith. Note the two circular scales exhibiting the same patterning as the distal surface of the scales of Coccolithus pelagicus. (See Manton & Leedale, 1963, figs. 6, 7.) Reversed print, x 23 800. Fig. 7. Longitudinal section through a group of coccoliths. Note the almost triangular appearance of some of the elements. The proximal and distal ornamentation of the organic matrix scale is visible (arrows), x 27200. Fig. 8. Detail of shadowed circular and smaller elliptical scales. Reversed print, x 25000.

Fig. 9. Section through one of the ' H '-shaped elements. One can distinguish between the upper region (u), lower region (/) and central cylinder (cy). x 57750. Fig. 10. An optical micrograph of an empty coccolith casing from which the contents have escaped. The coccoliths appear to be embedded in a mucilaginous sheath, x 1800.


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Fig. 11. Horizontal section of a mature coccolith. The ' H '-shaped elements (h) can be seen alternating with the rectangular (r) elements except at the longitudinal polar regions. The distal surface of the organic matrix scale does not show the definite ridged appearance, x 57750. Figs. 12, 13. Tangential sections of mature coccoliths. The organic matrix can still be seen in situ, x 77000. Fig. 14. Section through a coccolith to show the organic matrix scale and the elements of calcium carbonate. The upper and lower regions as well as the central cylinder can be distinguished, x 77000. Fig. 15. A stained circular scale as seen in thin section. Note the absence of the concentric ridges, and the radiating ridges extending beyond the margin of the scale, x 5775cFig. 16. A thick section of a recently divided cell to illustrate the positions of the unmineralized scales and coccoliths. x 21725. Fig. 17. A group of stained scales as seen in thin section. The two types of scales can be distinguished. The small elliptical scale exhibits the same characteristics as the circular scale, x 42 700.


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