Observation on the Breeding and Developmentof 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.

B r e e d i n g . — H e t e r a n d r i a formosa, one of the small-est viviparous fish, has been known for many years as a commonspecies in tropical aquaria. It is a member of the familyPoeciliidae (Garman, 1895-7; Began, 1913; Hubbs, 1924,1926),and a native of fresh-water streams from South Carolina toFlorida. Although some records as to its breeding are available,no details of its development or mode of viviparity are knownbeyond the fact that the egg has little or no yolk and theembryo does not leave the follicle until ready for birth. Wehave now bred the fish successfully in the aquarium at Univer-sity College, London, for four and a haK years in order to obtainexact information of its habits and life-history.

The fish have been kept in small rectangular tanks holdingfrom 3 to 4 litres of water with a layer of coarse sand at thebottom, and in each were usually one adult female and twomales. The tanks were placed in a specially heated room whichvaried in temperature from 20° to 26° 0. H e t e r a n d r i a arehardy and easy to keep; they can stand quite a large rangeof temperature, but seem to thrive best in water between 22°and 26° C. They are of a placid temperament and rarely fight, andhave never been known to eat their young during the four anda half years that they have been under observation.1 Their foodhas consisted of Enchytraeid worms, finely minced, or smallD a p h n i a , and the young were fed on newly hatched Ar-

1 A fairly large female since bought (1939) has been unusual andhabitually swallows her young as soon as they are born.

NO. 324 I i

480 ELIZABETH A. FRASER AND RACHEL M. EENTON

temia s a l i n a . It was never found possible to induce thesefish to eat anything but live food, although many of the usualdried fish foods on the market were tried at different i imes, butall without success. On the whole they were remarkablyhealthy and there was little disease; it was found, however, thatif 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 followsthe female unceasingly, and it is quite probable that it takesplace at night. The birth of young has frequently been watched,the young being born tail first. Usually they are strong andactive almost at once, and after resting on the sand for a fewminutes they swim to the top to fill the air bladder. Occasion-ally a fish1 (usually a very young female) will give birth to oneor two weaklings, which would appear to be born prematurely.These weak young have great difficulty in reaching the surfaceof the water, they lie on the sand and make repeated effortsto rise. Unless successful soon after birth they do not live long.

The life of a female appears to vary considerably withdifferent individuals, but lasts from two to three years. Themales have a shorter span. L e b i s t e s , the millions fish, andmany other viviparous forms give birth to a large number ofyoung at one time, with an interval of three or four weeksbetween each delivery. H e t e r a n d r i a , on the other hand,though much less prolific, breeds fairly continuously fromFebruary to September or October, with intervals between thebirths, 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. Veryoccasionally an exceptional female will give numbers in excessof this, one individual having had ten at one time on more thanone occasion. A female bought in November 1933, of unknownage, gave rise to fifty-one young before she died in June 1935,apparently of old age. Females, when at their prime, usuallymeasure about 25 to 30 mm. from the tip of the snout to theend of the tail, whereas adult males do not usually exceed17-5 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 exactmeasurement of her maximum length was not possible, for, whendestroyed in May 1936, she was sick and dying and considerableshrinkage had taken place. During this period she gave birthto 170young, of which 150 were born in eight months; she showedsigns of diminishing fertility only during the last few monthsof life, and after death her ovary was found to be a mass of de-generating tissue. Such fertility seems to be very exceptional.

The newly born young measure on an average 7-5 mm. Atabout four weeks old the females can be distinguished by theappearance of a dark spot at the base of the anal fin, but themales cannot be recognized until the anal fin becomes modifiedinto the long copulatory organ, a transformation which takesplace quite suddenly at a later date, at an age of about eightweeks. Whilst the males are not capable of copulating beforethis change is complete, the females may receive the sperma-tozoa from an adult male before the dark spot appears andwhen they are still quite small. Some young which had beenleft in the parents' tank until the characteristic spot had becomevisible, were found to contain embryos a few weeks afterremoval. Fertilization in this case must have taken placethrough the male parent. By isolating the young shortly afterbirth, and separating the females as soon as the dark spot isperceptible, a number of virgin females can be secured. A fewweeks later these may be mated with a male and some idea canthus be obtained of the time necessary for fertilization to takeplace. Accurate data are, however, difficult to obtain, foralthough the spermatozoa may be introduced almost im-mediately by the male, fertilization does not occur for somedays owing to disintegration of ripe ova. Degeneration of theeggs appears to be quite common in viviparous fish, and hasbeen observed in several forms, as well as in other Teleosts; inH e t e r a n d r i a it is especially marked in young females thathave not been fertilized. The nearest approach one can makeis that fertilization in isolated females seems to ensue in ap-proximately 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 ayoung female was isolated in a separate tank. Fifty-one dayslater she was mated, and the first young were bor:: fifty-sixdays afterwards. Assuming fertilization does not take placefor about three weeks, the gestation period would be aboutthirty-five days or about five weeks. This isolated female hashad fifty-four young from January 1935 to May 1936, and shedied the following July, having attained a length of 38 mm.Females in which the first young are developing measure about15 to 17 mm., and at first only one or occasionally two are bornat a time. As the mother increases in size the number of youngalso increases. It seems to be the rule for an actively breedingfish to have one to three at a delivery, though the young are notnecessarily born on consecutive days, and intervals of one totwo weeks may even occur; but this number may be greatlyexceeded in exceptional cases. For example, the very largefemale measuring 40 mm. has brought forth no less than nineyoung between the hours of twelve and four in the afternoon.

It must be borne in mind that these data are for fish bredexclusively in an aquarium under a necessarily artificial en-vironment, and how far they conform to natural conditions wehave had no opportunity of ascertaining.

Some years ago Seal (1911) published a few observations onthe breeding habits of H e t e r a n d r i a formosa, and as faras they go these tally with ours. Recently Turner (1937a), ina paper on reproductive cycles in Poeeiliid fishes, contributedsome further details for H e t e r a n d r i a . The numbers ofyoung at a birth and the intervals between the broods corre-spond in general with the average recorded by us. His fish,however, begin to breed in December, and continue until theend of May or July. The resting period is therefore ratherlonger than in our aquarium, where it begins in October orNovember and lasts until near the end of January.

Technique.—The fish were chloroformed and the ovarywas then cut out, and either fixed entire or the larger embryoswere removed and preserved separately. The fixation of laterstages and isolated embryos presented no difficulty, the usualfixatives, such as Bouin and picro-nitro-osmic, giving satis-

DEVELOPMENT OF HETEBANDEIA 483

factory results; but for the earlier stages where the zona isthick and firm, penetration was no easy matter, and moreoverthe fluid-filled vesicle readily collapses. The best results wereobtained 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 in5 per cent, neutral formalin. Before sectioning, the materialcan be immersed in a saturated solution of corrosive sublimatein absolute alcohol, or in Carnoy, for several hours, whichenables the tissues to stain more effectively. Sections were cutat 5, 6, or 7-5/t, but for some older stages thick sections of 20/xwere used. Various stains were tried, but Ehrlich's haematoxylincounterstained with eosin or Heidenhain's iron haematoxylinwas quite adequate.

Ovary and Ova.—The ovary (ovisac) of H e t e r a n d r i a(figs. 1, 2, PI. 26) is a single oval structure much distended whenfull of young at various stages of development. It is covered bythin peritoneal epithelium, immediately within which is a verysparse coat of connective tissue; the general histology is similarto that described in other viviparous fish. The ovarian cavity(fig. 3, ov.c, PL 26) is lined by a single layer of cubical cells andlies along the dorsal side. From this cavity in the young fisha canal extends ventrally into the ovary along the entire lengthof the latter, and from it narrow passages run out towards theova. Later, with the development of the embryos and conse-quent enlargement of the whole ovary, this ventral extensionbecomes irregular and branching. The structure of the ovaryof H e t e r a n d r i a appears to be very comparable to that ofG i r a r d i n u s described and figured by von Ihering (1883).

In the embryo two genital ridges are developed and in newlyborn fish, both male and female, the gonads are still paired so thattheir union into a single structure does not occur until afterbirth. In the adult there are no indications of a double origin.

The ripe egg (fig. 5, PI. 26; fig. 6, PI. 27) which after preserva-tion has a diameter of approximately 0-33 mm.,1 is enclosed by

1 Turner (1937 a) states that the largest unfertilized 'cells' measured17 mm. My measurements never reached this figure even when madeon the living egg.

484 ELIZABETH A. FKASER AND RACHEL M. EENTON

a clear homogeneous zona round which lies the usual follicularlayer, here composed of a single row of cells, cubical or somewhatcolumnar in shape with fairly prominent nuclei (fig. 5, PI. f.S; fig.6,/.ej?., PL 27). Outside the follicle cells is a very thin connectivesheath. Within the zona in immature eggs is acytoplasmicreticu-lum filled with fine yolk granules (fig. 3, PL 26), near the centreof which lies the nucleus (fig. 3, n., PL 26). Only a few scatteredpatches of solid yolk are present, and these completely disappearduring maturation and early cleavage stages. Patches are seenin fig. 10 at y., PL 28. As the egg ripens the nucleus migratesto the periphery, and at the same time spaces arise in the net-work of fine yolk which appears to contract until only a narrowfringe of fine yolk granules remains round the circumferenceof the egg, except below the nucleus, where there is a deeperzone (fig. 6, PL 27; figs. 12, 13, PL 28). The central spaceapparently contains a fluid of some kind which may show upafter staining (fig. 9, coag., PL 27); it cannot, however, be a veryviscid fluid, for the vesicle collapses readily on fixation.

Degeneration.-—In all young females, whether mated ornot, but more extensively in those which have had no contactwith a male, ripe and unripe ova are continually degenerating.During this process the nucleus becomes unrecognizable, thefollicular cells multiply and begin to penetrate into the egg, thezona thinning out at these places. As degeneration proceeds,the follicle cells entirely lose their epithelial character, and thezona gradually disappears with the further infiltration of cellsinto the egg. The whole becomes transformed into a mass ofcells, in many of which vacuoles arise; the lining of the centrallumen of the ovary breaks away and the disintegrated cells andsolid clumps of tissue stream into the ovarian cavity and downinto the oviduct. The cytology of this process is not consideredin this paper, and is similar to that described by Liu in L e -b i s tes (not yet published).

Immediately behind the ovary the lining of the oviduct isproduced into a number of well marked villi usually containingblood-vessels. As the detritus passes into this portion of the ductthe epithelium of the villi lose their epithelial character, andsolid particles of the debris together with entangled spermatozoa

DEVELOPMENT OF HETEBANDRIA 485

are ingested. Text-fig. 1 is a transverse section through theoviduct showing this absorption taking place in the swollenvilli. This is also seen in fig. 1 at v., PL 26. The greater part ofthe degenerated ovum is reabsorbed in this way; but perhaps

s p . -

Imm.TEXT-FIG. 1.

Drawing of section of the oviduct immediately behind the ovaryshowing 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 reachthe exterior by the genital aperture. Some part of the dis-integrated egg apparently remains behind in its original situa-tion, and becomes once more completely cut off from theovarian lumen as a more or less solid mass of cells within which

4 8 6 ELIZABETH A. FBASER AND EACHEL M. RENTON

patches of yellow or brown matter are present. Such patchesmay be seen for a time between developing ova.

Degenerating ova are also present in large femrles with manymaturing embryos within the ovary. Degeneration here is veryprobably due to lack of space, and to pressure from the growingyoung surrounding them.

E a r l y Development.—Spermatozoa introduced into theoviduct at copulation collect in small pockets which projectoutwards from the cavity of the ovary and are lined by theovarian epithelium. Close to the follicle of each ripening egga tubular extension of one of these pockets is present. Thedistal end presses against the follicular epithelium and widensout as a circular disc, its epithelium at the same time becomingthinner (Text-fig. 2). When the ovum is ready for fertilizationthe attenuated epithelium of the pocket disappears, whilst theegg sends out a spherical protuberance towards the ovariancavity; the zona and follicle cells become stretched and vanishcompletely over this area. The spherical protuberance is thenonly separated from the ovarian cavity by a very thin mem-brane; round this membrane spermatozoa swarm in largenumbers (fig. 7, sp., PL 27); one of them must penetrate throughit to the nucleus of the egg. It must, however, be pointed outthat such a protuberance from the egg was very rarely observed,so that its formation and withdrawal must occur with greatrapidity; or possibly spermatozoa, after passing through theattenuated walls of the pocket, may be able to penetratethrough the cells of the follicular epithelium and zona into theegg. The egg nucleus at this time passes to that side where theprotuberance forms and becomes difficult to distinguish withcertainty; fertilization was not observed. After the entranceof the spermatozoon the protuberance is withdrawn, the zonabecomes once more intact, whilst the epithelium of the follicleand that of the pocket close round and unite together so as toform a solid plug of cells which projects as a thick wedge intothe ovarian cavity (Text-fig. 3). Within the centre of the pluga narrow lumen usually persists for a short period, but this issoon obliterated and its place is filled by cells which are elon-gated 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, tomultiplication of the cells, for mitosis can frequently be ob-served (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 thefollicle wall (f.ep.). Masses of spermatozoa (sp.) are seen both inthe pocket and in the ovarian cavity, y.g., fine granules ofyolk. x520.

After fertilization the nucleus divides into two, four, eight(fig. 8, PL 27), and sixteen cells, in a manner typical of thedevelopment in Teleosts, until a cap of cells is formed on oneside 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 theunion of the follicular epithelium (f.ep.) and that of the ovarianpocket after fertilization. The zona (z.) has separated from thefollicle during fixation. Part of the wall (/.ep.1) of another ovum isalso seen, c.t., connecting tissue sheath of ovary; mit., cell under-going mitosis; ov.c, ovarian cavity; sp., spermatozoa; unil.ect.,unilaminar ectoderm. X530.

very limited, and the reticulum of cytoplasm containing fineyolk-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 verysoon begin to divide, so that the cells become multmuelear, andat least four nuclei may be seen within a single eel. At thisstage round the upper margin of the blastodisc a few cells passoutwards below the zona (fig. 10, uniLed., PL 28). Fig. 10,PL 28, is a drawing of an early blastodisc, the cells of which areactively dividing; from the upper side of the disc a few cells(unil.ect.) are growing outwards beneath the zona. This out-growth of cells proceeds rapidly, partly by addition of more cellsfrom the cap and partly by mitosis, so that very soon a con-tinuous sheet has spread completely round the egg closelyadjacent to the zona (fig. 9, unil.ect., PL 27). The cells of thissheet are at first large and somewhat flattened, but by furtherdivision they thin out and give rise to a very flattened layerwhich gradually increases in circumference as the egg grows.Its cells pass into those covering the embryonic dise whichrepresent the future ectoderm, now separated off as a distinctlayer (fig. 12, PL 28). Figs. 12, 13, PL 28, show a rather laterstage than fig. 10, PL 28, with an embryonic disc lying at oneside of the egg occupying about one-quarter of the whole; fromthe upper side of the disc a layer of cells (unil.eet.) is seenextending over about three-fifths of the circumference of theegg. In H e t e r a n d r i a , therefore, a unilaminar ectodermcompletely encircles the egg at a much earlier stage than inother Teleosts, and the blastopore is closed before any differ-entiation into endoderm and mesodenn has occurred in theembryonic region. This is doubtless due to the almost completeabsence of yolk, and the necessity for a firm cellular coat rounda vesicle which contains some fluid (coagulating after fixation)and a very small amount of granular yolk. Peripheral mesendo-derm, always very small hi quantity in Teleostei, is quite absent,and no invagination or thickening occurs round the envelopingmargin as it closes over the yolk. Apparently, again from thefact that no yolk is present, a definite yolk syncytium or peri-blast is never formed. A few irregular masses of protoplasm,however, separate off from the growing margin and apparentlyalso 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 ir-regular (fig. 13, fix., PI. 28). They become the so-called peri-blast cells. At first they are not always easy to distinguish fromthe other cells, but they are usually larger and their contoursmuch more uneven. Soon each one is completely detached andcomes to lie within the yolk as a mass of protoplasm with largeirregular nuclear material. Originally there are ten to eighteensuch cells; later they attain a considerable size and probablyfragment before disintegrating. A few periblast cells can stillbe seen within the diminishing yolk until the embryo is quitelarge and ready to hatch.

Meanwhile the division of the nuclei of the embryonic areaand subsequent separation into cells occurs very quickly, thisarea coming to consist of a mass of small cells amongst which,but chiefly collected together at one side, are a number of largerspherical cells. The latter stand out on account of their largesize, rather larger nuclei, their cytoplasm staining pinker witheosin, 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 thusseparate off at a very early period; they can be followed subse-quently throughout development. When first recognized theirnumber is about twenty to twenty-five, but they undergo con-tinuous division and, when the rudiment of the embryo arisesas an elongated undifferentiated mass of small cells, they haveincreased to fifty or over, the majority being now situated atthe posterior end of the embryonic rudiment. When mesodermfirst becomes apparent and a solid neural wedge is recogniz-able, the primitive germ cells still number about fifty, so thatmultiplication is arrested for some time.

After the first early cleavage stages the cubical cells of thefollicle increase in size, and by the time the zona has a con-tinuous epithelial layer below it they are considerably enlargedwith swollen elongated nuclei. The large nuclei are due to multipledivision before separation into constituent cells. Under theaction of most fixatives the follicle cells swell up abnormally andcontain large vacuoles, a feature characteristic of eggs whichare 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 thefollicle is rather a matter of chance, although, as already stated,hot potassium bichromate and formalin, four parts to two, andhot Bles's fluid, show the least distortion. As development of theembryo proceeds the follicle cells gradually flatten and continueto divide, eventually forming a flattened lining outside the zona.

In general outline the later development of the embryo ofH e t e r a n d r i a is similar to that of other Teleosts, and thiscommunication gives an account of only certain outstandingfeatures which are of exceptional interest.

P e r i c a r d i u m and Circulat ion.—In embryos withthirteen to fourteen somites where the optic vesicle is connectedwith the brain by a narrowing stalk and there is no lens, thepericardium has already developed. It consists of a mesodermalsac 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 slightlyexpanded tube which passes forwards into the ventral aorta,and is continued postero-ventrally along the inner boundaryof the yolk as a somewhat indefinite vessel which eventuallyunites with the ventral wall of the pericardium rather towardsthe right side. This vessel later becomes what may be calledthe viteUine vein. The first blood-corpuscles are visible asclumps of cells below the yolk posteriorly; they are alreadybecoming enclosed by mesenchyme cells which have penetratedfrom the pericardial walls between the yolk and the ectodermallayer within the zona. The yolk thus becomes encircled bymesoderm enclosing it in a sac, the delicate mesothelium ofthe pericardial wall covering one side and a vascular networksurrounding the lateral and posterior sides.

In embryos with sixteen to eighteen somites, where the opticcup is still united with the brain cavity and there is a lens, thepericardial sac is considerably expanded; moreover, it nowextends upwards on either side of the head from behind theauditory vesicle as far forwards as the eye which it partiallyor 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 anddorsally into the heart.

The upward growth of the pericardium continues rapidlyuntil in embryos with about thirty somites the head becomes

C.

v.v.

TEXT-FIG. 4.

Diagram of an embryo with about sixteen to eighteen somitesshowing the growth upwards of the pericardial sac (per.h.) whichcovers the auditory vesicle (aud.) and the eye (e.). The yolk (y.g.)lies behind the pericardium and the viteUine vein (v.v.) leaves thepericardial 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 reachesposteriorly beyond the ear region (Text-figs. 5, 6, per.h.). Themesodermal lining of the hood next to the body-wall adjoinsthe ectoderm covering the roof and sides of the brain, andalong 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 formsa 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

a.

y-9

parw.

v.v.

TEXT-JIG. 5.

Diagram of an embryo of approximately thirty somites. A peri-cardial hood (per.h.) now completely surrounds the head. Otherlettering as in Text-fig. 4. X100.

side beneath the external wall of the hood. Above the head theectoderm of this outer wall unites with that of the oppositeside forming a continuous sheet of ectoderm over the meso-dermal strand. The outer wall of the hood is thus analogousto the chorion of higher vertebrates. Posteriorly, below theauditory 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 / L 7 / I0?-L

ura

rra.c.

aud.

o.pt.c,

y-9

8

TEXT-FIG. 6.

Diagram of an older embryo from the right side. The pericardialhood has now reached its maximum development and en-circles the entire head, and the mesothelial walls of either sideare united to form a mesodermal strand {m.str.) from the eyeanteriorly to behind the auditory region (B to A). The amount ofyolk {y.g.) is reduced. The bladder (ur.bl.) has begun to expandand lies within the yolk; it opens to the exterior (ur.ap.) justbehind the anus (a.) and the Wolffian duct (W.D.) runs into itdorsally. The vessel from the right anterior cardinal vein (r.a.c.)is shown; it meets the pericardium at o.a.c. The posterior cardinalvein (pt.c.) comes from the tail and runs between the Wolffianducts meeting the pericardium at o.pt.c. h., heart; arrow op., posi-tion of operuculum. Other lettering as in Text-fig. 4. x 75.

m.str

DEVELOPMENT OF HETEBANDSIA 495

body-wall corves forwards; this bay marks the position of theoperculmn beneath which is a passage through the gils intothe pharynx (Text-figs. 6, 7 A, op.). The bay is commonlymore accentuated than is shown in Text-fig. 6.

msfcrm be

pane.

y-s-

urap.

TEXT-EIG. 7 (cp. Text-fig. 6).

A. Composite drawing from sections through, the posterior half of thebody of an embryo at about the same stage as Text-fig. 6 viewedfrom behind, the tail being removed. The cut edge of the peri-cardium and its hood is indicated by circles; between the two isa bay (cp. Text-fig. 6) in which lies the operculum (op.), internalto which is a passage from the cavity of the follicle between thegill 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 thepericardial wall at o.pt.c. The rectum (r.) opens to the exteriorclose 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 ofthe same embryo viewed from in front of the auditory regionforwards. 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 thewalls 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. Fourvessels connect the embryo with this network: (1) the largevitelline vein comes from the ventro-lateral, some vhat right,side near the lower end of the yolk-mass and runs up into theheart (Text-figs. 4-6, 7 A, 8, h.); (2) a vessel runs from theanterior 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 outwardsimmediately in front of the liver (I.) from which it receives ashort branch before joining the pericardial wall; (3) a thirdvessel runs down the tail and between the primordium of themesonephric tubules as the posterior cardinal (Text-figs. 6,7 A, pt.c.) which curves round ventrally below the rectum andthen backwards to join the vascular wall on the posterior-dorsalside of the yolk (Text-figs. 6, 7 A, o.pt.c); (4) a small vasculartwig running between the two layers of the mesodermal strandnear the level of the auditory region connects the vessels overthe brain with those on the pericardial wall. Some time beforebirth the pericardial hood begins to recede (Text-fig. 8, per.h.),so that shortly before the escape of the embryo from the follicleonly a small tubular portion still remains stretching above overthe hinder part of the eye (Text-figs. 9 A, 9 B) on each side. Afterbirth the pericardium has assumed the normal appearance ofa vesicle lying on the ventral side surrounding the heart, theyolk-granules having disappeared by this time.

E e s p i r a t i o n and Nutri t ion.—The enormous enlarge-ment of the pericardium with its extension round the head isa very striking feature, and is without doubt due to the need forincreased respiratory surface within the follicle. The entire wall,including that of the hood (figs. 14, 15, emb.c, PI. 29), istraversed by a rich vascular network, somewhat less welldeveloped over the area covered by the tail which curves roundeither to the right or left side of the body. Immediately outsidethe ectodermal wall of the pericardium is the zona, close to whichlie the swollen flattened cells of the follicular epithelium. Asdevelopment proceeds a fine network of maternal capillariesruns alongside the follicular wall (Text-figs. 10, 11, mate).

DEVELOPMENT OF HETEBAMJBIA

urap.497

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 themost anterior portion of the ear [mid.). The bladder (ur.bl.) hasattained its maximum development, filling almost the entire peri-cardium, and has pressed the last remaining yolk-granules (y.g.) tothe antero-ventral side of the pericardium. Other lettering as inText-figs. 4 and 6. x 75.

During later stages the maternal capillaries become greatly-enlarged and protrude between the cells of the follicle which arethen only united together by a fine strand of protoplasmstretched 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 thepericardial hood has begun to recede. Meanwhile, withTthe

TEXT-FIGS. 9 A, 9 B.

Drawing of an embryo removed from the follicle shortly before birthand measuring about 5 mm. in length, showing the further reduc-tion of the pericardial hood which now only covers the posteriorhalf of the eye. A, View of right side; B, ventral view. In B theheart is seen as a darker area through the transparent pigmentedwall of the pericardium from which many blood channels branchout over the wall.

growth of the embryo the zona becomes attenuated and, fora short period before hatching, is hardly visible. Oxygen forthe embryo has thus at first to penetrate two layers of cells,that of the ectoderm round the pericardium and that of thefollicle, 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 shrunkaway from the follicle during fixation. The follicular epithelium(f.ep.1) of an adjacent embryo is also seen, and between the two isconnective 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 proto-plasm separates the maternal capillaries from the wall of theembryo. 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. Thegill-slits break through at an early stage and communicatebehind the operculum (Text-figs. 6, 7 A, op.) with the follicularcavity posteriorly to the hinder boundary of the pericardialhood. The mouth does not develop for some time afterwards,when the pericardial hood has receded sufficiently, and then itopens 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 ofthe pericardium at a later stage than Text-fig. 10 showing theenlarged maternal capillaries (mate.) protruding between thefollicle cells (f.ep.). Other lettering as in Text-fig. 10. X 600.

derived from the parent through the follicle cells and laterdirectly 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 stagesby the mouth also. That a fluid of some kind exists is indicatedby the presence of a coagulum which may sometimes be seenafter fixation within the follicle outside the pericardium, andan apparently similar coagulum can occasionally be observed•within the stomach of older embryos and also passing out ofthe anus. Moreover, embryos extracted from the follicle areenveloped by viscous matter. As the tail grows out and in-creases in length and breadth the capacity of the follicular cavityincreases also, and there is more room for follicular fluid. Themature egg has an average diameter of 0-33 mm. after fixation.There is very little increase in size until after the vesicle isencircled by a cellular layer, at which time the diameter is stillonly about' 0-34 or 0*35 mm. When the embryonic rudimentbegins to develop both vesicle and follicle gradually expand(cp. Text-figs. 5 to 8), and shortly before the embryo hatchesthe follicle may have a diameter of 2 mm. or more, but usuallyat this late stage it is rather oval in shape, attaining as muchas 2-5 mm. across the longer diameter.

U r i n a r y Bladder.—In embryos of about eighteensomites the two archinephric ducts are connected with thedorsal wall of the posterior part of the gut; soon they pass intoa small vesicular swelling which opens to the exterior just behindand above the anus (Text-figs. 6, 7 A, ur.ap.). Originally quitesmall the vesicle increases rapidly in volume and finally ex-pands into a urinary bladder of enormous dimensions whichreaches its maximum size when the two sides of the pericardiumhave begun to recede from the dorsal side of the head (Text-fig. 8; fig. 4, ur.bl., PL 26). At first the bladder grows forwardsthrough the centre of the yolk-sac, where yolk-granules arescarce or absent (Text-fig. 7 B, ur.U.), and as the yolk becomesused up it enlarges and subsequently far exceeds the areaformerly occupied by the latter, eventually spreading acrossthe entire breadth of the pericardium and reaching forwards infront of the heart (fig. 4, PI. 26, and Text-fig. 8, ur.bl). As thebladder 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 withthe network of blood-vessels formerly lining the pericardium andthe external surface of the yolk (fig. 4, PL 26). This vascularnetwork, however, soon becomes reduced over the area coveredby the bladder.

At birth the bladder collapses suddenly, and in the newly bomyoung it is merely a small thick-walled swelling into which theWolffian ducts enter, and which opens to the exterior immedi-ately behind the anus.

A short time before the embryo has finished its developmentwithin the mother, the cells of the solid fertilization plugbecome absorbed; the protoplasm becomes vacuolated, thenuclei disappear, and only a network of vaeuolated tissueremains (figs. 14,15, PL 29, and fig. 3, PL 26, pi). This partlyoozes out into the ovarian cavity and partly thins out in s i t u ,and through the gap of degenerated tissue thus created theembryo escapes into the cavity and down the oviduct immedi-ately to the exterior. Simultaneously, the cells of the emptyfollicle enlarge, their chromosomes elongate, they becomevaeuolated and send out long pear-shaped processes into theempty follicle. These disintegrate together with the blood-vessels below them and flow into the cavity, the latter becomingquickly filled with a disintegrating mass of follicle cells, blood-corpuscles, and even sheath cells. Erom the follicle masses ofdecomposing tissue travel down the oviduct, which at thisperiod is much widened and consists of a thin layer of epi-thelium surrounded by a thick layer of longitudinal muscles,villi being no longer present. It is by the contraction of thislayer of muscles that the embryo is forced down the oviductto the exterior. Contractions of the oviduct were clearly seenduring the birth of an embryo immediately after the death ofthe parent. As the duct during birth and immediately after-wards is very expanded and consequently without villi, thedebris from the burst follicle must go down the duct and directlyto the exterior, and no ingestion by the cells of the wall wasobserved 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 ofviviparous fishes, Eigenmann in 1892 was the first to go intothe iife-history and development in detail when hi studied theBmbiotieid fish Oymatogas t e r . Eigenmann states thatviviparity 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 theovary. In the first type the egg remains in the follicle untilnear the end of gestation (Poeci l ia , Gambus ia , Scor-poenidae) and fertilization is affected within the follicle; thenumber of young bom at one time occasionally reaches manythousands, a i in Sebas tomus (Eigenmann), but is usuallyconsiderably less. In the second type (Zoarces, B lenn ius ,Anahlepg., Embiotooidae, also Goodeidae and J e n y n s i a(Turner, 1088-4)), the length of time th© embryo remains in thefollicle varies, as also does the time spent in the ovarian cavity;the egg may be fertilized within the follicle or in the ovariancavity. The number of young is here much smaller, althoughthey may be larger in size. In Oymatogas te r Eigenmannnotes that the number of young is directly proportional to thente® of the fish, the average number being about twelve, varyingaccording to size of parent; if the fish is small then the firstyoung are also of small size, and this is the case in H e t e r a n -dria and others, as for example in Zoarees (Stuhlmann,.1887; Wallace, 1908-4; Kolster, 1905). The conditions inH e t e r a n d r i a , however, correspond to neither of Eigen-mann's types, and is unique in that the embryo remains withinthe 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 Oymatogas te r resembles thatof H e t e r a n d r i a , and therefore a comparison between thetwo is of interest. In Oymatogas t e r (Eigenmann) copula-tion takes place in June or beginning of July, at a time when onegeneration of young are being set free (April to June) andspermatozoa remain in the ovary until the next series of eggsbecomes mature at about the month of December, when theyare then fertilized. In Oymatogas te r the eggs in one

DEVELOPMENT OF HETEBANDRIA 505

generation are all born at one time, and this holds for themajority of viviparous fish. In contrast to H e t e r a n d r i athe egg is freed from the follicle before cleavage begins; al-though fertilization was not observed it probably occurs justbefore or just after the egg escapes; for in a single case thesecond polar body was extruded, and a male pronucleus seenin an egg still enclosed within the follicle. The amount of yolkis much reduced, and the dividing cells of the egg encircle itat an early stage during the end of the eleventh cleavage (1,700cells) before the ectoderm is differentiated. In H e t e r a n d r i athe exact moment at which the encirclement of the yolk takesplace is difficult to determine as it occurs with great rapidity;between the seventh and eighth cleavage the cells surroundthree-fifths of the egg, and therefore the period at which acomplete lining is established probably corresponds to aboutthe eleventh cleavage stage as in Oymatogas te r as cal-culated by Eigenmann. In the latter, however, the yolk is notsurrounded by a single layer of cells but by several layers and,moreover, the edges of the blastoderm are separated for a shortperiod by a kind of yolk-plug called by this author the yolk-nucleus. The appearance of the egg at this stage will be madeclear by Text-fig. 13 which shows photographs of Eigenmann'sfigs. 85, 38. In this manner the young embryo at an early stagelies around the entire yolk-sac, a condition that never occursin H e t e r a n d r i a .

The periblast cells in Oymatogas te r are reduced tobetween ten to eighteen cells, averaging twelve in number, andin no case exceeding twenty, a condition comparable to thatin H e t e r a n d r i a , where these cells also number from twelveto eighteen. Without doubt this reduction is due to the scarcityof yolk in each case; a continuous layer of periblast cells as inother Teleosts would thus be superfluous.

The primitive germ-cells in Cymatogas t e r (Eigenmann,1891 and 1896-7) can be seen at a very early stage before theprotovertebrae are formed, when an average of twelve (9-23,the latter figure only once) were counted. On one occasion acell resembling a germ-cell was observed at the fifth segmenta-tion, but this needs further confirmation. In H e t e r a n d r i a

'•yk.pr.

TEXT-FIG. 13.

Reproduction of embryos of C y m a t o g a s t e r from Eigenmann(PL 96, 1892).

A.—Tenth section through early gastrula and blastopore. Theegg contained about 3,000 nuclei; twelfth segmentation. Thesection cuts through the embryonic axis obliquely. The entodermis 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 segmenta-tion, slightly older than the one figured in A. The yolk-nucleusis bluntly conical, forming a plug in blastopore. ril.per., periblastnuclei.

DEVELOPMENT OF HBTBEANDBIA 507

they are first visible at a slightly earlier period; immediatelyafter the unilamimr ectoderm has grown round the yolk-vesicle the underlying cells of the embryonic disc divide upinto a number of larger, very conspicuous germ-cells and intoa mass of smaller endo-mesodenn cells. At first about twenty-five to thirty primitive germ-cells can be counted, but theyundergo continuous division until between fifty to sixty arepresent. In L e b i s t e s r e t i c u l a t u s Goodrich, Dee, Flynn,and Mercer (1934) state that when the germ-cells are firstrecognizable they are scattered in the mesendodennal layerof blastoderms with a diameter of 1-3 mm. and an embryonicshield measuring 0-5 mm. along its longitudinal axis; hereforty were estimated. To what stage in H e t e r a n d r i a thiscorresponds it is impossible accurately to determine, but it maybe about the same time, and if so, in these two viviparousgenera we have the earliest record of the appearance of the germ-cells in the Teleostei. In later stages during their migrationinto the genital ridge the original number of primitive germ-cells is not exceeded. Such a resting period is present in otherTeleosts, as for example in Loph ius (Dodds, 1910-11), wherethe average number is thirty-seven and they undergo no furtherdivisions until the ridge is attained. The early history and fateof the germ-cells in viviparous fishes I hope to consider morefully in a future paper.

The spermatozoa in many viviparous fishes collect in pocketsof the ovarian epithelium, and it has already been noted thatin H e t e r a n d r i a they serve for the fertilization of manyseries of ova. Tubular prolongations from the ovarian cavityin which spermatozoa collect have been described by variousworkers. Lane (1909 and 1903-4) figured them in the Brotulidfish Luci fuga and S tyg i co l a , where they are exactlysimilar to those of H e t e r a n d r i a , and it was suggested thattheir function might be to afford an entrance for the sperma-tozoa. In Leb i s t e s also, both Thirumalacher (not yet pub-lished) and Liu (not yet published) have recently noted homo-logous pockets of the ovarian wall lying close to those of thefollicles. Identical structures were observed by Phillippi (1908-9) in G l a r i d i c h t h y s 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 occasionsonly, he noticed a small process from the follicle on the floor ofthe pocket which was calculated to be about five tc six timesthe head of a spermatozoon, and within the follicular epitheliumintercellular spaces were visible. This process he called thepropyle, and it may very possibly be the initiation of a pro-tuberance from the egg such as that figured in H e t e r a n d r i a(fig. 7, PI. 27). Such a prominent outgrowth as is found in thelatter genus has not previously been seen, very possibly owingto its transitory nature, as already indicated. It may, however,not be so well developed in all viviparous fish or may even beabsent, in which case the spermatozoa would have to penetratethrough the follicular epithelium.

In Zoarces (Stuhlmann, 1887) the follicles are situated onpapillae which project into the ovarian cavity; at the distalend of each papilla is a circular hollow ('Delle') over which thefollicle cells and those of the ovarian epithelium are closelyapposed. This formation is shallow, and through it the eggbursts into the cavity where it is fertilized. Tubular pockets suchas are present in H e t e r a n d r i a have been observed in someother Viviparous fish and erroneously compared with the' Delle'(i.e. Lane, 1903-4), but they are quite different both in structureand function, here serving merely as an area through which thespermatozoa can more easily reach the egg.

Degeneration in H e t e r a n d r i a occurs very extensively inmature 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 H e t e r a n d r i a it appears togo on to some extent throughout life; in older females, as alreadymentioned, this is partly due to compression of the young ovaby growing embryos. The process apparently takes place muchas in L e b i s t e s (Liu), except that in the latter much yolk ispresent which has to be absorbed by the proliferating cells.The material from the disintegration of the egg is used as foodfor the further growth of the ova in both fishes; but, whereas inLeb i s t e s it is carried by the epithelial cells and the blood toneighbouring regions but mainly to the stroma, in H e t e r a n -dr ia 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. Nosuch absorption has been observed in L e b i s t e s , but inGambus ia H o l b r o o k i i Thirumallacher (not yet pub-lished) has noted the presence of longitudinal ridges in theoviduct which contained ' masses of cells with granules of yellowpigment'; in some places these masses appeared to be 'burstinginto the lumen of the ovary', but very probably it is ingestionwhich is in reality going on here. A small part of the disin-tegrating material may be left in s i t u as a mass of yellowgranules. Yellow masses of this kind have been noted inZoarces by Wallace (1903-4), in Lueifuga and Stugi -cola by Lane (1903-4), and in Leb i s t e s amongst others.

The spent follicle has been described and figured by variousinvestigators and calls for no special discussion. Cunningham(1893-4 and 1897-8) puts forward the suggestion that theepithelium of a spent follicle in the plaice, here opening on tothe external surface of the ovary, becomes restored and oncemore forms a part of the germinal epithelium. In viviparousfishes the spent follicle is related to the wall of the ovariancavity and there is no direct evidence in H e t e r a n d r i a , or inL e b i s t e s (Liu), that it becomes again a part of this epithelium,although such a suggestion has been put forward by Thiru-malacher for L e b i s t e s . The ultimate fate of the spentfollicle in H e t e r a n d r i a was not determined.

At an early cleavage stage the egg of H e t e r a n d r i a withits limited yolk is comparable to the blastocyst of mammals.Blastomeres grow out from the peripheral portion of the blasto-disc when composed of only a few cells (195 or less), and theblastocyst very quickly comes to be surrounded by a unilaminarectoderm which is closely applied to the zona. In the Teleosteias in the Mammalia the development of such a blastodermicvesicle is due to loss of yolk. Until the vesicle is completelyenclosed by cells the diameter of the egg increases only slightly,but afterwards growth is very rapid. The unilaminar ectodermis analogous to the trophoblast of mammals and has a com-parable destiny, for it later forms the ectodermal wall of theyolk-sac and eventually the bladder, and gives rise to the ecto-dermal 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 chorionis of course restricted in H e t e r a n d r i a , for it only envelopsthe anterior portion of the animal, as far as the a; us posteriorlyand up dorsally to behind the ear, and does not include the tailwhich lies curved round free in the cavity of the follicle. Theouter layer of the hood or chorion is composed of two layers,ectoderm externally and an inner lining of mesoderm closelyadjacent to it. Mesoderm also covers the ectoderm of the sidesand roof of the head of the embryo, and the cavity within thehood is extra-embryonic coelom. The chorionic ectoderm be-comes continuous over the head, and from the eye to behindthe auditory region over the mid-dorsal line the mesodermforms a two-layered mesothelial membrane. There is then noectomesodermal amnion as present in the Amnio ta ; theectoderm takes no part in the fold on the side next to the head,the additional protection of an amniotic cavity being apparentlyunnecessary for development within the follicle.

The pericardium in many viviparous fish often reaches alarge size, and behind it lies the yolk. Its structure has beenwell described in J e n y n s i a by Scott (1928), and Eigenmann(1892) has figured the same condition in C y m a t o g a s t e r ,where a pericardium is developed many times larger than thevery small yolk-sac. In Gambus ia p a t r u e l i s Eyder(1885) described and figured a tubular 'process of the yolk-bag'which "is prolonged upwards over the head behind the eye tomeet its fellow of the opposite side; he thinks the same structureis present i n P u n d u l u s , where he noticed a vascular membraneon either side of the head. Eecently Turner (1937), in hisobservations on the breeding of H e t e r a n d r i a , noticed thatin the immature fish extracted from the follicle a structurewhich he terms the 'yolk-sac' surrounded the head. Its wallwas vascular and was in contact with the follicle up to the timeof birth. Such a remarkable extension has never previouslybeen recorded in any other fish. In the Goodeidae Turner(1933-4) also speaks of a 'yolk-sac', which at its maximumdevelopment partly envelops the anterior end of the head, andin a transverse section of L e r m i c h t h y s m u l t i r a d i a t u she 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 hatchmuch earlier than H e t e r a n d r i a and the later developmenttakes place in the ovarian cavity. It appears, therefore, as ifthe pericardial sac in this group has not had the time to reachthe same degree of development in the follicle, and once in theovarian lumen it is no longer required. The early developmentof the egg in the Goodeidae and the origin of the pericardiumis not described by Turner, and there is ambiguity between theterms' yolk-sac' and' pericardium'. The pericardium is a cavitylined by mesoderm cells; as it grows it comes to lie in front ofthe yolk which is finally surrounded by a vascular network.The yolk-sac and the pericardium are not synonymous struc-tures and it is incorrect to speak of the yolk-sac as being filledby the hypertrophied pericardium. In Teleostei the yolk isusually enclosed by periblast cells, in H e t e r a n d r i a meso-derm alone forms the wall of the sac. Turner does not givedetails of the cell-layers, nor are these shown in his figures; noperiblast layer is mentioned but presumably it is formed, forthe amount of yolk, although not large and absorbed by thetime of hatching, is much more abundant than in H e t e r a n -d r i a ; Turner compares the conditions as regards yolk withthose in J e n y n s i a where a definite layer of periblast cellsdoes surround the yolk (Scott).

In the wall of the pericardium and extending round the pos-terior side of the yolk runs a vascular network. The blood-vessels which connect this network with those in the body ofthe embryo are described in Gambusia by Ryder (1885), inAnab leps by Garman (1895-7), but rather more fully inJ e n y n s i a by Scott (1928), where they correspond very closelywith those in H e t e r a n d r i a . A detailed account of thedevelopment of these vessels in viviparous fish and an accuratedescription of the changes undergone during young stages upto the condition in the adult have never been undertaken. Thevascular area over the pericardium in viviparous fishes providesa respiratory surface by which the embryo may obtain oxygen,whilst nutriment is supplied by the yolk in those fish in whichit is present; when the yolk later diminishes or is much reducedfrom the first as in C y m a t o g a s t e r (Eigenmann), food is

NO. 324 L1

512 ELIZABETH A. FEASBE AND KACHEL M. RENTON

obtained by various methods from the ovary. In forms suchas Zoarces which undergo their whole development in theovarian cavity the epithelial lining of the wall degt nerates andmaterial is discharged into the cavity. The nature of theepithelium and the material ejected, such as lymphocytes, con-nective tissue, erythrocytes, fat, &c, change with the growthof the embryo, a process which has been explained and analysedin detail by Kolster (1905). The yolk in Zoarces is notabundant and is insufficient. The hind-gut is greatly hyper-trophied, filling the abdomen, and its inner epithelium providedwith vascular processes. Into these the red blood corpusclesengulfed by the mouth from the ovarian fluid, or so-calleduterine milk, are absorbed and from them Stahlmann (1887)suggests that oxygen may be obtained, respiration being adifficult process where, as in this fish, large numbers of youngare crowded together in the ovary.

In J e n y n s i a (Scott) the embryo is nourished entirely bythe yolk until it hatches into the ovarian cavity, when the longvascular villi of the ovarian epithelium then penetrate betweenthe gills into the pharynx. In some genera the whole embryomay lie closely apposed to these villi. In some of the Embioto-cidae, as in A n a b l e p s , respiration is effected by the dorsal,caudal, and anal fins which are provided with long delicatevascular filaments with a specially developed blood supply(Eyder, 1885). An interesting adaptation is present in the familyGoodeidae (Turner, 1933-4, 1937), where elongated vascularprocesses arise from the body-wall around the anal opening.These are for nutrition and possibly also for respiration, bothin the follicle where they first develop and later in the ovariancavity. Anab leps Grovnoi i (Wyman, 1864; Eyder, 1885;Garman, 1895-7, Turner, 1938) loses its yolk while still in thefollicle, and albuminous matter is taken up from the follicularcavity by rows of vascular papillae which develop in the wallof the yolk-sac; this modification may serve for respirationalso.

In H e t e r a n d r i a the entire development takes placewithin the follicle, but on the other hand there is little yolk andboth respiration and nutrition must be effected through the

DEVELOPMENT OF HETEEANDBIA 513

parent. The expansion of the pericardium and the developmentof a pericardial hood are doubtless correllated with the needfor an extension of the vascular area for respiration, and thisis accomplished by the close apposition of the pericardial andchorionic ectoderm to the follicle cells, which last take on therole of the uterine epithelium in higher vertebrates. The modi-fication of the follicle cells with their slender junctions over thematernal capillaries, as shown in Text-fig. 12, demonstrates astriking resemblance to the uterine epithelium in the matureallantoplacenta of Lygosoma o cellatum figured by Weekes(1930, Text-fig. 5 a), where the uterine epithelium is extremelythin over the maternal capillaries which are thus exposed andare in close apposition to the chorionic ectoderm below whichrun the embryonic capillaries. In the ease of the lizard thelatter capillaries are allantoic. My Text-fig. 10, where thematernal capillaries have begun the invasion of the folliclecells, also corresponds to an early stage in the development ofthe placenta in E g e r n i a Gunninghami (Weekes, 1930, Text-fig. 1), where the chorionic membrane is established but theallantois is only a small vesicle at the posterior end of the embryo.In H e t e r a n d r i a similar conditions are met with round theyolk, so that in this viviparous fish we have the simplest typeof placenta, established in relation to the yolk and to the outerwall 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 forthe passage of the food into the follicular cavity into whichopen the gills and later the mouth. This follicular fluid is themain source of nutrition for the growing embryo.

The presence of an enlarged urinary bladder in the embryoof H e t e r a n d r i a is a surprising feature. Some adult Teleostspossess a large urinary bladder, a description of which has beengiven by Hyrtl (1851). It is interesting to find that Duvernoy(1844) noted a thick-walled bladder in the adult of Poeci l ias u r i n a m e n s i s and drew attention to a dilated bifurcatedbladder in two preserved foetuses which he figured; as sucha unique condition had not been observed before he regrettedthat 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 organin the embryo has never been noticed in any Teleost.

The urinary bladder in the Teleostei (according to Felix, inSalmo, 1897) arises as an outgrowth from the dorsal wall of thehind-gut at the place of union of the two archinephric ducts. Aslong ago as 1866 Kupffer studied the development and comparedthe bladder to the allantois of higher vertebrates. The allantoisgrows out from the v e n t r a l side of the same portion of thegut and this difference in origin does not seem to me to be afundamental one. In the Eeptilia where it exists and the originis known, a large part of the bladder is derived from the stalkof the allantois. In the Marsupialia the main portion is formedfrom a part of the intra-abdominal allantoic stalk, and in highermammals the proximal end of the stalk may share in itsdevelopment.

In H e t e r a n d r i a the pronephros is well developed in theembryo and the function of the bladder is presumably for thestorage of urine. A similar function has been ascribed to theallantois in those mammals where there is an active excretoryorgan in the foetus (marsupials, pig, sheep, eat). In H e t e r a n -dr ia the bladder communicates with the exterior by a smallaperture and some of the fluid excreted can escape by thisroute. There is the probability also that the bladder is respira-tory, taking over the function of the pericardium of earlierstages, for at its maximum development it completely fills thearea formerly occupied by the latter except where the vitellinevein passes up from the pericardial wall into the heart; it neverinvades the hood which has begun to recede at this time. Inviviparous lizards, according to Weekes (1935), the allantois doesnot grow round the yolk-sac but only expands as the sac dimin-ishes in size just as it does in H e t e r a n d r i a . During its earlydevelopment when it pushes up into the centre of the yolk-sacbetween the yolk-granules the ventral wall comes to be in closecontact with the vascular network formerly over the yolk, andtherefore it would seem that, for a brief period, a structurethat simulates an allantoic placenta is present. Soon, however,the vessels of this vascular network are apparently much re-duced in calibre.

DEVELOPMENT OP HETERANDEIA 515

The brief period during which the embryo is retained in thefollicle in the Goodeidae is supposed by Turner (1933-4) to bedue to the small amount of yolk. This supposition cannot bethe correct one, for H e t e r a n d r i a is able to obtain sufficientnourishment from the parent until birth, a period of perhapsfive weeks. This author attempts to differentiate between vivi-parity and ovoviviparity in these fishes. He defines viviparityas a process by which the embryo is not only retained internallybut is dependent on the ovary for nutrition, whereas ovovivi-parity is understood as the process in which the embryo isretained internally but is dependent on yolk for sustenance.The family Embiotocidae is thus viviparous whilst the familyPoeciliidae is ovoviviparous. In the Goodeid type which ishatched relatively early, ovoviviparity is said to be super-imposed by viviparity. Such a definition cannot hold for thecase of H e t e r a n d r i a , a member of the Poeciliidae, in whichthe whole of development takes place in the follicle but nutri-ment and respiration are obtained almost entirely from the parentthrough the vascularized follicular wall. The condition inH e t e r a n d r i a may be a further development of that begunby the Goodeidae, but more details of the early developmentof other forms are needed for adequate comparison. It is morerational to reject the term ovoviviparous altogether, especiallywith reference to Teleostei, and to regard all the Teleosts whichbring forth living young as viviparous.

To Professor J. P. Hill I am much indebted for his valuablecriticism. Mr. J. E. Norman has given me assistance withregard to correct nomenclature. I wish to express my gratefulthanks to Miss Joyce Townend for the drawings reproducedas Text-figs. 4 to 9. For the microphotographs I have to thankMr. J. E. Thomas of this department.

SUMMARY.

1. A short account is given of the breeding habits of He t e r -a n d r i a formosa in an aquarium.

2. The ovary and the mature ovum are briefly described, thenoteworthy feature of the ripe ovum being the small quantityof yolk.

5 1 6 ELIZABETH A. FBASEB AND RACHEL M. EENTON

8. Degeneration of ova is found to be a common occurrence inunfertilized females, and to a less extent in those full of develop-ing embryos.

4. The method for ensuring the fertilization of the egg withinthe follicle is portrayed. Over the area where the spermatozoahave entered the cells of the ovarian epithelium and those of thefollicle form a solid plug which eventually disrupts to enablethe fully developed embryo to escape into the cavity of theovary.

5. It is characteristic of early development that the egg isencircled by a unilaminar ectoderm before there is any visibledifferentiation into endoderm and mesoderm.

6. Owing to the scarcity of yolk only a few periblast cells ariseand no syncytial layer is formed.

7. The primitive-germ cells are visible at an early stage withinthe apparently undifferentiated mesendoderm cells.

8. A striking feature is the large size of the pericardium and itsgrowth upwards as a pericardium hood which completely sur-rounds the head region of the embryo. Over the walls runs a net-work of blood-vessels from which the maternal capillariesbecome eventually separated by only an attenuated layer ofprotoplasm. Both respiration and nutrition are effected throughthe follicle.

9. A remarkable specialization is the development of a urinarybladder which expands into a thin-walled vesicle of enormousdimensions, finally occupying almost the entire area formerlyfilled by the pericardium and the yolk-granules.

10. The development of H e t e r a n d r i a is compared withthat of other viviparous fishes.

11. The significance of the unusual features in early and latedevelopment is discussed and some comparisons are made withthe 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 atthe Plymouth Laboratory. II . Development of the Egg in Flat Fishesand Pipe Fishes", 'Journ. Mar. Biol. Ass.', 3.

1897-8.—"Histology of the Ovary and Ovarian Ova in certainMarine 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 (Poeciliasurinamensis 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 NorthAmerica", '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 BlindFishes, 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 TeleosteerGlaridichthys 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 theSub-family Poeeiliinae", 'Proo. zool. Soc. Lond.', 2.

Ryder, J. A., 1885.—"Development of Viviparous Osseous Fishes and ofthe Atlantic Salmon", 'Proc. U.S. nat. Mus.'

Scott, M. I. H., 1928.—"Sobre el Desarrollo Intraovarial de Fitzroyialineata Berg.", 'An. Mus. nac. B. Aires.', 34.

Seal, W. P., 1911.—"Breeding Habits of the Viviparous Fishes GambusiaHolbrookii andHeterandriaformosa", 'Proc. Biol. Soc. Washington', 24.

Stuhlmann, F., 1887.—'Z. K. des Ovariums der Aalmutter (Zoarces vivi-parus 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-viviparityin the Goodeidae", 'Journ. Morph.', 55.

—1936.—"Absorbtive Processes in the Embryos of Parabrotuladentens, 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 ofCymatogaster aggregatus during Gestation", ibid., 62.

Wallace, W., 1903-4.—"Observ. on Ovarian Ova and Follicles in CertainTeleostei and Elasmobranch Fishes", 'Quart. Journ. Micr. Sci.', 47.

Weekes, H. C, 1930.—"Placentation in Retiles. I I " , '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, periblastcell; per.c, pericardial cavity; per.h., pericardial hood; per.w., wall ofpericardium; 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 5 1 9

PLATE 26.

Fig. 1.—Microphotograph of a longitudinal section through an ovary-containing three embryos (emb. 1, 2, 3) of different ages and showing theovarian cavity (ov.c.) along the dorsal side running into the oviductposteriorly. 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 containingtwo large embryos (emb. 1, 2) and one smaller one (emb. 3). The ovariancavity (ov.c.) lies at the dorsal side and is surrounded by ova (ov.) ofvarious sizes. X 35.

Fig. 3.—Part of fig. 2 enlarged to show the ovarian cavity (ov.c.) sur-rounded by developing ova. The smaller ova contain a reticulum with fineyolk-granules; in the larger one the contraction of these towards the peri-phery 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 anembryo shortly before birth, comparable with that in Text-fig. 8, showingthe anterior end of the expanded urinary bladder (ur.bl.) occupying theplace formally filled by the pericardium and yolk and extending forwardsas far as the ventral aorta (aort.) (cf. fig. 15, PI. 29). The section is some-what oblique and passes through the eye (e.) on the one side and thethymus (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 showingthe 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 thenucleus (n.) beneath which is a deeper zone of yolk than round the circum-ference of the egg. x 200.

Fig. 7.—^Microphotograph of a section through a spherical protuberancefrom an egg into the ovarian cavity (ov.c.) at the time of fertilization.Crowds of spermatozoa (sp.) are congregated over the thin membrane ofthe protuberance. The zona (2.) and the follicular epithelium (f.ep.) axeabsent beneath the protuberance, but round the proximal part of the latterthe 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 thevesicle is completely encircled by a unilamellar ectoderm (unU.ect.). Theembryonic disc is seen at one side and among them the larger primitivegerm-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 coagulablefluid, (coag.). The zona has shrunk considerably during fixation and isseparated from the cells of follicle. X 200.

PLATE 28.

Fig. 10.—Drawing of a section through a blastodisc of approximately194 cells. The cells of the disc are multinuclear. A few cells (unil.ect.) havebegun to migrate from the periphery outwards beneath the zona and thesealtogether encircle approximately three-eighths of the circumference of theegg at thin stage. X 400.

Fig. 11.—Drawing of a section through the blastodiso of a stage com-parable to flg. 0 showing the primitive germ-cells (g.e.) now distinguishablefrom 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 aslightly older atage. Mitosis can be seen in some of the cells of thedisc. The superficial cells are now separated off as the future ectodermallayer 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 theegg. Note the jaggod edges of the follicle cells where the zona has shrunkduring 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 isa periblast cell (pb.c). The section has passed through the inner portionof the fertilization plus (pi.). X300.

PLATE 29.

Fig. 14.—Mierophotograph of a section passing through the eyes andheart of the embryo in Text-fig. (5 showing the pericardial hood (per.h.)which has grown dorsally round tho brain. The mesodermal lining of thehood of eaoh side meets dorsally as the mesodermal strand (m.slr.). Theyolk lies beneath the heart. U, tail. X 133 (approx.)

Fig. 15.—Miorophotograph of a portion of the right side of fig. 14enlarged to show the vacuolated plug (pi.), the pericardial in which runthe embryonic capillaries (emb.c), and the mesodermal strand (m.str.).X266 (approx).

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