Studies in Spicule Formation. : VII.—The Sclerohlastic Development of the Plate-and-Anchor Spicules of Synapta, and of the Wheel Spicules of the Auricularia Larva.

THE complete morphogenesis of the curious aggregate (8) plate-and-anchor spicules of Synapta inhærens has been previously described and figured by Semon (6), but, with the exception of my description of the disposition of the scleroplasm associated with the adult spicules (7), no account of the part played by the living tissues of the organism in the production of these wonderful structures has hitherto been published.

These plate-and-anchor spicules are so well known that it is not necessary for me to do more than briefly mention their more obvious characteristics. Each spicule, as seen in the integument of Synapta inhærens, e.g., consists of two parts—the anchor and the plate—and these two parts are quite separate from each other. The anchor consists of a shaft, a bow (with its two arms), and a handle—to use the current terminology; the plate, on the other hand, has several large perforations, and is somewhat more pointed in shape where it comes into apposition with the handle of the anchor than at the opposite side (fig. 23). This more pointed region of the plate I shall term the base, and the opposite, more obtuse region the apex. In side aspect it can be seen that the handle of the anchor and the base of the plate form a distinct joint, the one with the other (fig. 25), and the anchor and the plate rotate upon each other at this joint, the angle included between the two in consequence varying in magnitude (Ostergren, 4). It can also be observed in side view that the arms of the anchor bow do not he in the same plane as the shaft, but in a plane slightly more inclined towards the dermal layer, i. e. more nearly parallel with the plane of the plate (^{text-fig 1}).

Respecting the general arrangement of the spicules, it is an obvious and noteworthy fact that the major axes of these plate-and-anchor spicules, as they lie in the body-wall, are disposed transversely to the long axis of the animal, so that in a transverse section of Synapta the spicules are seen in side aspect (^{text-fig. 1}). Further, although the spicules never assume, as regards their-major axis, other than a transverse, or approximately transverse, disposition in the body-wall, it is quite a matter of chance as to whether the bow of the anchor and apex of the plate of each spicule lie to the right cm to the left of the observer; in other words, they are disposed pretty equally in both positions.

A few words concerning the preparation of Synapta material for’ the study of the spicule development are necessary. My material was obtained from Naples, and consisted of specimens of the two species—Synapta inhærens and S. digitata. My specimens of S. inhærens averaged 1 cm. to 1·5 cm. in length, and they were specially prepared for me by the osmic acid and picro-carmine method described in Study IV, e. g 1

I may mention that after fixation it is well to slit open the wall of the Synapta so as to stain the integument from the inner side as well as the outer. The integument is subsequently cut into convenient pieces before mounting, and, like the Cucumarias of Study IV, immersed for about five minutes in a saturated solution of lichtgrun ¹ in absolute alcohol, then washed well in absolute, cleared in xylol, and mounted in balsam, the inner side of the wall being placed uppermost on the slide. To observe the spicules in side view, I cut transverse sections of the undecalcified² body-wall about 12 μ to 20 μ in thickness or slightly thicker. Numerous sections must be cut, since the majority of spicules are fractured during the process of cutting, and it is only occasionally that the knife happens to fall just on each side of the (usually young) spicule instead of on to it.

My specimens of S. digitata were considerably older, none being less than 5 cm. in length; nevertheless, I have been able to observe all stages in development (though naturally a far greater amount of material had to be examined) excepting the initial granule. I may here remark that the numerous specimens labelled “ Synapta. digitata “ which have been forwarded to me differ considerably in. their spiculation—in the forms and sizes of the spicules—and I cannot help suspecting that the S. digitata examined by me includes several distinct species, or at least sub-species. The specimens of S. inhærens and S. hispida, on the other hand, have always been constant in their spicule characters. In most specimens of S. digitata the spicules differ considerably in size and form in the same animal—a feature I have not noticed in the other two species.

The first sign of the future spicule is the multiplication of the cells of the dermal or outer layer at one point. I say “cells,” but, strictly speaking, it is a multiplication of nuclei to form a syncytium, since cell-outlines are rarely, if ever, distinguishable. This preliminary formation of a syncytium is best seen in a section of the body-wall (fig. 1). It will be observed in fig. 1 that in these young Synaptas the dermal layer is already quite separate from the circular muscle layer, the intervening space containing, at this stage, irregular strands of nerve- and muscle-fibres. These initial syncytia are easily distinguishable in section from the numerous thickenings of the dermal layer which form the sense-organs, since the elongated cell-outlines and pigment deposits are conspicuous features in these latter. It is also easy to distinguish them in surface view under a low magnification after a little practice. The sense-orggn thickenings are, needless to say, much the more numerous.

The actual spicule first appears asa small spherical granule situated in the centre of the syncytium, but more internally than externally, so that the majority of the nuclei are situated on the other side of the spicule when this is viewed from the internal side of the body-wall (fig. 2). This spherical granule next elongates on one side (figs. 3 and 4) in a direction transverse to the long axis of the animal. Thus, remarkable as it may seem, the minute rod which the granule gives rise to, and which, in its turn, becomes the anchor of the adult spicule, is from the very first orientated in the direction assumed by the full-grown structure. The process formed from the side of the initial granule (situated to the right or to the left of the observer, as the case may be) continues to grow (figs. 5-10) until there is produced a stick-like structure, equal in length to the future anchor. One end of this stickspicule is somewhat swollen to form a knob, and represents the unmodified half of the original granule—the other half having grown out to form the stick. The stick is also usually of slightly greater diameter mid-way in its length than towards its extremities; in other words, the stick tapers somewhat, both towards the knobbed and the pointed ends. Also it possesses, at this stage, a distinct axial thread. The nuclei of the syncytium, up to this period of growth, and until the knobbed end of the stick has produced the recurved arms of about half the adult length, remain, for the most part, on the outer side of the stick—i. e. towards the external dermal layer—although one or two may be situated more internally (figs. 9, 10, 11). I may also mention that the syncytium surrounding the spicule usually remains in connection with the epithelium from which it originated by means of a few irregular protoplasmic strands.

The next stage in the development of the anchor spicule is the protrusion of both sides of the knobbed extremity of the stick or shaft to form the arms of the future bow of the anchor (fig. 10). These incipient arms elongate, and very soon become recurved ; at the same time this lateral extension of the knobbed extremity stretches the substance of the syncytium enveloping the spicule so as to produce the appearance shown iu figs. 16 and 18.

When the arms of the anchor have become half-grown a very remarkable phenomenon occurs in connection with the syncytial nuclei, situated a little bow-ward of the middle of the shaft. A number of these (usually from six to ten), which up to the present have been situated either at the sides of the shaft or on its external aspect, now migrate on to its internal side, and form a small cluster in that position (fig. 12). This fact seems to me to be most remarkable and I am quite unable to account for it, at least in an adequate manner. This cluster of nuclei is always easily distinguishable from the rest of the syncytium, and it is this specialised part which gives rise to the plate spicule. Thus, although the anchor and plate portions of the entire aggregate spicule are quite separate, and without doubt equivalent to two spicule individuals—to two echinoderm plate-spicules, to be precise— yet they are both developed from the same syncytium, though in distinct parts of it.

The plate-spicule, like other spicules, first originates as a granule, and this is situated in the centre of the internal cluster of nuclei (fig. 13). This granule next elongates on opposite sides and at right angles to the length of the anchor-shaft (cf. the development of the plate-and-anchor spicule of Synapta digitata described below) to form a rod or rhabdus (figs. 14, 15, 16) which is at first pointed at both ends. The nuclei of the cluster are situated on both sides of this rod, and in fact the entire development of the plate spicule is identical with that of the plate spicules of the Cucumaridæ described by me in Study IV, with the exception that the number¹ of nuclei initially concerned is greater in the present instance. The rod thickens at its extremities (fig. 16), then bifurcates (fig. 18), these primary bifurcations elongate and themselves bifurcate (fig. 20), and the processes of bifurcation and fusion of the extremities continue (figs. 21, 22), as in the Cucumarian plate-spicule, until the adult plate is produced (fig. 24). The Synapta plate-spicule is obviously different in several respects from the Cucumarian platespicule, but the plan of structure is the same. The Synapta plate possesses extremely large perforations, which diminish in size towards the more pointed “ basal “ extremity, and the edges of these perforations develop small processes which add considerably to the picturesque effect of the whole. As the spicule increases in size, the nuclei of the portion of syncytium concerned in its formation distribute themselves more uniformly over its area. Apparently very few, if any, nuclei are subsequently added to those initially present in the internal cluster which gives rise to the plate-spicule. It must also be remarked that the special internally-situated portion of the syncytium which produces the plate becomes almost quite separated from the rest of the syncytium enveloping the anchor as the growth of the plate proceeds, since the anchor and the plate gradually diverge to include the angle previously mentioned (text-fig. 1) ; the syncytia of the anchor and of the plate in fact alone remain continuous at the joint formed between the handle of the anchor and the base of the plate, and this continuity of the two syncytia in this region probably serves to some extent both to keep the two structures in apposition and to render the joint a true joint, i. e. a mutual centre of rotation (see fig. 44, in Study IV, 7).

To return to the later development of the anchor. Up to the time when the arms of the anchor-bow are but half-grown, the syncytium in this region is, as before mentioned, stretched to form two “ patagia,” so to speak, on the two sides of the shaft (figs. 16, 18). But on further elongation of the arms the central portions of the patagia apparently give way, with the result that the scleroplasm remains in the neighbourhood of the shaft as a thin layer, and away from the shaft as “ two elongated strands of protoplasm containing many nuclei which run on either side from the arms of the bow to the handle “ (Study IV). When the arms are fully formed, those two strands (figs. 20-24) are thickened at the bow end, forming “ blobs,” and the nuclei at this end of the anchor are almost entirely collected in these two thickenings. There is also in each strand another such collection of nuclei situated in the vicinity of the handle. Nuclei do occur in other parts of the strand, but they are principally aggregated in these two regions ; they also occur, of course, on the shaft, bow, and handle, though not in clusters. Thus the formation of these conspicuous strands of protoplasm joining the extremities of the arms with the handle is due to the presence of these arms and not vice versa, as I suggested when I first observed the scleroplasm associated with the fully-formed spicule (7). The strands are not muscular’ and exercise no “ tractive action” which can account for the recurved shape of the arms, i. e. they do not exert an active pull producing this effect, though it is possible that they have a slight passive influence in this connection.

As might be anticipated, the development of the plate-and-anchor spicules of Synapta digitata proceeds on very much the same lines as that of Synapta inhærens, but there is one difference which, though slight, is yet remarkable on account of its striking incomprehensibleness. In the last section I described the plate of the spicule as arising in the form of a rod disposed transversely to the length of the anchor-shaft (figs. 16, 18, e.g.). In S. digitata the plate also arises as a rod, but curiously enough it is disposed parallel with the anchor-shaft and not transversely to it (figs. 17, 19). I have observed scores of young plates of both species of Synapta, but I have never observed a single exception to this r-ule, that the plate-rod of S. inhærens is disposed at right angles to, and the plate-rod of S. digitata parallel with, the length of the shaft.

The distribution and disposition of the plate-and-anchor spicules in, the body-wall of Synapta are subjects which first demand our attention. Respecting the former there is little to say, since the spicules are uniformly spread over the entire area of the body-wall. However, it is worth remarking that the production of these relatively few but huge spicules in Synapta involves the concentration of numerous scleroblasts in a limited number of centres—i. e. involves the formation of syncytia, whereas in the Oucumariidæ, e. g. in which the spicules are relatively numerous but small, the scleroblasts either remain independent or only associate in pairs. In other words, given a certain number of scleroblasts in the organism, these can either aggregate in some degree to produce a relatively few huge spicules, or can remain independent and produce numerous small spicules. What factor determines the choice of these alternatives in any given instance I do not pretend to know, and I can only remark, on Spencerian grounds, that the Synapta condition is the more highly evolved.

With regard to the conspicuously definite disposition of the spicules in the body-wall, the explanation of this feature is, I believe, not difficult to find. In the first place it must be remembered that the body-wall of Synapta is highly contractile, the creature being able by means of its powerful longitudinal muscles to contract itself with ease to a considerable fraction of its normal length. This longitudinal contraction must and does involve a transverse wrinkling of the body-wall, and this contraction not being an infrequent expression of the animal’s activity, it seems clear that the initial granule of the spicule will elongate in the transverse grooves formed in the body-wall during such contractions. It is only necessary to mount and examine a contracted portion of the body-wall of Synapta to see that rigid structures like the spicules must lie in the grooves so formed, and no objection can be taken to this view on the ground that the outgrowth of the initial granule is appropriately orientated from the very first, since the grooves may be assumed to be quite capable of determining the direction of the initial as well as of the later growth of the spicule. Whether the granule elongates on one side or the other I believe to be purely a matter of chance, the spicules being, as before-mentioned, pretty equally disposed in both directions, as they should be, in the absence of any determining factor. Why the granule only elongates on one side and not on both I am unable to say. The contractility of the animal is then in all probability the cause of the definite transverse disposition in the bodywall of the long axes of the anchors.

The shape of the anchor can also, I believe, be associated to some extent with the contractility of the body-wall. The body-wall of Synapta not only possesses bands of longitudinal muscle, but also a continuous sheet of circular muscle, the presence of which latter implies that the body-wall can diminish in diameter. Now the body-wall consists in the main of two layers : the external thin dermal layer and the internal, comparatively thick, sheet of circular muscle, and between these two layers the elongated spicules lie, on a bed of fibres, iu a transverse position. If the circular musclelayer, be imagined to contract, then it is evident that the dermal layer, which at first is uniformly attached to the muscle-layer, will be thrown into longitudinally-disposed folds (see text-fig. 1), and that the position of these longitudinal folds will, to some extent, be determined by the spicules. In other words, the dermal layer will adhere to the contracted muscle-layer in those parts of the circumference which are devoid of spicules, but that, where spicules are present, the dermal layer will, by the inevitable protrusion of elongated spicules situated transversely on a diminished circumference, be separated from the muscle-layer in the form of pockets pushed out by the anchors. Consider the mechanical aspect of the matter. Rods lying between two connected layers forming the wall of a cylinder and situated transversely with regard to its long axis will, if their length be adapted to the circumference of the cylinder, not project on the surface at their extremities in any appreciable degree (textfig. 2). If, now, the inner layer only of the cylinder contract in a considerable degree, it is evident that the extremities of the rods will project on the exterior and that the outer layer will be thrown into pocket like folds enveloping the extremities of the rods, but that in the portions of the circumference situated between the rods it will still remain more or less attached to the contracted inner layer, though in a longitudinally-wrinkled condition (text-fig. 3). If, further, we suppose that, in some way, the rods are caused to project on the exterior by one extremity only, the pocket-like folds will be rendered still more conspicuous (text-fig. 4). These conditions are those found in the case of the adult spicules of Synapta, the last possibly being brought about by the continued attachment of the knob end of the primary anchor rod to the dermal epithelium from which it originated, and by the connection of the handle extremity of the shaft with the plate, which lies internally and parallel with the muscular layer (see text-fig. I).¹ The knobbed extremity of the anchor supporting a pocket-like protrusion of the dermal epithelium, it seems probable that the lateral extension of the knob to form the recurved arms of the anchor-bow is, after all, but an illustration of the deposition of skeletal matter in the direction of least resistance. It is an elementary fact that the anchor-bow does lie in a closely-enveloping dermal pocket, which is identical in general outline with the bow, and as fresh calcareous matter has for some reason to be deposited at the knobbed extremity of the anchor-shaft, the suggestion that the form which this additional deposit of calcareous matter takes is largely determined by the enveloping dermal pocket is not a very bold one.

The objection that, were the form of this further deposit of calcareous matter merely determined by mechanical pressure this calcareous matter would simply assume a more or less spatulate form and not the arms of an anchor, may be met by the reply that the bow-form, which the calcareous matter does assume, is not supposed to be solely due to the mechanical contact of the dermal pocket. As is well known, rod-structures in echinoderms generally have, for some unknown reason, a tendency to bifurcate terminally, and though the shaft of the Synapta anchor is abnormally large owing to the large syncytium concerned in its production, yet this is no reason why this rod should prove an exception to the general rule. If this be the case, then the two arms of the anchor-bow of Synapta must be regarded as the echinoderm rod-bifurcations, which in this case have been secondarily reflected and otherwise modified by the special conditions obtaining. Similarly, the handle of the anchor may be regarded on this hypothesis as an abortive attempt of the shaft to bifurcate at its internal extremity.

It seems useless to attempt any further explanations in connection with the form of the Synapta spicule, since such explanations must necessarily, in our present state of knowledge, be vague and unsatisfactory. There is, of course, one obvious question which every intelligent observer of these spicules has asked himself, viz. why are the plates and anchors developed in connection with each other¹ ? But to assume that this question is capable of being answered implies that we know the answers to several questions which naturally precede it. Grant that the huge anchor-shaft owes its size, shape, and disposition to the factors already mentioned, grant also that the relatively small rod of the incipient plato is small because of the small syncytium concerned, yet we are quite ignorant either as to why the six to ten nuclei should migrate in a bunch from the mass of the syncytium on to the internal side of the shaft, or as to why this cluster of nuclei when produced should give rise to a separate plate-spicule. Why should not the internal cluster of nuclei merely deposit an outgrowth of the shaft on its internal aspect in the same manner as the gastral actinoblast of the calcareous sponge deposits the gastral ray on the triradiate basis ? Personally, I am as yet quite unable to suggest solutions to these problems.

Before concluding I may mention that occasionally no plate is developed in connection with the anchor, the anchor remaining solitary (fig. 24). For some reason or other, the migration of nuclei on to the internal surface of the shaft has (presumably) not taken place. Whether plate structures are ever developed in Synapta, apart from the anchor, I cannot say for certain. I have observed several fully-formed solitary plates, but, although no signs of disturbance were visible, yet I suspect that the anchors had, in every case, been detached. It is, indeed, hard to suppose that the plates would assume the normal shape (which is modified in connection with the anchor¹) when developed by themselves. I have also observed two or three instances of what appear to be young plates developing on their own account, but, again, I cannot be quite certain that they had not become detached from anchor counterparts. Anchors can develop without plates (I think the instances are too numerous to suppose that the plates had merely become detached), but it is very doubtful if normal plates can develop apart from anchors.

While searching the internal surface of the body-wall of S. inhærens for young spicules, I not infrequently encountered the curious bodies depicted in fig. 26. They were stained a dark green, and were apparently in every case enclosed each in a single cell, which was one of a cluster. The surrounding cells (which often contained large vacuoles not shown in the figures) did not appear to be in any way connected with the cell containing the body, and, so far as I observed, only one of these bodies was contained in each cluster. I did not detect any internal structure in the interior of these bodies. Beyond offering the somewhat trite suggestion that they may be parasites, I am unable to explain their nature.

(1) The first sign of the future spicule is the multiplication of the nuclei of the dermal epithelium at one point to form a syncytium.

(2) In this syncytium a calcareous granule is deposited on its internal aspect.

(3) This granule elongates on one side either to the right or to the left of the long axis of the animal to form the shaft of the future anchor ; the other side of the granule persists for a while as the knobbed extremity. During development tho knobbed extremity remains in apposition with the dermal epithelium, but the opposite end comes into connection with the subjacent fibrous layer, so that the shaft is not situated tangentially in the body-wall but is inclined. The shaft, like the initial granule, lies on the internal aspect of the syncytium, i. e., nearly all the nuclei lie on the external side of the shaft.

(4) When the shaft has attained its full length, and knobbed extremity has commenced by lateral growth to give rise to the arms of the anchor-bow, six to ten nuclei of the syncytium travel round in a cluster to the internal side of the shaft about mid-way in its length, and there produce, quite independently of the rest of the syncytium, a granule which elongates and bifurcates and finally becomes the plate much in the same way as that already described for Cucumarian spicules (Study IV). The shape of the Synapta plate is modified in connection with the anchor. Also the portion of the original syncytium devoted to the production of the plate becomes entirely separate from the rest of the syncytium except in the region of the anchor-handle and plate-base, where the anchor and the plate are in contact and include a joint.

(5) In Synapta inhærens the rod producing the plate lies transversely to the length of the anchor, whereas in S. digitata it as constantly lies parallel. No explanation is offered in connection with this curious fact.

(6) The arms of the anchor-bow are perhaps to be homologised with the almost universal terminal bifurcations of the echinoderm rod-spicule, and their recurved form is possibly due to contact with the dermal epithelium which forms a pocket for their reception. The syncytium in connection with the anchor is stretched by the formation of the arms into “patagia,” which subsequently form the conspicuous strands of protoplasm joining the extremities of the arms with the handle. These strands are, as shown by their development, not muscular, and exercise no tractive function which can account for the recurved shape of the arms.

(7) The transverse disposition of the Synapta spicules is attributed to longitudinal contractions of the body-wall.

(8) The shape of the anchor is to be largely attributed to contact with the body-wall, which, it must be remembered, is contractile transversely as well as longitudinally.

(9) The very usual association of anchor and plate is a physiological problem at present insoluble.

Part 2.—The Spicules of the Auricularia Larva.

The morphogenesis of the Auricularian wheel-spicule has been described and figured by Semon (5). An unillustrated account of the scleroblastic development of the Auricularian wheel has also been published by Chun (1) in 1892, but, as I surmised in Study IV, Chun’s remarkable statements concerning this subject are totally misleading, owing to the fact that he unconsciously studied decalcified material. As will be seen in the quotation given below, Chun described the spicule as being deposited within a mould which is formed for its reception, so to speak, in the substance of the sclero-plasm. I am able to say that this supposed mould is nothing but the space contained within the scleroplasm which was occupied by the spicule before it was dissolved away by acid reagents. I have recently observed many of these “ moulds” in improperly preserved (decalcified) material, and I am able to make the above statement concerning their real nature because I also possess properly preserved (undecalcified) material.

My material consisted of numerous Auricularia larvæ in different stages of growth obtained at Naples and specially fixed with absolute alcohol. I stained them for a week in a saturated solution of Griibler’s safranin in absolute alcohol, and washed out the superfluous stain by keeping the larvæ in warm 90 percent, alcohol¹ (several changes) for a week -, they were then dehydrated, cleared in cedar-wood oil and mounted in balsam in the ordinary way.

The wheels and “ globes,” as I shall term the other class of calcareous deposits found in the Auricularia larva, are found in the pair of processes situated at the ana], i. e. lower, extremity of the larva. Both wheels and globes abut against the ectoderm of the larva, though they arise from cells internally situated, i. e. from the mesenchyme cells. But before describing the development of the spicules I will quote the relevant passages of Chun’s account of this subject, which, though incorrect as regards the main point, yet contains many true observations. “ At the time of the appearance of the first calcareous wheels the cellular elements of the gelatinous substance (filling the interior of the larva) are sharply differentiated into skeletogenous and connective-tissue cells. The latter possess several long processes, which are much ramified, and are interwoven almost after the manner of felt; the skeletogenous cells, on the contrary, are spherical and surrounded by a distinct membrane, in consequence of which they emit no pseudopodia. The sharp histological differentiation of the mesoderm cells, which was certainly preceded by an indifferent stage, may be essentially due to the fact that the calcareous bodies originate at a remarkably late period in comparison with what is found to be the case in other Echinoderm larvae. The skeletogenous cells accumulate … close beneath the ectodermal epithelium.” “ A richly vacuolate plasma at once distinguishes the skeletogenous cells, the average size of which is 0·01 mm. They rapidly grow to twice and thrice this bulk, while simultaneously the number of the cell-nuclei increases. In the same Auricularia we meet with all intermediate stages between uni- and multinucleate cells, which at first still retain a rounded contour, but subsequently flatten out on one side and become cup-shaped. The nuclei measure from 0·003 mm. to 0·004 mm. in length, and originally (so long as only from two to four are present) occupy a peripheral position; they afterwards increase to from six to eight in the case of the Mediterranean Auriculariæ, and to from twelve to eighteen in that of those from the Canary Islands, and form a central nuclear cluster. When the cells have attained a size of 0·03 mm. there appears within the old cell-membrane a new one, which has an undulating outline towards the circular margin and speedily assumes a star-shaped form. The tubular rays of the star which grow out are equal in calibre and meet the external membrane, arching forwards somewhat at the points of contact. The longitudinal extension of the radially-arranged outgrowths keeps pace with the increase in the size of the cell, and finally, when the cell attains a size of from 0·06 mm. to 0·07 mm., the rays become united by a peripheral membranous ring. It is now impossible to mistake the mould of the subsequent calcareous wheel, prepared as it is by the complex folds of an internal membrane ; the central portion with the cluster of nuclei corresponds to the nave, the tubes running out like the rays of a star represent the spokes, and the peripheral ring takes the place of the circumference (the felly) of the future calcareous wheel. Moreover the calx is actually secreted into this organic matrix formed by the skeletogenous cell, as into a mould, and in such a way that (as the older accounts already teach us) calcification takes place first in the nave, then in the spokes, and finally in the felly of the wheel. It is likewise in accordance with the theories which have recently been formulated as to the share of the nuclei in the vital processes of the cell that, corresponding with the centrifugal progress of the calcification, the majority of the cell-nuclei also separate from one another in a centrifugal direction, and in the case of the Auriculariæ from the Canary Isles come to lie in the acute angles between the spokes. In rare instances they advance as far as the middle of the spokes, or even to the periphery. No secondary multiplication of the spokes of the wheel takes place; their number corresponds exactly with that of the undulating évaginations of the newly-formed internal membrane, which develop into radiating tubes. As is well known, the number of the spokes varies; in the case of the Auriculariæ from the Canaries, we find from thirteen to eighteen. Since the diameter of the fully-formed calcareous wheels is found to be from 0·09 mm. to 0T mm., it follows that a ten-fold enlargement of the diameter of the skeletogenous cells takes place, since the latter in the stage with a single nucleus only measure 0·01 ram. Nevertheless, after the secretion of the calcareous wheels they expand still further ; for if we examine the wheels in alcoholic preparations … we can distinguish a distinct periphery formed by a delicate membrane, from which, alternating with the spokes and almost equalling them in length, membranous tubes arranged in the shape of a star run to the periphery of the wheel, where they usually exhibit flask-shaped expansions. On careful decalcification of the wheels by means of weak chromic acid it is easy to show the nuclei and the contour of the wheel in the shape of a delicate membranous envelope within the skeletogenous cell. The above statements as to the formation of the wheels in the Auricularia reveal a mode of development which at first appears to be unique. While the skeletal pieces of Echinoderms were hitherto essentially regarded as intercellular structures, the formation of which was due to several mobile amoeboid cells (I am well aware that more recent observers are inclined to attribute the shape of the skeletal elements without hesitation to directly mechanical influences), we now find that the form of the calcareous wheel is traced out within a multinucleate cell by means of an organic membrane which assumes complex folds, and that in this definitely circumscribed mould the casting of the hard parts ensues.”

I have quoted Chun at length because his remarks show how carefully most of his observations were made and how purely accidental his mistakes were. I will now describe the mode of formation of the wheels and globes in the Auricularia larva as it really occurs. I am unable to say for certain as to whether the syncytium which deposits the spicule arises from one cell solely by the simple multiplication of its nucleus as Chun describes, or as to whether it is also formed in part by the fusion of originally separate scleroblasts. I have certainly seen cells containing two and three nuclei, but I have also seen numerous clusters of separate scleroblasts which, unless they take on some other function, must coalesce with each other to produce the few spicules found ; probably both processes occur. The syncytium of the Auricularia larva differs from the syncytium of Synapta in that in the former each nucleus is almost entirely surrounded by a distinct layer of protoplasm, i. e. cell-outlines are to a large extent visible, each nucleus occupying a distinct subspherical portion of the syncytium, whereas in the latter the individual cells are so fused together as to constitute merely a mass of protoplasm containing numerous nuclei. The syncytium of the Auricularian spicule contains, in the specimens I studied, from five to nine nuclei.

The spicule first appears in this syncytium as an endoplastic spherical granule (figs. 27, 28), which is usually situated on the side of the syncytium next to the adjacent body-wall (not indicated in most of the figures). This granule flattens and gradually becomes disc-shaped with a slightly concave internal surface, the centre of which at first bears a slight eminence, and a convex external surface (figs. 31, 32) which is in contact with the body-wall. All the scleroblasts (i. e. the nuclei with their subspherical masses of protoplasm) are situated on the concave side of the disc, i. e. remote from the body-wall, and are clustered together (figs. 31, 32, 34), though of course the entire disc (and all later stages of the spicule) is enveloped by the scleroplasm. The next step in the development is the formation of processes (variable in number, ranging from about fourteen to eighteen) at the margin of the disc or nave of the future wheel (fig. 35), and these processes, which vary in number in different spicules, elongate, and ultimately form the spokes (figs. 36-38). By the development of these spokes (the exact shape of which can be seen in fig. 41) the convexo-concave disc assumes more the shape of a cup. Finally these spokes, by lateral extension, join up to form the felly of the wheel (figs. 39—41). The adult structure then, as shown by the figures, forms a cup-shaped structure, in the concavity of which are lodged the scleroblasts—somewhat like eggs in a basket (fig. 41).

In every case the extension of the scleroplasm which is necessary for the envelopment of the entire spicule is effected by the growth of the spicule itself, the spokes, e. g. pushing out the peripheral scleroplasm before them as they increase in length. The syncytium no more enlarges itself for the accommodation of the spicule, as Chun imagines, than an adipose tissue corpuscle swells out to make room for the secreted oil.

Unlike Chun I have not observed that “ corresponding with the centrifugal process of the calcification, the majority of the cell-nuclei also separate from one another in a centrifugal direction.’” On the contrary, in the Auriculariæ observed by me, they do not desert the nave portion of the wheel. In short, the development of the Auricularian wheel resembles that of all other echinoderm spicules in that they are endoplastic deposits, the form of which bears little or no relation to the disposition of the nucleus or nuclei. The study of spicule formation clearly shows that the proximity of a nucleus is not essential to the deposition of skeletal material at any given point, and that deposition goes on in any given region of a cell independently of the distance of the nucleus or nuclei from that region. Nuclear material is in all probability a sine quâ non where spicular deposition is concerned, but, as in the political constitution, the exact position of the governing centre is of little orno account in regulating the activities of the various areas governed—work proceeds in all quarters, and not merely in the vicinity of the nucleus or parliament as the case may be.

The development of the calcareous globes of the Auricularia is quite simple. The globe originates as a granule in the syncytium and simply increases in diameter by the uniform deposition of calcareous matter on its surface. The globe protrudes on the surface of the body-wall, at least half of its area being in contact with the wall, and the nuclei of the syncytium, probably in consequence of the contact, become restricted to the surface of the internal hemisphere (fig. 42).

With regard to the cause or causes determining the form of the Auricularian spicules, little can be said. It seems probable that the flattened disc- and later cup-forms are at least in part due to contact of the spicule with the body-wall, unless these are indeed to be attributed to the presence of the nuclei on the inner side of the plate only, the nuclei, contrary to the usual assumption, preventing deposition in their immediate vicinity.¹

But whatever forms the spicules may assume, it is clear that one of their inevitable attributes, viz. weight, may play an important part in the life of the larva. In the Auriculariæ I have studied, the wheels and globes are all situated,as before stated, at the lowest extremity of the larva, and it seems certain that this extremity is lowest because of the presence of the relatively weighty spicules in this region. In other words, the larva maintains a certain position in the water because it is appropriately weighted. The so-called baguettes de corps of the pluteus larva doubtless serve a similar function.

(1) The spicule first appears as a granule contained in a syncytium, in which, however, the scleroblasts retain their individuality to some extent, which is not the case in the syncytium of Synapta.

(2) The spicule becomes disc- and then cup-shaped, develops the spokes as outgrowths from the margin of the disc, and finally forms the felly of the adult wheel, the spicule during the whole of its development being enclosed by the syncytium in which all the nuclei (scleroblasts) are situated on its internal side, i. e. away from the body-wall against which the spicule lies.

(3) The extension of the scleroplasm depositing the spicule is determined by the growth of the spicule itself, and is not the result of a mould-forming tendency on the part of the protoplasm, as Chun asserted.

(4) The situation of the heavy wheel and globe spicules at one (the lower) extremity of the larva determines the position which the larva assumes in the water—the spicules weight the larva.

The curious spicular structure represented in text-fig. 6 was found inserted for a short distance into the body of one of the Auricularia larvæ which I examined. As it is not doubly refractive under the polariscope, I conclude that it is siliceous. It is well clothed in a thick layer of scleroplasm containing numerous nuclei. Being quite unable to identify this spicule I should say that it is probably a freak.

Illustrating Mr. W. Woodland’s “Studies in Spicule Formation. VII.—The Scleroblastic Development of the Plate- and-Anchor Spicules of Synapta, and of the Wheel Spicules of the Auricularia Larva.”

Figs. I, 9-25 X 800 diameters; Figs. 2-8, 20-42 × 1000 diameters.

The Development of the Synapta Plate-and-Anehor Spicules.

FIG. 1.—Portion of a transverse section through tho body-wall of Synapta i nhærens showing the multiplication of nuclei at one centre on the internal side of the dermal epithelium to form a syncytial mass. To the right lies the circular muscle layer and between this and the dermal epithelium some musclc- and nerve-fibres.

FIG. 2.—A syncytium viewed from the internal side of the body-wall. The initial granule is deposited on its internal aspect—i.e. the majority of nuclei are situated on its external side. These syncytia have very definite outlines, being quite distinct and usually distant from all surrounding structures. It must also be remarked that, owing to difficulties of observation, the exact number of nuclei figured cannot be guaranteed as having been the actual number present. It can, however, be guaranteed that every nucleus figured was carefully observed and correctly placed relative to the spicule, but, as is self-evident, it is impossible to be certain that every nucleus present was observed.

FIG. 3.—The granule has elongated slightly towards the observer’s left. It is possible that the syncytium is in part formed by the fusion of at-first-separate scleroblasts as well as by the multiplication of nuclei ; this latter, however, is the principal mode of formation of the syncytium. Some of the nuclei are larger than others, a feature probably denoting approaching nuclear division.

FIG. 4.—The initial granule has here elongated to the observer’s right hand.

FIGS. 5—8 illustrate the further elongation of the granule to one side or the other. The axis of the future shaft is first discernible when the full length is nearly attained. The majority of the nuclei, it will be noticed, lie on the external side of the shaft.

FIG. 9.—The nearly adult shaft.

FIG. 10.—The fully-elongated shaft. The terminal knob (representing the unelongated portion of the initial granule) is now extending laterally. The axis is conspicuous at this stage.

FIG. 11.—The fully-elongated shaft seen in a transverse section of the bodywall. The external position of the majority of the nuclei relative to the shaft is well shown.

FIG. 12.—Adult shaft, in which the arms of the anchor are about half formed, seen in a transverse section of the body-wall. In this figure there is shown in the clearest manner the external position of the majority of the nuclei, and the internal position of the half-dozen or so nuclei which have migrated internally to form a separate cluster in this position. All stages of this migration can be seen in the actual preparations.

FIG. 13.—Spicule viewed from the internal aspect. A granule has been deposited in the internally-situated cluster of nuclei. The formation of the arms of the bow is distending the general syncytium in this region (formation of the “ patagia”).

FIG. 14.—The granule is elongating on both sides in a direction transverse to the length of the anchor-shaft.

FIG. 15.—The same stage of growth of another spicule viewed in a transverse section of the body-wall.

FIG. 16.—Spicule viewed from the internal aspect. The granule has become a distinct rod with rounded extremities and swollen centre. In this species— S. inhærens—it is, as remarked in the text, situated transversely to the length of the anchor-shaft.

FIG. 17.—The same stage in S. digitata. It will be noticed that in this species the rod is situated parallel with the shaft and not at right angles to it. This difference between the two species of Synapta is quite constant.

FIG. 18.—S. inhærens. The extremities of the transverse rod have bifurcated. The “ patagia “ are well shown here.

FIG. 19.—S. digitata. The extremities of the parallel rod have bifurcated.

PLATE 30.

FIG. 20.—S. inhærens. The bifurcated extremities of the transverse rod have themselves bifurcated. The “ patagia “ are giving place to the “ strands “ uniting the apices of the anchor arms and the handle.

FIGS. 21—23 illustrate the further formation of the plate and the protoplasmic strands with their aggregates of nuclei.

FIG. 24 illustrates an anchor, in connection with which no plate has been formed.

FIG. 25.—Side aspect of the anchor-handle and plate-base to show the joint formed between the two.

FIG. 26.—The problematic bodies found in the body-wall.

The Development of the Auricularian Wheel-Spicule.

FIG. 27.—The initial granule in the syncytium. Its proximity to the bodywall is indicated.

FIGS. 28—30 illustrate the growth of this granule (surface aspect).

FIG. 31.—The plate-like form which this granule assumes and the clustering of the nuclei on its inner aspect (the aspect remote from the wall).

FIG. 32.—The plate or dish in side view. The inner position of the cluster of nuclei is well shown.

FIG. 33.—Plate viewed from its inner aspect.

FIG. 34.—Lateral aspect of full-sized plate.

FIG. 35.—Plate viewed from its outer aspect (nuclei situated on its remote side). The edge of the plate is crenate—the first indication of the spokes.

FIGS. 36—38.—Plate in all cases viewed from its inner aspect (nuclei on the near side). The formation of the spokes and the corresponding distension of the scleroplasm of the syncytium is shown. The nuclei with their accompanying scleroplasm lie in the concavity of the plate.

FIG. 39.—The spokes extend circumferentially.

FIG. 40.—The felly of the wheel formed. The entire spicule is enveloped by the scleroplasm.

FIG. 41.—The wheel viewed from the side in optical section showing the “ eggs-in-a-basket “ appearance.

FIG. 42.—One of the calcareous globes of the Auricularia larva with its enveloping syncytium situated in a shallow pocket of the body-wall.