Observation on the Breeding and Development of the Viviparous Fish, Heterandria Formosa. by Elizabeth A

Home , Heterandria, Heterandria formosa, Viviparity

Observation on the Breeding and Development of the viviparous fish, Heterandria formosa. By Elizabeth A. Fraser, D.Se., Reader, assisted, by Rachel M. Eenton, Curator of the Aquarium. Department of Zoology, University of London, University College.

Witt Plates 26-29, and 13 Text-fignres.

Breeding.—Heterandria formosa, one of the small- est viviparous fish, has been known for many years as a common species in tropical aquaria. It is a member of the family Poeciliidae (Garman, 1895-7; Began, 1913; Hubbs, 1924,1926), and a native of fresh-water streams from South Carolina to Florida. Although some records as to its breeding are available, no details of its development or mode of viviparity are known beyond the fact that the egg has little or no yolk and the embryo does not leave the follicle until ready for birth. We have now bred the fish successfully in the aquarium at Univer- sity College, London, for four and a haK years in order to obtain exact information of its habits and life-history. The fish have been kept in small rectangular tanks holding from 3 to 4 litres of water with a layer of coarse sand at the bottom, and in each were usually one adult female and two males. The tanks were placed in a specially heated room which varied in temperature from 20° to 26° 0. Heterandria are hardy and easy to keep; they can stand quite a large range of temperature, but seem to thrive best in water between 22° and 26° C. They are of a placid temperament and rarely fight, and have never been known to eat their young during the four and a half years that they have been under observation.1 Their food has consisted of Enchytraeid worms, finely minced, or small Daphnia, and the young were fed on newly hatched Ar- 1 A fairly large female since bought (1939) has been unusual and habitually swallows her young as soon as they are born. NO. 324 I i 480 ELIZABETH A. FRASER AND RACHEL M. EENTON temia salina. It was never found possible to induce these fish to eat anything but live food, although many of the usual dried fish foods on the market were tried at different i imes, but all without success. On the whole they were remarkably healthy and there was little disease; it was found, however, that if several males were kept in a tank together without a female, there was quite a heavy mortality among them. Copulation was never observed although the male follows the female unceasingly, and it is quite probable that it takes place at night. The birth of young has frequently been watched, the young being born tail first. Usually they are strong and active almost at once, and after resting on the sand for a few minutes they swim to the top to fill the air bladder. Occasion- ally a fish1 (usually a very young female) will give birth to one or two weaklings, which would appear to be born prematurely. These weak young have great difficulty in reaching the surface of the water, they lie on the sand and make repeated efforts to rise. Unless successful soon after birth they do not live long. The life of a female appears to vary considerably with different individuals, but lasts from two to three years. The males have a shorter span. Lebistes, the millions fish, and many other viviparous forms give birth to a large number of young at one time, with an interval of three or four weeks between each delivery. Heterandria, on the other hand, though much less prolific, breeds fairly continuously from February to September or October, with intervals between the births, but produces on an average only one to three at a time. The numbers tend to decrease towards the end of the summer, when a resting period sets in until the following February. Very occasionally an exceptional female will give numbers in excess of this, one individual having had ten at one time on more than one occasion. A female bought in November 1933, of unknown age, gave rise to fifty-one young before she died in June 1935, apparently of old age. Females, when at their prime, usually measure about 25 to 30 mm. from the tip of the snout to the end of the tail, whereas adult males do not usually exceed 17-5 mm. The largest females examined by Eegan (1913) measured 30 mm., whilst the males varied from 15 to 20 mm. DEVELOPMENT OF HETEEANDEIA 481 A remarkably large female adult, when acquired in April 1934, must have measured at least as much as 40 mm.; the exact measurement of her maximum length was not possible, for, when destroyed in May 1936, she was sick and dying and considerable shrinkage had taken place. During this period she gave birth to 170young, of which 150 were born in eight months; she showed signs of diminishing fertility only during the last few months of life, and after death her ovary was found to be a mass of degenerating tissue. Such fertility seems to be very exceptional. The newly born young measure on an average 7-5 mm. At about four weeks old the females can be distinguished by the appearance of a dark spot at the base of the anal fin, but the males cannot be recognized until the anal fin becomes modified into the long copulatory organ, a transformation which takes place quite suddenly at a later date, at an age of about eight weeks. Whilst the males are not capable of copulating before this change is complete, the females may receive the spermatozoa from an adult male before the dark spot appears and when they are still quite small. Some young which had been left in the parents' tank until the characteristic spot had become visible, were found to contain embryos a few weeks after removal. Fertilization in this case must have taken place through the male parent. By isolating the young shortly after birth, and separating the females as soon as the dark spot is perceptible, a number of virgin females can be secured. A few weeks later these may be mated with a male and some idea can thus be obtained of the time necessary for fertilization to take place. Accurate data are, however, difficult to obtain, for although the spermatozoa may be introduced almost immediately by the male, fertilization does not occur for some days owing to disintegration of ripe ova. Degeneration of the eggs appears to be quite common in viviparous fish, and has been observed in several forms, as well as in other Teleosts; in Heterandria it is especially marked in young females that have not been fertilized. The nearest approach one can make is that fertilization in isolated females seems to ensue in approximately three weeks after the introduction of the males, for embryonic stages of a few cells have been observed about 482 ELIZABETH A. FBASEE AND RACHEL M. BENTON this time. In order to obtain more accurate information a young female was isolated in a separate tank. Fifty-one days later she was mated, and the first young were bor:: fifty-six days afterwards. Assuming fertilization does not take place for about three weeks, the gestation period would be about thirty-five days or about five weeks. This isolated female has had fifty-four young from January 1935 to May 1936, and she died the following July, having attained a length of 38 mm. Females in which the first young are developing measure about 15 to 17 mm., and at first only one or occasionally two are born at a time. As the mother increases in size the number of young also increases. It seems to be the rule for an actively breeding fish to have one to three at a delivery, though the young are not necessarily born on consecutive days, and intervals of one to two weeks may even occur; but this number may be greatly exceeded in exceptional cases. For example, the very large female measuring 40 mm. has brought forth no less than nine young between the hours of twelve and four in the afternoon. It must be borne in mind that these data are for fish bred exclusively in an aquarium under a necessarily artificial en- vironment, and how far they conform to natural conditions we have had no opportunity of ascertaining. Some years ago Seal (1911) published a few observations on the breeding habits of Heterandria formosa, and as far as they go these tally with ours. Recently Turner (1937a), in a paper on reproductive cycles in Poeeiliid fishes, contributed some further details for Heterandria. The numbers of young at a birth and the intervals between the broods correspond in general with the average recorded by us. His fish, however, begin to breed in December, and continue until the end of May or July. The resting period is therefore rather longer than in our aquarium, where it begins in October or November and lasts until near the end of January. Technique.—The fish were chloroformed and the ovary was then cut out, and either fixed entire or the larger embryos were removed and preserved separately. The fixation of later stages and isolated embryos presented no difficulty, the usual fixatives, such as Bouin and picro-nitro-osmic, giving satis- DEVELOPMENT OF HETEBANDEIA 483 factory results; but for the earlier stages where the zona is thick and firm, penetration was no easy matter, and moreover the fluid-filled vesicle readily collapses. The best results were obtained with 3 per cent, potassium bichromate (four parts) and 40 per cent, formalin (two parts), heated to boiling-point, and then poured over the ovary, followed by preservation in 5 per cent, neutral formalin. Before sectioning, the material can be immersed in a saturated solution of corrosive sublimate in absolute alcohol, or in Carnoy, for several hours, which enables the tissues to stain more effectively. Sections were cut at 5, 6, or 7-5/t, but for some older stages thick sections of 20/x were used. Various stains were tried, but Ehrlich's haematoxylin counterstained with eosin or Heidenhain's iron haematoxylin was quite adequate. Ovary and Ova.—The ovary (ovisac) of Heterandria (figs. 1, 2, PI. 26) is a single oval structure much distended when full of young at various stages of development. It is covered by thin peritoneal epithelium, immediately within which is a very sparse coat of connective tissue; the general histology is similar to that described in other viviparous fish. The ovarian cavity (fig. 3, ov.c, PL 26) is lined by a single layer of cubical cells and lies along the dorsal side. From this cavity in the young fish a canal extends ventrally into the ovary along the entire length of the latter, and from it narrow passages run out towards the ova. Later, with the development of the embryos and conse- quent enlargement of the whole ovary, this ventral extension becomes irregular and branching. The structure of the ovary of Heterandria appears to be very comparable to that of Girardinus described and figured by von Ihering (1883). In the embryo two genital ridges are developed and in newly born fish,bot h male and female, the gonads are still paired so that their union into a single structure does not occur until after birth. In the adult there are no indications of a double origin. The ripe egg (fig. 5, PI. 26; fig. 6, PI. 27) which after preservation has a diameter of approximately 0-33 mm.,1 is enclosed by 1 Turner (1937 a) states that the largest unfertilized 'cells' measured 17 mm. My measurements never reached this figure even when made on the living egg. 484 ELIZABETH A. FKASER AND RACHEL M. EENTON a clear homogeneous zona round which lies the usual follicular layer, here composed of a single row of cells, cubical or somewhat columnar in shape with fairly prominent nuclei (fig. 5, PI. f.S; fig. 6,/.ej?., PL 27). Outside the follicle cells is a very thin connective sheath. Within the zona in immature eggs is acytoplasmicreticu- lum filled with fine yolk granules (fig. 3, PL 26), near the centre of which lies the nucleus (fig. 3, n., PL 26). Only a few scattered patches of solid yolk are present, and these completely disappear during maturation and early cleavage stages. Patches are seen in fig. 10 at y., PL 28. As the egg ripens the nucleus migrates to the periphery, and at the same time spaces arise in the network of fine yolk which appears to contract until only a narrow fringe of fine yolk granules remains round the circumference of the egg, except below the nucleus, where there is a deeper zone (fig. 6, PL 27; figs. 12, 13, PL 28). The central space apparently contains a fluid of some kind which may show up after staining (fig. 9, coag., PL 27); it cannot, however, be a very viscid fluid, for the vesicle collapses readily on fixation. Degeneration.-—In all young females, whether mated or not, but more extensively in those which have had no contact with a male, ripe and unripe ova are continually degenerating. During this process the nucleus becomes unrecognizable, the follicular cells multiply and begin to penetrate into the egg, the zona thinning out at these places. As degeneration proceeds, the follicle cells entirely lose their epithelial character, and the zona gradually disappears with the further infiltration of cells into the egg. The whole becomes transformed into a mass of cells, in many of which vacuoles arise; the lining of the central lumen of the ovary breaks away and the disintegrated cells and solid clumps of tissue stream into the ovarian cavity and down into the oviduct. The cytology of this process is not considered in this paper, and is similar to that described by Liu in L e - bistes (not yet published). Immediately behind the ovary the lining of the oviduct is produced into a number of well marked villi usually containing blood-vessels. As the detritus passes into this portion of the duct the epithelium of the villi lose their epithelial character, and solid particles of the debris together with entangled spermatozoa DEVELOPMENT OF HETEBANDRIA 485 are ingested. Text-fig. 1 is a transverse section through the oviduct showing this absorption taking place in the swollen villi. This is also seen in fig. 1 at v., PL 26. The greater part of the degenerated ovum is reabsorbed in this way; but perhaps

sp.-

Imm. TEXT-FIG. 1. Drawing of section of the oviduct immediately behind the ovary showing the villi (v.), some of which are ingesting detritus (d.) derived from a disintegrated egg. sp., spermatozoa, x 240.

a small portion, probably only a very small portion, may reach the exterior by the genital aperture. Some part of the disintegrated egg apparently remains behind in its original situa- tion, and becomes once more completely cut off from the ovarian lumen as a more or less solid mass of cells within which 486 ELIZABETH A. FBASER AND EACHEL M. RENTON patches of yellow or brown matter are present. Such patches may be seen for a time between developing ova. Degenerating ova are also present in large femrles with many maturing embryos within the ovary. Degeneration here is very probably due to lack of space, and to pressure from the growing young surrounding them. Early Development.—Spermatozoa introduced into the oviduct at copulation collect in small pockets which project outwards from the cavity of the ovary and are lined by the ovarian epithelium. Close to the follicle of each ripening egg a tubular extension of one of these pockets is present. The distal end presses against the follicular epithelium and widens out as a circular disc, its epithelium at the same time becoming thinner (Text-fig. 2). When the ovum is ready for fertilization the attenuated epithelium of the pocket disappears, whilst the egg sends out a spherical protuberance towards the ovarian cavity; the zona and follicle cells become stretched and vanish completely over this area. The spherical protuberance is then only separated from the ovarian cavity by a very thin membrane; round this membrane spermatozoa swarm in large numbers (fig. 7, sp., PL 27); one of them must penetrate through it to the nucleus of the egg. It must, however, be pointed out that such a protuberance from the egg was very rarely observed, so that its formation and withdrawal must occur with great rapidity; or possibly spermatozoa, after passing through the attenuated walls of the pocket, may be able to penetrate through the cells of the follicular epithelium and zona into the egg. The egg nucleus at this time passes to that side where the protuberance forms and becomes difficult to distinguish with certainty; fertilization was not observed. After the entrance of the spermatozoon the protuberance is withdrawn, the zona becomes once more intact, whilst the epithelium of the follicle and that of the pocket close round and unite together so as to form a solid plug of cells which projects as a thick wedge into the ovarian cavity (Text-fig. 3). Within the centre of the plug a narrow lumen usually persists for a short period, but this is soon obliterated and its place is filled by cells which are elongated towards the ovarian cavity, parallel to the lumen of the DEVELOPMENT OF HETERANDBIA 487 former pocket whose place they now occupy (cf. Text-figs. 2, 3). The formation of the plug appears due, at least in part, to multiplication of the cells, for mitosis can frequently be observed (Text-fig. 3, mit.).

/ov.p.

•05 mm. TEXT-HG. 2. Drawing of a section through a pocket (ov.p.) of the ovarian cavity (ov.c), the expanded distal wall of which is pressed against the follicle wall (f.ep.). Masses of spermatozoa (sp.) are seen both in the pocket and in the ovarian cavity, y.g., fine granules of yolk. x520. After fertilization the nucleus divides into two, four, eight (fig. 8, PL 27), and sixteen cells, in a manner typical of the development in Teleosts, until a cap of cells is formed on one side of the egg. As already mentioned the amount of yolk is 488 ELIZABETH A. FRASER AND RACHEL M. RENTON

uniLect

05 mm.

TEXT-HG. 3. Drawing of a section through the solid plug (pi.) formed from the union of the follicular epithelium (f.ep.) and that of the ovarian pocket after fertilization. The zona (z.) has separated from the follicle during fixation. Part of the wall (/.ep.1) of another ovum is also seen, c.t., connecting tissue sheath of ovary; mit., cell undergoing mitosis; ov.c, ovarian cavity; sp., spermatozoa; unil.ect., unilaminar ectoderm. X530. very limited, and the reticulum of cytoplasm containing fine yolk-granules is only present as a thin layer below the zona; DEVELOPMENT OF HETEBANDBIA 489 underneath the embryonic disc it is rather deeper (figs. 12, 18, PL 28, and fig. 9, PL 27, y.g.}. The nuclei of the cap very soon begin to divide, so that the cells become multmuelear, and at least four nuclei may be seen within a single eel. At this stage round the upper margin of the blastodisc a few cells pass outwards below the zona (fig. 10, uniLed., PL 28). Fig. 10, PL 28, is a drawing of an early blastodisc, the cells of which are actively dividing; from the upper side of the disc a few cells (unil.ect.) are growing outwards beneath the zona. This outgrowth of cells proceeds rapidly, partly by addition of more cells from the cap and partly by mitosis, so that very soon a continuous sheet has spread completely round the egg closely adjacent to the zona (fig. 9, unil.ect., PL 27). The cells of this sheet are at first large and somewhat flattened, but by further division they thin out and give rise to a very flattened layer which gradually increases in circumference as the egg grows. Its cells pass into those covering the embryonic dise which represent the future ectoderm, now separated off as a distinct layer (fig. 12, PL 28). Figs. 12, 13, PL 28, show a rather later stage than fig. 10, PL 28, with an embryonic disc lying at one side of the egg occupying about one-quarter of the whole; from the upper side of the disc a layer of cells (unil.eet.) is seen extending over about three-fifths of the circumference of the egg. In Heterandria, therefore, a unilaminar ectoderm completely encircles the egg at a much earlier stage than in other Teleosts, and the blastopore is closed before any differentiation into endoderm and mesodenn has occurred in the embryonic region. This is doubtless due to the almost complete absence of yolk, and the necessity for a firm cellular coat round a vesicle which contains some fluid (coagulating after fixation) and a very small amount of granular yolk. Peripheral mesendoderm, always very small hi quantity in Teleostei, is quite absent, and no invagination or thickening occurs round the enveloping margin as it closes over the yolk. Apparently, again from the fact that no yolk is present, a definite yolk syncytium or periblast is never formed. A few irregular masses of protoplasm, however, separate off from the growing margin and apparently also from below the embryonic disc; each mass contains darker 490 ELIZABETH A. FEASER AND RACHEL M. EENTON patches of nuclear material often elongated and always irregular (fig. 13, fix., PI. 28). They become the so-called periblast cells. At first they are not always easy to distinguish from the other cells, but they are usually larger and their contours much more uneven. Soon each one is completely detached and comes to lie within the yolk as a mass of protoplasm with large irregular nuclear material. Originally there are ten to eighteen such cells; later they attain a considerable size and probably fragment before disintegrating. A few periblast cells can still be seen within the diminishing yolk until the embryo is quite large and ready to hatch. Meanwhile the division of the nuclei of the embryonic area and subsequent separation into cells occurs very quickly, this area coming to consist of a mass of small cells amongst which, but chiefly collected together at one side, are a number of larger spherical cells. The latter stand out on account of their large size, rather larger nuclei, their cytoplasm staining pinker with eosin, and their nuclei paler with Ehrlich's haematoxylin than . those of the smaller cells (fig. 9, PI. 27; fig. 10, PI. 28, g.c). These large cells are the primitive germ cells which thus separate off at a very early period; they can be followed subsequently throughout development. When first recognized their number is about twenty to twenty-five, but they undergo continuous division and, when the rudiment of the embryo arises as an elongated undifferentiated mass of small cells, they have increased to fifty or over, the majority being now situated at the posterior end of the embryonic rudiment. When mesoderm first becomes apparent and a solid neural wedge is recognizable, the primitive germ cells still number about fifty, so that multiplication is arrested for some time. After the first early cleavage stages the cubical cells of the follicle increase in size, and by the time the zona has a continuous epithelial layer below it they are considerably enlarged with swollen elongated nuclei. The large nuclei are due to multiple division before separation into constituent cells. Under the action of most fixatives the follicle cells swell up abnormally and contain large vacuoles, a feature characteristic of eggs which are undergoing degeneration, and at the same time the zona DEVELOPMENT OF HETERANDBIA 491 shrivels and becomes torn away from the follicle (fig. 9, PL 27). The retention of the stiff zona in its normal position close to the follicle is rather a matter of chance, although, as already stated, hot potassium bichromate and formalin, four parts to two, and hot Bles's fluid, show the least distortion. As development of the embryo proceeds the follicle cells gradually flatten and continue to divide, eventually forming a flattened lining outside the zona. In general outline the later development of the embryo of Heterandria is similar to that of other Teleosts, and this communication gives an account of only certain outstanding features which are of exceptional interest. Pericardium and Circulation.—In embryos with thirteen to fourteen somites where the optic vesicle is connected with the brain by a narrowing stalk and there is no lens, the pericardium has already developed. It consists of a mesodermal sac stretching across from side to side in front of the yolk- granules and extending down anteroventrally below the latter. The heart lies in the pericardium and has the form of a slightly expanded tube which passes forwards into the ventral aorta, and is continued postero-ventrally along the inner boundary of the yolk as a somewhat indefinite vessel which eventually unites with the ventral wall of the pericardium rather towards the right side. This vessel later becomes what may be called the viteUine vein. The first blood-corpuscles are visible as clumps of cells below the yolk posteriorly; they are already becoming enclosed by mesenchyme cells which have penetrated from the pericardial walls between the yolk and the ectodermal layer within the zona. The yolk thus becomes encircled by mesoderm enclosing it in a sac, the delicate mesothelium of the pericardial wall covering one side and a vascular network surrounding the lateral and posterior sides. In embryos with sixteen to eighteen somites, where the optic cup is still united with the brain cavity and there is a lens, the pericardial sac is considerably expanded; moreover, it now extends upwards on either side of the head from behind the auditory vesicle as far forwards as the eye which it partially or entirely covers. This dorsal extension is shown in Text-fig. 4. The viteUine vein (v.v.) is now a well marked vessel running 492 ELIZABETH A. FEASER AND EACHEL M. EENTON from the right ventral side of the pericardial wall inwards and dorsally into the heart. The upward growth of the pericardium continues rapidly until in embryos with about thirty somites the head becomes

v.v.

TEXT-FIG. 4. Diagram of an embryo with about sixteen to eighteen somites showing the growth upwards of the pericardial sac (per.h.) which covers the auditory vesicle (aud.) and the eye (e.). The yolk (y.g.) lies behind the pericardium and the viteUine vein (v.v.) leaves the pericardial wall at O. a., position of anus; /., follicle; I., liver; per.w., pericardial wall; t., tail, x about 110. completely enveloped by a pericardial hood which reaches posteriorly beyond the ear region (Text-figs. 5, 6, per.h.). The mesodermal lining of the hood next to the body-wall adjoins the ectoderm covering the roof and sides of the brain, and along the mid-dorsal line of the head from the fore-brain to DEVELOPMENT OF HETEEANDBIA. 493 behind the ear the mesothelium of either side meets and forms a double strand (Text-figs. 6 A-B, 7; and figs. 14, 15, m.str., PL 29). The two layers again separate to run round on either

y-9

parw.

v.v.

TEXT-JIG. 5. Diagram of an embryo of approximately thirty somites. A pericardial hood (per.h.) now completely surrounds the head. Other lettering as in Text-fig. 4. X100.

side beneath the external wall of the hood. Above the head the ectoderm of this outer wall unites with that of the opposite side forming a continuous sheet of ectoderm over the mesodermal strand. The outer wall of the hood is thus analogous to the chorion of higher vertebrates. Posteriorly, below the auditory vesicle, the line of attachment of the hood to the 494 ELIZABETH A. FKASBE AND RACHEL M. EBNTON o.a.c. pt.c. WD.v / LL7 / I0?- ura rra.c. aud.

o.pt.c,

m.str

y-9

TEXT-FIG. 6. Diagram of an older embryo from the right side. The pericardial hood has now reached its maximum development and encircles the entire head, and the mesothelial walls of either side are united to form a mesodermal strand {m.str.) from the eye anteriorly to behind the auditory region (B to A). The amount of yolk {y.g.) is reduced. The bladder (ur.bl.) has begun to expand and lies within the yolk; it opens to the exterior (ur.ap.) just behind the anus (a.) and the Wolffian duct (W.D.) runs into it dorsally. The vessel from the right anterior cardinal vein (r.a.c.) is shown; it meets the pericardium at o.a.c. The posterior cardinal vein (pt.c.) comes from the tail and runs between the Wolffian ducts meeting the pericardium at o.pt.c. h., heart; arrow op., position of operuculum. Other lettering as in Text-fig. 4. x 75. DEVELOPMENT OF HETEBANDSIA 495 body-wall corves forwards; this bay marks the position of the operculmn beneath which is a passage through the gils into the pharynx (Text-figs. 6, 7 A, op.). The bay is commonly more accentuated than is shown in Text-fig. 6. msfcr m be

pane.

y-s-

urap.

TEXT-EIG. 7 (cp. Text-fig. 6). A. Composite drawing from sections through, the posterior half of the body of an embryo at about the same stage as Text-fig. 6 viewed from behind, the tail being removed. The cut edge of the pericardium and its hood is indicated by circles; between the two is a bay (cp. Text-fig. 6) in which lies the operculum (op.), internal to which is a passage from the cavity of the follicle between the gill bars (g.) into the pharynx (ph.). The vessels (r.a.c. and l.a.c.) are seen running round into the pericardial wall, the left one re- ceiving a branch from the liver (I.). The posterior cardinal (pt.c.) runs forwards between the Woman ducts (W.D.) and joins the pericardial wall at o.pt.c. The rectum (r.) opens to the exterior close below the opening of the bladder (wr.ap.). ch., chorion; h.br., hind-brain; prom,., pronephros. B. Composite drawing from sections through the anterior half of the same embryo viewed from in front of the auditory region forwards. The cavity of the anterior portion of the bladder (ur.bl.) is seen; around and in front of it lies the yolk (y.g.). m., region of mouth; m.br., mid-brain. Other lettering as in Text-figs. 4 and 6. Meanwhile, a network of blood-vessels has arisen all over the walls of the pericardium, the hood and the external surface of HO. 324 K k 496 ELIZABETH A. FRASER AND RACHEL M. RENTON the yolk, and a definite circulatory system develops. Four vessels connect the embryo with this network: (1) the large vitelline vein comes from the ventro-lateral, some vhat right, side near the lower end of the yolk-mass and runs up into the heart (Text-figs. 4-6, 7 A, 8, h.); (2) a vessel runs from the anterior cardinal on either side (Text-fig. 7 A), the two anasto- mosing across the middle line; whilst the right (Text-fig. 7 A, r.a.c.) one passes directly laterally round to the pericardial wall (Text-fig. 6, o.a.c), the left (Text-fig. 7 A, l.a.c.) runs outwards immediately in front of the liver (I.) from which it receives a short branch before joining the pericardial wall; (3) a third vessel runs down the tail and between the primordium of the mesonephric tubules as the posterior cardinal (Text-figs. 6, 7 A, pt.c.) which curves round ventrally below the rectum and then backwards to join the vascular wall on the posterior-dorsal side of the yolk (Text-figs. 6, 7 A, o.pt.c); (4) a small vascular twig running between the two layers of the mesodermal strand near the level of the auditory region connects the vessels over the brain with those on the pericardial wall. Some time before birth the pericardial hood begins to recede (Text-fig. 8, per.h.), so that shortly before the escape of the embryo from the follicle only a small tubular portion still remains stretching above over the hinder part of the eye (Text-figs. 9 A, 9 B) on each side. After birth the pericardium has assumed the normal appearance of a vesicle lying on the ventral side surrounding the heart, the yolk-granules having disappeared by this time. Eespiration and Nutrition.—The enormous enlargement of the pericardium with its extension round the head is a very striking feature, and is without doubt due to the need for increased respiratory surface within the follicle. The entire wall, including that of the hood (figs. 14, 15, emb.c, PI. 29), is traversed by a rich vascular network, somewhat less well developed over the area covered by the tail which curves round either to the right or left side of the body. Immediately outside the ectodermal wall of the pericardium is the zona, close to which lie the swollen flattened cells of the follicular epithelium. As development proceeds a fine network of maternal capillaries runs alongside the follicular wall (Text-figs. 10, 11, mate). DEVELOPMENT OF HETEBAMJBIA 497 urap. W.D. I

ur.bL

TEXT-HG. 8. Diagram of an embryo at a stage when the pericardial hood (per.h.) has begun to recede and now partly covers the eye and only the most anterior portion of the ear [mid.). The bladder (ur.bl.) has attained its maximum development, fillingalmos t the entire pericardium, and has pressed the last remaining yolk-granules (y.g.) to the antero-ventral side of the pericardium. Other lettering as in Text-figs. 4 and 6. x 75.

During later stages the maternal capillaries become greatly- enlarged and protrude between the cells of the follicle which are then only united together by a fine strand of protoplasm stretched over the inner side of the capillaries (Text-fig. 12, 498 ELIZABETH A. FRASER AND RACHEL M. RENTON mate). This condition is very marked at the time when the pericardial hood has begun to recede. Meanwhile, withTthe

TEXT-FIGS. 9 A, 9 B. Drawing of an embryo removed from the follicle shortly before birth and measuring about 5 mm. in length, showing the further reduction of the pericardial hood which now only covers the posterior half of the eye. A, View of right side; B, ventral view. In B the heart is seen as a darker area through the transparent pigmented wall of the pericardium from which many blood channels branch out over the wall.

growth of the embryo the zona becomes attenuated and, for a short period before hatching, is hardly visible. Oxygen for the embryo has thus at first to penetrate two layers of cells, that of the ectoderm round the pericardium and that of the follicle, between which is the membranous zona; later a more mate.

TEXT-KG. 10. Drawing of a section through the wall of the pericardium (per.w) and of the follicle (f.ep.) showing the embryonic vessels (emb.c.) and the fine maternal capillaries (mate). The embryo has shrunk away from the follicle during fixation. The follicular epithelium (f.ep.1) of an adjacent embryo is also seen, and between the two is connective tissue (c.t.). unil.ect., unilaminar ectoderm; z., zona. X532. 500 ELIZABETH A. FEASER AND EACHEL M. EENTON intimate relation is established, and only a fine strand of protoplasm separates the maternal capillaries from the wall of the embryo. Nutrient material must also reach the embryo, for

Z. -^

cmb.c. \

TEXT-ITO. 11. Drawing of a section through the wall surrounding the yolk (y.g.) and of the follicle (f.ep.) at the same stage as Text-fig. 10. Letter- ing as in Text-fig. 10. X 600. the minute sparse granules of yolk are quite insufficient. The gill-slits break through at an early stage and communicate behind the operculum (Text-figs. 6, 7 A, op.) with the follicular cavity posteriorly to the hinder boundary of the pericardial hood. The mouth does not develop for some time afterwards, when the pericardial hood has receded sufficiently, and then it opens into the follicular cavity. Thus nourishment must be DEVELOPMENT OF HETERANDRIA 501

uniLect.

cmb.C.

TEXT-BIG. 12. Drawing of a section through the wall of the follicle (f.ep.) and of the pericardium at a later stage than Text-fig. 10 showing the enlarged maternal capillaries (mate.) protruding between the follicle cells (f.ep.). Other lettering as in Text-fig. 10. X 600. derived from the parent through the follicle cells and later directly from the maternal blood-vessels; this passes into the 502 ELIZABETH A. FEASEB AND BACHBL M. BENTON cavity and is imbibed by way of the gills, and in older stages by the mouth also. That a fluid of some kind exists is indicated by the presence of a coagulum which may sometimes be seen after fixation within the follicle outside the pericardium, and an apparently similar coagulum can occasionally be observed •within the stomach of older embryos and also passing out of the anus. Moreover, embryos extracted from the follicle are enveloped by viscous matter. As the tail grows out and increases in length and breadth the capacity of the follicular cavity increases also, and there is more room for follicular fluid. The mature egg has an average diameter of 0-33 mm. after fixation. There is very little increase in size until after the vesicle is encircled by a cellular layer, at which time the diameter is still only about' 0-34 or 0*35 mm. When the embryonic rudiment begins to develop both vesicle and follicle gradually expand (cp. Text-figs. 5 to 8), and shortly before the embryo hatches the follicle may have a diameter of 2 mm. or more, but usually at this late stage it is rather oval in shape, attaining as much as 2-5 mm. across the longer diameter. Urinary Bladder.—In embryos of about eighteen somites the two archinephric ducts are connected with the dorsal wall of the posterior part of the gut; soon they pass into a small vesicular swelling which opens to the exterior just behind and above the anus (Text-figs. 6, 7 A, ur.ap.). Originally quite small the vesicle increases rapidly in volume and finally expands into a urinary bladder of enormous dimensions which reaches its maximum size when the two sides of the pericardium have begun to recede from the dorsal side of the head (Text- fig. 8; fig. 4, ur.bl., PL 26). At first the bladder grows forwards through the centre of the yolk-sac, where yolk-granules are scarce or absent (Text-fig. 7 B, ur.U.), and as the yolk becomes used up it enlarges and subsequently far exceeds the area formerly occupied by the latter, eventually spreading across the entire breadth of the pericardium and reaching forwards in front of the heart (fig. 4, PI. 26, and Text-fig. 8, ur.bl). As the bladder grows, its thin endothelial wall (fig. 4, bl.w., PL 26) pushes upwards the mesoderm of the pericardium (fig. 4, per.w., PI. 26) which previously lined that part of the yolk next to the DEVELOPMENT OF HETEBANDSIA 503 pericardial cavity, whilst its external wall lies in contact with the network of blood-vessels formerly lining the pericardium and the external surface of the yolk (fig. 4, PL 26). This vascular network, however, soon becomes reduced over the area covered by the bladder. At birth the bladder collapses suddenly, and in the newly bom young it is merely a small thick-walled swelling into which the Wolffian ducts enter, and which opens to the exterior immediately behind the anus. A short time before the embryo has finished its development within the mother, the cells of the solid fertilization plug become absorbed; the protoplasm becomes vacuolated, the nuclei disappear, and only a network of vaeuolated tissue remains (figs. 14,15, PL 29, and fig. 3, PL 26, pi). This partly oozes out into the ovarian cavity and partly thins out in situ, and through the gap of degenerated tissue thus created the embryo escapes into the cavity and down the oviduct immediately to the exterior. Simultaneously, the cells of the empty follicle enlarge, their chromosomes elongate, they become vaeuolated and send out long pear-shaped processes into the empty follicle. These disintegrate together with the blood- vessels below them and flowint o the cavity, the latter becoming quickly filled with a disintegrating mass of follicle cells, blood- corpuscles, and even sheath cells. Erom the follicle masses of decomposing tissue travel down the oviduct, which at this period is much widened and consists of a thin layer of epithelium surrounded by a thick layer of longitudinal muscles, villi being no longer present. It is by the contraction of this layer of muscles that the embryo is forced down the oviduct to the exterior. Contractions of the oviduct were clearly seen during the birth of an embryo immediately after the death of the parent. As the duct during birth and immediately afterwards is very expanded and consequently without villi, the debris from the burst follicle must go down the duct and directly to the exterior, and no ingestion by the cells of the wall was observed as in the case of detritus from degenerated ova. Eemnants of the zona also can be seen passing down the duct. Discussion.—Although Eyder in 1885 made interesting 604 EMZABBTH A. FRABHB AKD BAOHBIi M. RBNTON observations on the habits and appearance of the embryos of viviparous fishes, Eigenmann in 1892 was the first to go into the iife-history and development in detail when hi studied the Bmbiotieid fish Oymatogaster. Eigenmann states that viviparity in fishes may be divided into at least two types: (a) in which the yolk furnishes all the intra-ovarian food, and (b) in which the greater part of the food is furnished by the ovary. In the first type the egg remains in the follicle until near the end of gestation (Poecilia, Gambusia, Scor- poenidae) and fertilization is affected within the follicle; the number of young bom at one time occasionally reaches many thousands, aiin Sebastomus (Eigenmann), but is usually considerably less. In the second type (Zoarces, Blennius, Anahlepg., Embiotooidae, also Goodeidae and Jenynsia (Turner, 1088-4)), the length of time th© embryo remains in the follicle varies, as also does the time spent in the ovarian cavity; the egg may be fertilized within the follicle or in the ovarian cavity. The number of young is here much smaller, although they may be larger in size. In Oymatogaster Eigenmann notes that the number of young is directly proportional to the nte® of the fish, the average number being about twelve, varying according to size of parent; if the fish is small then the first young are also of small size, and this is the case in Heteran- dria and others, as for example in Zoarees (Stuhlmann,. 1887; Wallace, 1908-4; Kolster, 1905). The conditions in Heterandria, however, correspond to neither of Eigen- mann's types, and is unique in that the embryo remains within the follicle until birth and yet the amount of yolk is very small, and quite insufficient for the needs of growth. In some respects, especially on account of the scarcity of yolk,, the early development of Oymatogaster resembles that of Heterandria, and therefore a comparison between the two is of interest. In Oymatogaster (Eigenmann) copulation takes place in June or beginning of July, at a time when one generation of young are being set free (April to June) and spermatozoa remain in the ovary until the next series of eggs becomes mature at about the month of December, when they are then fertilized. In Oymatogaster the eggs in one DEVELOPMENT OF HETEBANDRIA 505 generation are all born at one time, and this holds for the majority of viviparous fish. In contrast to Heterandria the egg is freed from the follicle before cleavage begins; although fertilization was not observed it probably occurs just before or just after the egg escapes; for in a single case the second polar body was extruded, and a male pronucleus seen in an egg still enclosed within the follicle. The amount of yolk is much reduced, and the dividing cells of the egg encircle it at an early stage during the end of the eleventh cleavage (1,700 cells) before the ectoderm is differentiated. In Heterandria the exact moment at which the encirclement of the yolk takes place is difficult to determine as it occurs with great rapidity; between the seventh and eighth cleavage the cells surround three-fifths of the egg, and therefore the period at which a complete lining is established probably corresponds to about the eleventh cleavage stage as in Oymatogaster as calculated by Eigenmann. In the latter, however, the yolk is not surrounded by a single layer of cells but by several layers and, moreover, the edges of the blastoderm are separated for a short period by a kind of yolk-plug called by this author the yolk- nucleus. The appearance of the egg at this stage will be made clear by Text-fig. 13 which shows photographs of Eigenmann's figs. 85, 38. In this manner the young embryo at an early stage lies around the entire yolk-sac, a condition that never occurs in Heterandria. The periblast cells in Oymatogaster are reduced to between ten to eighteen cells, averaging twelve in number, and in no case exceeding twenty, a condition comparable to that in Heterandria, where these cells also number from twelve to eighteen. Without doubt this reduction is due to the scarcity of yolk in each case; a continuous layer of periblast cells as in other Teleosts would thus be superfluous. The primitive germ-cells in Cymatogaster (Eigenmann, 1891 and 1896-7) can be seen at a very early stage before the protovertebrae are formed, when an average of twelve (9-23, the latter figure only once) were counted. On one occasion a cell resembling a germ-cell was observed at the fifth segmentation, but this needs further confirmation. In Heterandria '•yk.pr.

TEXT-FIG. 13. Reproduction of embryos of Cymatogaster from Eigenmann (PL 96, 1892).

A.—Tenth section through early gastrula and blastopore. The egg contained about 3,000 nuclei; twelfth segmentation. The section cuts through the embryonic axis obliquely. The entoderm is well separated from the ectoderm and contains smaller cells. The outermost layer of cells is continued beyond underlying layers, and nearly covers yolk-nucleus (yk.pr.). B.—A section through another gastrula of the twelfth segmentation, slightly older than the one figured in A. The yolk-nucleus is bluntly conical, forming a plug in blastopore. ril.per., periblast nuclei. DEVELOPMENT OF HBTBEANDBIA 507 they are first visible at a slightly earlier period; immediately after the unilamimr ectoderm has grown round the yolk- vesicle the underlying cells of the embryonic disc divide up into a number of larger, very conspicuous germ-cells and into a mass of smaller endo-mesodenn cells. At first about twenty- five to thirty primitive germ-cells can be counted, but they undergo continuous division until between fifty to sixty are present. In Lebistes reticulatus Goodrich, Dee, Flynn, and Mercer (1934) state that when the germ-cells are first recognizable they are scattered in the mesendodennal layer of blastoderms with a diameter of 1-3 mm. and an embryonic shield measuring 0-5 mm. along its longitudinal axis; here forty were estimated. To what stage in Heterandria this corresponds it is impossible accurately to determine, but it may be about the same time, and if so, in these two viviparous genera we have the earliest record of the appearance of the germ- cells in the Teleostei. In later stages during their migration into the genital ridge the original number of primitive germ- cells is not exceeded. Such a resting period is present in other Teleosts, as for example in Lophius (Dodds, 1910-11), where the average number is thirty-seven and they undergo no further divisions until the ridge is attained. The early history and fate of the germ-cells in viviparous fishes I hope to consider more fully in a future paper. The spermatozoa in many viviparous fishes collect in pockets of the ovarian epithelium, and it has already been noted that in Heterandria they serve for the fertilization of many series of ova. Tubular prolongations from the ovarian cavity in which spermatozoa collect have been described by various workers. Lane (1909 and 1903-4) figured them in the Brotulid fish Lucifuga and Stygicola, where they are exactly similar to those of Heterandria, and it was suggested that their function might be to afford an entrance for the spermatozoa. In Lebistes also, both Thirumalacher (not yet published) and Liu (not yet published) have recently noted homo- logous pockets of the ovarian wall lying close to those of the follicles. Identical structures were observed by Phillippi (1908- 9) in Glaridichthys and an expansion of their distal walls 508 ELIZABETH A. FRASER AND RACHEL M. RENTON •was figured; in a section through an ovum, on two occasions only, he noticed a small process from the follicle on the floor of the pocket which was calculated to be about five tc six times the head of a spermatozoon, and within the follicular epithelium intercellular spaces were visible. This process he called the propyle, and it may very possibly be the initiation of a protuberance from the egg such as that figured in Heterandria (fig. 7, PI. 27). Such a prominent outgrowth as is found in the latter genus has not previously been seen, very possibly owing to its transitory nature, as already indicated. It may, however, not be so well developed in all viviparous fish or may even be absent, in which case the spermatozoa would have to penetrate through the follicular epithelium. In Zoarces (Stuhlmann, 1887) the follicles are situated on papillae which project into the ovarian cavity; at the distal end of each papilla is a circular hollow ('Delle') over which the follicle cells and those of the ovarian epithelium are closely apposed. This formation is shallow, and through it the egg bursts into the cavity where it is fertilized. Tubular pockets such as are present in Heterandria have been observed in some other Viviparous fish and erroneously compared with the' Delle' (i.e. Lane, 1903-4), but they are quite different both in structure and function, here serving merely as an area through which the spermatozoa can more easily reach the egg. Degeneration in Heterandria occurs very extensively in mature ova of young females which have not been fertilized, as Barfurth (1886) has shown in the river trout and Turner (1933^4) in the Goodeidae. In Heterandria it appears to go on to some extent throughout life; in older females, as already mentioned, this is partly due to compression of the young ova by growing embryos. The process apparently takes place much as in Lebistes (Liu), except that in the latter much yolk is present which has to be absorbed by the proliferating cells. The material from the disintegration of the egg is used as food for the further growth of the ova in both fishes; but, whereas in Lebistes it is carried by the epithelial cells and the blood to neighbouring regions but mainly to the stroma, in Heteran- dria the greater part passes into the ovarian cavity and is DEVELOPMENT OP HETERANDRIA 509 reabsorbed by the villi in the proximal part of the oviduct. No such absorption has been observed in Lebistes, but in Gambusia Holbrookii Thirumallacher (not yet published) has noted the presence of longitudinal ridges in the oviduct which contained ' masses of cells with granules of yellow pigment'; in some places these masses appeared to be 'bursting into the lumen of the ovary', but very probably it is ingestion which is in reality going on here. A small part of the disintegrating material may be left in situ as a mass of yellow granules. Yellow masses of this kind have been noted in Zoarces by Wallace (1903-4), in Lueifuga and Stugi- cola by Lane (1903-4), and in Lebistes amongst others. The spent follicle has been described and figured by various investigators and calls for no special discussion. Cunningham (1893-4 and 1897-8) puts forward the suggestion that the epithelium of a spent follicle in the plaice, here opening on to the external surface of the ovary, becomes restored and once more forms a part of the germinal epithelium. In viviparous fishes the spent follicle is related to the wall of the ovarian cavity and there is no direct evidence in Heterandria, or in Lebistes (Liu), that it becomes again a part of this epithelium, although such a suggestion has been put forward by Thiru- malacher for Lebistes. The ultimate fate of the spent follicle in Heterandria was not determined. At an early cleavage stage the egg of Heterandria with its limited yolk is comparable to the blastocyst of mammals. Blastomeres grow out from the peripheral portion of the blastodisc when composed of only a few cells (195 or less), and the blastocyst very quickly comes to be surrounded by a unilaminar ectoderm which is closely applied to the zona. In the Teleostei as in the Mammalia the development of such a blastodermic vesicle is due to loss of yolk. Until the vesicle is completely enclosed by cells the diameter of the egg increases only slightly, but afterwards growth is very rapid. The unilaminar ectoderm is analogous to the trophoblast of mammals and has a comparable destiny, for it later forms the ectodermal wall of the yolk-sac and eventually the bladder, and gives rise to the ectodermal layer of the pericardial hood. The development of a 510 ELIZABETH A. FEASEE AND RACHEL M. BENTON pericardial hood whose outer wall is comparable to a chorion is of course restricted in Heterandria, for it only envelops the anterior portion of the animal, as far as the a; us posteriorly and up dorsally to behind the ear, and does not include the tail which lies curved round free in the cavity of the follicle. The outer layer of the hood or chorion is composed of two layers, ectoderm externally and an inner lining of mesoderm closely adjacent to it. Mesoderm also covers the ectoderm of the sides and roof of the head of the embryo, and the cavity within the hood is extra-embryonic coelom. The chorionic ectoderm becomes continuous over the head, and from the eye to behind the auditory region over the mid-dorsal line the mesoderm forms a two-layered mesothelial membrane. There is then no ectomesodermal amnion as present in the Amniota ; the ectoderm takes no part in the fold on the side next to the head, the additional protection of an amniotic cavity being apparently unnecessary for development within the follicle. The pericardium in many viviparous fish often reaches a large size, and behind it lies the yolk. Its structure has been well described in Jenynsia by Scott (1928), and Eigenmann (1892) has figured the same condition in Cymatogaster, where a pericardium is developed many times larger than the very small yolk-sac. In Gambusia patruelis Eyder (1885) described and figured a tubular 'process of the yolk-bag' which "is prolonged upwards over the head behind the eye to meet its fellow of the opposite side; he thinks the same structure is present inPundulus, where he noticed a vascular membrane on either side of the head. Eecently Turner (1937), in his observations on the breeding of Heterandria, noticed that in the immature fish extracted from the follicle a structure which he terms the 'yolk-sac' surrounded the head. Its wall was vascular and was in contact with the follicle up to the time of birth. Such a remarkable extension has never previously been recorded in any other fish. In the Goodeidae Turner (1933-4) also speaks of a 'yolk-sac', which at its maximum development partly envelops the anterior end of the head, and in a transverse section of Lermichthys multiradiatus he shows the pericardial cavity stretching up for a short distance DEVELOPMENT OF HBTBBANDBIA 511 on either side. It is worthy of note that the Goodeidae hatch much earlier than Heterandria and the later development takes place in the ovarian cavity. It appears, therefore, as if the pericardial sac in this group has not had the time to reach the same degree of development in the follicle, and once in the ovarian lumen it is no longer required. The early development of the egg in the Goodeidae and the origin of the pericardium is not described by Turner, and there is ambiguity between the terms' yolk-sac' and' pericardium'. The pericardium is a cavity lined by mesoderm cells; as it grows it comes to lie in front of the yolk which is finally surrounded by a vascular network. The yolk-sac and the pericardium are not synonymous structures and it is incorrect to speak of the yolk-sac as being filled by the hypertrophied pericardium. In Teleostei the yolk is usually enclosed by periblast cells, in Heterandria mesoderm alone forms the wall of the sac. Turner does not give details of the cell-layers, nor are these shown in his figures; no periblast layer is mentioned but presumably it is formed, for the amount of yolk, although not large and absorbed by the time of hatching, is much more abundant than in Heteran- dria; Turner compares the conditions as regards yolk with those in Jenynsia where a definite layer of periblast cells does surround the yolk (Scott). In the wall of the pericardium and extending round the posterior side of the yolk runs a vascular network. The blood- vessels which connect this network with those in the body of the embryo are described in Gambusia by Ryder (1885), in Anableps by Garman (1895-7), but rather more fully in Jenynsia by Scott (1928), where they correspond very closely with those in Heterandria. A detailed account of the development of these vessels in viviparous fish and an accurate description of the changes undergone during young stages up to the condition in the adult have never been undertaken. The vascular area over the pericardium in viviparous fishesprovide s a respiratory surface by which the embryo may obtain oxygen, whilst nutriment is supplied by the yolk in those fish in which it is present; when the yolk later diminishes or is much reduced from the first as in Cymatogaster (Eigenmann), food is NO. 324 L1 512 ELIZABETH A. FEASBE AND KACHEL M. RENTON obtained by various methods from the ovary. In forms such as Zoarces which undergo their whole development in the ovarian cavity the epithelial lining of the wall degt nerates and material is discharged into the cavity. The nature of the epithelium and the material ejected, such as lymphocytes, connective tissue, erythrocytes, fat, &c, change with the growth of the embryo, a process which has been explained and analysed in detail by Kolster (1905). The yolk in Zoarces is not abundant and is insufficient. The hind-gut is greatly hypertrophied, filling the abdomen, and its inner epithelium provided with vascular processes. Into these the red blood corpuscles engulfed by the mouth from the ovarian fluid, or so-called uterine milk, are absorbed and from them Stahlmann (1887) suggests that oxygen may be obtained, respiration being a difficult process where, as in this fish, large numbers of young are crowded together in the ovary. In Jenynsia (Scott) the embryo is nourished entirely by the yolk until it hatches into the ovarian cavity, when the long vascular villi of the ovarian epithelium then penetrate between the gills into the pharynx. In some genera the whole embryo may lie closely apposed to these villi. In some of the Embioto- cidae, as in Anableps, respiration is effected by the dorsal, caudal, and anal fins which are provided with long delicate vascular filaments with a specially developed blood supply (Eyder, 1885). An interesting adaptation is present in the family Goodeidae (Turner, 1933-4, 1937), where elongated vascular processes arise from the body-wall around the anal opening. These are for nutrition and possibly also for respiration, both in the follicle where they first develop and later in the ovarian cavity. Anableps Grovnoii (Wyman, 1864; Eyder, 1885; Garman, 1895-7, Turner, 1938) loses its yolk while still in the follicle, and albuminous matter is taken up from the follicular cavity by rows of vascular papillae which develop in the wall of the yolk-sac; this modification may serve for respiration also. In Heterandria the entire development takes place within the follicle, but on the other hand there is little yolk and both respiration and nutrition must be effected through the DEVELOPMENT OF HETEEANDBIA 513 parent. The expansion of the pericardium and the development of a pericardial hood are doubtless correllated with the need for an extension of the vascular area for respiration, and this is accomplished by the close apposition of the pericardial and chorionic ectoderm to the follicle cells, which last take on the role of the uterine epithelium in higher vertebrates. The modification of the follicle cells with their slender junctions over the maternal capillaries, as shown in Text-fig. 12, demonstrates a striking resemblance to the uterine epithelium in the mature allantoplacenta of Lygosoma o cellatum figured by Weekes (1930, Text-fig. 5 a), where the uterine epithelium is extremely thin over the maternal capillaries which are thus exposed and are in close apposition to the chorionic ectoderm below which run the embryonic capillaries. In the ease of the lizard the latter capillaries are allantoic. My Text-fig. 10, where the maternal capillaries have begun the invasion of the follicle cells, also corresponds to an early stage in the development of the placenta in Egernia Gunninghami (Weekes, 1930, Text- fig. 1), where the chorionic membrane is established but the allantois is only a small vesicle at the posterior end of the embryo. In Heterandria similar conditions are met with round the yolk, so that in this viviparous fish we have the simplest type of placenta, established in relation to the yolk and to the outer wall of the pericardial hood or chorion. It must be remembered, however, that here no entoderm ever covers the yolk. The follicle cells and the maternal capillaries serve also for the passage of the food into the follicular cavity into which open the gills and later the mouth. This follicular fluid is the main source of nutrition for the growing embryo. The presence of an enlarged urinary bladder in the embryo of Heterandria is a surprising feature. Some adult Teleosts possess a large urinary bladder, a description of which has been given by Hyrtl (1851). It is interesting to find that Duvernoy (1844) noted a thick-walled bladder in the adult of Poecilia surinamensis and drew attention to a dilated bifurcated bladder in two preserved foetuses which he figured; as such a unique condition had not been observed before he regretted that there had been no possibility of studying it in the living 514 ELIZABETH A. FEASEB AND RACHEL M. BBNTON foetus. Apart from this observation an expansion of this organ in the embryo has never been noticed in any Teleost. The urinary bladder in the Teleostei (according to Felix, in Salmo, 1897) arises as an outgrowth from the dorsal wall of the hind-gut at the place of union of the two archinephric ducts. As long ago as 1866 Kupffer studied the development and compared the bladder to the allantois of higher vertebrates. The allantois grows out from the ventral side of the same portion of the gut and this difference in origin does not seem to me to be a fundamental one. In the Eeptilia where it exists and the origin is known, a large part of the bladder is derived from the stalk of the allantois. In the Marsupialia the main portion is formed from a part of the intra-abdominal allantoic stalk, and in higher mammals the proximal end of the stalk may share in its development. In Heterandria the pronephros is well developed in the embryo and the function of the bladder is presumably for the storage of urine. A similar function has been ascribed to the allantois in those mammals where there is an active excretory organ in the foetus (marsupials, pig, sheep, eat). In Heteran- dria the bladder communicates with the exterior by a small aperture and some of the fluid excreted can escape by this route. There is the probability also that the bladder is respiratory, taking over the function of the pericardium of earlier stages, for at its maximum development it completely fills the area formerly occupied by the latter except where the vitelline vein passes up from the pericardial wall into the heart; it never invades the hood which has begun to recede at this time. In viviparous lizards, according to Weekes (1935), the allantois does not grow round the yolk-sac but only expands as the sac diminishes in size just as it does in Heterandria. During its early development when it pushes up into the centre of the yolk-sac between the yolk-granules the ventral wall comes to be in close contact with the vascular network formerly over the yolk, and therefore it would seem that, for a brief period, a structure that simulates an allantoic placenta is present. Soon, however, the vessels of this vascular network are apparently much reduced in calibre. DEVELOPMENT OP HETERANDEIA 515 The brief period during which the embryo is retained in the follicle in the Goodeidae is supposed by Turner (1933-4) to be due to the small amount of yolk. This supposition cannot be the correct one, for Heterandria is able to obtain sufficient nourishment from the parent until birth, a period of perhaps five weeks. This author attempts to differentiate between viviparity and ovoviviparity in these fishes. He defines viviparity as a process by which the embryo is not only retained internally but is dependent on the ovary for nutrition, whereas ovoviviparity is understood as the process in which the embryo is retained internally but is dependent on yolk for sustenance. The family Embiotocidae is thus viviparous whilst the family Poeciliidae is ovoviviparous. In the Goodeid type which is hatched relatively early, ovoviviparity is said to be superimposed by viviparity. Such a definition cannot hold for the case of Heterandria, a member of the Poeciliidae, in which the whole of development takes place in the follicle but nutriment and respiration are obtained almost entirely from the parent through the vascularized follicular wall. The condition in Heterandria may be a further development of that begun by the Goodeidae, but more details of the early development of other forms are needed for adequate comparison. It is more rational to reject the term ovoviviparous altogether, especially with reference to Teleostei, and to regard all the Teleosts which bring forth living young as viviparous. To Professor J. P. Hill I am much indebted for his valuable criticism. Mr. J. E. Norman has given me assistance with regard to correct nomenclature. I wish to express my grateful thanks to Miss Joyce Townend for the drawings reproduced as Text-figs. 4 to 9. For the microphotographs I have to thank Mr. J. E. Thomas of this department.

SUMMARY. 1. A short account is given of the breeding habits of Heter- andria formosa in an aquarium. 2. The ovary and the mature ovum are briefly described, the noteworthy feature of the ripe ovum being the small quantity of yolk. 516 ELIZABETH A. FBASEB AND RACHEL M. EENTON 8. Degeneration of ova is found to be a common occurrence in unfertilized females, and to a less extent in those full of developing embryos. 4. The method for ensuring the fertilization of the egg within the follicle is portrayed. Over the area where the spermatozoa have entered the cells of the ovarian epithelium and those of the follicle form a solid plug which eventually disrupts to enable the fully developed embryo to escape into the cavity of the ovary. 5. It is characteristic of early development that the egg is encircled by a unilaminar ectoderm before there is any visible differentiation into endoderm and mesoderm. 6. Owing to the scarcity of yolk only a few periblast cells arise and no syncytial layer is formed. 7. The primitive-germ cells are visible at an early stage within the apparently undifferentiated mesendoderm cells. 8. A striking feature is the large size of the pericardium and its growth upwards as a pericardium hood which completely surrounds the head region of the embryo. Over the walls runs a network of blood-vessels from which the maternal capillaries become eventually separated by only an attenuated layer of protoplasm. Both respiration and nutrition are effected through the follicle. 9. A remarkable specialization is the development of a urinary bladder which expands into a thin-walled vesicle of enormous dimensions, finally occupying almost the entire area formerly filled by the pericardium and the yolk-granules. 10. The development of Heterandria is compared with that of other viviparous fishes. 11. The significance of the unusual features in early and late development is discussed and some comparisons are made with the conditions in higher vertebrates.

EEFEEENOES. Bailey, R. J., 1933.—"Ovarian Cycle in the Viviparous Teleost Xipho- phorus Helleri", 'Biol. Bull. Woods', 64,. Barfurth, D., 1886.—"Biol. Unters. tt. d. Bachforelle", 'Arch. mikr. Anat.', 27. DEVELOPMENT OF HETERANDKIA 517

Buhler, A., 1902.—"Riickbildung der Eifollikel bei Wirbeltieren. I. Fische", 'Gegenbaurs Jb.', 30. Cunningham, J. T., 1893-1.—"Experiments and Observations made at the Plymouth Laboratory. II. Development of the Egg in Flat Fishes and Pipe Fishes", 'Journ. Mar. Biol. Ass.', 3. 1897-8.—"Histology of the Ovary and Ovarian Ova in certain Marine Fishes", 'Quart. Journ. Micr. Sci.', 40. Dodds, G. S., 1910-11.—"Segregation of the Germ-Cells of the Teleost, Lophius", 'Journ. Morph.', 21. Duvemoy, M., 1844.—"Developpement de la poecilie de Surinam (Poecilia surinamensis Val.)", 'Ann. Sci. nat.', 3, Ser. Zool. 1. Eigenmann, C. H., 1891.—"Precocious Segregation of Sex-Cells in Micro- metrus aggregatus Gibbons", 'Journ. Morph.', 5. 1892 (1894).—"Viviparous Fishes of the Pacific Coast of North America", 'Bull. U.S. Fish Comm.', 12. 1896-7.—"Sex-Differentiation in the Viviparous Teleost Cymato- gaster", 'Arch. Entw. Meek. Org.', 4. Felix, W., 1897.—"Beitr. z. Entwick. der Salmoniden", l.Teil. 'Ergebn. Anat. Entw.-Gesch. Wiesbaden', 8. Garman, S., 1895-7.—"The Cyprinodonts", 'Mem. Harv. Mus. Comp. Zool.', 19. Goodrich, Dee, Flynn, and Mercer, 1934.—"Germ Cells and Sex Differen- tiation in Lebistes reticulatus", 'Biol. Bull. Woods Hole', 67. Hubbs, C. L., 1924.—"Fishes of the Order Cyprmodontes", 'Misc. Publ. Mus. Zool. Univ. Mich.', Ho. 13. 1926.—Ibid., No. 16. Hyrtl, J., 1850.—"Beitr. z. Morph. der Urogenital-Organe der Fische. I. U. d. angehliche Fehlen der Harnblase bei mehreren Fischen", 'Denk- schr. Akad. Wiss. Wien', 1. 1851.—"Das uropoetische System der Knochenfische", ibid., 2. von Ihering, H., 1883.—"Z. K. der Gattung Girardinus", 'Z. wiss. Zool.', 38. Kolster, R., 1905.—"U. d. Embryotrophe, speciell bei Zoarces viviparus", 'Festschr. Palmen.', 1. KupfEer, C, 1866.—"Unters. ii. d. Entwiekl. des Harn und Geschlechts- systems", 'Arch. mikr. Anat.', 2. 1868.—"Beob. ii. d. Entwiekl. der Knochenfische", ibid., 4. Lane, H. H., 1903-4.—"Ovarian Structures of the Viviparous Blind Fishes, Lucifuga and Stygieola", 'Biol. Bull. Woods Hole', 6. 1909.—"Ovary and Ova in Lucifuga and Stygieola", 'Cave Verta- brates of America (Eigenmann), Washington.' Liu, Fah-Hsuen.—' Ovarian Eggs of a Tropical Fish, Lebistes reticulatus.' Thesis for Ph.D. degree, 1937 (not yet published). Philippi, E., 1908-9.—"Fortpflanzungsgeschichte der viviparen Teleosteer Glaridichthys januarius und G. decem-maeulatus", 'Zool. Jb.', 27. 518 ELIZABETH A. FBASEB AND EACHEL M. BENTON

Regan, C. Tate, 1913.—"A Revision of the Cyprinodont Fishes of the Sub-family Poeeiliinae", 'Proo. zool. Soc. Lond.', 2. Ryder, J. A., 1885.—"Development of Viviparous Osseous Fishes and of the Atlantic Salmon", 'Proc. U.S. nat. Mus.' Scott, M. I. H., 1928.—"Sobre el Desarrollo Intraovarial de Fitzroyia lineata Berg.", 'An. Mus. nac. B. Aires.', 34. Seal, W. P., 1911.—"Breeding Habits of the Viviparous Fishes Gambusia Holbrookii andHeterandriaformosa", 'Proc. Biol. Soc. Washington', 24. Stuhlmann, F., 1887.—'Z. K. des Ovariums der Aalmutter (Zoarces viviparus Cuv.).' Wurzburg. Thirumalacher, B.—' Viviparity in Teleost Fishes.' Thesis for Ph.D. degree (not yet published). Turner, C. L., 1933-4.—"Viviparity superimposed upon ovo-viviparity in the Goodeidae", 'Journ. Morph.', 55. —1936.—"Absorbtive Processes in the Embryos of Parabrotula dentens, a Viviparous Deep-sea Brotulid", ibid., 59. —— 1937.—"Trophotaeniae of Goodeidae. Viviparous Cyprinodonts", ibid., 61. • 1937a.—"Reproductive cycles and superfetation in Poeciliid Fishes", 'Biol. Bull. Woods Hole', 72. 1938.—"Adaptations for viviparity in embryos and ovary of Ana- blops Anableps", 'Journ. Morph.', 62. — 1938a.—"Histological and Cytological changes in the Ovary of Cymatogaster aggregatus during Gestation", ibid., 62. Wallace, W., 1903-4.—"Observ. on Ovarian Ova and Follicles in Certain Teleostei and Elasmobranch Fishes", 'Quart. Journ. Micr. Sci.', 47. Weekes, H. C, 1930.—"Placentation in Retiles. II", 'Proc. Linn. Soc. N.S.W.', 55. 1935.—"Review of Placentation among Reptiles", 'Proc. Zool. Soc. Lond.', 4. Wyman, J., 1857.—"Development of Anableps Gronovii", 'Boston Journ. Nat. Hist.', 6.

EXPLANATION OP PLATES 26-29 REFERENCE LETTEES. c.t., connective tissue; emb., embryo; emb.c, embryonio capillary; /.ep., epithelium of follicle; g.c, primitive germ-cell; h., heart; m.str., mesoderm strand; mat.c, maternal capillary; »., nuoleus; ov., ovum; ov.c, ovarian cavity; ov.p., ovarian pocket; ovd., oviduot; pb.c, periblast cell; per.c, pericardial cavity; per.h., pericardial hood; per.w., wall of pericardium; pi., plug; sp., spermatozoa; unil.ect., unilaminar ectoderm; ur.bl., urinary bladder; v., villus of oviduot; y.g., yolk-granules; z., zona. DEVELOPMENT OF HETERANDBIA 519

PLATE 26. Fig. 1.—Microphotograph of a longitudinal section through an ovary- containing three embryos (emb. 1, 2, 3) of different ages and showing the ovarian cavity (ov.c.) along the dorsal side running into the oviduct posteriorly. The oviduct (ovd.) shows a villus (v.) ingesting detritus. Developing ova (ov.) lie on the side of the ovary next to the cavity. X 35. Fig. 2.—Microphotograph of a transverse section of an ovary containing two large embryos (emb. 1, 2) and one smaller one (emb. 3). The ovarian cavity (ov.c.) lies at the dorsal side and is surrounded by ova (ov.) of various sizes. X 35. Fig. 3.—Part of fig. 2 enlarged to show the ovarian cavity (ov.c.) surrounded by developing ova. The smaller ova contain a reticulum with fine yolk-granules; in the larger one the contraction of these towards the periphery is seen. Through the vacuolated plug of tissue (pi.) the large embryo (cf. fig. 2, emb. 1) will escape from the follicle. X 87-5. Fig. 4.—Microphotograph of a section through the head region of an embryo shortly before birth, comparable with that in Text-fig. 8, showing the anterior end of the expanded urinary bladder (ur.bl.) occupying the place formally filled by the pericardium and yolk and extending forwards as far as the ventral aorta (aort.) (cf. fig. 15, PI. 29). The section is somewhat oblique and passes through the eye (e.) on the one side and the thymus (th.) and gill-bars on the other, bl., endothelial wall of bladder; r.per.h., receding tip of pericardial hood. X 50. Fig. 5.—Microphotograph of a section through two ripe eggs showing the yolk-granules round the circumference, leaving a central clear space. The smaller egg is cut tangentially. X 200.

PLATE 27. Fig. 6.—Microphotograph of a section through a ripe egg showing the nucleus (n.) beneath which is a deeper zone of yolk than round the circumference of the egg. x 200. Fig. 7.—^Microphotograph of a section through a spherical protuberance from an egg into the ovarian cavity (ov.c.) at the time of fertilization. Crowds of spermatozoa (sp.) are congregated over the thin membrane of the protuberance. The zona (2.) and the follicular epithelium (f.ep.) axe absent beneath the protuberance, but round the proximal part of the latter the follicular epithelium is thickened. X300. Fig. 8.—Microphotograph of a section showing a stage of eight cells. During fixation the zona has shrunk and torn away from the follicle cells (f.ep.) which consequently have jagged edges. X200. Fig. 9.—Microphotograph of a section through a stage at which the vesicle is completely encircled by a unilamellar ectoderm (unU.ect.). The embryonic disc is seen at one side and among them the larger primitive germ-cells (g.c.) are visible. The zone of fine yolk-granules (y.g.) is deeper 520 ELIZABETH A. FRA8ER AND RACHEL M. RENTON beneath the embryonic area. The central space is filled with a coagulable fluid, (coag.). The zona has shrunk considerably during fixation and is separated from the cells of follicle. X 200.

PLATE 28. Fig. 10.—Drawing of a section through a blastodisc of approximately 194 cells. The cells of the disc are multinuclear. A few cells (unil.ect.) have begun to migrate from the periphery outwards beneath the zona and these altogether encircle approximately three-eighths of the circumference of the egg at thin stage. X 400. Fig. 11.—Drawing of a section through the blastodiso of a stage comparable to flg. 0 showing the primitive germ-cells (g.e.) now distinguishable from the somatic cells of the embryo by their large size and staining pro- perties. Note the irregular periblast cell (pb.c.) within the yolk-granules. c,t,, connective tiswuo sheath, x 000. Fig. 12.—Drawing of an oblique section through a blastodisc of a slightly older atage. Mitosis can be seen in some of the cells of the disc. The superficial cells are now separated off as the future ectodermal layer and from it largo cells (unil.ect.) are passing out beneath the zona. These htivo now spread over about three-fifths of the circumference of the egg. Note the jaggod edges of the follicle cells where the zona has shrunk during fixation. X 300. Fig. 13.—Drawing of the same embryo showing the unilaminar cells (unil.ecl.) spreading out beneath the zona («.). At the growing edge is a periblast cell (pb.c). The section has passed through the inner portion of the fertilization plus (pi.). X300.

PLATE 29. Fig. 14.—Mierophotograph of a section passing through the eyes and heart of the embryo in Text-fig. (5 showing the pericardial hood (per.h.) which has grown dorsally round tho brain. The mesodermal lining of the hood of eaoh side meets dorsally as the mesodermal strand (m.slr.). The yolk lies beneath the heart. U, tail. X 133 (approx.) Fig. 15.—Miorophotograph of a portion of the right side of fig. 14 enlarged to show the vacuolated plug (pi.), the pericardial in which run the embryonic capillaries (emb.c), and the mesodermal strand (m.str.). X266 (approx). Quart. Journ. Micr. Set. Vol. 81, X. 8., PI. 26

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