The Reproductive System and Associated Organs of the Brittle-Star Ophiothrix Fragilis

Home , Ophiothrix, Ophiothrix fragilis

The Reproductive System and associated organs of the Brittle-star Ophiothrix fragilis. By J. B. Smith, M.A., PhJ)., Zoological Laboratory, Cambridge.

With 15 Text-figures.

CONTENTS. PAGE IHTKODTTCTION 267 METHODS 268 THE A-itTAT. OEGAN AXD BELATED SINUSES 269 The Left Axial Sinus and Organ ...... 279 The Right Axial Sinus and Organ ...... 280 THE GENITAL RACHIS 281 THE GONADS 287 Testis 288 Ovary 289 THE GoNODtrcrs 297 THE GENITAL BTTBSAE ...... 301 CoiTCLTrsiONs ...... 305 STTMMAEY 307 REFERENCES ...... 308 INTEODUCTION. The observations which are recorded in this paper are the outcome of an investigation directed, primarily, towards the interpretation of the morphology and histology of the nervous system of the ophiuroid Opldothrix fragilis, Abildgaard. During the course of the work opportunity was afforded for examination of large numbers of brittle-stars ranging from post- metamorphic individuals to fully mature specimens, the sizes being of a disc diameter of 0-4 to 16 mm. Prom observations made on living animals and by the examination of sectioned material it has been possible to obtain information relating to the morphology and development of the organs assoeiatedj either directly or indirectly, -with reproduction. The systems of organs described include the gonads, the gonodncts, the axial organ and related sinuses, the genital rachis and the 268 J. E. SMITH genital bursae. Some explanation of the inclusion of the axial organ complex, which is not primarily associated with the reproductive system, in this account is, perhaps, due. Two reasons may be given. The first is that the axial organ and the genital rachis, though of different origin, are in close association during development and throughout adult life, and the second is that the interpretation of the morphology of the axial organ complex, here given, is in close agreement with that of Fedotov (1924) who, in ascribing a double origin and structure to the axial organ and sinus system of Opbiuroids, is in disagreement with all earlier investigators of the problem. Much of the information recorded in this paper relating to oogenesis and to the number and disposition of the genital bursae in Ophiothrix fragilis is, I believe, of an original nature, and the same may be said of the account of the gonoducts which are here described for the first time. The germ-cells, during their development, are associated, at different stages, with different organs; and, in the account which follows, the organs concerned are described in the order in which this association occurs, namely, the axial organ complex and the genital rachis, the gonads, the gonoducts, and the genital bursae. METHODS. Material for sectioning was fixed in Heidenhain's 'Susa' fixative in sea-water, in strong Flemming without acetic or in Mann's fixative. Small Ophiothrix were fixed in to to, but with the larger specimens the arms were amputated at the margin of the disc prior to fixation of the latter. Decalcification in a mixture of 2 per cent, nitric acid in 70 per cent, alcohol was completed in 10-14 days, the fluid being renewed each day. After imbedding in paraffin wax of M.P. 52-54° C, the material was sectioned at 6-1 Op, and subsequently stained in Delafield's haematoxylin and eosin, Heidenhain's iron haematoxylin or Mallory's triple stain. In most instances the sections were cleared and mounted in Gurr's neutral mounting medium to which they were transferred from 95 per cent, alcohol. All drawings were made on squared paper with the aid of a squared net micrometer in the eye-piece. REPRODUCTIVE SYSTEM OF OPHIOTHRIX FRAGILIS 269

THE AXIAL OBGAH AHD BELATED SINUSES. Many diverse opinions have been expressed regarding the morphology, histology, and function of the axial organ in Ophiuroids. The close association of the primary germ-cells with the non-germinal tissue of the organ led Cuenot (1888) to suppose that the primary germ-cells were derived directly from the non-germinal tissue, a conclusion which MacBride (1892, 1907) showed, on embryological grounds, to be incorrect; for, in Amphiura squamata and Ophiothrix fragilis, the germ-cells are of independent origin. MacBride, moreover, disputes the earlier view held by Ludwig (1878, 1880), Cuenot (1888), and Hamann (1889), that the non-germinal tissue of the axial organ is composed of strands of tissue enclosing cavities filled with fluid and that it is of a vascular nature. More recently, however, Gemmill (1914) and Chadwick (1923) working on Asteroids, and Fedotov (1924) on Ophiuroids, have reverted to the original interpretation; and it is worthy of note, in support of their view, that rhythmic contractions of the organ—or of part of it—have been observed both in Asteroids; (Gemmill, 1914) and in Echinoids (Prouho, 1887; Gemmill, 1914). Furthermore, in the larval, and probably in the adult, Asteroid, Eehinoid, and Ophiuroid (Gemmill, 1914, 1919; Narasim- hamurti, 1932) the madreporic vesicle, which is a derivative of the wall of the right anterior coelom, undergoes periodic contractions which, since the vesicle becomes, in the adult, closely associated with the axial organ, would be in a position to assist in the circulation of the fluid contained in the lacunae of the latter. It is not the purpose of this paper to contribute to the dis- cussion of the function of the axial organ for, although the axial organ tissue of Ophiothrix fragilis has every appearance of being of a vascular nature, the true function of the organ, whether vascular, excretory, or endocrine, is hardly to be decided on morphological grounds alone. The second point on which there is a difference of opinion, however, is of primary interest to the morphologist since it concerns the actual form of the axial organ and related sinuses. In so far as the Ophiuroids are concerned, three distinct view- 270 J. B. SMITH points have arisen. The majority of workers (Ludwig, 1880; Hamann, 1889; Cuenot, 1888,1891; Beichensperger, 1908, ic.) regard the axial organ (Text-fig. 1 A, ax.org.) as a single structure surrounded by a sinus, the axial sinus (ax.sin.), the cavity of which is continuous orally with that of the ampulla (amp.). The stone canal (s.c.) and the ectodermal pore canal (p.c.) also open into the ampulla which is, therefore, in indirect communi-

l.axsin. ai.org.

r.ar.sm.

amp.

TEXT-ITC. 1. Diagrams of the axial organ complex of Ophiuroids according to different authors. For explanation see text, amp., ampulla; ax.org., axial organ; ax.sin., axial sinus; ax.sin.1, the false axial sinus (MacBride); gen.rach., genital rachis; gen.sin., genital sinus; l.ax.org., left axial organ; l.ax.sin., left axial sinus; mad.pore, madreporic pore; mad.ves., madreporic vesicle; p.c, pore canal; r.ax.org., right axial organ; r.ax.sin., right axial sinus; s.c, stone canal. cation with the outside world through the pore canal and madreporic pore (mad.pore). MacBride (1892, 1907) interprets the axial organ complex in a different way (Text-fig. 1 B). He, too, regards the axial organ as a single structure, but recognizes three* separate and dis- * The genital (aboral) sinus (Text-fig. 1 A, B, C, gen.sin.) derived from the left posterior coelom of the larva is, strictly speaking, a part of the axial organ complex, but is omitted from consideration in presenting the three view-points since it is recognized by all authors. • ; • REPRODUCTIVE SYSTEM OF OPHIOTHBIX FEAGILIS 271 tinct coelomic sinuses as against the one (axial sinus plus ampulla) quoted above. The cavity (ax-sin.1) which surrounds the axial organ is not, according to this author, homologous with a similarly placed cavity of other Echinoderms since it is derived from the left posterior coelom of the larva and not from the left anterior coelom. The ampulla (amp.), of left anterior coelom origin, is the true axial sinus and this is in no way connected with the first-named cavity. Finally, MacBride (1892) figures a third sinus which, in his paper on Amphiura, he designated' sinus B', but which was later shown by Narasim- hamurti (1933) to be the madreporic vesicle (mad.ves.) and to be derived from the right anterior coelom of the larva. As a third interpretation of the complex we have that of Fedotov (1924). The axial organ is figured (Text-fig. 1 c) as a double structure of which the one part (Lax.org.) which, to anticipate the general argument, we will call the left division, is surrounded by a sinus (l.ax.sin.) originating, in all probability, from the left anterior coelom of the larva, and the other, the right division (r.ax.org.), by a cavity which we will provisionally call the right axial sinus (r.ax.sin.). The left axial sinus com- municates with, and includes, the ampulla (amp.). The madreporic vesicle of MacBride no longer exists as a discrete cavity, although, again to anticipate matters, it is probably to be identified as the right axial sinus of Pedotov. Further consideration of these three views will be postponed until a description has been given of the axial organ complex of Ophiothrix fragilis. The axial organ (Text-fig. 2, l.ax.org., r.ax.org.), axial sinuses (l.ax.sin., r.ax.sin.), stone canal (s.c), ampulla (amp.), and pore canal (p.c.) encircle the distal border of the large external interradial muscle (ext.interr.musc.) and only encroach on its proximal border aborally, where the stone canal, which is contained within the axial organ tissue of the left axial sinus, runs into the water vascular ring-vessel (w.v.r.). The axial organ complex is shown in greater detail in Text-fig. 3. In well-fixed material it is possible to distinguish, even under low magnifica- tions, that the axial organ is composed of two parts which, though continuous one with the other and without boundaries of - pore l.ax.sin genital bursa; Lax.onj. g.b., r.ax.org. perivisc.coel. gen.sfn? nax.sin. P' am p.c inferior intervertebral muscle; l interradialmusole; stomach; sup.interv.nmsc, superior intervertebral water vascular ring. w.v.r., and the opposing arm of Ophiothrix fragilis. amp., TEXT-FIG. 2. left axial organ; l.ax.sin,, loft axial sinus; n.r., nerve-ring;p.c, right axial organ; r.ax.sin., right axial sinus; rad.n.c, radial nerve l.ax.org., r.ax.org., nc ^ ra muscle; t.pap., tooth papilla; 1.000^1. r epm.can. rad.perih.can.. buocal tube foot; epin.can,, opineural canal; ext.interr.musc, externa , radial portion of genital rachis; gen.sin., genital sinus; inf.interv.musc, 1 b.t.f., yen.rach cord; rad.perih.can., radial perihaemal canal; s.c, stone canal; st., canal;perivisc.coel., perivisceral coelom; gen.rach. ampulla; int.interr.musc, internal interradial muscle; Longitudinal section through the disc, inadreporic interradius, EEPEODUCTIVE SYSTEM OF OPHIOTHEIX FBA6ILIS 273 separation, are nevertheless histologically distinct. The part of the organ which is situated nearer to the aboral surface of the disc may be termed the left division (l.ax.org.) since, as will later be seen, it is associated with the left axial sinus, while the exb/mkemmusc

perhrisccoeL

TEXT-FIG. 3. A longitudinal section through the axial organ complex of Ophio- tjhrix fragilis. amp., ampulla; ezt.interr.musc., external interradial muscle; gen.rach., genital rachis; gen.sin., genital sinus; l.ax.org., left axial organ; l.ax.sin., left axial sinus; p.c, pore canal; perivisc.coel., perivisceral coelom; r.ax.org., right axial organ; r.ax.sin., right axial sinus; s.c, stone canal; St., stomach. right axial organ {r.ax.org.) is surrounded by its own sinus—- the right axial sinus {r.ax.sin.). The general form of the whole axial organ is best studied in horizontal sections through the disc which cut the organ transversely at different levels. Text- fig. 4 illustrates diagrammatically the appearance of the organ at the levels D-K of Text-fig. 3. At its oral extremity (Text-fig.4D) 274 J. E. SMITH the right axial organ (r.ax.org.) is tapered and is approximately circular in section. It surrounds, on its proximal side, the stone canal (s.c). As it extends in an aboral direction the right axial organ expands (E), and finally (F, G, H, J) becomes drawn out along a line transverse to the interradius. It will be noticed (H, J) that the left axial organ (l.ax.org.) has made its appearance as a large mass of tissue on the distal side of the right axial organ. The tapered aboral end of the latter is, eventually (K), completely surrounded and replaced by the left axial organ which continues to run in an aboral direction, being, at first, of an irregularly rounded outline but, later (L), strap- shaped with the long axis transverse to the interradius. In Ophiothrix fragilis the left axial organ is much more extensive than its right counterpart, having a volume approximately double that of the latter. Left and right divisions of the axial organ resemble each other in so far as each consists of a reticulum of cells in the interspaces of which is a coagulable fluid containing large numbers of cellular elements. The reticulum of the left (Text-fig. 5, l.ret.) is much denser than that of the right division (r.ret.) and the difference in finer structure is further accentuated by the greater numbers of cells contained in the lacunae of the former. These cellular elements are of two kinds. The more numerous of the two are amoebocytes (amoeb.) with an irregular cell outline and a vesicular nucleus, of about 3-5//, diameter, which includes a central nucleolus and extranucleolar chromatin. The second type of cell (c.) is characterized by the small amount of eyto- plasmie material present and by the nucleus, which is somewhat smaller than that of the amoebocyte and consists of a homogeneous deeply staining mass of chromatin. This second type is the more numerous in the right division of the organ even though the amoebocytes preponderate in the axial organ taken as a whole. It is possible that the two sorts of cell represent, in reality, active and degenerating amoebocytes, and that the differences in numerical distribution of the two forms in the left and right divisions of the axial organ are without significance. Nevertheless, it would seem reasonable to assume that the differences in form of the trabecular network and the inclusion EEPRODIXCTIVE SYSTEM OF OPHIOTHEIX FEAGILIS 275 of the two parts of the axial organ in separate divisions of the axial sinus are indicative of the morphological integrity of the two parts. The position and extent of the left and of the right axial sinus are best considered by reference to Text-figs. 3 and 4. A longitudinal section through the axial organ complex (Text- fig. 3) shows the left axial sinus (l.ax.sin.) to be a cavity extending the full length of the axial organ on its proximal side, while, distally, it descends oralwards only as far as the level of the genital sinus (gen.sin.). The right axial sinus (r.ax.sin.) which encloses the right axial organ (r.ax.org.) thus comes to be enclosed, proximally, by the left cavity. The interrelations of the left and right axial sinuses are, perhaps, best understood by reference to Text-fig. 4 (D-J), where the outlines of the sinuses, as seen in transverse section at different levels of the complex, are diagrammatically shown. There remain, as constituent parts of the complex, two additional coelomie cavities, the ampulla and the genital sinus, and the pore canal of ectodermal origin. The ampulla (Text-fig. 3, amp.) lies at the oral end of the axial organ complex. Its cavity is in direct communication with the left axial sinus (contra MacBride, 1892), and into it open, in addition, the stone canal (s.c), and the inner end of the pore canal (jp.c). A single longitudinal section through the complex, such as is illustrated in Text-fig. 3, does not adequately portray the relations of the ampulla, stone canal, and pore canal, but, on referring to Text-fig. 4 (A-C), which represents diagrammatically transverse sections at the levels A-C of Text- fig. 3, some conception of the form and position of the various parts may be obtained. The stone canal (Text-fig. 4 c, s.c.) opens into the ampulla (amp.) through its distal wall, almost in the mid-line of the interradius—indicated by the arrow. Orally, the ampulla expands in a horizontal plane transverse to the interradius (Text-fig. 4 B), and in so doing acquires an asymmetrical development about the mid-line. This asymmetry is further accentuated by the position of the pore canal and its openings. The pore canal (Text-fig. 4 B, p.c.) opens under the distal edge of the scutum buccale of the madreporic interradius NO. 326 T 276 J. B. SMITH and extends, in a proximal direction, into the substance of the ossicle. At the point where it comes to lie directly under the axial organ complex the canal expands in a direction transverse to the interradius and opens into the ampulla (Text-fig. 4 A, amp.) on the opposite side of the mid-line to that on which its T

s.c.

TEXT-FIG. 4. Diagrams of the axial organ complex of Ophiothrix fragilis as seen in transverse section at the levels A—K of Text-fig. 3. L, at a higher level, i.e. aboral to K, is not indicated in Text-fig. 3. The arrow is directed away from the mouth along the mid-line of the madreporic interradius. The divisions of the genital sinus are shown in F only. Black and white squares indicate germ- cells, amp., ampulla; gen.sin., genital sinus; l.ax.org., left axial organ; l.ax.sin., left axial sinus; p.c, pore canal; r.ax.org., right axial organ; r.ax.sin., right axial sinus; s.c, stone canal. external opening is situated. The pore canal is ciliated throughout, its cells being, except in regard to the length of their cilia, similar in form to those of the general ectoderm from which they are derived. The genital sinus (Text-figs. 3, 4F, gen. sin.) is best considered in connexion with the genital rachis, and will be dealt with in EEPBODUCTIVE SYSTEM OF OPHIOTHEIX FBAGHJS 277 the account of the course of the rachis and the form of the germ-cells. It will be seen from the above description of the axial organ and of the system of sinuses with which it is associated that the writer is in complete agreement with Fedotov (1924), whose interpretation of the complex in Ophiothrix fragilis and

rfcfc.

17-5 |i.

TEXT-FIG. 5. A section passing through the junction of the left and right divisions of the axial organ, amoeb., amoebooyte; c, ? degenerating amoebocyte; 1.ret., reticulum of the left division of the axial organ; r.ret., reticulum of the right division of the axial organ.

other Ophiuroids is illustrated by Text-fig. 1 c. In justification of the view that the complex is, in Ophiuroids, a double structure due consideration must be given, in the first place, to the inter- pretations of Ludwig and other workers on the one hand, and of MacBride on the other. Secondly, it would be of interest, if we are led to accept Fedotov's representation, to attempt an explanation of the origin of the various parts. Ludwig (1880), Hamann (1889), Cuenot (1888, 1891), and Eeichensperger (1908) based their observations on an examination of adult animals, so that in comparing their results "with those of Fedotov (1924) we are assessing the value of two series 278 J. E. SMITH of observations made on similar material and using siirilar methods. In so doing, the writer must confirm categorically the view of Fedotov; for, as has already been pointed out, there is no denying the presence of two divisions of the axial organ and of two sinuses in the ophiuroid complex. One must assume that the earlier workers missed seeing the wall of the right sinus and the obvious differentiation of the axial organ into two regions, either because their methods of fixation were inadequate or because shrinkage of the tissues caused the integrity of the sinuses to be obliterated. MacBride's opinions are, although they differ fundamentally from those of Fedotov, of greater intrinsic value than those just considered, since they are based on an examination of both adult and larval stages, by means of which latter he is able to trace the development of the organs of the complex. Never- theless, for reasons "which will now be given, we must conclude that he has erred in his interpretation of the complex, mainly because his observations of the adult system are open to question. The primary error into which he has fallen is revealed by his statement (1892) that the ampulla has no connexion with any other coelomic sinus. This is certainly not the ease in Ophiothrix as Text-fig. 3 clearly shows. The ampulla (amp.) is in direct communication with the left axial sinus (l.ax.sin.) and gives the appearance of being part of it. The second difficulty which arises on reading MacBride's account of the axial organ system of Amphiura is in placing the madreporic vesicle, which he figures as a discrete sinus situated (Text-fig. 1 B, mad.ves.) distal and oral to the ampulla. Of this sinus—at least in the position which MacBride figures— there is no trace in Ophiothrix. Finally, there is no indication in his account either of a right axial sinus or of the subdivision of the axial organ into two parts. There can be but little doubt that the picture which MacBride gives of the axial organ complex of Amphiura is incorrect in regard to these four points, and the explanation is probably to be found in the extremely small size of the animal on which he worked. Amphiura (Amphipholis) squamata, D. Chiaje, has a maximum size of 5 mm. disc diameter (Mortensen, EBPEODUCTIVE SYSTEM OF OPHIOTHKEX FRAGILXS 279 1927), while the specimens which MacBride figuresar e all under 1 mm. disc diameter. While, therefore, one feels confident, from the morphology of the complex in the adult Ophiothrix fragilis, in sup- porting Fedotov's conclusions in preference to those of earlier workers, the question of the homology of the sinuses is one of greater difficulty which must of course be settled ultimately by the embryologist. It is, however, possible to suggest homo- logies for the sinuses and divisions of the axial organ on the basis of the position of the adult structures and from such embryological evidence as is available. In the preceding description we have used the terms 'left' and 'right' axial sinus and 'left' and 'right' axial organ. This necessarily implies that the left sinus is derived, during development, from the left anterior coelom of the larva, and the right from, the right anterior coelom. The axial organ tissue would then have to be considered as being derived in part from the wall of the left anterior coelom and, in part, from the right. It is towards the justification of this view that both left and right halves of the larva contribute to the formation of the adult complex that the following argument is directed. The Left Axial Sinus and the Left Division of the Axial Organ. MacBride (1892,1907) states that the cavity which surrounds the axial organ of the adult Ophiuroid is derived from the left posterior coelom of the larva, and that the true axial sinus (i.e. the cavity derived from the left anterior coelom) is represented by the ampulla. While it must be admitted that a careful account of development of the adult sinus system, such as MacBride gives, far outweighs in value any speculation based on a study of adult morphology, there are, nevertheless, very cogent reasons for not accepting it in its entirety. We know, for instance (p. 275), that the ampulla is in direct communication with the cavity which we have termed the left axial sinus, just as it is in the Asteroidea, where the axial sinus and ampulla form one continuous cavity derived from the anterior coelom of the larva. On this evidence alone we should regard with a 280 J. E. SMITH certain amount of suspicion any suggestion that the axial sinus and ampulla were of separate origin. It seems much more reasonable to suppose that both are derived from the left anterior coelom of the larva as in the Asteroid. Furthermore, it may be noted that MacBride's interpretation of the complex allows, as did that of earlier workers, of only a single sinus surrounding a homogeneous axial organ. In view of the fact that there are, in reality, two sinuses and two divisions of the axial organ, how may these be accounted for on MacBride's hypo- thesis? Are they derived by a subdivision of his 'false axial sinus' (the left posterior coelom of the larva) ? No such subdivision has ever been demonstrated and there is no a priori reason why it should occur. The double nature of the complex is, however, readily understood if we suppose that it is of double origin. The morphology of the complex in the adult Ophio- thrix permits of this assumption since the left axial sinus and ampulla, being confluent, are to be interpreted as being of left anterior coelom origin, while the right axial sinus is, with some justification (vide infra), to be considered as being derived from the right anterior coelom of the larva. The Eight Axial Sinus and Eight Axial Organ. In so far as this sinus and division of the axial organ was described and figured for the first time by Fedotov in 1924 it is, perhaps, not surprising that embryologists, in work pub- lished prior to that date, give no indication that the right anterior coelom of the larva becomes, at metamorphosis, associated with the axial organ complex. The latest paper on the subject (Narasimhamurti, 1933) deals with the development of Ophiocoma nigra. Narasimhamurti is in agreement with previous authors in showing that the right anterior coelom of the larva disappears during development. This in itself would be taken as conclusive evidence that the right anterior coelom does not contribute to the adult axial organ system were it not for the fact that, prior to its obliteration, the wall of the right anterior coelom buds off tissue in which a cavity appears. This new sinus becomes the madreporic vesicle and, although it is not the anterior coelom itself, it is a derivative of it. What REPRODUCTIVE SYSTEM OF OPHIOTHEIX FRAGILIS 281 then is the ultimate fate of this sinus ? MacBride figures it as a cavity (sinus B, 1892) lying distally to the ampulla; but in Gphiothrix (p. 275) it does not occupy this position, and the only sinus with which it may be identified is the right axial sinus, as comparison of Text-figs. 1 B and 1 c will show. This identification may appear to rest on very flimsy evidence, but corroboration of the probable correctness of the view is given by Narasimhamurti when he states that the primitive germ- cells—proliferated from the wall of the left posterior coelom— project into the concavity of the madreporic vesicle as a solid nodule. If the madreporic vesicle does, indeed, become the right axial sinus we then have an explanation of the position the germ-cells come to occupy in the adult. The conclusion that the axial sinus and axial organ are of double origin in the Ophiuroid would be accepted the more readily if a comparable constitution could be demonstrated in other Echinoderms. While the writer is not in a position to present any evidence bearing on this point, attention may be drawn to Fedotov's (1924) paper in which he shows that the axial organ complex in Asteroids and Eehinoids does appear to be of a double nature. If this is indeed so there can be no further doubt that the ophiuroid complex is homologous in every way with that of the Asteroidea and Echinoidea.

THE GENITAL EACHIS. Ophiothrix fragilis is dioecious. The gonads (Text- fig. 6, gon.) which are ten in number—two in each of the five interradial pouches—are localized expansions of a string of genital cells, which, having its origin at the base of the stone canal, runs in conjunction with an extension of the right axial organ into the genital sinus, within which it is continued as a rachis encircling the margin of the disc. The course of the genital rachis during its progress round the disc is, as in all Ophiuroids, not confined to the horizontal plane, but is looped in a vertical direction so that it crosses the radii aborally (Text-fig. 2, gen.rack.1) and the interradii (with the exception of the madreporic interradius) orally, across the floor of the pouch. Text-fig. 2 does not illustrate this latter point as the pouch buccal •inberr aita;b.t.f., arm. g:b.2, g\b.1. gen.slib. l ossicle. genital slit; gon., gonad; inf.interv.iwusc, inferior gon. left axial organ; n.r., nerve-ring; st., stomach; sp., c ofOphiothrix fragilis. arm, 0. 1 and g.b. 2, the genital bursae; gd., gonoduot; gen.pl., fa.bf l.ax.org. TEXT-FIG. fc.pap. inberv.musc inf. ' > n'.r. r " verb : • " st. space left after decalciiication; t.pap., tooth papilla; vert., ambulacra genital plate j gen.sc, genital scale; gen.sin., genital sinus; gen.slit, intervertebral muscle; interr.pouch, interradial pouch; l.ax.org., tube foot; ext.interr.musc, external interradial muscle; g.b. eacb.inberr.musc. A horizontal and slightly oblique section through apart of the dis gen.sin REPRODUCTIVE SYSTEM OF OPHIOTHEIX FKAGIMS 288 section passes through the madreporic interradius in which the rachis has its origin. Figures showing the course of the racbis may be found in Delage and Herouard's volume on the Echino- dermata (PL 23). The primitive germ-cells which arise as a proliferation of the wall of the left posterior coelom (MacBride, 1907), later project into the right axial sinus (madreporic vesicle) (Narasirrthamurti, 1933), where they become associated with the tissue of the right axial organ. It is of interest to note that in the adult 0 p h i o - thrix certain branches of the proliferating germinal tissue fail to make contact with the axial organ. Text-fig. 7, which represents diagrammatically a series of sections transverse to the right axial organ at the level at which the genital rachis (gen.raeh.) arises, will illustrate this point. Text-fig. 7F-H shows the main branch of the genital rachis extending from its point of origin at the base of the stone canal (s.c.) into the axial organ (r.ax.org.), where it bifurcates to penetrate the left and right divisions of the genital sinus (gen.sin.l., gen.sin.r.). This is not, however, the only branch of the rachis, for genital cells (Text- fig. 7 A-L, g.c.) are also to be found in the wall of the right axial sinus where they are unattended by axial organ tissue. One must suppose that when the germ-cells invade the sinus some, at least, will inevitably become associated with axial organ tissue. These undergo their full development, pushing out into the genital sinus to be continued as the rachis. The germ-cells which remain independent of axial organ tissue are unable to proliferate to any great extent and remain localized probably because, in the absence of axial organ tissue, their supply of nutriment is inadequate for extensive growth. The two branches of the genital rachis proper enter the genital sinus, the two parts of which (Text-fig. 7 B-H, gen.sin.l. and gen.sin.r.) have an asymmetrical disposition, the left sinus (as viewed from the centre of the disc) being the more extensive. This asymmetry is not, however, apparent when the two parts of the sinus begin separately to extend into the interradial pouch (Text-fig. 7 J). Each of the two divisions of the sinus and rachis becomes applied to the lateral wall of the pouch proximally to the gonoduct (Text-fig. 6, gen.sin., gd.) along the side of 284 J. E. SMITH which it ascends to expand into the gonad at the point of entry of the gonoduct. Its course is indicated by the arrows in Text- fig. 15. The rachis leaves the gonad at a slightly higher level

TEXT-FIG. 7. Diagrams of the axial organ complex of Ophiothrix fragilis as seen in transverse section. The sections figured are at 16/i. intervals and occupy the interval ap of Text-fig. 3. g.c, genital cells; gen.rach., genital rachis; gen.sin.l., left division of the genital sinus; gen.sin.r., right division of the genital sinus; l.ax.sin., left axial sinus; r.ax.sin., right axial sinus; s.c, stone canal.

(B) than, and somewhat proximal to, its point of entry (A) and continues its ascent to the summit of the pouch. Here it bends at right angles to cross the arm where the latter is inserted into the disc to enter the gonad of the adjacent interradius, and is so REPRODUCTIVE SYSTEM OF OPHIOTHBIX FRAGIHS 285 continued as a series of vertical loops encircling the disc in a manner to which reference has already been made. The genital rachis (Text-fig. 8, gen.rach.) consists of a strand of germinal tissue, circular or semi-elliptical in section, which,

r.a:

sbrv

amoabr"!

r.ax.sx.orgo .

TEXT-EIG. 8. Transverse section through the stone canal and right axial organ near the origin of the genital rachis. amoeb., amoebocyte; c, ? degenerating amoebocyte; gen.rach., genital rachis; gen.sin.l., left division of the genital sinus; l.ax.sin., left axial sinus; lac., lacuna of axial organ; nuc.sh., nucleus of cell of rachis sheath; p.g.c, primary germ-cell; r.ax.org., right axial organ; r.ax.sin., right axial sinus; s.c, stone canal; sir., stranded tissue of right axial organ.

while preserving a uniformly small diameter throughout most of its course, is expanded enormously into the ten gonads. Smaller, but nevertheless well marked, expansions appear where the rachis pursues a horizontal path, that is to say where it 286 J. E. SMITH crosses the arm aborally and the interradial pouch orally Here, too, the genital sinus is correspondingly enlarged. The tissue which accompanies the genital rachis (Text-fig. 8, r.ax.org.) is histologically similar to that of the right axial organ from which it takes its origin, being composed of loosely woven fibres, with intervening spaces filled with a fluid containing amoebocytes. Text-fig. 8 represents a section taken transversely to the stone canal and right axial organ at the level of extension of the rachis (gen.rach.) into the left division of genital sinus {gen.sin.l.). The specimen sectioned was a mature female, but, since the primary germ-cells of the two sexes as they exist in the rachis are indistinguishable, any description of them may be taken to apply equally to both sexes. The rachis is invested by an indefinite sheath composed of flattened cells, the nuclei (nucsh.) of which are 3-5-4^. long, and stain readily and uniformly with the nuclear stains employed. They are easily distinguishable from the nuclei of the primary germ-cells (p.g-c) which are round, vesicular, and of a diameter varying between 4-5 and 1-O\L. The germ-cells may be so crowded together that the boundaries of the individual cells become obliterated; but it is possible, in many instances, to detect the irregular cell outline which Hamann (1887) describes. Hamann suggested, on this account, that the cells were capable of active migration along the rachis, a view which MacBride (1892) supports, adding the significant observation that the number of germ-cells in a given part of the rachis is subject to variation. It would be a matter of great difficulty to observe directly any migration of cells, and for that reason alone the following observations, made on preserved and sectioned material, are presented since they may be relevant to the point in question. The germ-cells in all parts of the rachis are of a comparable size, 7—10/x. in diameter, and they resemble precisely, in respect of size and form of nucleus, the smallest of the gametocytes in the gonad. Within the latter, by far the greatest number of the smallest germ-cells are congregated round the point of entrance of the rachis. Since there is no evidence of proliferation of germ-cells from germinal epithelium within the gonad, one BBPEODITCTIVB SYSTEM OF OPHIOTHEIX FKAGILIS 287 concludes that this mass of primary germ-cells has entered the gonad from the rachis. The gonad is not the place of origin of the gametocytes, but within it maturation and growth of the germ-cells take place.

THE GOJJADS. Each gonad (Text-figs. 6,15, gon.) is a sac, roughly ellipsoidal in shape, invested by a connective tissue sheath, to the outside of which and bordering the perivisceral eoelom is a thin layer of epithelial cells. The central lumen of the gonad is largely occluded by the germ-cells usually to be seen at various stages of development. In young specimens of Ophiothrix fragilis, both male and female, the lumen is undivided, but with increase in size and attainment of full sexual maturity, septa (Text-figs. 11, 14 B, sep.) develop which partition the gonad into numerous compartments. The septa are sheets of tissue erected in a vertical plane at right-angles to the interradius. They are formed for the most part (Text-fig. 11, sep.) of connective tissue fibres which, having their origin in the investing sheath, invade the central lumen. In addition to the connective tissue it is probable that the septa also contain axial organ tissue which enters the gonad from the rachis. Unfortunately it is not possible to be definite upon this point since axial organ and connective tissue have similar staining reactions. The immature gonad, while still unpartitioned, is without muscle-fibres, but, with the development of septa (Text-fig. 11, musc.fib.), muscle-fibres appear both in the cortical layer and in the septa. Fox (1924&) has described a similar development of muscle in the gonad of Echinoids and has shown that, on stimulation, the gonad contracts with consequent emission of ova or of sperm. The conditioning stimulus may be furnished by the presence of other spawning Echinoids or by direct contact with a brush or needle. The fact that the presence of an intrinsic musculature or even of connective tissue within the gonads of Ophiuroids has so frequently been overlooked is no doubt due to the fact that muscle and connective tissue reach their full development only in the more mature testis or ovary. Although Eox's work demonstrates without doubt that nervous 288 J. E. SMITH tissue is present in the Echinoid gonad its presence w^uld, in all probability, only be detected by the use of specific nerve stains, and it has not been seen during the course of the present work. Pigment is developed in both the ovary and testis of Ophio- t h r i x. In both cases the pigment is confined to the interstitial tissue, but while it is present in the ovary in sufficient quantity to impart to it an orangy-pink colour, the testis, on account of the very small amount of pigment present, is either white or pale yellow in colour. Testis.—Eipe, motile sperm may be obtained from specimens of Ophiothrix fragilis, above a minimum size, at all times of the year. The smallest animals in which ripe sperm can, with certainty, be seen are of a disc diameter of about 0-3 cm., although sperm in abundance is only to be found on attainment of full sexual maturity, i.e. animals with a disc diameter of 1-1-6 cm. Text-fig. 9 shows part of a section through the testis of a mature male. All stages of spermatogenesis are visible. Ad- jacent to the cortex of connective tissue (c.t.) in which are contained the muscle-fibres {musc.fib.) there is a layer of cells (AT. 1) each of a diameter of approximately 7fi. The large nucleus is vesicular and has a central nucleolus. These cells are the primary germ-cells (spermatogom'a), similar in all respects to the primitive germ-cells of the genital rachis from which they are probably derived directly without division. Inclusive of the spermatogonia, five stages are visible in the development of the flagellate sperm. These are designated N. 1-5 in the Text-figure and, although the nuclear divisions mitotic and meiotic have not been followed in detail, one may assume that they represent, in order, spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa. One observes a progressive diminution in the amount of cytoplasmic material surrounding the nucleus of successive phases, and each stage is readily recognized by the condition of the nucleus. The nucleus of the spermatocyte (N. 2) is rather smaller than that of the spermatogonium (N. 1) and has its chromatin arranged in threads. Meiosis is accompanied by a marked reduction in REPRODUCTIVE SYSTEM OP OPHIOTHEIX FRAGILIS 289 size of the nucleus (N. 3) and of quantity of stalnable chromatin. A further reduction in size occurs at the mitotie division which precedes the formation of the spermatids (N. 4). The sperma-

TEXT-FIG. 9. A section through a part of a testis of a mature male, c.t., connective tissue sheath; muse.fib., muscle-fibre; N.l-5, the nuclei of the spermatogonia, spermatocytes, and spermatozoa (for explanation see text). tozoa show the typical aggregation of chromatin into a deeply staining mass. They project into the lumen of the testis in roughly parallel rows prior to discharge into the sea-water through the gonoduct. Text-fig. 10 is of a section through the testis of a small male of 0-5 cm. disc diameter and shows the precocious development of sperm. Although the germ-cells are practically all in the 290 J- B. SMITH spermatogonium stage (N.I), some few have undergone maturation and have become spermatozoa (N. 5). The rapid but limited nature of spermatogenesis in the young Ophiothrix is reflected in the small number of spermatocytes (N. 3) present

N.5

TEXT-FIG. 10. A section through a part of a testis of a small male (disc diameter, 5 mm.), c.t., connective tissue sheath; N. 1, nucleus of spermatogonium; N. 3, nucleus of spermatocyte; N. 5, nucleus of sperma- tozoon. and in the absence of spermatids; the ability so to produce small quantities of sperm in this way is, no doubt, responsible for its appearance at all times of the year and is in marked con- trast to the periodic rhythm which attends oogenesis and which results in the extrusion, by the females, of ripe ova only at certain times of the year. Ovary.—In view of the fact that many Echinoids exhibit marked periodicity of spawning (Fox, 1924a, 1924&) a study REPRODUCTIVE SYSTEM OF OPHIOTHBIX FEAGILIS 291 of oogenesis is, in any Echinoderm, a matter of initial interest. Very little attention has been directed to the problems of oogenesis in Ophiuroids, our knowledge of the subject resting almost entirely on the work of Eusso (1881), Hamann (1887), and Cuenot (1888), who limit their accounts to descriptions of the cytology and histology of the ovary. That OpMuroids are not, at all times of the year, capable of the liberation of fertilizable and therefore fully matured ova is evident from the embryological studies of MacBride (1907) and Mortensen (1937) among others. Mortensen, in particular, refers to the difficulty with which artificial fertilizations are, at times, to be effected when he states (p. 9) that 'it seems to be a nearly constant rule that eggs taken out of the ovaries, even if they look ever so ripe, cannot be fertilized; there is always a distinct nucleus, and even when left for hours in the water they will not ripen5. He goes on to say that spawning may frequently be induced by placing a number of ripe males and females in aquaria. This latter observation would appear to indicate that the final stages of maturation are only completed under the influence of an appropriate chemical stimulus. While this is no doubt true, there is reason to believe, as will be shown, that the non- fertilizability of the ova, at certain times during the breeding season, may be due to periodic maturation and expulsion of the eggs during the intervals of which the ovary contains ooeytes which require to undergo further growth before they are capable of being fertilized, even though the stimulus adequate for the completion of maturation be present. During the course of this work the writer has had the opportunity of examining the developing ooeytes of Ophiothrix fragilis at different times of the year, and, although the observations were not made at as frequent intervals as one would have wished, the conclusions which it is possible to draw from them have some bearing on a possible periodicity of spawning of this species. It is at once evident on examining either living ovaries or sectioned material that the developing ooeytes are of different sizes, and that they fall into well-defined size groups (Text-fig. 11). As an example we may instance a count of ooeytes from the ovary of a female collected in August 1938. The largest NO. 326 U 292 ' J. E. SMITH number of oocytes in this specimen were of a diameter of "* 10[i; the germinal vesicle had broken down and the first polar spindle was found, on sectioning, to be clearly visible. These oocytes were obviously ready, or almost ready, for liberation,

perivisc.coel.. qd. , , J \ | -w.interr.pouchLL .

oocybi TBXT-ECG. 11. A section through a part of an ovary of a mature female (December specimen), gd., gonoduct; gen.rach., genital rachis; gen.sin., genital sinus; him,., lumen of the gonad; musc.flb., muscle-fibre; oocyt. 1-4, developing oocytes of four different size groups; p.g.c, primary germ-cell; perivisc.coel., perivisceral coelom; sept., septum of gonad; w.interr.pouch, wall of the interradial pouch. a conclusion which was borne out by the fact that many specimens of a like size had shot their largest oocytes. Other oocytes within the ovary were 60-70 \L in diameter. These were in large numbers, while smaller numbers of oocytes of 30 [L, 20p, and lOju, were also present. This observation, in itself, suggests that there will be a periodic ripening and liberation of the ova. Some estimate of the total duration of the breeding season and of the number of broods of eggs liberated during that time REPRODUCTIVE SYSTEM OP OEHIOIHBIX FBAGILIS 293 may be obtained on analysing the ovary content of numbers of animals of different sizes taken at different times of the year (Table I). It must be pointed out that the data are presented

TABLE I. December-January (1937-8). Size of Animal. Diam. of Diameter of Ooeytes in p disc in cm. 100+ 80 60 40 20 <20 10-15 | Few i .. Many Many ; Very many

5-10 •- i " • * • 55 55 \ ?» S» 5

March (1939). Size of Animal. Diam. of Diameter of Ooeytes in y disc in cm. 100+ 80 60 40 20 <20 10-15 Many or none I Very many | Many Many Many : Many (shot) 5-10 ] " " j " 5 •• •- 55 '•• Very many

September (1937).

Size of Animal. Diam. of Diameter of Ooeytes in y,. disc in cm. 100+ 80 60 40 20 <20 10-15 Many or none Very many Many Many Many (shot) 5-10 Many or none (shot) 5 Very many in a way which, while convenient for the purpose in view, does not adequately express the differentiation of the ooeytes of a single ovary into significantly different size groups, for 294 J. B. SMITH whereas the groups are, in a single gonad, quite distinct they tend to overlap when large numbers of specimens are included. In December and January the ovaries, even of the large specimens, do not contain any oocytes which, either in size or condition of the nucleus, are fertilizable. The gonads of the larger animals (5-15 cm. diameter of disc) contain a large reserve of oocytes of a size up to 40(t. By March (or perhaps a little earlier) of the following year the 40 /A group has become fully matured and is ready for liberation. The smaller size groups have also grown rapidly and are represented in the March specimens by oocytes of a diameter of SO/L and under. It is clear that the breeding season extends throughout the summer into September and possibly into October, for specimens collected during September contain two series of well-developed oocytes, one of which is shed in September and the other rather later. The data presented in the table allow certain generalizations regarding the breeding of Ophiothrix fragilis to be drawn. These may conveniently be summarized as follows: (a) Females may become sexually mature after attaining a size represented by a disc diameter of about 5 mm. (b) Since the males are capable of emitting sperm at all times of the year, the breeding season is limited to the period when ripe eggs are available, i.e. from about the beginning of March to the end of September. (c) A single female is capable of producing a number of batches of ripe eggs at intervals throughout the breeding season. There is no information available either in confirmation of, or contradictory to, the first of these points; but the second conclusion is borne out in a general way by the records of the time of breeding and of the occurrence of the larva of Ophio- thrix in the Plymouth area (Plymouth Marine Fauna, 1931). Eipe ova have been obtained from March to September and successful fertilizations made in July, August, and October. Ophioplutei have been recorded from the plankton in April and July. The question of periodic emission of eggs at intervals during REPRODUCTIVE SYSTEM OF OPHIOTHEIX FKAGIMS 295 the breeding season is one which, as far as the author is aware, has not been studied in detail in the Ophiuroidea. Fox (1924a and b) finds that Echinoids frequently exhibit a periodicity which may be lunar, regular but not lunar, or irregular. Morten- sen's (1937) observations have much the same import, but he adds that many species appear to have only one breeding season during the summer. The only reference which I have been able to find on periodicity of spawning in the Ophiuroidea occurs in this paper; at Ghardaqa, Mortensen says that Ophiocoma valenciae Mull and Trosch. and Qphiothrix hirsuta Mull, and Trosch. ' showed signs of becoming ripe by the end of September (probably by full moon), or perhaps not even till October'. It would be unwise, on the limited data available, to assume that the ova of Qphiothrix are shed at regular lunar periods. Nevertheless there is good reason for believing that spawning may take place at approximately monthly intervals. The following table (Table II) gives the oocyte size groups of mature April, September, and December specimens.

TABLE II. April 100' 80-90 80-70 50 40 30-20 20-10 <10 September 100 90 70 .. 40 30-20 20-10 <10 December .. 90, v. few .. .. 40 30-20 20-10 <10 Assuming that the April group has already spawned once, as the records (Plymouth Fauna, 1931) show they may, there are available, during that month, in the ovaries of mature females, six or seven distinct groups of ooeytes in addition to the primary germ-cells of less than 10/i diameter. If these ooeytes all matured in a single season one would anticipate six or seven spawning periods, a number corresponding roughly to the number of lunar months elapsing before the end of the breeding season. If we make this assumption the September and December figures become intelligible. In the former we see, in the 90JU, group, the developing ooeytes which in April had attained a size of 20/t only. The smaller ooeytes in the Septem- ber ovary would be developed from the primary germ-cells of March and would remain, during the winter months (see the December figures), in an arrested stage of development. If we 296 J. B. SMITH have interpreted the data correctly we are led to assume that there is a periodic discharge of ova during the breeding season, probably at monthly intervals, and that the winter marks a period of arrested growth to be followed, prior to March, by rapid growth of the oocytes so that the largest of them are mature in that month. Certain changes take place in the constitution of the nucleus and in the form of the cytoplasm during oogenesis to which brief reference may be made. In view of the fact that special cytological fixatives were not employed, it is not possible to give an account of the development of yolk or of the form and distribution of mitochondria and Golgi bodies. The youngest oocytes (Text-fig. 12 a) have a vesicular nucleus (nuc.) with a central or slightly exeentrie nucleolus (nuel.) surrounded by a colourless nuclear fluid. The nueleolus at first is dense, but when the oocyte attains a diameter of about 80/A (Text-fig. 12 c), it begins to exhibit vacuolation, a process which continues (Text-fig. 12(1) until the oooyte is about 90-100/j, in diameter (Text-fig. 12 e). The nueleolus of the germinal vesicle (g.v.) has the appearance of a sphere, the boundary of which stains with nuclear stains although its contents do not. It is probable that the germinal vesicle remains in the condition shown in Text-fig. 12 e for some time prior to maturation, for it is much more common to find fully grown oocytes with the nucleus in the resting condition than with a polar spindle as in Text-fig. 12 /. There are indications that some extrusion of nucleolar material into the karyolymph may occur during oogenesis, for whereas the karyolymph of the nucleus of the younger oocytes does not react with nuclear stains there are, in the fully grown oocyte (Text-fig. 12 e), small but distinct localizations of chromatin (chr.). It does not necessarily follow, however, that these extrusions of nucleolar material occur in the living oocyte. They may be a product of fixation, for, as Harvey (1981) has shown in the oocytes of Ante don, extrusion of nucleolar substance may occur on shrinkage and rupture of the nuoleolar membrane after treatment with acids. This frequently happens when the ovaries of Ophiothrix have been fixed in Bouin or in 'Susa' (Text-fig. 12g). The nucleolar fluid (nud.fl.) runs BEPRODUCTIVE SYSTEM OF OPHIOTHBIX FEAGILIS 297 into the karyolymph and even, in some instances, into the surrounding cytoplasm. In view of this possibility it would be inadvisable, solely from an examination of fixed material, to draw any definite conclusions regarding the extrusion of nucleolar chromatin in the living oocyte.

nue. b. c. d.

a-e,g. . 47 p. g.v f. .- TEXT-ITO. 12. Oogenesis of Ophiothrix fragilis. a-e., developing oocytes; /., the first polar spindle; g., germinal vesicle with ruptured nucleolus after staining with Heidenhain's iron haematoxylin. chr., extra-nucleolar chromatin; g.v., germinal vesicle; nuc., nucleus; nud., nucleolus.

THE GONODUCTS. The statement will be found in all the leading text-books of zoology in which accounts of the Echinodermata are given that the gonads of Ophiuroids shed their products through the genital bursae and that, in the few instances where bursae are absent, an opening is formed temporarily in the body-waE through which the sperm or ova pass. Delle Chiaje (1841) was 298 J. B. SMITH initially responsible for the statement and it has be< n per- petuated by Gregory (1900), Hamann (1901), MacBride (1906), Sedgwick (1909), and Mortensen (1927) among others. While this condition may be true of many Ophiuroids it certainly is not of Ophiothrix fragilis, where the gonads discharge through permanent ducts directly to the exterior. Each interradial pouch has a pair of ducts one to each gonad (Text-figs. 6, 14A, 14B, gd.). The gonoduct is situated proximally to the genital scale (gen.sc), an ossicle which lends support to the lateral wall of the pouch, and distal to the genital sinus (gen.sin.). Text-fig. 13, representing a part of a section transverse to the arm and through the interradial pouch at the level of the gonoduct, shows the entire course of the duct (gd.) from its external opening into the genital slit (gen.sl.) to its point of entrance into the gonad. The duct is about 200/*, long and is lined by ciliated cells. It penetrates the body-wall to enter the genital sinus (gen.sin.), in which cavity it ascends vertically and rather obh'quely to open into the lumen of the gonad. Text-figs. 14 A and B are of sections taken transversely to the gonoduct at different levels. In Text-fig. 14 A the duct (gd.) is seen to pass through the body-wall and to be distal to the genital sinus. The wall of the duct comprises a single layer of ciliated cells each with an oval darkly staining nucleus. The cells of the gonoduct, except for the possession of cilia, are remarkably similar to those of the general ectoderm where it borders the external opening of the duct, but no evidence as to the actual origin of the duct has been obtained. Attention has already (p. 284) been drawn to the fact that the position of the internal opening of the gonoduct coincides with the place of entry of the genital rachis (gen.rach.). Text-fig. 14 B illustrates this point. The gonoduct would appear to push against the wall of the genital sinus (gen.sin.) and must eventually perforate it since the primary germ-cells of the genital rachis completely invest the cells of the duct (nuc.gd.) which, as it penetrates further and further into the substance of the gonad, becomes increasingly ill-defined until, finally, it disappears altogether. There can be no doubt that the duct does function as a BBPBODUCTIVB SYSTEM OF OPHIOTHBIX FBAGIMS 299

gonoduct, for its lumen is, in mature males, frequently to be found packed with ripe sperm. Comparable sections of females with ova in the gonoduct have not been obtained, although this

inf.interv.musc

gan.slit penvisccoeL

TBXT-HG. 13. A part of a section transverse to the arm and through the interradial pouch of Ophiothrix fragilis, at the level of the gonoduot. g.b., genital bursa; gd., gonoduct; gon., gonad; gen.sin., genital sinus; gen.slit., genital slit; inf.interv.musc., inferior intervertebral muscle; perivisccod., perivisceral coelom; st., stomach.

is not altogether surprising considering the relatively short duration of the spawning periods. If, as one would expect, the ripe eggs are expelled through the duct, there must be considerable dilation of its walls, for the lumen, in the normal state of expansion, is of about 15/u, diameter only. 800 j. B. SMITH

d muse.fib. 9, - geai.rachr . w.inberr. pouch.

gzn.se.

108 u. A. gen.rach, nuc.gd.

gen.sin.

gen.se

TBXT-BTG. 14. Horizontal sections through a part of the wall of the interradial pouch of Ophiothrix fragilis. A, near the external opening of the gonoduct. B, near the internal opening of the gonoduct. gd., gonoduct; gen.rach., genital rachis; gen.se, genital scale; gen.sin., genital sinus; gon., gonad; musc.fib., muscle-fibre; nuc.gd., nucleus of a cell of the gonoduct wall; perivisc.cod., perivisceral coelom; sept, septum of gonad; st., stomach wall; w.interr.pouch, wall of the interradial pouch. BBPBODUCTIVE SYSTEM OF OEHIOTHBIX FKAGILIS 801 Sections through young Ophiothrix in which the gonads are as yet undeveloped fail to reveal the presence of gonodttcts. Their appearance coincides with the expansion of the raehis to form the gonads and they are completed at a stage when the gonad contains primary germ-cells only.

THE GENITAL BUESAE. The last of the series of organs to be described in this paper are the genital bursae. They are, essentially, sac-like imagina- tions of the ectoderm which project into the perivisceral coelom of the interradii of the brittle-star; the cavity of the bursa is in communication with the outside sea-water through apertures situated in the genital slits between the arm and the mterradial pouch. Some Ophiuroids (Hamann, 1901) are without bursae, but where they are present they are described (Cuenot, 1888; Hamann, 1901; MaeBride, 1906) as being ten in number, two to each mterradial pouch. Test-fig. 15 which illustrates, diagrammatically, the disposition of the bursae in Ophiothrix shows that there are, in this Ophiuroid, not one but two pairs of bursae to each interradius, i.e. twenty in all. One of the bursae (g.b. 1) of each pair opens to the exterior by a wide aperture (g.b.op.) situated at the level of, but proximal to the external opening of the gonoduct (gd.). The aperture leads internally into a horizontal sac which penetrates the substance of the external mterradial muscle (ext.interr.musc), where it expands into a vertical pouch. Each of the external mterradial muscle masses thus comes to include a pair of bursal sacs, the vertical portions of which are elliptical in section (Text-fig. 6, g.b. 1), devoid of ramifications, and extend almost throughout its substance in an oro-aboral direction. A bursa of the second pair {g.b. 2) has its opening aboral and distal to that of its partner. The position of the invaginated sac may be seen in the Text-figure. Distally, it expands into a wide pouch which abuts laterally against the gonad (gon.) and medially against the mterradial diverticulum of the stomach (not shown in the Text-figure). A proximal extension of the 302 J. E. SMITH bursa enfolds the aboral part of the external interradial muscle to end blindly, adjacent to its proximal border.

intern pouch.

9-b.Z pen vise, coel."

gon

ext.interr.musc. g'd. g.b.op. TEXT-PIG. 15. Diagrammatic figure of an interradial pouch of Ophiothrix fragilis showing the relations of the genital bursae, gonad, and external interradial muscle. The roof and part of the lateral wall of the pouch have been removed. The arrows indicate the course of the genital rachis and sinus along the lateral wall of the pouch. A is the point of entrance of the rachis and gonoduct into the gonad; the rachis leaves the gonad at B. ext.interr.musc, external interradial muscle; g.b.l and g.b. 2, the genital bursae; g.b.op., openings of the bursae into the genital slit; gd., gonoduct; gon., gonad; interr.pouch, interradial pouch; perivisc.coel., perivisceral coelom.

The wall of the genital bursa consists of a layer of cells, bearing cilia some 15/A in length, lining its cavity, a middle layer of connective tissue, and a thin covering of coelomic epithelium. Muscle-fibres do not appear to be present in any part of the wall. EEPHODUCTIVB SYSTEM OF OPHIOTHBIX FEAGILIS 803 Ludwig (1878a) ascribed to the bursae a reproductive and a respiratory function, a conclusion with which subsequent authors are in agreement, although Cuenot (1891) believes that they may, in addition, subserve excretion. While it is not proposed to offer any evidence relating to Cuenot's findings, some remarks on the part played by the bursae in respiration and reproduction may conveniently be included at this juncture. The name 'genital' bursae has been applied to the sacs on the assumption that the gonads open directly into them, the ova and sperm, in consequence, being liberated into the sea- water through the external openings of the bursae. As we have seen, this does not apply in the case of Gphiothrix fragilis, and the use of the word 'genital' would, therefore, in the sense indicated, be unwarranted. Nevertheless, the bursae are associated with reproduction in this species, for small Ophio- thrix are often to be found within the bursal sacs of large mature specimens, an association which has frequently been taken as indicative of a viviparous habit, llortensen (1932-3) has pointed out, however, that young specimens may be found in the sacs of oviparous forms, the larvae of which are free- swimming prior to metamorphosis. In a recent note (Smith, 1938) I had occasion to describe how this may come about in Ophiothrix fragilis. In this species it is the invariable rule that the young intrabursal specimens are post-metamorphic. Their position within the bursae is a secondary one in so far as they tend, after settling on the arm spines of the adults, to creep into the bursae where they find convenient temporary shelter. This conclusion has been borne out by a subsequent observation that males of a sufficiently large size may also house young Ophiothrix within their bursae. In speaking of the bursae of Ophiothrix as 'genital' bursae we are using the term in a secondary sense and it would be a matter of interest to discover to what extent, if any, the original applica- tion, based on the assumption that the bursae receive the germ- cells directly from the gonads, should be retained for the Ophiuroidea as a whole. The respiratory significance of the bursae may be implied from direct observation of the behaviour of the animal and from 304 J. E. SMITH the morphology of the sacs and of the musculature of the disc. The long cilia of the cells of the bursa wall create an inhalent stream of water (Hamann, 1889), the contained oxygen of which could diffuse—although there is no experimental evidence bearing on this point—through the thin wall into the fluid of the perivisceral coelom. In order to ensure an adequate supply of oxygen, periodic renewal of the sea-water within the bursae would be necessary, a circumstance which would necessitate the expulsion of the deoxygenated water from time to time. This could be effected either by a reversal of beat of the cilia or by contraction of the bursa. There is good reason for believing that the latter is the method employed and that, since the bursa is without any intrinsic or extrinsic musculature, the contraction is passive and consequent on a pressure applied to the external wall of the sac. The pressure required for evacuation of the bursae is developed through the perivisceral fluid and depends, initially, on the contraction of the musculature of the disc. MacBride (1933) has observed a periodic raising and lowering of the roof of the disc of Ophiothrix fragilis. This movement is controlled by a series of ten muscles each attached, aborally, to one of the radial plates of the disc and, orally, to an ossicle in the lateral wall of the interradial pouch. Contraction of the muscles would cause pressure to be applied through the perivisceral fluid to the wall of the bursa, and an expulsion of the fluid contained in the latter would follow. Although it is possible that, under conditions of low oxygen tension in the sea-water, water would be forced in and out of the sacs solely by the rapid contraction and relaxation of the muscles involved, it is probable that, under more normal circumstances, the muscles relax slowly and water is renewed by ciliary action. Finally, in the case of the bursa (Text-figs. 6,15, g.b. 1) which enters the substance of the external interradial muscle, one cannot overlook the possibility of evacuation of water being assisted very materially by a direct squeezing of the sac on contraction of the muscle-fibres which surround it. The bursae develop at a comparatively late stage. Small Ophiothrix of a disc diameter of 0-4 mm. taken from the REPRODUCTIVE SYSTEM OF OPHIOTHBIX FBAGILIS 305 bursa of an adult showed, on sectioning, no trace of bursae. Oxygen intake, in young specimens, must be limited to the podia and perhaps also the alimentary canal; in this connexion it is not without interest that the young should seek the bursa where not only is shelter afforded but an ample supply of well oxygenated sea-water is assured.

CONCLUSIONS. It is evident, from a survey of the literature, that there is a considerable diversity of opinion as to the morphology, histology, and function of the axial organ complex of Qphiu- roids. In the foregoing account observations on the morphology of the complex, as it exists in Ophiothrix fragilis, are presented in favour of the view formulated by Fedotov (1924) that the axial organ complex consists, not of a single organ and sinus (Text-fig. 1A) as Ludwig (1878, 1880) and other workers supposed, but is of composite structure and origin. The organ is divisible into two parts—left and right divisions —which, while showing no morphological separation, are, nevertheless, histologically distinct and are further to be differen- tiated in that each is surrounded by a separate sinus (the left and right axial sinus respectively). The relations of the parts, as they occur in Ophiothrix fragilis, are shown diagrammatically in Text-fig, lc and, as they occur in actual longitudinal section, in Text-fig. 3. Eeasons are given (pp. 279-281) in support of the supposition that the left axial sinus and ampulla are together derived from the left anterior coelom of the larva, and the right sinus from the right anterior coelom. In this latter instance the sinus represents not the right anterior coelom itself but a derivative of it—the raadreporie vesicle. No support can be given to MacBride's (1892) conclusions that the axial organ and sinus of Ophiuroids are unpaired structures or that the sinus being, as MacBride states, derived from the left posterior coelom of the larva is not homologous with the axial sinus of Asteroids. The germ-cells, which have their origin in a proliferation of the wall of the left posterior coelom of the larva (MacBride, 306 J. B. SMITH 1892), become, after metamorphosis, disposed around, the base of the stone canal (Text-fig. 7). They extend (Text-fig. 8) from this point as a string of cells, the genital rachis, which is invested by axial organ tissue and enclosed in the genital sinus, to the interradial pouch of the madreporic interradius. From here they are continued around the entire periphery of the disc as a band which, since it crosses the radii aborally and the interradii orally is thrown into a series of vertical loops. The details of the course of the rachis are well known from previous accounts and are only briefly redescribed in so far as they apply specifi- cally to Ophiothrix fragilis (pp. 283-286). Ophiothrix fragilis is dioecious. The gonads, ten in number and situated two in each of the interradial pouches, are localized expansions of the rachis. Existing evidence favours the view that the primitive germ-cells migrate from the rachis into the gonad, there to mature, rather than that they proliferate from germinal tissue situated within the gonad itself (pp. 286-287). The salient features of spermatogenesis and of oogenesis are described (pp. 288-292 and Text-figs. 9, 10,11,12). It would appear that the males are capable of producing sperm at all times of the year and that the production of ripe sperm is not limited to the larger and older specimens since limited amounts have been found in the testis of young individuals of a disc diameter of 0-3 cm. only (Text-fig. 10). Females, on the other hand, mature at a more advanced stage (diameter of disc greater than 0-5 cm.). The ovary contains oocytes which fall into well- defined size groups. These mature in succession, probably at monthly intervals from March to October (pp. 295-296 and Text-figs. 11 and 12). Contrary to all previous accounts the ova and sperm are discharged into the surrounding sea-water, in Ophiothrix at least, not through the genital bursae nor through temporary perforations in the body-wall, but through ciliated gonoducts, one to each gonad; each has its external opening in the genital slit between the arm and interradial pouch (Text-figs. 13 and 15). The duct is developed at a time when the gonad contains primary germ-cells only, but it has not been found possible to BEPEODtJCTIVB SYSTEM OF OPHIOTHBIX FBAGILIS 307 obtain any information as to the actual mode of origin of the duct. The ciliated genital bursae, of eetodermal origin, project into the perivisceral coelom of the interradii, and open to the outside in the genital slits at points proximal to the external opening of the gonoducts. Each interradius has four bursae arranged in two pairs (Text-fig. 15). The bursae are, primarily, respiratory in function. Water enters the sac by the action of the cilia of the cells which line its cavity. Expulsion of fluid from the bursa is effected by pressure developed through the perivisceral fluid as the result of the contraction of part of the disc musculature and consequent depression of the roof of the disc. In addition, it is probable that contraction of the external interradial musele assists in the evacuation of one of the pair of sacs by causing pressure to be applied directly to its walls (Text-figs. 6 and 15). Young Ophiothrix are frequently to be found within the bursae of mature specimens, both male and female. They are always post-metamorpbic individuals which creep into the bursae after a period of free-swimming life. Presence of the young within the bursae is not, therefore, necessarily indicative of a viviparous habit. SUMMARY. 1. The organs associated, either directly or indirectly, with reproduction in the Ophiuroidea are the axial organ and related sinuses (axial organ complex), the genital raehis, the gonads (localized expansions of the raehis), the gonoducts, and the genital bursae. 2. Evidence is presented in favour of the view of Fedotov (1924) that the axial organ of Ophiuroids is made up of two closely associated parts each surrounded hj its own sinus from the wall of which it is, during development, proliferated. The left axial sinus (aboral in the adult) is derived from the left anterior coelom of the larva, the right axial sinus (oral in the adult) from the madreporie vesicle which itself is a derivative of the right anterior coelom of the larva. 3. The ampulla of the stone canal is continuous with, and is part of, the left axial sinus. NO. 326 X 308 J. E. SMITH 4. An account is given of the morphology and histology of the genital rachis and sinus. 5. Examination of the gonads of female Ophiothrix indicate that the breeding season extends from about March to October and that, during this time, there is periodic emission of ova, probably at monthly intervals. Males, on the other hand, produce sperm all the year round. 6. The genital bursae number two pairs to each interradial pouch. They serve, primarily, as organs of respiration. Special mechanisms, which are described, are concerned in the intake and expulsion of water. 7. The gonads do not discharge their products directly into the genital bursae nor through temporarily formed pores in the body-wall but through specially developed and permanent gonoducts, one to each of the ten gonads. 8. Young specimens found in the genital bursae have attained their position only after a period of free-swimming larval life. After settling and metamorphosing, some of the young individuals crawl into the bursae. 9. As a consequence of the previous observation it is pointed out that the presence of the young within the genital bursae of the adult is by no means an indication of a viviparous habit.

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