Some notes on the fine structure of Arcella shells (and an answer to a question nobody asked)
Amoebae in the family Arcellidae make their shells from a self-produced organic substance, without incorporating any other building materials (diatoms, hard scales, bits of quartz etc.). Although described in older sources as “chitinous,” and in newer ones as “keratin-like”, the precise composition of this substance is still unknown. The stuff is not laid down as a continuous sheet, but is assembled from small “thecagenous granules” produced in the Golgi apparatus of the cell (Porfirio-Sousa et al., 2020).
On the surface of the shell these building units form a field of tesselated hexagons with little perforations at their vertices:
A few weeks ago, I was measuring a small morphotype of Arcella spectabilis, and ran across a damaged specimen, which provided a more intimate view of the shell’s structure. The images I made of them show the outer surface of the shell, with its distinctive honeycomb pattern; and beneath that, the walls that make up the individual hexagonal units of the shell:
The broken edge also reveals something I find interesting. At several points, the breakage has occurred directly through the pores at each corner of the hexagonal units, giving us a very nice cross-section of the open, vertical shafts (or sub-triangular “intercalary wells”, as Mignot and Raikov call them) which pass through the shell.
The actual width of these shafts is hard to discern from the outside of the shell, because the opening is reduced by a layer of organic cement, which partially bridges the triangular gaps.
Viewed in cross-section, we see the neatly walled chambers of Arcella‘s hexagonal prisms as they actually are: discrete structures, though fused together at their sides and covered with a bridging layer of cement at either end.
The separability of these units has been known for a long time. As early as 1963, Roger Cambar and colleagues managed, “by destruction”, to isolate “prismatic elements” in the shell of Galeripora discoides“. They recorded these with electron microscopy, showing them scattered loosely on the background like a handful of hex nuts.
I’ve destroyed quite a few arcellid shells, over the years, but have never been lucky enough to see them come apart as nicely as that. What I have seen has sometimes been hard to interpret. Peering closely at SEMs of Arcella spectabilis in recent weeks, I realized I still had some unanswered question about their geometry.
The basic structure of the test–hexagonal prisms, sandwiched between two plates–has been known since the 19th century, although not all authors have agreed about the details.
The first to illustrate and describe the “hexagonal prisms” that make up Arcella shells was George Charles Wallich, an amusingly grumpy British doctor who did pioneering work on freshwater amoebae. I’ve mentioned Wallich before, in connection with his eccentric notion that all testate amoebae were varieties of Difflugia, whose distinct shapes were caused by movements of the water in which they were formed. He had some quirky fixations, but he was a careful observer, and his description of the organism he called “Difflugia Arcella” is largely correct.
In 1864, he was the first to recognize that the primary building units of Arcella tests were approximately hexagonal in outline and prismatic in form.
Ten years later, the German researchers Richard von Hertwig and Edmund Lesser confirmed Wallich’s observations, noting as well that the shell had “two plates,” one facing the outside of the shell, and the other facing the inside. Between these plates, was a framework of hexagonal prismatic cells, each of which they believed to be filled with fluid.
To test their interpretation, they soaked an Arcella shell in sodium carbonate, then added vinegar, forcing C02 into the cavities within the walls of the shell. Bubbles of the gas were observed in the microscope, trapped within the spaces, confirming that the hexagonal prisms were indeed hollow cavities, or cells.
Having established their approximate shape, they went on to propose that the presence of these hexagonal units should serve as a defining character of the genus Arcella. From their genus diagnosis:
According to its finer structure, the shell consists of two plates, an outer and an inner one, which are arranged parallel to each other and are united by a honeycomb-like framework forming hexagonal figures. (Translated from the original German)
20 years later, Ludwig Rhumbler agreed with this description, and added a proposed mechanism for the production of the hexagons. Concerning the “small plates” of Arcella, he writes:
They appear as tall, hexagonal, small prisms filled with a fluid (cf. Butschli, “Protozoa,” p. 20-n. 21). I believe they originate from small, spherical droplets, such as phaeosomes, which, upon reaching the outside of the shell, solidify, perhaps due to direct contact with the water on their surface, or secrete a precipitating membrane, and thereby flatten into hexagonal prisms through mutual pressure, somewhat like the cells of a columnar epithelium. As far as I know, hexagonal reserve plates have not yet been found in the Arcella soft body; therefore, their formation could very well have occurred during the budding process of the Arcella. (translated from German).
Rhumbler’s view–that the building units of Arcella begin as soft spherules, forced into hexagonal shapes by “mutual pressure”–is largely consistent with our current understanding of shell construction.
In 1906, Joseph A. Cushman and William P. Henderson were the first to document the fine structure of the Arcella shells with microphotography. In their brief paper, the authors describe a simple experiment demonstrating that they could easily introduce air bubbles into the hexagonal “cancelli” of the shell, simply by withdrawing water from their samples.
These air pockets seemed to form in cavities on the outer surface of the test. From this, the authors concluded that Hertwig and Lesser had been mistaken in their opinion that the hexagonal cells were enclosed between two membranes. They asserted that there was only a single membrane, which had to be on the interior surface of the shell. From this membrane, the hexagonal “cancelli” projected outward, and were open on their outer sides.1
Cushman & Henderson also noted that the pattern formed by the hexagonal cells seemed to be quite different from that illustrated in earlier works, which typically showed a structure resembling that of a honeycomb. According to Cushman and Henderson:
In the honeycomb arrangement the hexagons have sides in common. In the Arcella test the hexagons have no sides in common. Instead, the hexagonal areas are so placed that the three adjacent sides of three neighboring areas enclose a small triangular space. Just here we find a further complication of the structure. These interpolated triangles are not solid portions of the network,but themselves contain areoles of subtriangular outline.
In support of this view, they supply several photomicrographs–remarkably good ones, considering they were made in 1906. Their images do seem to show–in contradiction of earlier observations–that individual hexagonal units did not share sides with one another, but were arranged with their vertices touching. Between the sides of the hexagons were small empty triangular spaces, open at the top, and generally similar in construction to the large hexagonal cavities they had observed on the outside of the shell.
They supplied a diagram showing what they believed to be the true arrangement: hexagons meeting at their vertices, with hollow triangular spaces between the sides:
These observations are somewhat perplexing, not only because they contradict those of earlier researchers, but also because they contradict high-magnification images produced with the use of a scanning electron microscope, such the close view of Arcella spectabilis I inserted at the beginning of this post. As far as the authors were concerned, they had clearly demonstrated that the pattern of the network was, as they put it, “radically unlike the honeycomb structure heretofore assigned to it”; and indeed, their microphotographs seems to support that conclusion.
It’s all a bit confusing! However, I think I’ve worked out the reasons for these apparent contradictions.
To begin with, the lack of a double membrane, which Cushman and Henderson demonstrated in their air-bubble experiment, is likely the result of examining specimens with “collapsed areoles”, in which the “lids” that normally cover the hexagonal units have caved in.
This is a very common occurrence in arcellid shells, particularly when they are old. It often seems to occur as shells are dried and prepared for observation in the microscope: essentially, the outer face of the hexagonal units sags, or collapses entirely, leaving a pattern of empty holes dotted over the outer surface of the shell. In some cases, the whole shell surface is a meshwork of these collapsed areoles.
Collapsed areoles like these would allow the entry of air bubbles, exactly as shown in Cushman & Henderson’s Fig. 1, and the authors could hardly be blamed for supposing that this was the natural condition of their Arcella.
So, that would explain the single, interior membrane they detected; but what about their claim to have disproven the honeycomb pattern recorded by Wallich and others? That is a bit more difficult. It is certainly incorrect, given what we know from SEM images of the shells, but it is not immediately obvious where they have gone astray. So, what is going on, there?
The problem there does not lie in their imaging or sample preparation. The triangular openings the authors found are not an artifact, but a typical permanent feature of arcellid shells. In some individuals, these perforations appear as small holes positioned at the vertex of each hexagon, and in others they appear as larger, roughly triangular openings like these ones, from the interior surface of Arcella spectabilis:
In the pictures above, the building units appear roughly circular, or vaguely octagonal, because the vertices of each hexagon are truncated by those relatively large triangular openings. In the second image, I’ve superimposed lines, similar to those in Cushman and Henderson’s diagram, which give the impression that the hexagonal units meet only at their vertices. However, that is little more than a trick of the eye, caused by our tendency to read the discontinuous spaces between the triangular apertures as a series of intersecting lines. There are no natural structures corresponding to these line, simply the negative space between the triangles. Whereas, on the outer surface of the shell, where the perforations are much smaller (partly occluded by organic cement), we read the geometry in the usual way, as a field of tesselated hexagons with a small open hole at each vertex.
To my mind, that resolves this discrepancy. The controversy, such as it is, could not be less important. In the 120 years since Cushman and Henderson published their paper, it has been cited only five or six times, and I don’t think anyone but me has been particularly bothered by their description arcellid shell geometry.
Anyway, twelve decades later, there it is: my long-winded answer to a question nobody asked.
REFERENCES:
Cambar, R., Thomas, R., Leblanc, M., 1963, Recherches sur la constitution de la theques des Arcelles (Genre Arcella, Rhizopoda testacea) observations au microscope electronique: Compte Rendu Hebdomadaire des Seances de l’Académie des Sciences, ser. D, v. 256, p. 1364-1366.
Cushman, Joseph A., and William P. Henderson. “A preliminary study of the finer structure of Arcella.” The American Naturalist 40, no. 479 (1906): 797-802.
von Hertwig, Richard. Ueber Rhizopoden und denselben nahestehende Organismen: Morphologische Studien von Richard Hertwig und E. Lesser. Vol. 10. Max Cohen & Sohn, 1874.
Moraczewski, J. “La composition chimique de la coque d’Arcella discoides Ehrbg.” Acta Protozoologica 8 (1971): 423-437.
Mignot, Jean-Pierre, and Igor B. Raikov. “New ultrastructural data on the morphogenesis of the test in the testacean Arcella vulgaris.” European journal of protistology 26, no. 2 (1990): 132-141.
Penard, Eugène. Faune rhizopodique du bassin du Léman. H. Kündig, 1902.
Porfírio-Sousa, Alfredo L., and Daniel JG Lahr. “Current knowledge and research perspectives of the shell formation process in the genus Arcella (Arcellinida: Amoebozoa).” Protistology 14, no. 1 (2020): 3-14.
Rhumbler, L. “Beitrage zur Kenntnis der Rhizopoden.” Beitrag III, IV und V (1895).
Wallich, George Charles. “XXVI.—On the extent, and some of the principal causes, of structural variation among the Difflugian Rhizopods.” Annals and Magazine of Natural History 13, no. 75 (1864): 215-245.
- This was similar to the opinion of Eugene Penard, who had suggested in 1902 that, contrary to Hertwig and Lesser, the Arcella shell had just one membrane. However, according to Penard, that membrane was on the outside of the hexagonal mesh, and the hexagons themselves were open on the inside of the shell! ↩︎