Studies in Spicule Formation. VI.—The Scleroblastic Development Of

STUDIES IN SP10ULE FORMATION. 45

Studies in Spicule Formation.

VI.—The Scleroblastic Development of the Spicules in some Mollusca and in one Genus of Colonial Ascidians.

\V. Woodland, Demonstrator of Zoology, Kiug's College, London.

Witli Plate 5.

SPICULES IN NUDIBEAKCH MOLLUSCA.

The nudibranchs examined, all obtained from Plymouth, consisted of three genera—G-oniodoris castanea, Archi- doris tuberculata, and Lamellidoris bilamellata. Most of my specimens were simply fixed and preserved in absolute alcohol, and were, in consequence, much contracted; others were fixed with 1 per cent, osmic acid, opened to givo admission to the stain, and stained as usual with picro-carmine (three hours), and I received them in this condition. In both cases I divided the animals horizontally into halves (with scissors), and in each instance scraped away with a scalpel the viscera and most of the musculature of the body-wall, until the portion of integument left looked to some extent translucent (whether unstained or stained). I usually found that the integument of the foot showed the spicules better than that of the dorsum. The unstained portions of integument (alcohol specimens) I stained in a saturated solution of 46 W. WOODLAND. safranin (nigrosin is also good) in absolute alcohol for a fort- night; washed thoroughly in warm absolute for a day or so, stained for a few minutes in a saturated solution of lichtgriin in absolute alcohol, again washed well in absolute, cleared in xylol and finally mounted in balsam. In most cases the picro- carmine specimens became too dark if also stained with lichtgriin. The spicules of these three genera are all somewhat irregular monaxons with a concentric structure when viewed in section, aud containing a larger or smaller amount of oi'ganic matter, but they differ slightly both in size and shape accord- ing to the genus. In Archidoris the spicules are much more slender than in the other two genera,, and do not attain such a great length; moreover, they do not possess a sudden thickening situated midway in the length of the spicule like those of the other two genera, and the nucleus of the scleroblast is much smaller both relatively to the size of the spicule and absolutely, is spherical instead of oval and possesses a distinct nncleolus. The spicules ofi both Goniodoris and Lauiellidoris are, as just mentioned, thickened at the middle and in the latter genus this midway thickening (which is not so prominent as in Goniodoris) often possesses one or two large spikes. The spicules of Lamellidoris attain a much greater size than those of Goniodoris, and are much smoother in general outline. Corroded spicnles exhibit straight-sided regular outlines at the exposed edges, whence we may suppose that nudibranch spicules, like most other calcareous spicules, are essentially aggregates of calcite crystals. The nearest approach to the spherical concretion stage of the spicule which I have observed is that represented in fig. 1. Without doubt the spicule originates, as in every other case of simple spicules, in this form, but unfortunately my specimens are not young enough to show this, although the stages figured afford sufficient proof that this is the case. The granule becomes a rod and the rod assumes the form of the adult monaxon, growing over its entire surface (but, of course, chiefly at the extremities) by the deposition of cal- STUDIES IN SPIOULE FORMATION. 47 careous matter derived from the scleroblast cytoplasm which entirely surrounds the spicule. The median portion of the adult spicule is formed first (evident when this is thickened), the tapering extremities growing out from this. The scleroblast, i. e. single nucleus, never divides, so that the spicules are purely unicellular products, and in most cases the body of the scleroblast (the small mass of protoplasm immediately surrounding the nucleus) is constantly situated midway in the length of the spicule, i. e. in the vicinity of the thickening in those spicules possessing this feature.

Spicules in Aplacophorous Mollusca. My material consisted of specimens of Proneomenia aglaophenite and Dondersia banyulensis, specially prepared at Plymouth by the osmic acid and picro-carmine method which gave excellent results. So much has been previously written and so many good figures provided in connection with the development of these characteristic calcareous spicules of the Aplacophora that my sole excuse for re-considering the subject is the uncertainty which still prevails as to whether these spicules are unicellular (Thiele [8], Wiren [9]) or multicellular (Heuscher [3], Hubrecht [4], Kowalevsky and Marion [5], Pruvot [7]) in growth. There is also a misapprehension to correct, which is that the spicules embedded iu the cuticle of Proneomenia are "in relation internally with epithelial papillee" (Sedgwick's ' Text-book of Zoology,' p. 353, likewise Pelseneer's 'Mollusca' in Lankester's 'Treatise on Zoology '). To state the results of my direct observations and inquiries sis briefly as possible, I may say that it is now quite certain that the spicules of the Aplacophora, like those of the Poly- placophora (Pelseneer), all arise individually in a single cell of the hypodermis (figs. 7—13). It is also certain that the spicules in Proneomenia (and other Aplacophora which possess an integument of similar type) do not in the majority of cases bear any relation to the hypodermal papillae, and that 48 W. WOODLAND.

when they do the relation is purely an accidental one. This occasional accidental relation apparently originates thus : the young spicules arise each in a single cell situated in the hypodermis, and in the majority of cases the portion of the hypodermis containing this spiculiferous cell remains in its ordinary position, but if, as sometimes happens (say in 20 per

Semi-diagrammatic drawing of Hie hypodermis and cuticle of Pro- neomenia aglaopheniee, illustrating the "carrying up" of the spiculiferous cells and spicules by the hypodermal papillae. The figure (X 400 diam.) is composed of drawings of the actual objects brought together into one field. In A the spicule is in its normal position ; in B the scleroblast is being detached from the hypodermis; in C the scleroblast has lost its spicule, this lying free as at K; in D a scleroblast with a well-grown spicule has been caught up in the young papillary elevation; in E a fairly young spicule has been carried up some distance by a papilla; in E the spicule has similarly been carried up, but is older; G and H represent young papillse; J a full grown papilla, with its pigmenled swollen extremity lying just below. Hie outer limit of the cuticle. It must be understood that normally the papiLlte and spicules are quite distinct, not being in any way associated. L represents debris ou the exterior of the thick cuticle; M is the hypodermis. cent, of cases), it becomes raised up into a papilla, the spiculiferous cell is inevitably carried up with it, and then the spicule superficially appears to be a product of the papilla (see the accompanyiug text-figure). The majority of the spicules are quite separate from the papillae, but in those cases in which they are associated the purely accidental nature of this association is proved by the various positions which the spicule assume relative to the papilla, these illustrating the various stages of " carrying up " referred to above. STUDIES IN SP10ULE FORMATION. 49 The spicules of Proneoinenia, from their first a'ppearance each as a small needle contained within a single cell (figs. 7, 8), grow in a more or less vertical direction, and soon burst through the cell-membrane (fig. 9). From this stage onward the further growth of the spicule is confined to the basal extremity (so resembling the growth of a hair), which alone is enveloped by the cell-substance (fig. 10). An axial cavity appears in the spicule substance before the spicule is half grown, but closes up proximally before the full size is attained. Many of the scleroblasts attached to the larger spicules become more or less withdrawn from the hypodermis (text-figure, B, C), and subsequently also lose connection with the spicules themselves, which, perhaps owing to contrac- tions of the integument, often come to lie, when full grown, near the outer limit of the cuticle (K in text-figure). In Dondersia the monaxon spicule, instead of being vertical in position as in Proneomeniaj is disposed more or less horizontally from the first (figs. 11, 12) in correspondence with the thin cuticle (thin as compared with that of Pro- neoraenia, being little more than the thickness of the hypodermis). These spicules also when adult become separated from the hypodermis, and lose their scleroblasts. I may add that the figures and statements of Heuscher and Hubrecht, affirming the multicellular papillary origin of the spicules of Proneomenia, are misleading, though superficially they appear to be correct. They have solely resulted from insufficient attention being paid to detail, and, bearing in mind the facts described above, it will easily be seen how the mistake has arisen. It may also be mentioned that the hypodermis contains many gland-cells with mucilaginous contents, which have been,, on at least one occasion, figured as young spicules! Needless to say the two are readily distinguishable. With reference to the mode of growth of these spicules of the Aplacophora, I may here point out that the production of a straight "finished" mineral structure by terminal accretion due solely to the activity of a single scleroblats VOL. 51, PART 1.—NEW SEEIES. 4 50 W. WOODLAND. under undisturbed conditions is here clearly demonstrated; there is in these aplacophore spicules no question whatever as to whether or no the symmetrical form is due to crystal- lisation or the like. And, bearing this example in mind, it further cannot be denied that the straightest rays of the triradiates of clathrinid Calcarea in all probability owe their symmetry to the same cause. Crystalline matter is deposited by the terminal cell in a similar manner in both cases, and, so long as the conditions remain undisturbed, the growth of the spicule must continue in a straight line. " Bio-crystal- lisation " and the rest are here at least superfluous. On the other hand, introduce disturbed conditions and, as might be expected, the more irregular spicules of Leucosoleniidte and Sycons are the result. Further, granting placid conditions, bring three cells into such close apposition that their inner surfaces become adpressed into the outline of a triradiate and let each of these cells divide centripetally, the distal cell in each case producing, in relation with the fixed proximal cell, a straight monaxon,1 and personally I can see no reason why cumbrous hypotheses should be invented in order to explain why the three contained angles of such a triradiate spicule (found in most calcarea) should in almost all cases be equal. Surely the equiaugularity is the direct result of the apposition of the three scleroblasts, as I have previously contended [10]. No such explanations, however, apply to the young triradiate " stars" of most echinoderms described in the last Study, which, however, differ, like most other spicules, from the calcareous spicules of sponges and Aplacophora in that they are entirely invested by the formative protoplasm.

THE SPICULES IN THE ASCIDIAN GENUS LEPTOCLINUM. I have examined three species of Leptoclinum—L. commune, L. maculosum, and L. sp.— all obtained from 1 This monaxon is, under these conditions, pointed at both ends. In Aplacophora each spicule, corresponding to its one-celled basal origin, is truncated at its proximal end. STUDIES IN SPIOULE FOEMATION. 51 Naples and preserved in alcohol. In all cases I embedded portions of the colony in paraffin wax and cut thin free- hand sections with a razor; I then stained these sections in the manner described below. The spicules are identical in the three species. Staining methods which do not differ- entiate the nuclei very prominently do not give good results for ascertaining the number of scleroblasts in connection with the adult stellate spicules, chiefly because the mass of the spicule more or less effectually hides all objects situated underneath, and the conical protuberances largely obscure objects situated to the side. The use of picro-carmine would doubtless give better results, but I was unable to employ this stain. The only effectual method which I employed was the ordinary borax-carmine method, the differentiation with acid- alcohol dissolving the spicules sufficiently to render the scleroplasm apparent. This method merely revealed one scleroblast (nucleus) in connection with each spicule, the layer of scleroplasm closely investing the entire spicule and following all its outlines, and the large nucle as being situated in a mound of protoplasm at the periphery (figs. 17, 18). The stellate spicule originates in a scleroblast as a spherical granule (fig. 14; these early stages are quite visible in the non-decalcified safranin and lichtgriin-stained preparations) which later acquires spines on its surface as it increases in size (fig. 16), and which spines ultimately become the conical processes of the adult spicule. Previous literature dealing with the scleroblastic development of Ascidian spicules is very small in amount (see Herdman [2] for the literature relating to Ascidian spicules up to 1885, since which year, I believe, no literature on the present subject has appeared). Loewig and Kolliker [6] in 1846 provided very poor figures of the stellate " cellules incrustees" in Didemnum, and illustrated one of these " cellules " partly decalcified, showing the cell-wall spherical in outline. Giard [1] in 1872 figured stellate spicules of Euccelium and Didemnum also situated each within a circle which is supposed to represent the cell-wall; he figured as 52 W. WOODLAND. well several small spicules situated within one cell. No nuclei are shown in any of these figures. In the first place I may say that I do not remember ever having observed more than one spicule contained within a single cell; and secondly, that the above authors are greatly mistaken in supposing the outline of the scleroblast containing the adult spicule to be spherical. A scleroblast containing a spicule only remains spherical when the contained spicule is small compared with the original size of the cell (microscleres of some siliceous sponges and the young stages of larger spicules e.g.); when as in Leptoclinum or Didemnum, the spicule is many times larger than the original scleroblast, this latter necessarily becomes distended and envelops the entire spicule with a thin layer of scleroplasra, which of course assumes the shape of the spicule. If the spicules are too hastily decalcified it is quite conceivable that the evolution of gas, being coincident with the disappearance of the mass of the spicule, inflates the scleroblast, and thus causes it to artificially assume a spherical shape; indeed, I have some evidence that this is liable to occur. Decalcification by means of the acid-alcohol used in the borax-carmine method is very slow, and the scleroplasm retains its stellate form, though, the spicule has largely or wholly disappeared.

LITERATURE. 1. GIAKD, A.—" Recherches sur les Asoidies Composes ou Synascidies," 'Archiv Zool. expdr.,' t. i, 1872. 2. HEIIDMAN, W. A.—"The Presence of Calcareous Spicula in the Tuni- cata," 'P. Geol. Soc. Liverpool,' 1884-85. 3. HEBSCHEK, J.—"Zur Anatomie und Histologie der Proneomenia sluiteri, Hubrecht," 'Jena Zeitschr.,' Bd. xxvii, 1892. 4. HTJBMECHT, A. A. W.—"Proneomenia sluiteri; with Remarks upon the Anatomy and Histology of the Amphineura," ' Archiv fur Zoologie," Suppl., Bd. i, 1881. 5. KOWALEVSKY, A. 0., and MAHION, A. P.—" Contributions a l'Histoire des Solenogastres ou Aplacophores," 'Annales du Mus6e d'Histoire Naturelle de Marseille,' "Zoologie," t. iii, 1887- STUDIES IN SPICULE FORMATION. 53

6. LOEWIG and K6LHK.EE.—"De la composition et de la Structure des En- veloppes des Tuniciers," 'Ann. Sci. Nat.,' 3rd ser., t. v, 1846. 7. PEUVOT.—" Sur l'organization de quelques Ne'omeniens des cotes de Trance," 'Arcb. Zool. Exp.' (2), ix, 1891. 8. TIIIELE, J.—" Beitrage zur Vergleichenden Anatomie der Amphineuren," 'Zeit. f. wiss. Zoologie,' JBd. lviii, 1894. 9. WIKEN.—" Studien iib. die Solenogastres," I and II. ' Svenska vet. Akad. Handl.,' xxiv, xxv, 1892-93. 10. WOODLAND, W. N. F.—" Studies in Spicule Formation. I. Sycon Sponges," 'Quart. Journ. Micr. Sci.,' vol. 49, 1905. 11. " Studies in Spicule Formation," V. 'Quart. Journ. Micr. Sci.,' vol. 51,1907.

EXPLANATION OF PLATE 5,

Illustrating Mr. W. Woodland's " Studies in Spicule Formation" (VI).

Figs. 1 and 2, x 640; Figs. 3—6, x 192 ; Figs. 7—18, x 1280. All drawings were made with the camera lucida. FIG. 1.—Young stages in the development of the monaxon spicules of Goniodoris castanea. The thick median portion is the first to be formed. FIG. 2.—Apparently an abnormal spicule of G. castanea possessing two thickened club-like extremities and two nuclei, and so somewhat resembling a young alcyonaiian spicule. FIGS. 3 and 4.—Older spicules of G. castanea, each entirely enveloped iu cell-substance and with one nucleus. FIG. 5.—Small monaxon spicules of Archidoris tuberculata. These spicules possess no central thickening and have small spherical nuclei. FIG. 6.—Small (young) spicule of Lamellidoris bilamellata; large type of spicule, and usually possessing a median spine. FIGS. 7 and 8.—Young spicules of Proneomenia aglaophenise. FIGS. 9 and 10.—Older spicules of the same. FIGS. 11 and 12.—Young spicules of Dondersia banyulensis. FIG. 13.—Older spicule of the same. FIGS. 14—18.—Stages in development of the stellate spicules of Lepto- clinum commune. 14. l5- 16. 17. WW del

5PICULES OF M0LLU5CA AND TUN1CATA.