L^A^ A TREATISE ON COMPARATIVE EMBRYOLOGY. A TREATISE ON COMPARATIVE EMBEYOLOGY BY FRANCIS M. BALFOUR, LL.D., F.R.S., I'ELLOW AND LECTUBER OF TRINITY COLLEGE, CAMBRIDGE. IN TWO VOLUMES. VOL. II. MACMILLAN AND CO. 1881 [The Right of Translation is reserved. . papilla for attachment. VI. Body and anterior part of the tail of a two days' larva, klm. atrial aperture; en. endostyle; ks. branchial sack; lAs, 2/i«. branchial slits; hh. branchial vessel between them ; ch. axial portion of notochord ; chs. peripheral layer of cells. Other reference letters as before. 12 THE TEST. During the above changes the tail becomes considerably elongated and, owing to the larva being still in the egg-shell, is bent over to the ventral side of the trunk. The larva at this stage is represented in a side view in fig. 8 iv. The epidermis is formed throughout of a single layer of cells. In the trunk the mesenteron is shewn at dd and the dilated part of the nervous system, no longer communicating with the exterior, at 7i. In the tail the notochord is shewn at ch, the muscles at m, and the solid remnant of the ventral wall of the archenteron at dd'. The delicate continuation of the neural canal in the tail is seen above the notochord at n. An optical section of the tail is shewn in fig. 6. It is worthy of notice that the notochord and muscles are formed in the same manner as in Amphioxus, except that the process is somewhat simplified. The mode of disappearance of the archen- teric cavity in the tail, by the employment of the whole of its walls in the formation of various organs, is so peculiar, that I feel some hesitation in accepting Kowalevsky's statements on this head\ The larva continues to grow in length, and the tail becomes further curled round the ventral side of the body within the egg- membrane. Before the tail has nearly reached its full length the test becomes formed as a cuticular deposit of the epiblast cells (O. Hertwig, No. 13, Semper, No. 37). It appears first in the tail and gradually extends till it forms a complete investment round both tail and trunk, and is at first totally devoid of cells. Shortly after the establishment of the test there grow out from the anterior end of the body three peculiar papillye, developed as simple thickenings of the epidermis. At a later stage, after the hatching of the larva, these papillae develop glands at their extremities, secreting a kind of glutinous fluids After these papillae have become formed cells first make their appearance in the test ; and there is simultaneously formed a fresh inner cuticular layer of the test, to which at first the cells are confined, though subsequently they are found in the outer layer also. On the appearance of cells in the test the latter must be regarded as a form, though a very abnormal one, of connective tissue. When the tail of the larva has reached a very considerable length' the egg-membrane bursts, and the larva becomes free. The hatching takes place in Asc. canina about 48 — CO hours after impregnation. The free larva (fig. 8 V.) has a swollen trunk, and a very long tail, which soon becomes straightened out. It has a striking resemblance to a tadpole {vide fig. 10). In the free larval condition the Ascidians have in many respects a higher organization than in the adult state. It is accordingly 1 It is more probable that this part of the alimentary tract is equivalent to the post-anal gut of many Vertebrata, which is at first a complete tube, but disappears later by the simple absorption of the walls. * It is probable that these papillaj are very primitive organs of the Chordata. Structures, which are probably of the same nature, are formed behind the mouth in the larvas of Amphibia, and in front of the mouth in the larvae of Ganoids (Acipenser, Lepidosteus), and are used by these larvc-e for attaching themselves. UROCHORDA. 13 convenient to divide the subsequent development into two periods, the jfirst embracing the stages from the condition represented in fig. 8 V. up to the full development of tlie free larva, and the second the period from the full development of the larva to the attainment of the fixed adult condition. Growth and Structure of the free larva. The nervous system. The nervous system was left as a closed tube consisting of a dilated anterior division, and a narrow posterior one. The former may be spoken of as the brain, and the latter as the spinal cord ; although the homologies of these two parts are quite uncertain. The anterior part of the spinal cord lying within the trunk dilates somewhat (fig. 8 V. and vi. Rg) and there may thus be distinguished a trunk and a caudal section of the spinal cord. The original single vesicle of the brain becomes divided by the time the larva is hatched into two sections (fig. 9) — (1) an anterior vesicle with, for the most part, thin walls, in which unpaired auditory and optic organs make their appearance, and (2) a posterior nearly solid cephalic ganglion, through which there passes a narrow continuation of the central canal of the nervous system. This ganglion consists of a dorsal section formed of distinct cells, and a ventral section formed of a punctated material with nuclei. The auditory organ ^ consists of a ' crista acustica ' (fig. 9), in the form of a slight promi- nence of columnar cells on the ventral side of the anterior cerebral vesicle ; to the summit of which a spherical otolith is attached by fine hairs. In the crista is a cavity containing clear fluid. The dorsal half of the otolith is pigmented : the ventral half is without pigment. The crista is developed in situ, but the otolith is formed Fig. 9. Labva of Ascidia mentula. (From Gegenbaur ; after Kupffer.) Only the anterior part of the tail is represented. N'. anterior swelling of neural tube; N, anterior swelling of spinal portion of neural tube; n. hinder jmrt of neural tube; ch. notochord; K. branchial region of alimentary tract ; d. oesophageal and gastric region of alimentary tract ; 0. eye ; a. otolith; o. mouth; s. papilla for attachment. from a single cell on the dorsal side of the cerebral vesicle, which forms a projection into the cavity of the vesicle, and then travels (in ^ For a fuller account of the organs of sense inde the chapters on the eye and ear. U F.YE. a manner not clearly made out) round the right side of the vesicle till it comes to the crista ; to which it is at first attached by a narrow pedicle. The fully developed eye (figs. 8 vr. and 9, 0) consists of a cup-shaped retina, which forms a prominence slightly on the right side of the posterior part of the dorsal wall of the anterior cerebral vesicle, and of refractive media. The retina is formed of columnar cells, the inner ends of which are imbedded in pigment. The refractive media of the eye are directed towards the cavity of the cerebral vesicle, and consist of a biconvex lens and a meniscus. Half the lens is imbedded in the cavity of the retina and surrounded by the pigment, and the other half is turned toward a concavo-convex meniscus which corresponds in position with the cornea. The de- velopment of the meniscus and lens is unknown, but the retina is formed (fig. 8 V. a) as an outgrowth of the wall of the brain. At the inner ends of the cells of this outgrowth a deposit of pigment appears. The trunk section of the spinal cord (fig. 9, N) is separated by a sharp constriction from the brain. It is formed of a superficial layer of longitudinal nervous fibres, and a central core of ganglion cells. The layer of fibres diminishes in thickness towards the tail, and finally ceases to be visible. KupfFer detected three pairs of nerves passing off from the spinal cord to the muscles of the tail. The foremost of these arises at the boundary between the trunk and the tail, and the two others at regular intervals behind this point. The mesoblast and muscular system. It has already been stated that the lateral walls of the archenteron in the tail give rise to muscular cells. These cells lie about three abreast, and appear not to increase in number ; so that with the growth of the tail they grow enormously in length, and eventually become imperfectly striated. The mesoblast cells at the hinder end of the trunk, close to its junction with the tail, do not become converted into muscle cells, but give rise to blood corpuscles ; and the axial remnant of the archenteron undergoes a similar fate. According to Kowalevsky the heart is formed during larval life as an elongated closed sack on the right side of the endostyle. The notochord. The notochord was left as a rod formed of a single row of cells, or in As. canina and some other forms of two rows, extending from just within the border of the trunk to the end of the tail. According to Kowalevsky, KupfFer, Giard, etc. the notochord under- goes a further development which tinds its only complete parallel amongst Chordata in the doubtful case of Amphioxus. There appear between the cells peculiar highly refractive discs (fig. 8 v. Chs). These become larger and larger, and finally, after pushing the remnants of the cells with their nuclei to the sides, coalesce together to form a con- tinuous axis of hyaline substance. The remnants of the cells with their nuclei form a sheath round the hyaline axis (fig. 8 vi. ch.). Whether the axis is to lie regarded as formed of an intercellular substance, or of a differ- UROCHORDA. 15 entiatioii of pai-ts of the cells is still doubtful. Kupffer inclines to the lat- ter view : the analogy of the notochord of higher types appears to me to tell in favour of the former one. The alimentary tract. The anterior part of the primitive ar- chenteron alone retains a lumen, and from this part the whole of the permanent alimentary tract (mesenteron) becomes developed. The anterior part of it grows upwards, and before hatching an involu- tion of the epiblast on the dorsal side, just in front of the anterior extremity of the nervous system, meets and opens into this upgrowth, and gives rise to the permanent mouth (fig. 8 V. o). Kowalevsky states that a pore is formed at the front end of the nervous tube leading into the mouth (tig. 8 v. and vi. y*) which eventually gives rise to the ciliated sack, which lies in the adult at the junction between the mouth and the branchial sack. Kupifer however was unable to find this opening; but Kowalevsky's observations are confirmed by those of Salensky on Salpa. From the hinder end of the alimentary sack an outgrowth directed dorsalwards makes its appearance (figs. 8 V. and 9, d), from which the oesophagus, stomach and intestine become developed. It at first ends blindly. The remainder of the primitive alimentary sack gives rise to the branchial sack of the adult. Just after the larva has be- come hatched, the outgrowth to form the stomach and oesophagus, etc. bends ventralwards and to the right, and then turns again in a dorsal and left direction till it comes close to the dorsal surface, somewhat to the left of and close to the hinder end of the trunk. The first ventral loop of this part gives rise to the oesophagus, which opens into the stomach; from this again the dorsally directed intestine passes off. On the ventral wall of the branchial sack there is formed a narrow fold with thickened walls, which forms the endostyle. It ends ante- riorly at the stomodseum and posteriorly at the point where the solid remnant of the archenteron in the tail was primitively continuous with the branchial sack. The whole of the alimentary wall is formed of a single layer of hypoblast cells. A most important organ connected with the alimentary system still remains to be dealt with, viz. the atrial or peribranchial cavity. The first rudiments of it appear at about the time of hatching, in the form of a pair of dorsal epiblastic involutions (fig. 8 v. kl), at the level of the junction between the brain and the spinal cord. The^e involutions grow inwards, and meet corresponding outgrowths of the branchial sack, with which they fuse. At the junction between them is formed an elongated ciliated slit, leading from the branchial sack into the atrial cavity of each side. The slits so formed are the first pair of branchial clefts. Behind the first pair of branchial clefts a second pair is formed during larval life by a second outgrowth of the branchial sack meeting the epiblastic atrial involutions (fig. 8 vi. Iks and 2^'s). The intestine at first ends blindly close to the left atrial involution, but the anus becomes eventually formed by an opening 16 MET A MO R Pilosis. being established between the left atrial involution and the intes- tine. During the above described processes the test remains quite intact, and is not perforated at the oral or the atrial openings. The retrogressive metamorphosis of the larva. The development of the adult from the larva is, as has already been stated, in the main a retrogressive metamorphosis. The stages in this metamorphosis are diagrammatically shewn in figs. 10 and 11. It commences with the attachment of the larva (fig. 10 A) which takes place by one of the three papillue. Simultaneously with the attach- ment the larval tail under- goes a complete atrophy (fig. 10 B), so that nothing is left of it but a mass of fatty cells situated close to the point of the previous inser- tion of the tail in the trunk. The nervous system also undergoes a very rapid re- trogressive metamorphosis ; and the only part of it which persists would seem to be the dilated portion of the spinal cord in the trunk (Kupffer, No. 28). The three papillae, in- cluding that serving for at- tachment, early disappear, and the larva becomes fixed by a growth of the test to foreign objects. An opening appears in the test some time after the larva is fixed, leading into the mouth, which then becomes functional. The bran- chial sack at the same time undergoes important changes. In the larva it is provided with only two ciliated slits, which open into the, at this stage, paired atrial cavity (fig. 10). The openings of the atrial cavity at first are shut off from communication with the exterior by the test, but not long after the larva becomes fixed, two perforations are formed in the test, which lead into the openings of the two atrial cavities. At the same time the atrial cavities dilate so as gradually to embrace the whole branchial sack to which their inner walls attach themselves. Shortly after this the branchial clefts rapidly increase in number S 1 The account of the multiplication of the branchial clefts is taken from Krohn's paper on Phallusia mammillata (No. 24), but there is every reason to think that it holds true in the main for simple Ascidians. Fig. 10. Diagram shewing the mode op attachment and subsequent retrogressive metamorphosis of a larval ascidian. (from Laukester. ) UROGHORDA. 17 B^IN Fig. 11. DiAGEAM of a very young AsciDiAN. (From Lankester.) The increase of the branchial clefts is somewhat complicated. Between the two primitive clefts two new ones appear, and then a third appears behind the last cleft. In the interval between each branchial cleft is placed mojith a vascular branchial vessel (fig. 8 vi. bb.). Soon a great number of clefts become added in a row on each side of the branchial sack. These clefts are small ciliated openings placed ti'ans- versely with reference to the long axis of the branchial sack, but only oc- cupying a small part of the breadth of each side. The intervals dorsal and ventral to them are soon filled by series of fresh rows of slits, separated from each other by longitudinal bars. Each side of the branchial sack be- comes in this way perforated by a number of small openings arranged in rows, and separated by transverse and longitudinal bars. The whole struc- ture forms the commencement of the branchial basketwork of the adult ; the arrangement of which differs con- siderably in structure and origin from the simple system of branchial clefts of normal vertebrate types. At the junction of the transverse and longitu- dinal bars papillae are formed projecting into the lumen of the branchial sack. After the above changes are far advanced towards completion, the openings of the two atrial sacks gradually approximate in the dorsal line, and finally coalesce to form the single atrial opening of the adult. The two atrial cavities at the same time coalesce dorsally to form a single cavity, which is continuous round the branchial sack, except along the ventral line where the endostyle is present. The atrial cavity, from its mode of origin as a pair of epi- blastic involutions^ is clearly a structure of the same nature as the branchial or atrial cavity of Amphioxus ; and has nothing whatever to do with the true body cavity. It has already been stated that the anus opens into the original left atrial cavity ; when the two cavities coalesce the anus opens into the atrial cavity in the median dorsal line. Two of the most obscure points in the development are the origin of the mesoblast in the trunk, and of the body cavity. Of the former subject we know next to nothing, though it seems that the cells ^ In the asexually produced buds of Ascidians the atrial cavity appears, with the ex- ception of the external opening, to be formed from the primitive branchial sack. In the buds of Pyrosoma however it arises independently. These peculiarities in the buds cannot weigh against the embryonic evidence that the atrial cavity arises from in- volutions of the epiblast, and they may perhaps be partially explained by the fact that in the formation of the visceral clefts outgrowths of the branchial sack meet the atrial involutions. B. E. II. 2 18 MOLGULA. resulting from the atrophy of the tail are employed in the nutrition of the mesoblastic structures of the trunk. The body cavity in the adult is well developed in the region of the intestine, where it forms a wide cavity lined by an epithelioid mesoblastic layer. In the region of the branchial sack it is reduced to the vascular channels in the walls of the sack. Kowalevsky believes the body cavity to be the original segmenta- tion cavity, but this view can hardly be regarded as admissible in the present state of our knowledge. In some other Ascidian types a few more facts about the mesoblast will be alluded to. With the above changes the retrogressive metamorphosis is com- pleted ; and it only remains to notice the change in position under- gone in the attainment of the adult state. The region by which the larva is attached grows into a long process (fig. 10 B), and at the same time the part carrying the mouth is bent upwards so as to be re- moved nearly as far as possible from the point of attachment. By this means the condition in the adult (fig. 11) is gradually brought about; the original dorsal surface with the oral and atrial openings becoming the termination of the long axis of the body, and the nervous system being placed between the two openings. The genus Molgula i)resents a remarkable exception amongst the simple Ascidians in that, in some if not all the species belonging to it, develop- ment takes place (Lacaze Duthiers 29 and 30, Kupffer 28) quite directly and without larval metamorphosis. The ova are laid either singly or adhering together, and are very opaque. The segmentation (Lacaze Duthiers) commiences by the formation of four equal spheres, after which a number of small clear spheres are formed which envelope the large spheres. The latter give rise to a closed enteric sack, and probably also to a mass of cells situated on the ventral side, which appear to be mesoblastic. The epiblast is constituted of a single layer of cells which completely envelopes the enteric sack and the mesoblast. While the ovum is still within the chorion five peculiar processes of epiblast grow out; four of which usually lie in the same sectional plane of the embryo. They are contractile and contain prolongations of the body cavity. Their relative size is very variable. The nervous system is formed on the dorsal side of the embryo before the above projections make their appearance, but, though it seems probable that it originates in the same manner as in the more normal forms, its develop- ment has not been worked out. As soon as it is formed it consists of a nervous ganglion similar to that usually found in the adult. The history of the ma.-is of mesoblast cells has been inadequately followed, but it con- tinuously disappears as the heai't, excretory organs, muscles, etc. become formed. So far as can be determined from Kupffer's descriptions the body cavity is jirimitively i)arenchymatous — an indication of an abbrevi- ated development — and does not arise as a definite split in the mesoblast. The primitive enteric cavity becomes converted into the branchial sack, and from its dorsal and posterior corner the oesophagus, stomach and intestine grow ont as in, the normal forms. The mouth is formed by the UnOCHORDA. 19 invagination of a disc-like thickening of the epidermis in front of the nervous system on the dorsal side of the body; and the atrial cavity arises behind the nervous system by a similar process at a slightly later period. Tlie gill clefts opening into the atrial cavity are formed as in the type of simple Ascidians described by Krohn. The embiyo becomes hatched not long after the formation of the oral and atrial openings, and the five epiblastic processes undergo atrophy. They are not employed in the attachment of the adult. The larva when hatched agrees in most important points with the adult ; and is without the characteristic provisional larval organs of ordinary forms; neither organs of special sense nor a tail becoming developed. It has been suggested by Kupfier that the ventrally situated mesoblastic mass is the same structure as the mass of elements which results in ordinary types from the degeneration of the tail. If this suggestictfi is true it is difiicult to believe that this mass has any other than a nutritive function. The larva of Ascidia ampulloides described by P. van Beneden is regarded by KupiFer as intermediate between the Molgula larva and the normal type, in that the larval tail and notochord and a pigment spot are first developed, while after the atrophy of these organs peculiar processes like those of Molgula make their appearance. Sedentaria. The develoj)ment of the fixed composite Ascidians is, so far as we know, in the main similar to that of the simple Ascidians. The larvsB of Botryllus sometimes attain, while still in the free state, a higher stage of development with reference to the number of gill slits, etc. than that reached by the simple Ascidians, and in some instances (Botryllus auratus Metschnikoff) eight conical processes are found springing in a ring- like fashion around the trunk. The presence of these processes has led to somewhat remarkable views about the morphology of the group ; in that they were regarded by Kolliker, Sai-s, etc. as separate individuals, and it was supposed that the product of each ovum was not a single individual, but a whole system of individuals with a common cloaca. The researches of Metschnikoff (No. 32), Krohn (No. 25), and Giard (No. 12), etc. demonstrate that this paradoxical view is untenable, and that each ovum only gives rise to a single embryo, while the stellate systems are subsequently formed by budding. Natantia. Our knowledge of the development of Pyrosoma is mainly due to Huxley (No. 16) and Kovvalevsky (No. 22), In each individual of a colony of Pyrosoma only a single egg comes to maturity at one time. This egg is contained in a capsule formed of a structureless wall lined by a flattened epithelioid layer. From this capsule a duct passes to the atrial cavity, which, though called the oviduct, functions as an afferent duct for the spermatozoa. The segmentation is meroblastic, and the germinal disc adjoins the opening of the oviduct. The segmentation is very similar to that which occurs in Teleostei, and at its close the germinal disc has the form of a cap of cells, without a trace of stratification or of a seg- mentation cavity, resting upon the surface of the yolk, which forms the main mass of the ovum. After segmentation the blastoderm, as v^e may call the layer of 2—2 20 PYROSOMA. cells derived from the germinal disc, rapidly spreads over the sur- face of the yolk, and becomes divided into two layers, the epiblast and the hypoblast. At the same time it exhibits a distinction into a central clearer and a peripheral more opaque region. At one end of the blastoderm, which jfor convenience sake may be spoken of as the posterior end, a disc of epiblast appears, which is the first rudiment of the nervous system, and on each side of the middle of the blastoderm there arises an epiblastic involution. The epiblastic involutions give rise to the atrial cavity. These involutions rapidly grow in length, and soon form longish tubes, opening at the surface by pores situated not far from the poste- rior end of the blastoderm. The blastoderm at this stage, as seen on the surface of the yolk, is shewn in fig. 12 A. It is somewhat broader than long. The nervous system is shewn at n, and at points to an atrial tube. A transverse sec- tion, through about the middle of this blastoderm, is represented in fig. 1 2 B. The epiblast is seen above. On each side is the sec- tion of an atrial tube {at). Below is the hypoblast which is separated from the yolk especially in the middle line ; at each side it is beginning to grow in below, on the surface of the yolk. The space below the hypoblast is the ali- mentary cavity, the ven- tral wall of which is form- ed by the cells growing in at the sides. Between the epiblast and hypoblast are placed scattered meso- blast cells, the origin of which has not been clearly made out. In a later stage the openings of the two atrial tubes gradually travel backwards, and at the same time approximate, till finally they meet and coalesce at the posterior end of the blastoderm behind the nervous disc (fig. 13, cl). The tubes themselves at the same time become slightly constricted not far from their hinder extremities, and so divided into a posterior region nearly coterminous with the nervous system (fig. 13), and an anterior region. These two regions have very different histories in the subsequent development. A. Fig. 12. Surface vikw of the ovum of Pyrosoma NOT FAR ADVANCED IN DEVELOPMENT. The embry- onic structures are developed from a disc-like blas- toderm. B. Transverse section through the middle PART OF the same BLASTODERM. at. atrial cavity; hy. hypoblast; n. nervous disc in the region of the future Cyathozooid. UROGHORDA. 21 The nervous disc has during these changes become marked by a median furrow (fig. 13, ng), which is soon converted into a canal by the same process as in the simple Ascidians. The closure of the groove commences posteriorly and travels forwards. These pro- cesses are clearly of the same nature as those which take place in Chor- data generally in the formation of the central nervous system. In the region of the germinal disc which contains the anterior part of the atrial tubes, the alimentary cavity becomes, by the growth of the layer of cells described in the last stage, a complete canal, on the outer wall of which the endostyle is formed as a median fold. The whole anterior part of the blastoderm becomes at the same time gradually constricted off from the yolk. The fate of the anterior and posterior parts of the blastoderm is very different. The anterior part becomes segmented into four zooids or individuals, called by Huxley Ascidiozooids, which give rise to a fresh colony of Pyrosoma, The posterior part forms a rudimentary zooid, called by Huxley Cyathozooid, which eventually atrophies. These five zooids are formed by a process of embryonic fission. This fission commences by the appearance of four transverse constrictions in the anterior part of the blastoderm; by which the whole blastoderm becomes imperfectly divided into five regions, fig. 14 A. The hindermost constriction (uppermost in my figure) lies just in front of the pericardial cavity; and separates the Cyathozooid from the four Ascidiozooids. The three other constrictions mark off the four Ascidiozooids. The Cyathozooid remains for its whole length attached to the blastoderm, which has now nearly enveloped the yolk. It contains the whole of the nervous system {ng), which is covered behind by the opening of the atrial tubes {cl). The alimentary tract in the Cyathozooid forms a tube with very delicate walls. The pericardial cavity is completely contained within the Cyathozooid, and the heart itself {ht) has become formed by an invo- lution of the walls of the cavity. The Ascidiozooids are now completely separated from the yolk. They have individually the same structure as the undivided rudiment from which they originated ; so that the organs they possess are simply two atrial tubes, an alimentary tract with an endost}'le, and undifferentiated mesoblast cells. Fig. 13. Blastoderm of Pyrosoma shortly before its division into cy- ATHOZOOID AND Ascidiozooids. (After Kowalevsky.) cl. cloacal (atrial) opening; en. en- dostyle; at. atrial cavity; ngf. nervous groove. The heart and pericardial cavity are seen to the left. 22 PYROSOMA. In the following stages the Ascidiozooids grow with great rapidity. They soon cease to lie in a straight line, and eventually form a ring round the Cyathozooid and attached yolk sack. While these changes are being accomplished in the external form of the colony, both the Cyathozooids and the Ascidiozooids progress considerably in development. In the Cyathozooid the atrial spaces gradually atrophy, with the exception of the external opening, which becomes larger and more conspicuous. The heart at the same time comes into full activity and drives the blood through the whole colony. The yolk becomes moi^e and more enveloped by the Cyatiiozooid, and is rapidly absorbed; while the nutriment derived from it is transported to the Ascidiozooids by means of the vascular connection. The nervous system retains its previous condition ; and round the Cya- thozooid is formed the test into which cells migrate, and arrange themselves in very conspicuous hexagonal areas. The delicate ali- mentary tract of the Cyathozooid is still continuous with that of the first Ascidiozooid. After the Cyathozooid has reached the development just described it commences to atrophy. The changes in the Ascidiozooids are even more considerable than those in the Cyathozooid. A nervous system appears as a fresh Fig. 14l. Two stages in the development of Pyeosoma in which the Cyathozooid AND FOUR Ascidiozooids ake already distinctly formed. (After Kowalevsky.) cy. cyathozooid; an. ascidiozooid; ng. nervous groove ; ht. heart of cyathozooid ; cl. cloacal opening. formation close to the end of each Ascidiozooid turned towards the Cyathozooid. It forms a tube of which the open front end eventually develops into the ciliated pit of the mouth, and the remainder into the actual nervous ganglion. Between the nervous system and the endostyle an involution appears, which gives rise to the mouth. On each side of the primitive alimentary cavity of each Ascidiozooid branchial slits make their appearance, leading into the atrial tubes; so that the primitive alimentary tract becomes converted into the branchial sacks of the Ascidiozooids. The remainder of the alimen- tary tract of eacli zooid is formed as a bud from the hind end of the UROCHORDA. 23 branchial sack in the usual way. The alimentary tracts of the four Ascidiozooids are at first in free communication by tubes opening from the hinder extremity of one zooid into the dorsal side of the branchial sack of the next zooid. At the hinder end of each Ascidio-(1 zooid is developed a mass of fatty cells known as the elffioblast,! which probably represents a rudiment of the larval tail of simple, Ascidians. (Cf. pp. 25 and 26.) The further changes consist in the gradual atrophy of the Cyatho- zooid, which becomes more and more enclosed within the four Asci- diozooids. These latter become completely enveloped in a common test, and form a ring round the remains of the yolk and of the Cyathozooid, the heart of which continues however to beat vigor- ously. The cloacal opening of the Cyathozooid persists through 1 all these changes, and, after the Cyathozooid itself has become com-1 pletely enveloped in the Ascidiozooids and finally absorbed, deepens/ to form the common cloacal cavity of the Pyrosoma colony. The main part? of the Ascidiozooids were already formed during the last stage. The zooids long remain connected together, and united by a vascular tube with the Cyathozooid, and these connec- tions are not severed till the latter completely atrophies. Finally, after the absorption of the Cyathozooid, the Ascidiozooids form a rudimentary colony of four individuals enveloped in a common test. The two atrial tubes of each zooid remain separate in front but unite posteriorly. An anus is formed leading from the rectum into the common posterior part of the atrial cavity ; and an opening is estab- lished between the posterior end of the atrial cavity of each Ascidio- zooid and the common axial cloacal cavity of the whole colony. The atrial cavities in Pyrosoma are clearly lined by epiblast, just as in simple Ascidians. When the young colony is ready to become free, it escapes from the atrial cavity of the parent, and increases in size by budding. DollolldSB. The sexually developed embryos of Doliolum have been observed by Krohn (No. 23), Gegenbaur (No. 10), and Keferstein and Ehlers (No. 17); Vjut the details of the development have been very im- perfectly investigated. The yoiuigest embryo observed was enveloped in a large oval transparent covering, the exact nature of which is not clear. It is perhaps a larval rudiment of the test which would seem to be absent in the adult. Within this covering is the larva, the main organs of which are already developed ; and which primarily differs irom the adult in the possession of a larval tail similar to that of simple Ascidians. In the body both oral and atrial openings are present, the latter on the dorsal surface ; and the alimentajy tract is fully established. The endostyle is already formed on the ventral wall of the branchial sack, but the branchial slits are not present. Nine muscular rings are already visible. The tail, though not so developed as in the simple Ascidians, contains an axial notochord of the usual structure, and lateral muscles. It is inserted on the ventral side, and by its slow movements the larva progresses. 24 SALPA. In succeeding stages the tail gradually atrophies, and the gill slits, four in number, develop ; at the same time a process or stolon, destined to give rise by budding to a second non-sexual generation, makes its appear- ance on the dorsal side in the seventh inter-muscular space. This stolon is comparable with that which appears in the embryo of Salpa, When the tail completely atrophies the larva leaves its transparent covering, and becomes an asexual Doliolum with a dorsal stolon. SalpidSB. -A.S is well known the chains of Salpa alone are sexual, and from each individual of the chain only a single embryo is produced. The ovum from which this embryo takes its origin is visible long before the separate Salps of the chain have become completely developed. It is en- veloped in a capsule continuous with a duct, which opens into the atrial cavity, and is usually spoken of as the oviduct. The capsule with the ovum is enveloped in a maternal blood sinus. Embryonic development commences after the chain has become broken up, and the spermatozoa derived from another individual would seem to be introduced to the ovum through the oviduct. At the commencement of embryonic development the oviduct and ovi- capsule undergo peculiar changes; and in part at least give rise to a structure subservient to the nvitrition of the embryo, known as the placenta. These changes commence with the shoi'tening of the oviduct, and the dis- appearance of a distinction between oviduct and ovicapsule. The cells lining the innermost end of the capsule, i.e. that at the side of the ovum turned away from the atrial cavity, become at the same time very columnar. Tlie part of the oviduct between the ovum and the atrial cavity dilates into a sack, communicating on the one hand with the atrial cavity, and on the other by a very narrow opening with the chamber in which the egg is contained. This sack next becomes a prominence in the atiial cavity, and eventvTally constitutes a brood-pouch. The prominence it forms is covered by the lining of the atrial cavity, immediately within which is the true wall of the sack. The external opening of the sack becomes gradually narrowed and finally disappears. In the meantime the chamber in which the embryo is at first placed acquires a larger and larger opening into the sack ; till finally the two chambers unite, and a single brood-pouch contain- ing the embryo is thus produced. The inner wall of the chamber is formed by the columnar cells already spoken of. They form the rudiment of the placenta. The double wall of the outer part of the brood-pouch becomes stretched by the growth of the embryo; the inner of its two layers then atrophies. The outer layer subsequently gives way, and becomes rolled back so as to lie at the inner end of the embryo, leaving the latter projecting freely into the atrial cavity. While these changes are taking place the placenta becomes fully developed. The first rudiment of it consists, according to Salensky, of the thickened cells of the ovicapsule only, though this view is dissented from by Brooks, Todaro, etc. Its cells soon divide to form a largish mass, which becomes attached to a part of the epiblast of the embryo. On the formation of the body cavity of the embryo a central axial portion of the placenta becomes separated from a peripheral layer; and a channel is left between them which leads from a maternal blood sinus into the embryonic body cavity. The peripheral layer of the placenta is formed of cells continuous with the epiblast of the embryo; while the UROCHORDA. 25 axial portion is constituted of a disc of cells adjoining the embryo, with a column of fibres attached to the maternal side. The fibres of this column are believed by Salensky to be products of the original rudiment of the placenta. The placenta now assumes a more spherical form, and its cavity becomes shut off from the embryonic body cavity. The fibrous column breaks up into a number of strands perforating the lumen of the organ, and the cells of the wall become stalked bodies projecting into the lumen. When the larva is nearly ready to become free the placenta atrophies. The placenta functions in the nutrition of the embryo in the following way. It projects from its first formation into a maternal blood sinus, and, on the appearance of a cavity in it continuous with the body cavity of the embryo, the blood of the mother fully intermingles with that of the embryo. At a later period the communication with the body cavity of the embryo is shut off, but the cavity of the placenta is supplied with a continuous stream of maternal blood, which is only separated from the foetal blood by a thin partition. It is now necessary to turn to the embryonic development about which it is unfortunately not as yet possible to give a completely satisfactory account. The statements of the different investigatoi-s contradict each otiier on most fundamental points. I have followed in the main Salensky (No. 34), but have also called attention to some points where his obser- vations diverge most from those of other writers, or where they seem unsiftisfactory. The development commences at about the period when the brood-pouch is becoming formed ; and the ovum passes entirely into the brood-pouch before the segmentation is completed. The segmentation is regular, and the existence of a segmentation cavity is denied by Salensky, though affirmed by Kowalevsky and Todaro'. At a certain stage in the segmentation the cells of the ovum become divided into two layers, an epiblast investing the whole of the ovum with the exception of a small area adjoining the placenta, where the inner layer or hypoblast, Avhich forms the main mass of the ovum, projects at the surface. The epiblast soon covers the whole of the hypoblast, so that there would seem (according to Salensky's observations) to be a kind of epibolic invagination: a conclusion supported by Todaro's figures. At a later stage, on one side of the free apex of the embryo, a meso- blastic layer makes its appearance between the epiblast and hypoblast. This layer is derived by Salensky, as it appears to me on insufficient grounds, from the epiblast. Nearly at the same time there arises not far from the same point of the embryo, but on the opposite side, a solid thickening of epiblast which foi'ms the rudiment of the nervous system. The nervous system is placed close to the front end of the body; and nearly at the opposite ])ole, and therefore at the hind end, there appears immedi- ately below the epiblast a mass of cells forming a provisional organ known as the elseoblast. Todaro regaids this organ as mesoblastic in origin, and Salensky as hypoblastic. The organ is situated in the position which would be occupied by the larval tail were it developed. It may probably be regarded (Salensky) as a disappearing rudiment of the tail, and be 1 From Todaro's latest paper (No. 39) it would seem the segmentation cavity has very peculiar relatious. 26 SALPA. compared in this respect with the more or less similar mass of cells described by Kupffer in Molgula, and with the eljeoblast in Pyrosoma. After the differentiation of these organs a cavity makes its appearance between the epiblast and hypoblast, which is regai'ded by Sale n sky as the body cavity. It appears to be equivalent to the segmentation cavity of Todaro. According to Todaro's statements, it is replaced by a second cavity, which appears between the splanchnic and somatic layers of mesoblast, and constitutes the true body cavity. The embryo now begins to elongate, and at the same time a cavity makes its appearance in the centre of the hypoblast cells. This cavity is the rudiment of the branchial and alimentary cavities: on its dorsal wall is a median projection, the rudiment of the so- called gill of Salpa. At two points this cavity comes into close contact with the external skin. At one of these, situated immediately ventral to the nervous system, the mouth becomes formed at a later period. At the other, placed on the dorsal svirface between the nervous system and the elseoblast, is formed the cloacal aperture. By the stage under consideration the more important systems of organs are established, and the remaining embryonic history may be very briefly narrated. The embryo at this stage is no longer covered by the walls of the brood-pouch but projects freely into the atrial cavity, and is only attached to its parent by means of the placenta. The epiblast cells soon give rise to a deposit which forms the mantle. The deposit appears however to be formed not only on the outer side of the epiblast but also on the inner side ; so that the epiblast becomes cemented to the subjacent parts, branchial sack, etc., by an intercellular layer, which would seem to fill up the primi- tive body cavity with the exception of the vascular channels (Salensky). The nervous system, after its separation from the epiblast, acquires a central cavity, and subsequently becomes divided into three lobes, each with an internal pi-otuberance. At its anterior extremity it opens into the branchial sack ; and from this part is developed the ciliated pit of the adult. The nervous ganglion at a later period becomes solid, and a median eye is subsequently formed as an outgrowth from it. According to Todaro there ai'e further formed two small auditory C? olfactory) sacks on the ventral surface of the brain, each of them placed in communication with the branchial cavity by a narrow canal. The mesoblast gives rise to the muscles of the branchial sack, to the heart, and to the pericardium. The two latter are situated on the ventral side of the posterior extremity of the branchial cavity. Branchial sack and alimentary tract. The first development of the enteric cavity has already been described. The true alimentary tract is formed as a bud from the hinder end of the primitive cavity. The remain- der of the primitive cavity gives rise to the branchial sack. The so-called gill has at first the form of a lamella attached dorsally to the walls of the branchial sack; but its attachment becomes severed except at the two ends, and it then forms a band stretching obliquely across the branchial cavity, which subsequently becomes hollow and filled with blood corpuscles. The whole structure is probably homologous with the peculiar fold, usually pro- longed into numerous processes, which normally projects fz'om the dorsal wall of the Ascidian branchial sack. UROCHORDA. 27 On the completion of the gill the branchial sack becomes divided into a region dorsal to the gill, aiid a region ventral to it. Into the former the single atrial invagination opens. No gill slits are formed comparable with those in simple Ascidians, and the only representative of these structures is the simple communication which becomes established between the dorsal division of the branchial sack and the atrial opening. The whole branchial sack of Salpa, including both the dorsal and ventral divisions, corresponds with the branchial sack of simple Ascidians. On its ventx'al side the endostyle is formed in the normal way. The mouth arises at the point already indicated near the front end of the nervous system', 1 Brooks takes a very different view of the nature of the parts in Salpa. He says, No. 7, p. 322, " The atrium of Salpa, when first observed, was composed of two broad " lateral atria within the body cavity, one on each side of the branchial sack, and a very " small mid-atrium.... The lateral atria do not however, as in most Tunicata, remain " connected with the mid-atrium, and unite with the wall of the branchial sack to " form the branchial slits, but soon become entirely separated, and the two walls of " each unite so as to form a broad sheet of tissue, which soon splits up to form the " muscular bands of the branchial sack." Again, p. 'd2i, "During the changes which " have been described as taking place in the lateral atria, the mid-atrium has increased " in size.... The branchial and atrial tunics now unite upon each side, so that the " sinus is converted into a tube which communicates, at its posterior end, with the " heart and perivisceral sinus, and at the anterior end with the neural sinus. This " tube is the gill.... The centres of the two regions upon the sides of the gill, where " these two tissues have become united, are now absorbed, so that a single long and " narrow branchial slit is produced on each side of the gill. The branchial cavity is " thus thrown into communication with the atrium, and the upper surface of the lat- " ter now unites with the outer tunic, and the external atrial opening is formed by " absorption." The above description would imply that the atrial cavity is a space lined by meso- blast, a view which would upset the whole morphology of the Ascidians. Salensky's account, which implies only an immense redueticm in the size of the atrial cavity as compared with other types, appears to me far more probable. The lateral atria of Brooks appear to be simply parts of the body cavity, and have certainly no connection with the lateral atria of simple Ascidians or Pyrosoma. The observations of Todaro upon Salpa (No. 38) are very remarkable, and illustrated by beautifully engraved plates. His interpretations do not however appear quite satis- factory. The following is a brief statement of some of his results. During segmentation there arises a laj'er of small superficial cells (epiblast) and a central layer of larger cells, which becomes separated from the former by a segmen- tation cavity, except at the pole adjoining the free end of the brood-pouch. At this point the epiblast cells become invagiiaated into the central cells and form the alimentary tract, while the primitive central cells remain as the mesoblast. A fold arises from the epiblast which Todaro compares to the vertebrate amnion, but the origin of it is un- fortunately not satisfactorily described. The folds of the amnion project towards the placenta, and enclose a cavity which, as the folds never completely meet, is permanently open to the maternal blood sinus. This cavity corresponds with the cavity of the true amnion of higher Vertebrates. It forms the cavity of the placenta already described. Between the two folds of the amnion is a cavity corresponding with the vertebrate false amnion. A structure regarded by Todaro as the notochord is formed on the neck, connecting the involution of the alimentary tract with the exterior. It has only a very transitory existence. In the later stages the segmentation cavity disappeai'S and a true body cavity is formed by a split in the mesoblast. Todaro's interpretations, and in part his descriptions also, both with reference to the notochord and amnion, appear to me quite inadmissible. About some other parts of his descriptions it is not possible to form a satisfactory judgment. He has recently published a short paper on this subject (No. 39) preliminary to a larger memoir, which is very difficult to understand in the absence of plates. He finds however in the placenta various parts which he regards as homologous with the decidua vera and reflexa of Mammalia. 28 APPENDICULARIA. Development of the chain of sexual Salps. My description of the embryonic development of Salpa would not be complete without some reference to the development of the stolon of the solitary generation of Salps by the segmentation of which a chain of sexual Salps originates. The asexual Salp, the embryonic development of which has just been described, may be compared to the Cyathozooid of Pyrosoma, from which it mainly differs in being fully developed. While still in an embryonic condition it gives rise to a process or stolon, which becomes divided into a number of zooids by transverse constrictions, in the same manner that part of the germ of the ovum of Pyrosoma is divided by transverse constrictions into four Ascidiozooids. The stolon arises as a projection on the right side of the body of the embryo close to the heart. It is formed {Salensky, No. 35) of an outgrowth of the body wall, into which there gi'ow the following structures : (1) A central hollow process from the end of the respiratory sack. (2) A right and left lateral prolongation of the pericardial cavity. (3) A solid process of cells on the ventral side derived from the same mass of the cells as the elteoblast. (4) A ventral and a dorsal blood sinus. Besides these parts there appears on the dorsal side a hollow tube, the origin of which is unknown, which gives rise to the nervous system. The hollow pi-ocess of the respiratory sack is purely provisional, and disa[)pears without giving rise to any permanent structure. The right and left prolongations of the pericardial cavity become solid and eventually give origin to the mesoblast. The ventral process of cells is the most important structure in the stolon in that it gives rise both to the alimentary and respiratory sacks, and to the genei'ative organs of the sexual Salps. The stolon containing the organs just enumei'ated becomes divided by transverse constrictions into a number of rings. These rings do not long remain complete, but become interrupted dorsally and ventrally. The imper- fect rings so formed soon overlap, and each of them eventually gives rise to a sexual Salp. Although the stolon arises while the asexual Salp is still in an embryonic condition, it does not become fu.lly developed till long after the asexual Salp has attained maturity. Appendicularia. Our only knowledge of the development of Appen- dicularia is derived from Fol's memoir on the group (No. 8). He simply states that it develops, as far as he was able to follow, like other Ascidians ; and that the extremely minute size of the e^g prevented him from pursuing the subject. He also states that the pair of pores leading from the bran- chial cavity to the exterior is developed from epiblastic involutions meeting outerrowths of the wall of the branchial sack. Metagenesis. One of the most remarkable phenomena in connection with the life liistory of many Ascidians is the occurrence of an alternation of sexual and gemmiparous generations. This alternation appears to have originated from a complication of the process of reproduction by budding, which is so common in this group. The mode in which this very probably took place will be best understood by tracing a series UROCHORDA. 29 of transitional cases between simple budding and complete alternations of generations. ■ In the simpler cases, which occur in some Composita Sedentaria, the process of budding commences with an outgrowth of the body wall into the common test, containing a prolongation of part of the alimentary tract \ Between the epiblastic and hypoblastic layers of the bud so formed, a mesoblastic and sometimes a generative outgrowth of the parent also appears. The systems of organs of the bud are developed from the corre- sponding layers to those in the embryo^. The bud eventually becomes detached, and in its turn gives rise to fresh buds. Both the bud and its parent reproduce sexually as well as by budding : the new colonies being derived from sexually produced embryos. The next stage of complication is that found in Botryllus (Krohn, Nos. 25 and 26). The larva produced sexually gives rise to a bud from the right side of the body close to the heart. On the bud becoming detached the parent dies away without developing sexual organs. The bud of the second generation gives rise to two buds, a right one and a left one, and like the larva dies without reaching sexual maturity. The buds of the third generation each produce two buds and then suffer the same fate as their parent. The buds of the third generation arrange themselves with their cloacal extremities in contact, and in the fourth generation a common cloaca is formed, and so a true radial system of zooids is established; the zooids of which are not however sexual. The buds of the fourth generation in their turn produce two or three buds and then die away. Fresh systems become formed by a continuation of the process of budding, but the zooids of the secondary systems so formed are sexual. The ova come to maturity before the spermatozoa, so that cross fertilization takes place. In Botryllus we have clearly a rudimentary form of alternations 1 It is not within tlie scope of this work to enter into details with reference to the process of budding. The reader is referred on this head more especially to the papers of Huxley (No. 16) and Kowalevsky (No. 22) on Pyrosoma, of Salensky (No. 35) on Salpa, and Kowalevsky (No. 21) on Ascidians generally. It is a question of very great interest how budding first arose, and then became so prevalent in these degenerate types of Chordata. It is possible to suppose that budding may have commenced by the division of embryos at an early stage of development, and have gradually been carried onwards by the help of natural selection till late in life. There is perhaps little in the form of budding of the Ascidians to support this view — the early budding of Didemnum as described by Gegenbaur being the strongest evidence for it — but it fits in very well with the division of the embryo in Lumbricus trapezoides described by Kleinenberg, and with the not unfrequent occurrence of double monsters in Vertebrata which may be regarded as a phenomenon of a similar nature (Eauber). The embryonic budding of Pyrosoma, which might perhaps be viewed as supporting the hypothesis, appears to me not really in favour of it; since the Cyathozooid of Pyrosoma is without doubt an extremely modified form of zooid, which has obviously been specially developed in con- nection with the pecuUar reproduction of the Pyrosomidffi. * The atrial spaces form somewhat doubtful exceptions to the rule. 30 METAGENESIS. of generations, in that the sexually produced larva is asexual, and, after a series of asexual generations, produced gemmiparously, there appear sexual generations, which however continue to reproduce themselves by budding. The type of alternations of generations observable in Botryllus becomes, as pointed out by Huxley, still more marked in Pyrosoma. The true product of the ovum is here {vide p. 21) a rudimentary individual called by Huxley the Cyathozooid. This gives rise, while still an embryo, by a process equivalent to budding to four fully developed zooids (Ascidiozooids) similar to the parent form, and itself dies away. The four Ascidiozooids form a fresh colony, and repro- duce (1) sexually, whereby fresh colonies are formed, and (2) by ordinary budding, whereby the size of the colony is increased. All the individuals of the colony are sexual. The alternation of generations in Pyrosoma widely differs from that in Botryllus in the fact of the Cyathozooid differing so markedly in its anatomical characters from the ordinary zooids. In Salpa the process is slightly different \ The sexual forms are now incapable of budding, and, although at first a series of sexual individuals are united together in the form of a chain, so as to form a colony like Pyrosoma or Botryllus, yet they are so loosely connected that they separate in the adult state. As in Botryllus, the ova are ripe before the spermatozoa. Each sexual individual gives rise to a single offspring, which, while still in the embryonic condition, buds out a 'stolon' from its right ventral side. This stolon is divided into a series of lateral buds after the solitary sexual Salp has begun to lead an independent existence. The solitary sexual Salp clearly corresponds with the Cyathozooid of Pyrosoma, though it has not, like the Cyathozooid, undergone a retrogressive metamorphosis. By far the most complicated form of alternation of generations known amongst the Asciclians is that in Doliolum. The discovery of this metamorphosis was made by Gegenbaur (No. lo). The sexual form of Doliolum is somewhat cask-shaped, with ring-like muscular bands, and the oral and atrial apertures placed at opposite ends of the cask. The number of gill slits varies according to the species. The ovum gives rise, as already described, to a tailed embryo which subsequently develops into a cask -shaped asexual form. On attaining its full size it loses its branchial sack and alimentary tract. While still in the embryonic condition, a stolon grows out from its dorsal side in the seventh intermuscular space. The stolon, like that in Salpa, contains a prolongation of the branchial sack^ On this stolon there develop two entirely dififerent types of buds, (1) lateral buds, (2) dorsal median buds. 1 Vide p. 28. 2 I draw this conclusion from Gegenbaur's fig. (No. lo), PI, xvi., fig. 15. The body (x) in the figure appears to v^e \\{j hypoblast is com- EL A SMOBRANCHII. 43 pleted, since all intermediate gradations between complete separation and complete attachment are to be seen. Shortly after the separation takes place, a fairly thick bridge is found connecting the two lateral halves of the hypo- blast, but this bridge is an- teriorly excessively delicate and thin, and in some cases is barely visible except with high powers. In some sec- tions I have observed possi- ble indications of the pro- cess like that described by Calberla for Petronyzon, by which the lateral parts of the hypoblast grow in under- neath the axial part, and so isolate it bodily as the noto- chord. It is not absolutely clear whether the noto- cliord is to be regarded as an axial differentiation of the hypoblast, or as an axial differentiation of the lower layer cells. The facts of develop- ment both in Amphioxus and Elasmobranchii tend towards the former view ; but the nearly simultaneous differentiation of the notochord and the mesoblastic plates lends some support to the supposition that the notochord may be merel)' a median plate of mesoblast developed slightly later than the two lateral plates. The alimentary canal or mesenteron was left as a space between the hypoblast and the yolk, ending blindly in front, but opening behind by a widish aperture, the blastopore or anus of Rusconi {vide fig. 19 B). The conversion of this irregular cavity into a closed canal com- mences first of all at the anterior extremity. In this conversion two distinct processes are concerned. One of these is a process of folding off of the embryo from the blastoderm. The other is a simple growth of cells independent of any fold. To the first of these processes the depth and narrowness of the alimentary cavity is due ; the second is concerned in forming its ventral wall. The process of the folding off of the embryo from the blastoderm resembles exactly the similar process in the embryo bird. The fold is a perfectly continuous one round the front end of the embryo, but may be conveniently spoken of as composed of a head-fold and two lateral folds. Fig. 25. Thkee sections of a Pristiukxjs embryo slightly older than fig, 28 b. The sections shew the development of the noto- chord. C/t. notochord; Ch'. developing notochord ; mg. medullary groove; Ip. lateral plate of mesoblast; ep. ei)iblast; hy. hypoblast. 44 THE HYPOBLAST. Fig. 26. Section through the ANTERIOR PART OF A PrISTIURUS EMBRYO TO SHEW THE FORMATION OF THE ALIMENTARY TRACT. Gh. notochord; hy. hypoblast al. alimentary tract ; 7ia. cells passing in from the yolk to form the ventral wall of the alimentary tract. Of far greater interest than the nature of these folds is the forma- tion of the ventral wall of the alimentary canal. This originates in a growth of cells from the two sides to the middle line (fig. 26). I The cells for it are not however mainly derived from pre-existing hypoblast cells, but are formed de novo around the nuclei of the yolk which have already been spoken of (fig. 26, na). The ventral wall of the mesenteron is in fact, to a large extent at any rate, formed as a differentiation of the primitive yolk floor. The folding off and closing of the alimentary canal in the anterior part of the body proceeds rapidly, and not only is a considerable tract of the alimentary canal formed, but a great part of the head is completely folded off from the yolk before the medullary groove is closed. The posterior part of the alimentary canal retains for a longer time its primitive condition. Finally however it also becomes closed in, by the lips of the blastopore at the hind end of the embryo meeting and uniting. The peculiarity of the closing in of the posterior part of the alimentary canal consists in the fact that a similar continuity to that in Amphioxus obtains between the neural and alimentary canals. This is due to the medullary folds being continuous at the end of the tail with the lips of the blastopore, which close in the hind end of the alimentary canal ; so that, when the medullary folds unite to form a canal, this canal becomes continuous with the alimentary canal, which is closed in at the same time. In other words, the me- dullary folds assist in enveloping the blas- topore which does not therefore become ab- solutely closed, but opens into the floor of the neural canal. It will afterwards be shewn that it is only the posterior part of the blastopore that becomes closed during the above process, and that the anterior and ventral part long remains open. The general arrangement of the parts, at the time when the hind end of the mesenteron is first closed, is shewn in fig. 27. The same points may be seen in the diagrammatic longitudinal section fig. 19 C. The middle portion of the alimentary tract is the last to be closed in, since it remains till late in embryonic life as the umbilical or Fig. 27. LoNGiTUDiNAii VERTICAL SECTION OF AN EM- BRYO SLIGHTLY YOUNGER THAN THAT IN FIG. 26 D. The section shews the communication which exists between the nem-al and ali- mentary canals. nc. neural canal; aL ali- mentary tract; Ch. noto- chord; Ts. tail swelling. ELASMOBRANGHII. 45 t/i';P vitelline canal, connecting the yolk sack with the alimentary cavity. The umbilical canal falls into the alimentary tract immediately behind the entrance of the hepatic duct. At a fairly early stage of development a rod is constricted off from the dorsal wall of the alimentary canal (figs. 27* and 23 x), which is known as the subnotochordal rod. It is placed immediately below the notochord, and disappears during embryonic life. General features of the Elasmohranch embryo at successive stages. Shortly after the three germinal layers be- come definitely established, the rudiment of the embryo, as visible from the surface, consists of an oblong plate, which extends inwards from the periphery of the blastoderm, and is bound- ed on its inner side by a head-fold and two lateral folds (fig. 28 B). This plate is the me- dullary plate ; along its axial line is a shallow groove — the medullary groove {mg). The ru- diment of the embryo rapidly increases in length, and takes a spatula-like form (fig. 28 C). The front part of it, turned away from the edge of the blastoderm, soon becomes dilated into a broad plate, — the cephalic plate (h) — while the tail end at the edge of the blastoderm is also enlarged, being formed of a pair of swellings — the tail swellings (ts) — derived from the lateral parts of the original embryonic rim. By this stage a certain number of mesoblastic somites have become formed but are not shewn in my figure. They are the foremost somites of the trunk, and those behind them continue to be added, like the segments in Chaetopods, between the last formed somite and the end of the body. The increase in length of the body mainly takes place by growth in the region between the last mesoblastic somite and the end of the tail. The anterior part of the body is now completely folded off from the blastoderm, and the medullary groove of the earlier stage has become converted into a closed canal. By the next stage (fig. 28 D) the embryo has become so much folded off from the yolk both in front and behind that the separate parts of it begin to be easily recognizable. The embryo is attached to the yolk by a distinct stalk or cord, which in the succeeding stages gradually narrows and elongates, and is known as the umbilical cord (so. s.). The medullary canal has now Fig. 27*. Teans- verse section through the tail region of a PrISTIURUS EMBRYO OF THE SAME AGE AS FIG. 28 E. df. dorsal fin; sp.c. spinal cord; pp. body- cavity; sp. splanchnic layer of mesoblast; so. somatic layer of meso- blast ; mp. commencing differentiation of mus- cles; ch. notochord; x. subnotochordal rod aris- ing as an outgrowth of the dorsal wall of the alimentary tract; al. ali- mentary tract. <^ 46 GENERAL GROWTH OF THE EMBRYO. become completely closed. The anterior region constitutes the brain ; and in this part slight constrictions, not perceptible in views of the embryo as a transparent object, mark off three vesicles. These vesicles are known as the fore, mid, and hind brain. From the fore- brain there is an outgrowth on each side, the first rudiment of the optic vesicles [op). The tail swellings are still conspicuous. The tissues of the body have now become fairly transparent, and there may be seen at the sides of the body seventeen mesoblastic Fig. 28. Views of Elasmobeanch embryos. A — F. Pkistiurtts. G. and H. Scylliom. A. A blastoderm before the formation of the medullary plate, sc. segmentation cavity; es. embryonic swelling. B. A somewhat older blastoderm in which the medullary groove has been es- tablished, mg. medullary groove. C. An embryo from the dorsal surface, as an opaque object, after the medullary groove has become posteriorly converted into a tube. mg. medullary groove: the reference line points very nearly to the junction between the open medullary groove with the medullary tube ; h. cephalic plate ; ts. tail swelling. D. Side view of a somewhat older embryo as a transparent object, ch. notochord; op. optic vesicle; I.v.c. 1st visceral cleft; al. alimentary tract; so.s. stalk connecting the yolk-sack with the embryo. E. Side view of an older embryo as a transparent object, mp. muscle-plates; au.v. auditory vesicle ; vc. visceral cleft; Jit. heart; m. mouth invagination ; an. anal diverticulum; al.v. posterior vesicle of post-anal gut. F. G. H. Older embryos as opaqiie objects. ELASMOBRANCHII. 47 somites. The notochord, which was formed long before the stage repre- sented in figure 28 D, is now also distinctly visible. It extends from almost the extreme posterior to the anterior end of the embryo, and lies between the ventral wall of the spinal canal and the dorsal wall of the intestine. Round its posterior end the neural and alimen- tary tracts become continuous with each other. Anteriorly the termination of the notochord cannot be seen, it can only be traced into a mass of mesoblast at the base of the brain, which there separates the epiblast from the hypoblast. The alimentary canal {al) is completely closed anteriorly and posteriorly, though still widely open to the yolk-sack in the middle part of its course. In the region of the head it exhibits on each side a slight bulging outwards, the rudiment of the first visceral cleft. This is represented in the figure by two lines (i. v.c). The embryo represented in fig. 28 E is far larger than the one just described, but it has not been convenient to represent this increase of size in the figure. Accompanying this increase in size, the folding off from the yolk has considerably progressed, and the stalk which unites the embryo with the yolk is proportionately narrower and longer than before. The brain is now very distinctly divided into the three lobes, the rudiments of which appeared during the last stage. From the fore- most of these the optic vesicles now present themselves as well- marked lateral outgrow^ths, towards which there has appeared an involution from the external skin (op) to form the lens. A fresh organ of sense, the auditory sack, now for the first time becomes visible as a shallow pit in the external skin on each side of the hind-brain {au.v). The epiblast which is involuted to form this pit becomes much thickened, and thereby the opacity, indicated in the figure, is produced. The mesoblastic somites have greatly increased in number by the formation of fresh somites in the tail. Thirty-eight of them were present in the embryo figured. The mesoblast at the base of the brain is more bulky, and there is still a mass of unsegmented mesoblast which forms the tail swellings. The first rudiment of the heart Qit) becomes visible during this stage as a cavity between the mesoblast of the splanchnopleure and the hypoblast. The fore and hind guts are now longer than they were. An inva- gination from the exterior to form the mouth has appeared (m) on the ventral side of the head close to the base of the thalamence- phalon. The upper end of this eventually becomes constricted off as the pituitary body, and an indication of the future position of the anus is afforded by a slight diverticulum of the hind gut towards the exterior, some little distance from the posterior end of the embryo {an). The portion of the alimentary canal behind this point, though at this stage large, and even dilated into a vesicle at its posterior end {al.v), becomes eventually completely atrophied. It is known as the postanal gut. In the region of the throat the rudiment of a 48 GENERAL GROWTH OF THE EMBRYO. second visceral cleft has appeared behind the first ; neither of them is as yet open to the exterior. In a somewhat older embryo the first spontaneous movements take place, and consist in somewhat rapid excursions of the embryo from side to side, produced by a serpentine motion of the body. A ventral flexure of the prseoral part of the head, known as the cranial flexure, which commenced in earlier stages (fig. 28 D and E), has now become very evident, and the mid-brain^ begins to project in the same manner as in the embryo fowl on the third day, and will soon form the anterior termination of the long axis of the embryo. The fore-brain has increased in size and distinctness, and the anterior part of it may now be looked on as the unpaired rudi- ment of the cerebral hemispheres. Further changes have taken place in the organs of sense, especially in the eye, in which the involution for the lens has made considerable progress. The number of the muscle-plates has again increased, but there is still a region of unsegmented mesoblast in the tail. The thickened portions of mesoblast, which caused the tail swellings, are still to be seen, and would ■^ B seem to act as the reserve from which is drawn the matter for the rapid growth of the tail, which occurs soon after this. The mass of the mesoblast at the base of the brain has again increased. No fresh features of interest are to be seen in the notochord. The heart is very much more conspicuous than be- fore, and its commencing flexure is very apparent. It now beats actively. The post-anal gut is much longer than during the last stage ; and the point where the anus will ap- pear is very easilydetected by a bulging out of the gut towards the external skin. The alimentary vesicle at the end of the post-anal gut, first observ- FlG. 28*. FOUE SECTIONS THROUGH THE POST- ANAL PART OF THE TAIL OF AN EMBRYO OF THE SAME AGE AS FIG. 28 F. A. is the posterior section. nc. neural canal; al. post-anal gut; alv. caudal vesicle of post-anal gut ; x. subnotochord rod; vip. muscle-plate; ch. notochord; cl.al. cloaca; ao. aorta; v.cau. caudal vein. ^ The part of the brain which I have here called mid-brain, and which unquestion- ably corresponds to the part called mid-brain in the embryos of higher vertebrates, becomes in the adult what Milducho-Maclay and Gegenbaur called the vesicle of the third ventricle or thalamencephalon. ELA SMOBBA NGHII. 49 able during the last stage, is now a more conspicuous organ. There are three visceral clefts, none of which are as yet open to the exterior. Figure 28 F represents a considerably older embryo viewed as an opaque object, and fig. 29 A is a view of the head as a transparent object. The stalk connecting it with the yolk is now, comparatively speaking, quite narrow, and is of sufficient length to permit the embryo to execute considerable movements. The tail has grown immensely, but is still dilated terminally. The terminal dilatation is mainly due to the alimentary vesicle (fig. 28* alv)^ but the postanal section of the alimentary tract in front of this is now a solid cord of cells. Both the alimentary vesicle and this cord very soon disappear. Their relations are shewn in section in fig. 28*. The two pairs of limbs have appeared as differen- vqr gr_ auv tiations of a continuous but not very conspicuous epi- blastic thickening, which is probably the rudiment of a lateral fin. The an- terior pair is situated just at the front end of the umbilical stalk ; and the posterior pair, which is the later developed and less conspicuous of the two, is situated some little dis- tance behind the stalk. The cranial flexure has greatly increased, and the angle between the long axis of the front part of the head and of the body is less than a right angle. The conspicuous mid-brain (29 A, mh) forms the an- terior termination of the long axis of the body. The thin roof of the fourth ven- tricle {Jih) may be noticed in the figure behind the mid-brain. The auditory sack iau.V) is nearly closed, and its opening is not shewn in the figure. In the eye (op) the lens is completely formed. The olfactory pit [pV) is seen a little in front of the eye. B. E. II. Fig. 29. Views of the head op Elasmo- branch embryos at two stages as transparent OBJECTS. A. Pristiurus embryo of the same stage as fig. 28 F. B. Somewhat older Scyllium embryo. III. third laerve; V. fifth nerve; VII. seventh nerve; axi.n. auditory nerve ; gl. glossopharyngeal nerve; Vg. vagus nerve; fb. fore-brain; pn. pineal gland; mh. mid-brain; hh. hind-brain; iv.v. fourth ventricle ; ch. cerebellum ; ol. olfactory pit ; oj). eye; an.V. auditory vesicle ; m. mesoblast at bafe of brain; ch. notochord; lit. heart; Vc. visceral clefts; eg. external gills; j)p. sections of body- cavity in the head. 4 50 GENERAL GROWTH OF THE EMBRYO. Owing to the opacity of the embryo, the muscle-plates are only indistinctly indicated in fig. 28 F, and no other features of the meso- blast are to be seen. The mouth is now a deep pit, the hind borders of which are almost completely formed by a thickening in front of the first branchial or visceral cleft, which may be called the first branchial arch or mandibular arch. Four branchial clefts are now visible, all of which are open to the exterior, but in the embryo, viewed as a transparent object, two more, not open to the exterior, are visible behind the last of these. Between each of these and behind the last one there is a thicken- ing of the mesoblast which gives rise to a branchial arch. The arch between the first and second cleft is known as the hyoid arch. Fig. 29 B is a representation of the head of a slightly older embryo in which papillae may be seen in the front wall of the second, third, and fourth branchial clefts : these papillae are the commencements of filiform processes which grow out from the gill-clefts and form ex- ternal gills. The peculiar ventral curvature of the anterior end of the notochord {ch) both in this and in the preceding figure deserves notice. A peculiar feature in the anatomy makes its appearance at this period, viz. the replacement of the original hollow cesophagus by a solid cord of cells (fig. 23 A, ces) in which a lumen does not reappear till very much later. I have found that in some Teleostei (the Salmon) long after they are hatched a similar solidity in the cesophagus is present. It appears not impossible that this feature in the cesophagus may be connected with the fact that in the ancestors of the present types the cesophagus was perforated by gill slits ; and that in the process of embryonic abbrevia- tion the stage with the perforated cesophagus became replaced by a stage with a cord of indifferent cells (the cesopliagiis being in the embryo quite functionless) out of which the non-perforated cesophagus was directly formed. In the higher types the process of development appears to have become quite direct. By this stage all the parts of the embryo have become established, and in the succeeding stages the features characteristic of the genus and species are gradually acquired. Two embryos of Scyllium are represented in fig. 28 G and H, the head and anterior part of the trunk being represented in fig. G, and the whole embryo at a much later stage in fig. H. In both of these, and especially in the second, an apparent diminution of the cranial flexure is very marked. This diminution is due to the increase in the size of the cerebral hemispheres, which grow upwards and forwards, and press the original fore-brain against the mid-brain behind. In fig. G the rudiments of the nasal sacks are clearly visible as small open pits. The first cleft is no longer similar to the rest, but by the closure of the lower part has commenced to be metamorphosed into the spiracle. ELASMOBRAXCHII. 51 Accompanying the change in position of the first cleft, the man- dibular arch has begun to bend round so a;s to enclose the front as well as the sides of the mouth. By this change in the mandibular arch the mouth becomes narrowed in an antero-posterior direction. In fig. H are seen the long filiform external gills which now pro- ject out from all the visceral clefts, including the spiracle. They are attached to the front wall of the spiracle, to both walls of the next four clefts, and to the front wall of the last cleft. They have very possibly become specially developed to facilitate respiration within the egg ; and they disappear before the close of larval life. When the young of Scyllium and other Sharks are hatched they have all the external characters of the adult. In Raja and Torpedo the early stages, up to the acquirement of a shark-like form, are similar to those in the Selachoidei, but during the later embryonic stages the body gradually flattens out, and assumes the adult form, which is thus clearly shewn to be a secondary acquirement. An embryonic gill-cleft behind the last present in the adult is found (Wyman, No. 54) in the embryo of Raja batis. The unpaired fins are developed in Elasmobranchs as a fold of skin on the dorsal side, which is continued round the end of the tail along the ventral side to the anus. Local developments of this give rise to the dorsal and anal fins. The caudal fin is at first symmetrical, but a special lower lobe grows out and gives to it a heterocercal character. Enclosure of the yolk- sack and its relation to the embryo. The blastoderm at the stage represented in fig. 28 A and B forms a small and nearly circular patch on the surface of the yolk, composed of epiblast and lower layer cells. While the body of the embryo is gradually being moulded this patch grows till it envelopes the yolk ; the growth is not uniform, but is less rapid in tlie immediate neigh- bourhood of the embryonic part of the blastoderm than elsewhere. As a consequence of this, that part of the edge, to which the embryo is attached, forms a bay in the otherwise regular outline of the edge of the blastoderm, and by the time that about two-thirds of the yolk is enclosed this bay is very conspicuous. Jt is shewn in fig. 30 A, where hi points to the blastoderm, and yk to the part of the yolk not yet covered by the blastoderm. The embryo at this time is only connected with the yolk-sack by a narrow umbilical cord ; but, as shewn in the figure, is still attached to the edge of the blastoderm. Shortly subsequent to this the bay in the blastoderm, at the head of which the embryo is attached, becomes obliterated by its two sides coming together and coalescing. The embryo then ceases to be attached at the edge of the blastoderm. But a linear streak formed by the coalesced edges of the blastoderm is left connecting the embryo with the edge of the blastoderm. This streak is probably 4—2 52 FORMATION OF THE YOLK-SACK. analogous to (though not genetically related with) the primitive streak in the Amniota, This stage is represented in fig. 30 B. In this figure there is only a small patch of yolk {yk) not yet enclosed, which is situated at some little distance behind the embryo. Throughout all this period the edge of the blastoderm has remained thickened : a feature which persists till the complete investment of the yolk, which takes place shortly after the stage last described. In this thickened edge a circular vein arises which brings back the blood from the yolk-sack to the embryo. The opening in the blastoderm, exposing the portion of the yolk not yet covered, may be conveni- ently called the yolk blas- topore. It is interesting to notice that, owing to the large size of the yolk in Elasmobranchs, the poster! or part of the primitive blas- topore becomes encircled by the medullary folds and tail- swellings, and is so closed long before the anterior and more ventral part, which is represented by the uncover- ed portion of the yolk. It is also worth remarking that, owing to the embryo be- coming removed from the edge of the blastoderm, the final closure of the yolk blastopore takes place at some little distance from the embryo. The blastoderm enclos- ing the yolk is formed of an external layer of epiblast, a layer of mesoblast below in which the blood-vessels Fig. 30. Thkee views of the vitellus of AN ElASMOBBANCH, SHEWING THE EMBEYO, THE BLASTODERM, AND THE VESSELS OF THE YOLK-SACK. The shaded part {U) is the blastoderm; the white part the uncovered yolk. A. Young stage with the embryo still at- tached at the edge of the blastoderm. B. Older stage with the yolk not quite en- closed by the blastoderm. 0. Stage after the complete enclosure of the yolk. ?//c.yolk; bl. blastoderm; v. venous trunks of yolk-sack; a. arterial trunks of yolk-sack; y. point of closure of the yolk blastopore; x. por- tion of the blastoderm outside the arterial sinus terminalis. ELASMOBRANCHII. 53 are developed, and within this a layer of hypoblast, which is especially well marked and ciliated (Leydig, No. 46) in the umbilical stalk, where it lines the canal leading from the yolk-sack to the intestine. In the region of the yolk-sack proper the blastoderm is so thin that it is not easy to be quite sure that a layer of hypoblast is throughout dis- tinct. Both the hypoblast and mesoblast of the yolk-sack are formed by a differentiation of the primitive lower layer cells. Nutriment from the yolk-sack is brought to the embryo partly through the umbilical canal and so into the intestine, and partly by means of blood-vessels in the mesoblast of the sack. The blood- vessels arise before the blastoderm has completely covered the yolk. Fig. 30 A represents the earliest stage of the circulation of the yolk-sack. At this stage there is visible a single arterial trunk {a) passing forwards from the embryo and dividing into two branches. No venous trunk could be detected with the simple microscope, but probably venous channels were present in the thickened edge of the blastoderm. In fig. 30 B the circulation is greatly advanced. The blastoderm has now nearly completely enveloped the yolk, and there remains only a small circular space {yk) not enclosed by it. The arterial trunk is present as before, and divides in front of the embryo into two branches which turn backwards and form a nearly complete ring round the embryo. In general appearance this ring resembles the sinus terminalis of the area vasculosa of the Bird, but in reality bears quite a different relation to the circulation. It gives off branches on its inner side only. A venous system of returning vessels is now fully developed, and its relations are very remarkable. There is a main venous ring in the thickened edge of the blastoderm, which is connected with the embryo by a single stem running along the seam where the edges of the blastoderm have coalesced. Since the venous trunks are only developed behind the embryo, it is only the posterior part of the arterial ring that gives off branches. The succeeding stage (fig. 30 C) is also one of considerable interest. The arterial ring has greatly extended, and now embraces nearly half the yolk, and sends off trunks on its inner side along its whole cir- cumference. More important changes have taken place in the venous system. The blastoderm has now completely enveloped the yolk, and the venous ring is therefore reduced to a point. The small veins which originally started from it may be observed diverging in a brush-like fashion from the termination of the unpaired trunk, which originally connected the venous ring with the heart. At a still later stage the arterial ring embraces the whole yolk, and, as a result of this vanishes in its turn, as did the venous ring- before it. There is then present a single arterial and a single venous trunk. The arterial trunk is a branch of the dorsal aorta, and the venous trunk originally falls into the heart together with the sub- intestinal or splanchnic vein. On the formation of the liver the 54 INTERNAL YOLK-SACK. proximal end of the subintestinal vein becomes the portal vein, and it is joined just as it enters the liver by the venous trunk from the yolk-sack. The venous trunk leaves the body on the right side, and the arterial on the left. The yolk-sack persists during the whole of embryonic life, and in the majority of Elasmobranch embryos there arises within the body walls an outgrowth from the umbilical canal into which a large amount of the yolk passes. This outgrowth forms an internal yolk- sack. In Mustelus vulgaris the internal yolk-sack is very small, and in Mastelus laevis it is absent. The latter species, which is one of those in which development takes place within the uterus, presents a remarkable peculiarity in that the vascular surface of the yolk-sack becomes raised into a number of fedds, which fit into corresponding depressions in the vascular walls of the uterus. The yolk-sack becomes in this way firmly attached to the walls of the uterus, and the two together constitute a kind of placenta. A similar placenta is found in Carcharias. After the embryo is hatched or born^as the case may be, the yolk- sack becomes rapidly absorbed. Bibliography. (40) F. M.. Balfour. "A preliminary account of the development of the Elasmo- branch Fishes." Quart. J. of Micr. Science, \ol. xiY. 1876'. (41) F. M. Balfour. "A Monograph on the development of Elasmobranch Fishes." London, 1878. Reprinted from the Journal of Anat. and Physiol, for 1876, 1S77, and 1878. (42) Z. Gerbe. "Jiecherches sur la segmentation de la cicatrule et la formation des produit» adventifs de Vceuf des Plagiostomes et particvXierement des Raies." Vide also Journal de VAnatomie et de la Fliysiologie, 1872.. (43). W. His. "Ueb. d. Bildung v. Haifischenembryonen." Zeit. fur Anat. u. Entw'ick., Vol. II. 1877. (44) A. Kowale-vslsy. "Development of Aeanthias vulgaris and Mustelus Isevis." (Russian^) Transactions of the Kieio Society of Naturalists, Vol. i. 1870. (45) R. Leuckart. "Ueber die allmahlige Bildung d. Korpergestalt bei d. Rochen." Zeit. f. wiss. Zool., Bd. h., p. 258; (46) Fr. Leydig. Rochen u. Haie. Leipzig, 1852» (47) A. W. Malm. "Bidrag till kannedom om utvecklingen af Rajae." Konyl. retenskaps akademiens forhandlingar. Stockholm, 1876.. (48) Joh. Miiller. Glatter Haie des Aristoteles und iiher die VerscMedenheiten itnter den Haifischen und RacJien in der Entioiehlung des Eies. Berlin, 1 -;40. (49) S..L. S«henk. "Die Eier von Raja quadrimaculata innerhalb der Eileiter." Sitz. der k. Akad. Wien, Vol.. lxxiii. 1873. (50) Alex. Schultz. "'Zur Entwicklungsgeschichte des Selachiereies." Archiv fiir micro. Aimt., Vol. xi. 1875. (51) Alex. Schnltz. "Beitrag zur Entwixsklungsgeschichte d. Knorpelfische." Archiv fiir micro. Anat., Vol. xiii. 1877. (52) C. Semper. "Dfe Stammesverwandschaft d. Wirbelthiere u. Wirbellosen." Arbeit, a, d. zool.-zoot. Instit. Wiirzhurg, Vol. 11. 1875. (53) C. Semper.. "Das Urogenitalsystem d. Plagiostomen, etc." Arbeit, a. d. zool.-zoot. Initit. WUrzburg, Vol. 11.. 1875. (54) Wyman. "Observations on the Development of Raja batis." Memoirs of the American Academy of Arts and Sciences, Vol. ix. 1864. CHAPTER IV. TELEOSTEI. The majority of the Teleostei deposit their eggs before impregna- tion, but some forms are viviparous, e.g. Blennius viviparus. Not a few carry their eggs about ; but this operation is with a few exceptions performed by the male. In Syngnathus the eggs are carried in a brood-pouch of the male situated behind the anus. Amongst the Siluroids the male sometimes carries the eggs in the throat above the gill clefts. Ostegeniosus militaris, Arius falcarius, and Arius fissus have this peculiar habit The ovum when laid is usually invested in the zona radiata only, though a vitelline membrane is sometimes present in addition, e.g. in the Herring. It is in most cases formed of a central yolk mass, which may either be composed of a single large vitelline sphere, or of distinct yolk spherules. The yolk mass is usually invested by a granular protoplasmic layer, which is especially thickened at one pole to form the germinal disc. In the Herring's ovum the germinal disc is formed, as in many Crustacea, at impregnation ; the protoplasm which was previously diffused through the egg becoming aggregated at the germinal pole and round the periphery. Impregnation is external, and on, its occurrence a contraction of the vitellus takes place, so that a space is formed between the vitellus and the zona radiata, which becomes filled with fluid. The peculiarities in the development of the Teleostean ovum can best be understood by regarding it as an Elasmobranch ovum very much reduced in size. It seems in, fact very probable that the Teleostei are in reality -derived from a type of Fish with a much larger ovum^ The occurrence of a meroblastie segmentation, in spite of the ovum being usually smaller than that of Amphibia and Aci- penser, etc., in which the segmentation is complete, as well as the solid origin of many of the organs, receives its most plausible explanation ' on this hypothesis. The proportion of the germinal disc to the whole ovum varies considerably. In very small eggs, such as those of the Herring, the disc may form as much as a fifth of the whole. 56 SEGMENTATION. The segmentation, which is preceded by active movements of the germinal disc, is meroblastic. There is nothing very special to note with reference to its general features, but while in large ova like those of the Salmon the first furrows only penetrate for a certain depth through the germinal disc, in small ova like those of the Herring, they extend through the whole thickness of the disc. During the segmentation a great increase in the bulk of the blas- toderm takes place. In hardened specimens a small cavity amongst the segmentation spheres may be present at any early stage; but it is probably an arti- ficial product, and in any case has nothing to do with the true segmentation cavity, which does not appear till near the close of segmentation. The peripheral layer of granular matter, continuous with the germinal disc, does not undergo division, but it becomes during the segmentation specially thickened and then spreads itself under the edge of the blastoderm ; and, while remaining thicker in this region, gradually grows inwards so as to form a continuous sub- blastodermic layer. In this layer nuclei appear, which are equivalent to those in the Elasmobranch ovum. A considerable number of these nuclei often become visible simultaneously (van Beneden, No. 60) and they are usually believed to arise spontaneously, though this is still doubtful \ Around these nuclei portions of protoplasm are segmented off, and cells are thus formed, which enter the blasto- derm, and have nearly the same destination as the homologous cells of the Elasmobranch ovum. During the later stages of segmentation one end of the blastoderm becomes thickened and forms the embryonic swelling ; and a cavity appears between the blastoderm and the yolk which is excentrically situated near the non-embryonic part of the blastoderm. This cavity is the true segmentation cavity. Both the cavity and the embryonic swelling are seen in section in fig. 31 A and B. In Leuciscus rutilus Bambeke describes a cavity as appearing in the middle of the blastoderm during the later stages of segmentation. From his figures it might be sn])posed that this cavity was equivalent to the segmentation cavity of Elasmobranchs in its earliest condition, but Bam- beke states that it disappears and that it has no connection with the true segmentation cavity. Bambeke and other investigators have failed to recognize the homology of the segmentation cavity in Teleostei with that in Elasmobi-anchii, Amphibia, etc. With the appearance of the segmentation cavity the portion of the blastoderm which forms its roof becomes thinned out, so that the whole blastoderm consists of (1) a thickened edge especially prominent at one point where it forms the embryonic swelling, and (2) a thinner central portion. The changes which now take place result in the differentiation of the embryonic layers, and in the rapid 1 Vide Vol. I. p. 89. TELEOSTET. 67 (72) T. Oellacher. "Beitrage zur Entwicklungsgeschichte der Knoclienfische nach Beobachtungen am Bachforellenei." Zeit. f. wiss. Zool., Vol. xxii., 1872, and Vol. xxiii., 1873. (7a*) H. Eathke. Ahh. z. Bildung u. EnUcich. d. Menschen u. Thiere. Leipzig, 1832-3. Part ii. Blennius. (73) Bieneck. "Ueber die Schichtung des Forellenkeims." Archiv f. mikr. Anat., Bd. v. 1869. (74) S. Strieker. " Untersuchungen iiber die Entwicklung der Bachforelle." Sitzungsberichte der Wiener k. Akad. d. Wiss., 1865. Vol. li. Abth. 2. (75) Carl Vogt. "Embryologie des Salmones." Histoire Naturelle des Poissons de I Europe Centrale. L. Agassiz. 1842. (76) C. Weil. "Beitrage zur Kenntniss der Knochenfische." Sitzungsber. der Wiener kais. Akad. der Wiss., Bd. lxvi. 1872. )— 2 CHAPTER V. CYCLOSTOMATA\ Petromyzon is the only type of this degenerated but primitive group of Fishes the development of which has been as yet studied ^ The development does not however throw any light on the rela- tionships of the group. The similarity of the mouth and other parts of Petromyzon to those of the Tadpole probably indicates that there existed a common ancestral form for the Cyclostomata and Amphibia. Embryology does not however add anything to the anato- mical evidence on this subject. The fact of the segmentation being complete was at one time supposed to indicate an affinity between the two groups; but the discovery that the segmentation is also com- plete in the Ganoids deprives this feature in the development of any special weight. In the formation of the layers and in most other developmental characters there is nothing to imply a special relation- ship with the Amphibia, and in the mode of formation of the nervous system Petromyzon exhibits a peculiar modification, otherwise only known to occur in Teleostei and Lepidosteus. Dohrn^ was the first to bring into prominence the degenerate character of the Cyclostomata. I cannot however assent to his view that they are descended from a relatively highly-organized type of Fish. It appears to me almost certain that they belong to a group of fishes in which a true skeleton of branchial bars had not become developed, the branchial skele- 1 The following classification of the Cyclostomata is employed in the present chapter : I- Hyperoartia ex. Petromyzon. n. Hyperotreta ex. Myxine, Bdellostoma. '^ The present chapter is in the main founded upon observations which I was able to make in the spring of 1880 upon the development of Petromyzon Planeri. Mr Scott very kindly looked over my proof-sheets and made a number of valuable suggestions, and also sent me an early copy of his preliminary note (No. 87), which I have been able to make use of in correcting my proof-sheets. 3 Der Ursprung d. Wirbelthierc, etc. Leipzig, 1875. CYCLOSTOMATA, 69 ton they possess being simply an extra-branchial system ; while I see no c reason to suppose that a true branchial skeleton has disappeared. If the \ primitive Cyclostomata had not tiue branchial bars, they could not have had jaws, because jaws are essentially developed from the mandibular branchial bar. These considerations, which are supported by numerous other features of their anatomy, such as the character of the axial skeleton, the straightness of the intestinal tube, the presence of a subintestinal vein etc., all tend to prove that these fishes are remnants of a primitive and prsegnathostomatous group. The few surviving members of the group have probably owed their preservation to their parasitic or semiparasitic habits, while the group as a whole probably disappeared on the appearance of gnathostomatous Vertebrata. The ripe ovum of Petrorayzon Planeri is a slightly oval body of about 1 mm. in diameter. It is mainly formed of an opaque nearly white yolk, invested by a membrane composed of an inner perforated layer, and an outer structureless layer. There appears to be a pore perforating the inner layer at the formative pole, which may be called a micropyle (Kupffer and Benecke, No. 79). Enclosing the egg-membranes there is present a mucous envelope, which causes the egg, when laid, to adhere to stones or other objects. Impregnation is effected by the male attaching itself by its sucto- rial mouth to the female. The attached couple then shake together; and, as they do so, they respectively emit from their abdominal pores ova and spermatozoa which pass into a hole previously made \ The segmentation is total and unequal, and closely resembles that in the Frog's egg (Vol. i. p. 78). The upper pole is very slightly whiter than the lower. A segmentation cavity is formed very early, and is placed be- tween the small cells of the upper pole and the large cells of the lower pole. It is pro- portionately larger than in the -, ; Frog ; and the roof eventually thins out so as to be formed of a single row of small cells. At the sides of the segmenta- tion cavity there are always several rows of small cells, which gradually merge into the larger cells of the lower pole of the egg. The segmen- tation is completed in about fifty hours. The segmentation is fol- TnC. y Fig. 37. Longitudinal vertical section THROUGH AN EMBRYO OF PeTROMYZON PlANERI OF 136 HOURS. me. mesoblast; yk. yolk-cells; al. alimen- tary tract; hi. blastopore; s.c. segmentation cavity. ^ Artificial impregnation may be effected witiiout difficulty by squeezing out into tlie same vessel the ova and si)erniatozoa of a ripe female and male. The fertilized eggs are easily reared. Petromyzon Planeri breeds during tlie second half of April. 70 FORMATION OF THE LAYERS. lowed by an asymmetrical invagination (fig. 37) which leads to a mode of formation of the hypoblast fundamentally similar to that in the Frog, The process has been in the main correctly described by M. Schultze (No. 8i). On the border between the large and small cells of the embryo, at a point slightly below the segmentation cavity, a small circular pit appears ; the roof of which is formed by an infolding of the small cells, while the floor is formed of the large cells. This pit is the commencing mesenteron. It soon grows deeper (fig. 37, al) and extends as a well-defined tube (shewn in transverse section in fig. 88, al) in the direction of the segmentation cavity. In the course of the Fig. 38. Tuansvkusk section thkough a Petromyzox embryo 160 hours after impregnation. ep. epiblast; al. mesenterou; yk. yolk-cells; ms. mesoblast. formation of the mesenteron the segmentation cavity gradually becomes smaller, and is finally (about the 200th hour) obliterated. The roof of the mesenteron is formed by the continued invagination of small cells, and its floor is composed of large yolk-cells. The wide external opening is arched over dorsally by a somewhat prominent lip — the homologue of the embryonic rim. The opening persists \ till nearly the time of hatching ; but eventually becomes closed, I and is not converted into the permanent anus. On the f )rmation of 'the mesenteron the hypoblast is composed of two groups of cells, (1) the yolk-cells, and (2) the cells forming the roof of the mesen- teron. While the above changes are taking place, the small cells, or as they may now be called the epiblast cells, gradually spread over the large yolk-cells, as in normal types of epibolic invagination. The growth over the yolk-cells is not symmetrical, but is most rapid in the meridian opposite the opening of the alimentary cavity, so that the latter is left in a bay (cf. Elasmobranchii, p. 51). The epibolic invagination takes place as in Molluscs and many other forms, not eimply by the division of pre-existing epiblast cells, but by the forma- CYCLOSTOMATA. 71 tion of fresh epiblast cells from the yolk cells (fig. 87) ; and Xill after the complete enclosure of the yolk cells there is never present a sharp line of demarcation between the two groups of cells. By the time that the segmentation cavity is obliterated the whole yolk is enclosed by the epiblast. The yolk cells adjoining the opening of the mesen- teron are the latest to be covered in, and on their enclosure this opening constitutes the whole of the blastopore. The epiblast is composed of a single row of columnar cells. Mesoblast and notochord. During the above changes the meso- blast becomes established. It arises, as in Elasmobranchs, in the form of two plates derived from the primitive hypoblast. During the invagination to form the mesenteron some of the hypoblast cells on each side of the invaginated layer become smaller, and marked off as two imperfect plates (fig. 38, ms). It is difficult to say whether these plates are entirely derived from invaginated cells, or are in part directly formed from the pre-existing yolk cells, but I am inclined to adopt the latter view ; the ventral extension of the mesoblast plates undoubtedly takes place at the expense of the yolk cells. The meso- blast plates soon become more definite, and form (fig. 39, tns) well- defined structures, triangular in section, on the two sides of the ^<^ middle line. At the time the mesoblast is first formed the hypoblast cells, which roof the mesenteron, are often imperfectly two layers thick (fig. 38), They soon how- ever become constituted of a single layer only. When the mesoblast is fairly established, the lateral parts of the hypo- blast grow inwards underneath the axial part, so that the latter (fig. 89, ch) first becomes isolated as an axial cord, and is next in- closed between the medullary cord (nc) (which has by this time been formed) and a continuous sheet of hypoblast below (fig. 40). Here its cells divide and it becomes the notochord. The notochord is thus bodily formed out of the axial portion of the primitive hypoblast. Its mode of origin may be compared with that in Amphioxus, in which an axial fold of the archenteric wall is constricted off as the notochord. The above features in the development of the notochord were first established by Calberla^ (No. 78). ^ In Calberla's figure, sbewinp: the development of the notochord, the limits of mesoblast and hypoblast are wrongly indicated. Fig. 39. Tkansverse section through AN EMBUYO OF PETBOilYZON PlANEEI OF 208 HOURS. The figure illustrates the formation of the neural cord and of the notochord. ms. mesoblast; n.c. neural cord; ch. noto- chord; yk. yolk cells; al. alimentary canal. 72 GENERAL DEVELOPMENT. Fig. 40. Tkansverse section through PART OF AN EMBRYO OF PeTROMYZON PlANERI OF 2-36 HOURS. m.c. medullary cord; ch. notochord; ah alimentary caual; ms. mesoblastic plate. General history of the development. Up to about the time when the enclosure of the hypoblast by the epiblast is completed, no external traces are visible of any -^ ^ of the organs of the embryo ; but about this time, i.e. about 180 hours after impregnation, the rudiment of the medullary plate becomes established, as a linear streak extending forwards from the blastopore over fully one half the circumference of the embryo. The medullary plate first contains a shallow median groove, but it is con- verted into the medullary cord, not in the usual vertebrate fashion, but, as first shewn by Calberla, in a manner much more closely resembling the formation of the medullary cord in Teleostei. Along the line of the median groove the epiblast becomes thickened and forms a kind of keel projecting inwards towards the hypoblast (fig. 89, nc). This keel is the rudiment of the medullary cord. It soon becomes more prominent, the median groove in it disappears, and it becomes separated from the epiblast as a solid cord (fig. 40, mc). By this time the whole embryo has become more elongated, and on the dorsal surface is placed a ridge formed by the projection of the medullary cord. At the lip of the blastopore the medullary cord is continuous with the hypoblast, thus forming the rudiment of a neur- enteric canal. Calberla gives a similar account of the formation of the neural canal^ to that which he gives for the Teleostei (vide p. 59). He states that the epiblast becomes divided into two layers, of which the outer is involuted into the neural cord, a median slit in the involution representing the neural groove. The eventual neural canal is stated to be lined by the involuted cells. Scott (N"o. 87) fully confirms Calberla on this point, and, although my own sections do not clearly shew an involu- ti(m of the outer layer of epiblast cells, the testimony of these two observers must no doubt be accepted on this point. Shortly after the complete establishment of the neural cord the elongation of the embryo proceeds with great rapidity. The pro- cesses in this growth are shewn in fig. 41, A, B, and C. The cephalic portion (A, c) first becomes distinct, forming an anterior protuberance free from yolk. About the time it is formed the mesoblastic plates begin to be divided into somites, but the embryo is so opaque that this process can only be studied in sections. Shortly afterwards an axial lumen appears in the centre of the neural cord, in the same manner as in Teleostei. The general elongation of the embryo continues rapidly, and, as shewn in my figures, the anterior end is applied to the ventral CYCLOSTOMATA. 73 surface or of the yolk (B). With the growth of the embryo the yolk becomes entirely confined to the posterior part. This Fig. 41. Four stages in the development of Petromyzon. (After Owsjanuikoff.) c. cephalic extremity; hi. blastopore; op. optic vesicle; au.v. auditory vesicle; br.c. branchial clefts. part is accordingly greatly dilated, and might easily be mistaken for the head. The position of the yolk gives to the embryo a very peculiar appearance. The apparent diti'erence between it and the embryos of other Fishes in the position of the yolk is due in the main to the fact that the postanal portion of the tail is late in developing, and always small. As the embryo grows longer it becomes spirally coiled within the eggshell. Before hatching the mesoblastic somites become distinctly marked (C). The hatching takes place at between 13 — 21 days after im- pregnation ; the period varying according to the temperature. During the above changes in the external form of the embryo, the development of the various organs makes great progress. This is especially the case in the head. The brain becomes distinct from the spinal cord, and the auditory sacks and the optic vesicles of the eye become formed. The branchial region of the mesenteron becomes established, and causes a dilatation of the anterior part of the body, and the branchial pouches grow out from the throat. The anus becomes formed, and a neurenteric canal is also established (k^cott). The nature of these and^other changes will best be understood by a description of the structure of the just-hatched larva. The general appearance of the larva immediately after hatching is shewn in fig. 41, D. The body is somewhat curved; the posterior extremity being 74 GENERAL DEVELOPMENT. much dilated with yolk, while the anterior is very thin. All the cells still contain yolk particles, which render the embryo very opaque. The larva only exhibits slow movements, and is not capable of swim- ming about. The structure of the head is shewn in figs. 42 and 43. Fig. 42 is a section through a very young larva, while fig. 43 is taken from a larva three days after hatching, and shews the parts with considerably greater detail. On the ventral side of the head is placed the oral opening (fig. 43, in) leading into a large stomodseum which is still without a com- munication with the mesenteron. Ventrally the stomodseum is prolonged for a considerable distance under the anterior part of the mesenteron. Immediately behind the stomodseum is placed the bran- chial region of the mesenteron. Laterally it is produced on each side into seven or perhaps eight branchial pouches (fig. 43, hr.c), which extend outwards nearly to the skin but are not yet open. Between the successive pouches are placed mesoblastic segments, of the same nature and structure as the walls of the head cavities in the em- bryos of Elasmobrauchs, and like them enclosing a central cavity. A similar structure is placed behind the last, and two similar structures in front of the first persistent pouch. This pouch is situated in the same vertical line as the auditory sack (au.v), and would appear there- fore to be the hyo-branchial cleft ; and this identification is confirmed Fig. 42. Diagrammatic vertical section of a just-hatched larva of Petromyzon. (From Gegenbaur; after Calberla.) o. mouth ; o'. olfactory pit ; v. septum between stomodaeum and mesenteron ; ]i. thyroid involution ; n. spinal cord ; ch. notochord ; c. heart ; a. auditory vesicle. ^ by the fact of two head cavities being present in front of it. At the \ front end of the branchial region of the mesenteron is placed a thickened ^ ridge of tissue, which, on the opening of the passage between the stomodseum and the mesenteron, forms a partial septum between the two, and is known as the velum (fig. 43, tv). eculiar flattening out of the embryo over the yolk {vide pp. 8G and 87) is no doubt connected with the mode in which the yolk becomes enveloped by the hypoblast. As the posterior part of the trunk, containing the intestine, becomes formed, the yolk is gradually confined to the anterior part of the alimentary tract, which, as before stated, becomes the stomach. The epithelial cells of the stomach, as well as those of the intestine, ai'e enormously dilated with food yolk (fig. 57, st). Behind the stomach is formed the liver. The subintestinal vein bringing back the blood to the liver appears to have the same course as in Teleostei, in that the blood, after passing through the liver, is distributed to the walls of the stomach and is again collected into a venous trunk which falls into the sinus venosus. As the yolk becomes absorbed, the liver grows forwards underneath the stomach till it comes in close contact with the heart. The relative position of the parts at this stage is shewn diagrammatically in fig. 56. At the com->' mencement of the intestine there arises in the larva of about 14 mm. a great number of diverticula, which are destined to form the compact,] glandular organ, which opens at this spot in the adult. At this stage'/ there is also a fairly well developed pancreas opening into the duodenum at the same level as the liver. No trace of the air-bladder was present at the stage in question. The spiral valve is formed, as in Elasmobranchii, as a simple fold in the wall of the intestine. There is a well-developed subnotochordal rod (fig. 57), which, according!) to Salensky, becomes the subvertebral ligament of the adult; a statement n which confirms an earlier suggestion of Bridge. The pronephros (head- \\ kidney) resembles in the main that of Teleostei (fig. 57) ; while the front end of the mesonephros, which is developed considerably later than the pronephros, is placed some way behind it. In my oldest larva (14 mm.) the mesonephros did not extend backwards into the posterior part of the abdominal cavity. Bibliography. (88) Knock. "Die Beschr. d. Eeise z. Wolga Behufs d. Sterlettbefruchtnng. " Bull. Soc. Nat. Moscow, 1871. (89) A. Kowalevsky, Ph. Owsjannikoff, and N. Wagner. "DieEntwick. d. Store." Vorlauf. Mittheilung. Melanges Biologiques tires du Bulletin d. I'Acad. Zotjj. St Petersbourg, Vol. vii. 1870. (90) W. Salensky. "Development of the Sterlet (Acipenser ruthenus)." 2 Parts. Proceedings of the Society of Naturalists in the imperial Vniversiiy of Kasan. 1878 and 9 (Russian). Part I., abstracted in Hoffmann and Schwalbe's Juhresbcricht for 1878. (91) W. Salensky. "Zur Embryologie d. Ganoiden (Acipenser)." Zoologischer Anzeiger, Vol. i., Nos. 11, 12, 13. LefidosteusV The ova of Lepidosteus are spherical bodies of about 3 mm. in 1 Alexander Agassiz was fortunate enough to succeed in procuring and rearing a batch of eggs of this interesting form. He has given an adequate account of the external characters of the post-embryonic stages, and very liberally placed his preserved material of the stages both before and after hatching at Prof. W. K. Parker's and my disposal. The account of the stages prior to hatching is the result of investigations carried on by Professor Parker's son, Mr W. N. Parker, and myself on the material supplied to us by Agassiz. This material was not very satisfactorily preserved, but I trust that our results are not without some interest. 92 LEPIDOSTEUS. diameter. They are invested by a tough double membrane, composed of (1) an outer layer of somewhat pyriform bodies, radiately arranged, which appear to be the remains of the follicular cells ; and (2) of an inner zona radiata, the outer part of which is radiately striated, while the inner part is homogeneous. The segmentation, as in the Sturgeon, is complete, but approaches closely the meroblastic type. It commences with a vertical furrow at the animal pole, extending through {tbout one-fifth of the circum- ference. Before this furrow has proceeded further a second furrow is formed at right angles to it. The next stages have not been observed, but on the third day after impregnation (fig, 58), the animal pole is completely divided into small segments, which form a disc similar to the blastoderm of meroblastic ova; while the vegetative pole, which subsequently forms a large yolk-sack, is divided by a few vertical furrows, four of which nearly meet at the pole opposite the blastoderm. The majority of the vertical furrows extend only a short way from the edge of the small spheres, and are par- tially intercepted by imperfect equatorial furrows. The stages immediately fol- lowing the segmentation are still unknown, and in the next stage satisfactorily observed, on the fifth day after impregnation, the body of the embryo is distinctly differentiated. The lower pole of the ovum is then formed of a mass in which no traces of segments or segmentation furrows can be detected. The embryo (fig. 59) has a dumbbell-shaped outline, and is com- posed of (1) an outer area, with some resemblance to the area pellu- cida of an avian embryo, forming the lateral part of the body ; and (2) a central portion consisting of the vertebral plates and medullary plate. The medullary plate is dilated in front to form the brain {hr). Two lateral swellings in the brain are the commencing optic vesicles. The caudal extremity of the embryo is somewhat swollen. Sections of this stage (fig, GO) are interesting as shewing a re- markable resemblance between Lepidosteus and Teleostei. The three layers are fully established. The epiblast {ep) is formed of a thicker inner nervous stratum, and an outer flattened epidermic stratum. Along the axial line there is a solid keel-like thickening of the nervous layer of the epidermis, which projects towards the hypo- blast. This thickening {MG) is the medullary cord ; and there is no evidence of the epidermic layer being at this or any subsequent period Fig, 58, Surface view of the ovum OF Lepidosteus with the membranes re- moved ON THE third DAY AFTER IMPREG- NATION. GANOIDEI. 93 concerned in its formation {vide chapter on Teleostei, p. 58). In the region of the brain the medullary cord is so thick that it gives rise, ^ as in Teleostei, to a projection of the whole body of the embryo towards the yolk. Posteriorly it is flatter. The mesoblast {Me) in the trunk has the form of two plates, which thin out laterally. The hypoblast (%) is a single layer of cells, and is nowhere folded in to form a closed ali- mentary canal. The hypoblast is separated from the neural cord by the notochord (Gh), which throughout the greater part of the embryo is a distinct struc- ture. In the region of the tail, the axial part of the hypoblast, the notochord, and the neural cord fuse together, the fused part so formed is the homologue of the neurenteric canal of other types, embryo the mesoblastic plates cease to be separable from the axial structures between them. Fig. 59. Subface view of a Lepidos- teds embryo on the fifth day after impregnation. hr. dilated extremity of medullary plate which forms the rudiment of the brain. Quite at the hinder end of the Fig. 60. Section through an embryo of Lepidosteus on the fifth day after impregnation. MC. medullary cord; Ep. epiblast; Me. mesoblast; liy. hypoblast; Ch. notochord. In a somewhat later stage the embryo is considerably more elon- gated, embracing half the circumference of the ovum. The brain is divided into three distinct vesicles. Anteriorly the neural cord has now become separated from the epidermis. The whole of the thickened nervous layer of the epiblast appears to remain united with the cerebro-spinal cord, so that the latter organ is covered dorsally by the epidermic layer of the epiblast 94 LEFIDOSTEUS. only, sides. The nervous layer soon however grows in again from the two *? ^T* Where the neural cord is separated from the epidermis, it is already provided with a well-developed lumen. Posteriorly it re- mains in its earlier condition. In the region of the hind-brain traces of the auditory vesicles are present in the form of slightly involuted thicken- ings of the nervous layer of the epidermis. The mesoblast of the trunk is divided anteriorly into splanchnic and somatic layers. In the next stage, on the sixth day after impreg- nation (fig. 61), there is a great advance in develop- ment. The embryo is con- siderably longer, and a great number of mesoblastic so- mites are visible. The body is now laterally com- pressed and raised from the yolk. The region of the head is more distinct, and late- rally two streaks are visible (br.c), which, by comparison with the Sturgeon, would seem to be the two first visceral clefts*: they are not yet perforated. In the lateral regions of the trunk the two segmental ducts are visible in surface views (fig. 61, sd) occupying the same situation as in the Sturgeon. Their position in section is shewn in fig. 62, sg. With reference to the fea- tures in development, visible in sections, a few points may be alluded to. The optic vesicles are very prominent outgrowths of the brain, but are still solid, though the anterior cerebral vesicle has a well-developed lumen. The Fig. 61. Embryo of Lepidosteus on the sixth day aftee impeegnation. op. optic vesicles ; fer.c. branchial clefts (?); sd. segmental duct. N.B. The branchial clefts and segmental duct are somewhat too prominent. Fig. 62. Section through the trunk of A Lepidosteus embryo on the sixth day AFTER impregnation. mc. medullary cord; Tns. mesoblast; sg. segmental duct; ch. notochord; x. sub-noto- chordalrod; %. hypoblast. ^ I have as yet been unable to make out these structures in section. GANOIDEI. 95 auditoi-y vesicles are now deep pits of the uervoiis layer of the epiblast, the openincrs of which are covered by the epidermic layer. They are shewn for a slightly later stage in fig. 63 {au.v). There is now present a sub- notochordal rod, which develops as in other types from a thick- ening of the hypoblast (fig. 62, x). In an embryo of the seventh day after impregnation, the features of the preceding stage become generally more pro- nounced. Fig. 63. Section through the head of A Lepidosteus embryo on the sixth day AFTER impregnation. au.v. auditory vesicle; au.n. auditory nerve; ch. notochord ; hy. hypoblast. The optic vesicles are now provided with a lumen (fig. 64), and have approached close to the epidermis. Adjoining them a thickening [l) of the nervous layer of the epidermis has appeared, which will form the lens. The cephalic extremity of the segmental duct, which, as shewn in fig. 61, is bent inwards towards the middle line, has now become slightly convoluted, and forms the rudi- ment of a pronephros (head-kidney). During the next few days the folding off of the embryo from the yolk commences, and proceeds till the embryo acquires the form represented in fig. (So. Both the head and tail are quite free from the yolk ; and the embryo presents a general resemblance to that of a Tele- ostean. On the ventral surface of the front of the head there is a disc (figs. 65, 66, sd), which is beset with a number of processes, formed as thicken- ings of the epiblast. As shewn by Agassiz, these eventually become short suctorial pa- pill8e\ Immediately behind this disc is placed a narrow depression which forms the Fig. 64. Section through the front rudiment of the mouth. vkrt of the head of a Lepidosteus embryo rT\^ M' . -J. " on the SEVENTH DAY AFTER IMPREGNATION. The olfactory pits are now , ,. ^ . x ^x. *v, i i, , , , 111 al. alimentary tract; fo. tnalamencepna- developed, and are placed near j^j^. j ^^^g ^f gyg. ^^^^^ optic vesicle. The the front of the head. mesoblast is not represented. 1 These papillae are very probably sensitive structures ; but I have not yet investi- gated their histological characters. 96 LEPIDOSTEUS. U:-- A great advance has taken place in the development of the vis- ceral clefts and arches. The oral region is bounded behind by a well- marked mandibular arch, which is separated by a shallow depression from a still more prominent hyoid arch (fig. 65, hy). Between the hyoid and mandibular arches a double lamella of hypoblast, which represents the hyomandibular cleft, is continued from the throat to the external skin, but does not, at this stage at any rate, contain a lumen. The hyoid arch is prolonged backwards into a considerable oper- cular fold, which to a great extent overshadows the branchial clefts behind. The hyobranchial cleft is widely open. Behind the hyobranchial cleft are four pouches of the throat on each side, not yet open to the exterior. They are the rudiments of the four branchial clefts of the adult. The trunk has the usual compressed piscine form, and there is a well- developed dorsal fin con- tinuous round the end of the tail, with a ventral fin. There is no trace of the paired fins. The anterior and pos- terior portions of the ali- mentary tract are closed in, but the middle region is still open to the yolk. The circulation is now fully established, and the vessels present the usual vertebrate arrangement. There is a large subintestinal vein. The first of Agassiz' embryos was hatched about ten days after impregnation. The young fish on hatching immediately used its suctorial disc to attach itself to the sides of the vessel in which it was placed. The general form of Lepidosteus shortly after hatching is shewn in fig. 07. On the ventral part of the front of the head is placed the large suctorial disc. At the side of the head are seen the olfactory pit, the eye and the auditory vesicle ; while the projecting vesicle of the mid-brain is very prominent above. Behind the mouth follow the visceral arches. The mandibular arch {md) is placed on the hinder border of the mouth, and is separated by a deep groove from the hyoid arch {hy). This groove is connected with the hyomandibular cleft, but I have not determined whether it is now perforated. The posterior border of the hyoid arch is prolonged into an opercular fold. Behind the hyoid arch are seen the true branchial arches. Fig. 65. Embryo of Lepidosteus shortly before hatching. ol. olfactory pit ; sd. suctorial disc ; hy. hyoid arch. aJN'OIDEl. — -sA There is still a continuous dorso-ventral fin, in which there are as yet no fin-rays, and the anterior pairetl fins are present. The yolk-sack is very laroe, but its communication with the alimentary canal is confined to a narrow vitelline duct, which opens into the ' commencement of the intes- tine immediately behind the duct of the liver, which is now a compact gland. The) yolk in Lepidosteus thus be- haves very differently from that in the Sturgeon. In the first place it forms a special external yolk-sack, instead of an internal dilatation of part of the alimentary tract ; and in the second place it is placed behind instead of in front of the liver. I failed to find any trace of a pancreas. There is however, opening on the dorsal side of the throat, a well-developed appendage op Fig. 66. Ventral view of the head of a Lepidosteus embryo shortly before hatchin(j, TO shew the large suctorial disc. 111. mouth ; op. eye ; sd. suctorial disc. Fig. 67. Larva of Lepidosteus shortly after hatching. (After Parker.) ol. olfactory pit ; op. optic vesicle ; au v. auditory vesicle ; mb. mid-brain ; sd. suc- torial disc ; md. mandibular arch ; hy. hyoid arch with operculum ; br. branchial arches ; an. anus. continued backwards beyond the level of the commencement of the intestine. This appendage is no doubt the air-bladder. In the course of the further growth of the young Lepidosteus, the yolk-sack is rapidly absorbed, and has all but disappeared after three weeks. A rich development of pigment early takes place ; and the pigment is specially deposited on the parts of the embryonic fin which will develop into the permanent fins. The notochord in the tail bends slightly upwards, and by the special development of a caudal lobe an externally heterocercal tail like that of Acipenser is established. The ventral paired fins are first visible P.. E. IL 7 98 LFAUBOSTEUS. after about the end of the third week, and by this time the operculum has grown considerably, and the gills have become well developed. The most remarkable changes in the later periods are those of the mouth. The upper and lower jaws become gradually prolonged, till they eventually form a snout ; while at the end of the upper jaw is placed the suctorial disc, which is now considerably i^educed in size (fig. 68, sd). The "fleshy globular termination of the upper jaw of the adult Lepidosteus is the remnant of this embryonic sucking disc." (Agassiz, No. 92.) The fin-rays become formed as in Teleostei, and parts of the con- tinuous embryonic fin gradu- ally undergo atrophy. The dorsal limb of the embryonic tail, as has been shewn by Wilder, is absorbed in pre- "■ cisely the same manner as in Fig. 68. Head of an advanced larva op Tplpmtpi Ipavino- thp vpntral Lepidosteus. (After Parker.) leleostei leaving tne ventral 7 • r, ^t +1,^ ^ifonf^,.-, r^u. ow vn lobe to lorm the whole 01 the oh openings 01 the olractory pit; sd. re- mains of the larval suctorial disc. permanent taii-hn. Bibliography. (92) Al. Agassiz. "The development of Lepidosteus." Proc. Amer. Acad, of Arts and Sciences, Vol. xiii. 1878. General observations on the Emhryology of the Ganoids. The very heterogeneous character of the Ganoid group is clearly shewn both in its embryology and its anatomy. The two known types of formation of the central nervous system are exemplified in the two species which have been studied, and these two species, though in accord in having a holoblastic segmentation, yet difier in other important features of development, such as the position of the yolk etc. Both types exhibit Teleostean affinities in the character of the pronephros ; but as might have been anticipated Lepidosteus pre.'sents in the origin of the nervous system, the relations of the hypoblast, and other characters, closer approximations to the Teleostei than does Acipenser. There are no very prominent Amphibian characters in the development of either type, other than a general similarity in the segmentation and formation of the layers. In the young of Polypleius an interesting amphibian and dipnoid character is found in the pr-esence of a pair of true external gills covered by epiblast. These gills are attached at the hinder end of the operculum, and receive their blood from the hyoid arterial arch^ In the peculiar suctorial disc of Lepi- dosteus, and in the more or less similar sti'ucture in the Sturgeon, these fishes retain, I believe, a very primitive vertebrate organ, which has disappeared in the adult state of almost all the Vertebrata ; but it is probable that further investigations will shew that the Teleostei, and especially the Siluroids, are not without traces of a similar structure. ^ Vide Steindachner, Polypterus Laprodei, &c., and HjTtl, " Ueber d. Blutgefasse, &c." Sitz. Iflener Akad., Vol. lx. CHAPTER VI I. AMPHIBIA'. The eggs of most Ampiiibia^ are laid in water. They are smallish nearly spherical bodies, and in the majority of known Anura (all the European species), and in many XJrodela (Amblystoma, Axolotl, though not in the common Newt) part of the surface is dark or black, owing to the presence of a superficial layer of pigment, while the remainder is nnj igmented. The pigmented part is at the upper pole of the egg, and contains the germinal vesicle till the time of its atrophy; and the yolk-granules in it are smaller than those in the un- pigmented part. The ovum is closely surrounded by a vitelline mem- brane'', and receives, in its passage down the oviduct, a gelatinous investment of varying structure. In the Anura the eggs are fertilized as they leave the oviduct. In some of the Urodela the mode of fertilization is still imperfectly understood. In Salamanders and probably Newts it is ioternal*; ^ The following classification of the Amphibia is employed in the present chapter : J . { Aglossa. • ■^•1111^8'. (Phaneroglossa. fn \ Trachystomata. PeRENNIBRANCHIATA vr, , -'i IProteidae. TT TT J 1 in iAmvihiumidas. II. Urodela. ^. Cabucibranchiata JMenopomida.. n^ \ Amblystomidffi. Myctodeba ' q , ■' J ■ ^ ( oalamandridaB. III. Gymnophiona . 2 I am under great obligations to Mr Parker for having kindly supplied me, in answer to my questions, with a large amount of valuable information on the develop- ment of the Amphibia. 3 Within the vitelline membrane there appears to be present, in the Anura at any rate, a very delicate membrane closely applied to the yolk. * Allen Thomson informs me that he has watched the process of fertilization in the Kewt, and that the male deposits the semen in the water close to the female. From the water it seems to enter the female generative aperture. Von Siebold has shewn that there is present in female Newts and Salamanders a spermatic bursa. In this bursa the spermatozoa long (three months) retain their vitality in some Sala- manders. Various peculiarities in the gestation are to be explained by this fact. 100 FORMATION OF THE LAYERS. but in Amblystoma punctatum (Clark, No. 98), the male deposits the semen in the water. The eggs are laid by the Anura in masses or strings. By Newts they are deposited singly in the angle of a bent blade of grass or leaf of a water-plant, and by Amblystoma puncta- tum in masses containing from four eggs to two hundred. Salaman- dra atra and Salamandra maculosa are viviparous. The period. of gestation for the latter species lasts a whole year. A good many exceptions to the above general statements have been i-ecorded '. ( In Notodelpliis ovipara the eggs are transported (by the male?) into a / peculiar dorsal pouch of the skin of the female, which has an anterior I opening, but is continued backwai'ds into a pair of diverticula. The eggs ! are very large, and in this pouch, which they enormously distend, they \ undergo their development. A more or less similar pouch is found in Nototrema marsupiatum. ! In the Surinam toad (Pipa doraigera) the eggs are placed by the male on the back of the female. A peculiar pocket of skin becomes de- veloped round each egg, the open end of which is covered by a gelatinous t>perculum. The larvse are hatched, and actually undergo their metamor- phosis, in these pockets. The female during this period lives in water. Pipa Americana (if specifically distinct from P. dorsigera) presents nearly the same peculiarities. The female of a tree frog of Ceylon (Polypedates , leticulatus) carries the eggs attached to the abdomen. V Rhinoderma Darwinii^ behaves like some of the Siluroid fishes, in that I the male carries the eggs during their development in an enormously developed laryngeal pouch. Some Anura do not lay their eggs in water. Chiromantis Guineeusis attaches them to the leaves of trees ; and Cystignathus mystacius lays them in holes near ponds, which may become filled with water after heavy rains. 1 The eggs of Hylodes Martinicensis are laid under dead leaves in moist i situations. Formation of the layers. Anura. The formation of the germinal layers has so far only been studied in some Anura and in the Newt. The following descrip- tion applies to the Anura, and I have called attention, at the end of the section, to the points in which the Newt is peculiar. The segmentation of the Frog's ovum has already been described (Vol. I. p. 78), but I may remind the reader that the segmentation (fig. 69) results in the formation of a vesicle, the cavity of which is situated excentrically ; the roof of the cavity being much thinner than the floor. The cavity is the segmentation cavity. The roof is formed of two or three layers of smallish pigmented cells, and 1 For a summary of these and the literature of the subject vide "Amphibia," by C. K. Hoffmann, in Brouu's Classen uitd Ordnungen d. Thier-reichs. 2 Vide Spengel, "Die Fortpflanzung des llhinodermaDarwinii." Zeit.f. wiss. ZooL, Bd. XXIX., 1877. This paper contains a translation of a note by Jiminez de la Espada on the development of the species. AMPHIBIA. 101 the tioor of large cells, which form the greater part of the ovum. Fig. 69. Segmentation of Common Frog. Eana Tempokaria. (After Ecker. ) The numbers above the figures refer to the number of segments at the stage figured. These large cells, which are part of the primitive hypoblast, will be spoken of in the sequel as yolk-cells : they are equivalent to the food -yolk of the majority of vertebrate ova. The cells forming the roof of the cavity pass without any sharp boundary into the yolk-cells, there being at the junction of the two a number of cells of an intermediate character. The cells both of the roof and the floor continue to in- crease in number, and those of the roof become divided into two distinct strata (fig. 70, ep). The upper of these is formed of a single row of somewhat cubical cells, and the lower of several rows of more rounded cells. Both of these strata eventually become the epiblast, of which they form the epidermic and nervous layers. The roof of the segmentation cavity appears therefore to be entirely constituted of epiblast. The next changes which take place lead (1) to the formation of the mesenteron', and (2) to the enclosure of the yolk-cells by the epiblast. The mesenteron is formed as in Petromyzon and Lepidosteus by an unsymmetrical form of invagination. The invagination first com- mences by an inflection of the epiblast-cells for a small arc on the 1 Since the body-cavity is not developed as diverticula from the cavity of invagina- tion, the latter cavity may conveniently be called the mesenteron and not the archeu- teron. Fig. 70. Section through Frog's ovum at the close of segmentation. (After Gotte.) sg. segmentation cavity; II. large yolk- containing cells; ep. small cells at forma- tive pole (epiblast) ; X. point of inflection of epiblast; y. small cells close to junction of the epiblast and yolk. 102 FORMATION OF THE LAYERS. equatorial line which marks the junction between the epiblastic cells and the yolk-cells (fig. 70, x). The inflected cells become continuous with the adjoining cells ; and the region where the inflection is formed constitutes a kind of lip, below which a slit-like cavity is soon established. This lip is equivalent to the embryonic rim of the Elasmobianch blastoderm, and the cavity beneath it is the rudiment of the mesenteron. The mesenteron now rapidly extends by the invagination of the cells on its dorsal side. These cells grow inwards towards the segmen- tation cavity as a layer of cells several rows deep. At its inner end, this layer is continuous with the yolk-cells ; and is divided into two strata {fig. 71 A), viz. (Ij a stratum of several rows of cells adjoining the epiblast, which becomes the mesoblast {m), and (2) a stratum of a single row of more columnar cells lining the cavity of the mesenteron, which forms the hypoblast {hy). The growth inwards of the dorsal wall of the mesenteron is no doubt in part a true invagination, but it seems probable that it is also due in a large measure to an actual differentiation of yolk-cells along the line of growth. The mesenteron is at first a simple slit between the yolk and the hypoblast (fig. 71 A), but as the involution of the hypoblast and mesoblast extends further inwards, this slit enlarges, especially at its inner end, into a con- siderable cavity ; the blind end of which is separated by a narrow layer of yolk-cells from the segmentati(jn-cavity (fig. 71 B). In the course of the involution, the segmentation-cavity becomes gradually pushed to one side and finally obliterated. Before oblitera- tion, it appears in some forms (Pelobates fuscus) to become completely enclosed in the yolk-cells. While the invagination to form the mesenteron takes place as above described, the enclosure of the yolk has been rapidly proceed- ing. It is effected by the epiblast growing over the yolk at all points of its circumference. The nature of the growth is how^ever very different at the embryonic rim and elsewhere. At the embryonic rim it takes place by the simple growth of the rim, so that the point x in figs. 70 and 71 is carried further and further over the surface of the yolk. Elsewhere the epiblast at first extends over the yolk as in a typical epibolic gastrula, without being inflected to form a definite lip. While a considerable patch of yolk is still left uncovered, the whole of the edge of the epiblast becomes however inflected, as at the embryonic rim (fig. 71 A); and a circular blastopore is established, round the whole edge of which the epiblast and intermediate cells are continuous. From the ventral lip of the blastopore the mesoblast (fig. 71, m), derived from the small intermediate cells, grows inwards till it comes to the segmentation-cavity ; the growth being not so much due to an actual invagination of cells at the lip of the blastopore, as to a differentiation of yolk-cells in situ. Shortly after the stage repre- sented in fig. 71 B, the plug of yolk, which fills up the opening of the blastopore, disappears, and the mesenteron communicates freely AMPHIBIA. 103 with the exterior by a small circular blastopore (fig. 73). The position A. B. Ay Fig. 71. Diagrammatic longitodinai. sections thbough the embkyo of a Frog AT TWO stages, TO SHEW THE FORMATION OF THE GERMINAL LAYERS. (Modified from Gottc.) cp. ei^iblast; m. dorsal mesoblast; m. ventral mesoblast ; /(?/. hypoblast; yfc. yolk; X. point of junction of the epiblast and hypoblast at the dorsal side of the blastopore ; al. mesenteron ; s