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 gono- ducts 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 sup- port 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 struc- ture 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 madre- poric 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 con- tained 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.