W. f ^ *^'
J f i :
^^' K¥.I%H
:jf ji' i' t:
* i *: » f, ;»
. ._.. -. „. ^, ^j^ ,
1 1». >•, ;
^ t # 1^ if «
»;. ,f i. I? tl ^:, ^* % -m h ^^
■ ^^ I- i' )»> t ji
^- "' >*^ k? !'• V
m'i*iife-«i
* .1 r ,
*»%^:
% '0- M '0
> ;& ]» I
» Jt I > • > ,1 >
^ I » P ' > I ?
- ■'. f ^
i r
ir I > 7 \ I- >. 3,
- ,«. > I I
^ :^' » I i i
i * ?^ * ^ > >
i *' i#: *. ^^' *
■^*.».%'
* !■ * i '.
n ■•» I '
■ t > > ..
I I l»
'm % »■ ^' i
-. I. i. -. «
^ii> (« A i '^ ^ fit. ir ^ r
02671 050
mMm
( ^-v /
II
'UiHl» -"^t Jill'*'
BUDD^llii IN PEROPHORA
DISSERT AT I OK Presented to the Board of University Studies of tne Johns Hopkins University for the Degree of Doctor of Philosophy, -By-
— ^ 2. L ^ ^ h ^ L !^ L L ^ 1 8 9 6.
'r-m
buddtnct in pekophora
George Lefevre ,
Although the phenomenon of budding in the group of Tunicata has received the attention of numerous observers for a long time, during the past twelve or fifteen years investi- gation has been largely confined to the study of the process in the pelagic forms, Salpa, DoliolMm, and Pyrosoma . In the Ascidians, hov/ever, our knowledge of the budding, until re- cently, has been based almost entirely on the observations of earlier workers. Among the latter, Kowalewsky, (12 and IS) who studied the bud-development in Perophora, Didemnum, and Amaroucium, Delia Valle (4) in Didemnum, Distaplia , and Botryllus, and finally Seeliger (29) in Glavelina are espe- cially prominent.
Within the last two or three years, hov/ever, our knowledge has been greatly enriched by the researches of Pi- zon (22) and Hjort (8) on Botryllus, and of Salensky (27) on Distaplia.
Although the observations of these investigators
-1-
agree on many important points, still on others, notably the origin of the nervous system, they stand at the greatest va- riance.
While the llarine Laboratory of the Johns Hopkins University was stationed at Beaufort, N. C, during the sum- mer of 1H94, I collected material for the purpose of studying the development of the buds of Perophora viridis, Verrill, which was found there in great abundance. This material I supplemented the follov/ing summer at V/oods Holl, Mass. while working in the laboratory of the United States Pish Commis- sion.
My main object in undertaking the investigation was to compare the bud -development of Perophora with that of Bo- tryllus, as described by Hjort (8), and especially to deter- mine, if possible, the origin of the nervous system.
The material, which was easily obtained at both places in large quantities, proved to be most excellent for my purpose, as it contained an unlimited supply of buds in all stages of development.
The preserving fluids used were, 1, glacial acetic acid; 2, a mixture of eighty parts concentrated corrosive sublimate solution and twenty parts glacial acetic acid; and 3, Perenyi's fluid. The latter reagent gave perhaps the most satisfactory results, although very good preparations were obtained with the sublimate-acetic mixture, when the ob- jects were left in it not longer than ten minutes.
Mayer's Haemalum proved to be most serviceable.
while borax carmine gave an excellent stain when used after acetic acid or sublimate-acetic, but was of no value for ob- jects fixed in Perenyi's fluid.
I have made use of Patten's method of orientation (21) to great advantage, and have found it of invaluable as- sistance in manipulating very small buds, which I was enabled to cut with accuracy in any plane desired.
In studying the bud-development of Perophora I have employed serial sections of all the stages in the sagittal, frontal and transverse planes, and also a complete series, throughout the entire development, of buds mounted in various positions as total preparations.
The sections were cut from 3 to S/*- in thickness on a Thome microtome, and all the drawings were made with the aid of a Zeiss camera lucida.
I take much pleasure in acknowledging my indebted- ness to Prof. Brooks, for the kindly interest with which he has follov/ed my work, and for valiiable assistance given me. I also desire to express in this place my great appreciation of the many courtesies extended to me by the late Colonel Marshall MacDonald at the Station of the 'J. S. Pish Gommis-
A preliminary account of some of my results '.vas published in the Johns Hopkins University Circulars, No. 119, June, 1895.
PEROPHORA VIRIDIS, VERRILI,.
This Ascidian, which is the only species of Pero- phora known to occur on the Atlantic coast of North America, was first found in Vineyard Sound by Vcrrill (34), and des- cribed by him in 1871. A nev/ species. P.annectens, has re- cently been reported from the coast of California by Ritter, (23)
Perophora viridis grov/s luxuriantly below lov^-water mark on the wharf piles in Little Harbor, V'oods Holl, and Vineyard Haven, :iartha's Vineyard. I also found it to be equally abundant during the summer months in the harbor of Beaufort, N. C. The colonies form large thick clusters of a beautiful greenish-yellow color, and usually occur together with other Ascidians, and with Hydroids, Bryozca, Sponges and Barnacles, the delicate stolons creeping over and cover- ing the surfaces of everything within reach.
THE RUDIMENT OF THE BUD.
Budding in Perophora was first studied by Kowalew- sky (12), v/hose careful work on this form laid the foundation for all subsequent investigation of the process of budding in the Ascidians .
■■ietschnikoff (18) had already discovered in Bo try 1-
-4-
lus, that the bud-rudiment consists of two vesicles, one with- in the other, the outer being derived from the ectoderm, and the inner fron the peribranchial wall of the parent. He al- so observed the splitting up of the inner vesicle to form the median branchial sac and the lateral peribranchial spaces, but neither Metschnikoff nor Krohn (15) who confirmed these observations on the Botryllus bud, sav/ at all clearly the de- tails of the development. This was left for Kov/alewsky, who described many of the internal processes occurring in the bud-development of Perophora Lister i . He show^ed that in Pe- rophora also the young bud is composed of two vesicles, the outer one being derived from tr.e ectodem and the inner from the partition wall of the stolon. According to Kowalewsky's account the endodermal or inner vesicle becomes divided com- pletely into three portions, the two lateral fusing dorsally and form.ing the peribranchial cavity, and the median giving rise to the branchial sac. I shall try to show below that in Perophora viridls, at all events, the peribranchial cavity is formed by quite a different process.
The origin of the bud as a double-v.'alled vesicle has been verified by all subsequent investigators, and tho- roughly established as a type of development cormion to all Ascidians whose budding has been studied.
The outer wall of tne vesicle is directly derived from the ectodem of the parent animal, and becomes the ecto- dermal covering of the bud. According to the majority of investigators this outer layer takes no active part in tiie furtner development, but Salen,3ky (27) and Oka (20), as v;ill
be pointed out belov>f, inaijitain that the ectodem is concerned in the formation of the nervous system.
As to the derivation of the inner wall, of t.ie ves- icle, the case is not so simple, for in different Ascilians this layer arises from entirely different parts of the parent, coming in some forms from an endodermal, in o triers from an ectodermal structure. In Perophora and Glavelina it is de- rived from the parti t ion-v;all of the stolon, whicn in the latter, and presumably in the former, is of endodermal origin; in Didernnurn and Distaplia from the wall of the gut, and in the Polyclinidae from the endodermal wall of the post-abdomen. In all the above mentioned species, then, th« inner ves- icle of the bud-rudiment is derived from an endodermal struc- ture. In Botryllus , however, this inner vescicle is fonned directly as an evagination of the outer v^all of the peribran- chial space, whose origin in the Ascidian larva has been a question of much dispute. Kowalewsky (11) first showed that in the larva of simple Ascidians the peribranchial cavity is formed from two symmetrical ectodermal invaginations, which later fuse together dorsally and surround the branchial sac. Delia Valle (4), however, contradicted these results, and maintained an endodennal origin for the peribranchial sacs in both simple and compound Ascidians, and Pizon (22) has re- cently described the sacs as arising from two divertecula from the branchial sac in the larva of Botryllus. Kowalev/- sky's account is borne out by all the later investigation of the subject, with the exception of that of Pizon. Seeliger
(29: Clavelina) , 7Jilley (35: Ciena) , Salen.sky (28: Diploso- aa, Didemn-on) , and Caullery (1: Dlstaplla,Tjeptoellnum, Qlos- sophorum, CircinaliujTi) , agree in their statements that the peribranchial sacs arise in t"ne manner described by Kowalew- sky, and are therefore purely ectodermal. It is fairly safe to assume that Pizon is v.frong, and tnat in Botryllus, as in all other Ascidians studied, the sacs are ectodermal. Tf triis be the case, as the inner vesicle of the bud-rudiment is directly cut off from the outer peribranchial wall in both larva and buds, it follov;s that all the organs, including the peribranchial sacs, derived from this inner vesicle through- out the entire series of buds proceeding from the larva, are ultimately of ectodermal origin.
After this brief review of tne state of our knov;- ledge concerning trie origin of tne bud-rudiment in the Asci- dians, I shall now describe the process, as I have found it to occur in Perophora viridis .
The buds arise in a single rov/ along the course of the delicate stolons, which branch profusely and adhere on one side to the surface over which they creep. The branches come off approximately at right angles to the stems, from which they proceed, but aside from this char;icteris tic there is no regularity whatever in the method of branching; they arise at unequal intervals, and as each grows out, it gives off in its turn still younger shoots, the whole system be- coming much tangled and twisted to form a loose felt-work.
Proceeding towards the growing tips of the stolons, both buds and branches are seen to become successively young-
-7-
er .
In Fig. 6, Is sho-^n a transverse section of a sto- lon. The outer wall of trie hollow cylindrical tube consists of a flattened epitheliini, the ectoderm, ^ct., covered exter- nally by the transparent cellulose test, _t. Running from one side to the other the partition-wall, p_. t . , is seen in cross-section, completely dividing the tube into two com- partments, in which the free cells of the blood lie scattered about. The stolonic partition consists of a double lamella made up of flat, attenuated cells, and may be described as a collapsed cylinder, the walls of which are closely pressed together, and attached along the upper and lower borders to the inner surface of the ectodermal tube. It divides the stolon longitudinally into halves, and stretches entirely across the lumen, although in preserved material it is usual- ly more or less shrunken away from the outer wall. The two compartments of the stolon are in open communication with the body cavities of the animals, so that a free circulation of blood occurs from one individual to another.
As Kov;alev;sky (12) has pointed out, the partition- wall ( "Scheidewand ") does not reach quite to the extreme dis- tal end of the stolon, but there the two spaces or sinuses open into each other. The blood flows in opposite direction; in the two sides of the stolon, up one to the tip, around the free end of the partition, and down the otner. v/hen the heart-beat is reversed, of course the direction of the blood- flovf in tne stolon is changed.
•8-
The buds always arise in the plane of the partition wall, on the side of the stolon opposite to that which is at- tached to the underlying surface; the line of attachment is, therefore, parallel to and immediately beneath the lower bor- der of tne partition. It will be seen further on that the definitive s-agittal plane of the bud coincides with the plane of the partition-wall, and hence the latter structure divides the stolon into a right and left half in reference to the parts of tne bud, and not into a dorsal and ventral portion, as described by Kowalewsky (12), whose well-knov/n figure of the P^erophora stolon is consequently misleading. Looking at the lateral surface of the buds, as shown in that figure^, the partition-wall would be seen from the flat side and not on edge.
The first indication of tne appearance of a bud is a slight bulging out of tne ectoderm of the stolon at one point, and if a transverse section be taken at this spot, it will be seen that the two layers of the partition-wall have spread widely apart, giving the appearance of a more or less spherical vesicle. This is well shown in Pig. 7. The walls of the partition are seen to be no longer composed of flat cells, except on the lov/er side, that is, the side to- wards the surface of attachment, but have become very much thickened by active cell-multiplication. The cells, toe, of the ectoderm over the thickeiied portion of the partition have
-'■ This figure is reproduced in Korschelt and Heider's "Ent- wicklungsgeschichte",page 1366 .
-9-
increased in height and are now nearly cuboldal. The por- tion, where it is s-^rollen out into a vesicle, becomes drawn away from the outer wall of the stolon. I do not think that this is due to shrinkage, for it is invariably found, and moreover, beyond this region on ei trier side, where the partition is still flat, it is seen to stretch clear across the lumen of the stolon.
The ectoderm continues to bulge out more and more, until it forms a hemispherical protuberance on the surface of the stolon. The cells composing the raised portion of the ectoderm do not remain cuboidal, but from now on, as the swelling increases, become gradually flattened again. The thickened portion of the partition keeps pace with the ecto- dermal evagination, and grov/s out into the latter, but the thin walls below nov; come together, and close off tne upper portion as a thick-walled vesicle, without, however, severing the connection. This process is readily understood from Pig. 8, in which the walls of the lower part of the partition have become united again.
In Pig. 1, a bud is shown at tnis stage from the exterior.
As the ectoderm continues to push out, it becomes constricted where it passes over into the stolonic ectoderm, the constriction being greater at first before and behind than on the sides. The bud-rudiment, which by this time is almost spherical, is sharply marked off from the stolon, and stands eitner straight out as a round knob from the surface
• 10-
of the latter, or is slightly inclined towards its free tip; Pig. 2.
It novi represents the characteristic double vesicle of the young Ascidian bud; the outer or ectodermal vesicle is directly derived from the ectoderm of the stolon, and the inner or entodermal vesicle, v/hich has become a closed sac, arises by evagination of the thickened portion of the dilated stolonic partition. The connection between the partition and the inner vesicle is retained for a long time, and the cavity of the latter is to be regarded as being in communi- cation with the potential cavity of the partition.
A transverse section at this stage, Fig. 8, shows the still somewhat thickened cells of the ectoderm of the bud rudiment, and the endodermal vesicle, lying inside, v;-i th its much thickened v/alls. Active cell-multiplication has been going on in the latter, which are of uniform thickness, ex- cept below where they pass off gradually into the thin walls of the partition. The cells of the blood are found scatter- ed about betv/een the two vesicles, and it is to be especially noted that many are lying close against the inner surface of the ectodenn and outer surface of the endoderm at niimerous points .
The bud-rudiment does not long remain spherical, but soon becomes elongated by a grovTth towards tne apex of the stolon. It now assumes an oval shape, and one end lies free over the surface of the stolon beyond the portion which is directly connected with the latter, Pig. 3. It is the free end which is later to be distinguished as the anterior
-11-
portion of the bud, v/hile by this process of elongation an- teriorly the connection with the stolon comes to be left be- hind in the posterior region. The side next th^'e stolonic wall becomes the ventral surface of the bud, and that oppo- site it the dorsal.
The protuberances of the stolonic ectodenn, which Seeliger (29) describes as occurring at the base of the bud- rudiment in C_lavelina, are not present in Perophora ; they are merely simple ectodennal sacs, filled with blood-cells, and do not contain an evaginatlon of the partition-wall. Seeliger calls them "TIahrkanrnern", and regards them as res- ervoirs of food-material for the developing buds.
Before going on to describe the origin of the va- rious internal organs, some of which have by this time begun to make their appearance, I wish to say a word about the par- tition-wall of the stolon.
A.S the inner vesicle of the bud is formed entirely from this structure, it is a matter of importance to know from what it is derived in the larva.
It has never been observed in Perophora how the stolonic partition arises, although it is usually supposed to be of endodermal origin from its likeness to the similar structure in Clavelina.
Van Beneden and Julin (33) have shown that in the larva of Clavelina it is formed as a direct continuation of the epicardiim, which arises as a diverticulum from the pos- terior wall of the branchial sac, and is therefore entirely
■12.
endodermal.. During the month of August, 1895, I made an attempt to discover the ori;^in of the stolon in Perophora. Larvae were put into aquaria, through which water was kept constantly flowing, and although they settled down and under- went tiie metamorphosis, at the expiration of nearly four weeks, not the trace of a stolon sprouted from them. VJhen larvae were afterwards sectioned and studied, nothing like an epicardium, such as occurs in Clavelina, was found.
If then such a structure exists in the larvae of Perophora, it must be formed at a very late period. As I shall try to show further on, it is doubtful whether there is a true epicardium. in the buds of this Ascidian, and it is possible that it is also absent in the larvae.
The further Development of the Bud.
At the time when the bud-rudiment begins to e- longate, or very shortly after, the rudiments of several nev/ structures are laid down. These are the (1) pericardium; (2) the peribranchial sacs; (3) the d_orsal tube ; (4) the gut; and (5) the ganglion. These do not all arise sim.ultaneously and although the rudiment of the pericardium is the earliest to appear, it will be necessary to describe the formation of the peribranchial sacs first, in order to render intelligible certain relations between these and other structures.
Origen and Development of _th_e Peribranchial Cavity.
All investigators agree in deriving the peribran- -15-
chial cavity from the inner vesicle of the bud-rudiment, but the manner in which it arises is not the same in all species
of Ascidians.
e
In Perophora , Didennum, and Amarouium, according
to Kowalewsky (12 and 13) two parallel longitudinal furrows appear on the outside of the inner vesicle, and by gradually deepening finally divide the latter completely into three portions. The two lateral divisions which are thus cut off, grow up over the middle one, and fuse to form the median por- tion of the peribranchial cavity, which now surrounds the branchial sac dorsally and laterally.
Seeliger (29) has described a different method of formation of the peribranchial cavity in Glavelina. Accor- ding to him the inner vesicle is not divided into three por- tions, but into two, one of which, the posterior, gives rise to the branchial sac and the gut, while the other forms the whole peribranchial cavity. These results were contradicted by Van Beneden and Julin (33), who maintained that in Glave- lina, the process is the same as that described by Kowalev/sky-
As Seeliger's view was not founded on an investigation of an unterrupted series of stages, and as there were wide gaps in his observations at periods which are especially concerned in the formation of the peribranchial cavities, the supposi- tion that his results are wrong is very probable. In Dis- taplia Salensky (27) has shown, and his results have been confiiined by Hjort and Prl . Bonnevie (10), that the inner vesicle gives rise to two lateral evaginations , which become completely constricted off as separate vesicles, the peri-
-14-
branchial sacs, and gradually extending dorsally, fuse to- gether on the median line. These sacs are not formed at the same time, but one is given off from the inner vesicle before the other, so that at a very early stage an asymmetry of the bud is produced.
Delia Valle (4) described a sim.ilar method of form- ation of the peribranchial cavity for Botryllus , but both Pizon (22) and Hjort (8) have conclusively proven his obser- vations to be erroneous.
According to these authors both the lateral and me- dian portions of the peribranchial cavity arise at the same time as a saddle-shaped bag, which is cut off by two longi- tudinal furrov/s from a median vesicle, the later branchial sac. Hjort regards this process as a great curtailing of the embryonic development, such as often takes place in buds.
Salensky (27) confirms this conclusion, but goes a step further, saying that, "die Entwicklung der Peribranchialhohl- en des Botryllus eine Abkurzung nicht nur beziiglich der era- bryonalen Entwicklung, sondern auch beziiglich der Entwicklung dieser Organe in den Knospen anderer Ascidien darstellt."
My observations on the development of this struc- ture in Perophora viridis, although agreeing with those of Iijort, in so far as they show that the peribranchial sacs do
not arise separately as closed vesicles, which later unite to
the form clcacal cavity, indicate that the process is not so sim- ple as that which occurs in Botryllus .
If a transverse section of a bud be examined about
-15-
the time when the elongation spoken of above is just begin- ning, it will be found that the -vvall of the inner or endoder- mal vesicle is no longer of uniform thickness. ?ig. 9, is drawn from such a section. The ectoderm covering tne bud, although it is not shown in the figure, has again become flattened after its temporary thickening, and is nov/ like that of the stolon. ^ The figure clearly shov/s that the sto- lonic partition is made up of two lamellae which are contin- uous below Y/ith each other and pass over above into the walls of the inner vesicle. The important change to be noted, how- ever, is that the wall of the endodermal vesicle on one side, the left, is getting perceptibly thinner than elsewhere, and that the whole vesicle is no longer symmetrically placed in reference to the stolonic partition, but is bulging out slightly to the right. This is the first indication of a marked change -Thich is about to take place in the internal relations of the bud-rudiment.
By a peculiar process, which may be described as a transverse or rotatory growth affecting the inner vesicle, the thicker wall of the right side is carried or pushed down gradually until it comes to lie eventually on the ventral side, that is, the side next to the stolon.
The stolonic partition remains stationary, and the displacement or shifting around of the inner vesicle takes place on this as a fixed support. The process might be il- lustrated by the drooping of a flower to one side on its stem, although the change of position cannot be a purely passive falling over of the vesicle. In Fig. 9, a small
-16-
collection of cells, p c . , is seen adhering to the -.vail of the vesicle high up on the right side, which, as we shall see below, is the rudiment of the pericardium. This cell- mass remains fixed at the same place on the wall, and during the shifting of the vesicle is borne dov/n towards the ventral side, describing in its descent an arc of about 90 . It therefore furnishes a good register of the pi'ogress of the displacement of the vesicle.
As the turning proceeds, the difference in thick- ness between what was at first nearly the whole right side, and the rest of the vesicle, becomes more marked; conse- quently the cells composing the entire vesicle, except in the thicker region, are seen to be growing more and more flatten- ed.
The displacement is most probably brought about by a rapid growth and stretching of the cells composing the greater portion of the vesicle, whereby the actual right side which is morphologically the ventral side of the vesicle, is shifted or pushed ventrally through 90*^ .
This process is analogous, at all events, with the rotation or displacement of the pharynx of the Amphioxus lar- va from, right to left, although I am not prepared to claim any phylogenetic relation between the two.
By comparing Fig. 9, with Pigs. 10 and 11, the pro- cess can be readily understood. The shifting involves the anterior end of the vesicle only to a slight extent. In this region a difference in thickness of the walls is not ob-
■17-
served, and the rudiment of the dorsal tube, which defini- tively has a median dorsal portion in the anterior end of the bud, arises as a collection of cells, almost at the same time that the pericardial rudiment appears, lying a little to the left of the mid-dorsal line on the wall of the vesicle, d.t. Fig. 28. If the displacement took place to as great an ex- tent anteriorly as posteriorly, it is evident that this cell- mass would appear much further down on the left side; but that the anterior end is slightly rotated is shown by the fact that the rudiment appears not exactly in the median line where it is eventually brought through the shifting of the vesicle, but somewhat to the left of it.
The formation of the peribranchial cavity is asso- ciated with this change of position of the endodermal vesi- cle. In Figs. 10 and 11, it is seen that the lov/er portion of the vesicle at the point indicated by the line a_, is being bent in, with the result that the wall in this region makes two angles, one directed inward and the otner outward, a and _b, Fig. 11. The apex of the latter marks a point on the wall of the vesicle, which will have travelled through 90 , when the displacement is completed, as its final position will be in the mid-ventral line.
As the inv/ardly directed fold deepens, it gradually divides off a portion of the inner vesicle on tne left side, which is connected with the stolonic partition; this is the left peribranchial cavity, 1 .pbr . c . Pigs. 11 and 17. This fold begins somewhat in front of the middle of the vesicle.
•18-
and deepenirig rapidly in this region, gradually extends pos- teriorly.
As these changes are going on, the connection with the stolonic partition is gradually becoming co: stricted, and is now only present in the posterior half of the bud, while at the same time the ectodermal stalk is also getting narrov/- er. Ritter (24) in a preliminary note on the budding of Pe- rophora, says that, "When the differentiation of tne 'endo-
derm' into the branchial and two peribranchial sacs, takes
tnat the de\/elopJng- place, it does so in such a way blastozooid is connected v/ith
the double walled partition of the stolon, not by the bran- chial sac, as has been hitherto supposed, but by the left pe- ribranchial sac." He does not, however, describe how this comes about. Prom an examination cf Pigs. 10 and 11, it is readily understood. The communication of the body-cavity of the bud with the blood spaces of the stolon is never complete- ly closed, as there is always a free circulation of blood from the one to the other, but eventually the left peribran- chial cavity is entirely severed from the stolonic partition.
I cannot, hov/ever, confirm Ritter 's statement (I.e., page 367), that this connection is lost at an early stage, namely, "at a time v/hen the two peribranchial pouches have merely be- gun to envelop the branchial sac." I find that it persists for a very much longer time, and is still present, although greatly constricted, at a stage when some of the gill-slits have been formed and the peribranchial cavity has been wholly separated from the brancnial sac. Pig. 21, shov/s the con-
•19-
nection at such a stage.
The first indication of the right peribranchial cavity is a slight longitudinal folding-in of the wall of the inner vesicle, some distance up on the right side, v/hich ap- pears after the shifting of the vesicle has begun. This furrov/ starts a little in front of the anterior terrranation of the left peribranchial fold, and as it deepens and extends posteriorly, it is gradually carried down towards the ventral side, in the same way as the pericardial rudiment. It is already present at the stage represented in Fig. 11, but has not yet reached back far enough to appear in a section which shows the left fold. In Fig. 12, which is taken from the same series of sections, but a little further forward, it is v/ell marked, r . p hr.c .
As the shifting continues, the inner vesicle tends more and more to assume a symmetrical position. The two furrows, which deepen rapidly and run in obliquely to meet each other, do not come together on the dorsal surface of the vesicle, but some distance below it. The result of this is, that when the right and left peribranchial cavities are sep- arated from the inner vesicle, a median dorsal portion con- necting them, is constricted off at the same time. This me- dian piece, hence, does not arise, as Kov/alewsky (12 and 13) describes, from the fusion of the lateral sacs dorsally, but the three portions are cut off together. We now find a me- dian vesicle, the later pharynx, surrounded dorsally and lat- erally by a U-shaped bag, which consists of the dorsal or
-20-
cloacal and the lateral divisions of the peribranchial cavi- ty. This is essentially the same process as that which Pi- zon (22) and Hjort (8) have described for Botryllus .
In Perophora the folds which separate the peribran- chial cavity from the inner vesicle, do not involve the en- tire length of the latter, but leave nearly the whole of the anterior half undivided, as well as a short region at the posterior end of the vesicle. And further, the whole peri- branchial cavity is not constricted off at the same tir.e, but as stated above, the furrows begin anteriorly and extend gradually back, so that at any given stage the opening of the median vesicle into the peribranchial cavity is much wider in a posterior section than in one further forward.
V^hen the right peribranchial cavities are being separated off, as just described, a broad pouch or divesti- culum grov/s out from the anterior margin of each, and by de- grees spreads over the undivided portion of the inner vesi- cle. These pouches are direct continuations of the lateral Cavities, and later completely cover the sides of the ante- rior region of the peribranchial sac, but they never fuse dorsally.
Similar extensions are carried out from the poste- rior margin of the lateral cavities, and though not promi- nent at first, still, as the bud gets older and increases in length, they attain a considerable size and surround a part of the digestive tract.
The peribranchial cavity nov/ consists of two deep
-21-
lateral sacs, surrounding the spacious branchial sac, and connected dorsally bj'^ a median space, the cloacal cavity. The lateral sacs are unsymmetrical, however, until quite a late stage, for the anterior pouch of the right peribranchi- al sac grov/s more rapidly and extends further forward than the sirrilar pouch on the left side, while the posterior pouch of the left side extends further backv/ard than the corres- ponding one on the right side. Eventually the two sacs be- come symraetrical.
The formation of the peribranchial cavity is eas- ily understood from the series of sections represented in Pigs. 13--1S; these v/ill be rendered more intelligible by a comparison with Pig. 4, which is drawn from a total prepara- tion of a bud at the same stage of development. The sec- tions are taken respectively at the levels indicated by the parallel lines, a_, _b, _c, d, e^ and f_ of Pig. 4. In Pig. 13, line a of Pig. 4, the most anterior one of the series, the branchial sac, br, is seen by itself, for the extensions of the peribranchial sacs have liot re.ucle-ct far enoi.'gii forv/tird to appear in the section; the hypophyseal tube, d . t , is shoivn on the dorsal side of the branchial sac. Fig. 14, line _b, only includes the anterior extension of the right sac, for, as just stated above, the pouch on the opposite side lags be- hind, in its growth. Fig. 15, line c, represents a section taken just in front of trie anterior face of of the cloacal cavity, and shov/s both peribranchial sacs at the level where they are continued foTtfard into their anterior extensions.
In Pig. 16, line d., trie section passes through tiie anterior portion of the cloacal cavity, cl, which is seen to connect the lateral sacs; the constriction, which will untirnately completely cut off the U-shaped bag from the median vesicle has proceeded in this region to a considerable extent, and has greatly narrowed the opening between the peribranchial and branchial cavities. In a section further back. Fig. 17, line e, the folds which are foniing the peribranchial sacs, are much less deep and v/ider apart; the connection between the left sac and tne stolonic partition is present in this region. Finally, Fig. IS, line _f, represents a section be- yond the peribranchial sacs, the posterior pouches of which at this stage have not yet begun to grow out; the section passes behind the connection with the stolonic partition, but through the storaach, s^t^, which is seen on the left side.
The further development of the peribranchial cavity from this stage on merely consists in the completion of tne constriction, whereby the 'J-shaped bag is completely cut off from the branchial sac, and in the extension of tne anterior and posterior pouches of the peribranchial sacs, which final- ly surround the whole pharynx laterally. These relations are illustrated by the series of sections. Figs. 19--21, which are taken from the same bud. Fig. 19 is a section through the anterior end of the bud, and shows tne lateral extensions of the peribranchial sacs surrounding the pharynx. The peri- branchial cavity is now entirely cut off, and its lateral portions are united in the middle region of the bud by the
-23-
dorsal connecting piece or cloaca; this condition appears in Pig, 20. Beyond the cloaca, the posterior pouches of the lateral sacs, which like the anterior pouches are not united dorsally, are seen in Fig. 21; the connection of the left sac witn the stolonic partition in tnis figure has already been referred to. By this time the process of displacement is completed, and the definitive syminetrical arrangement of the pharynx and peribranchial cavity is reached. The con- nection between the left peribranchial sac and tae partition of the stolon is nearly severed; it is found in only two sections of this series, one of which is seen in Fig. 21. A total preparation of a bud at about this stage is shown in Pig. 5.
Epicardiam. This structure was first described by Van Beneden and Julin (33) in the buds and larvae of Clavelina, and was shown by these authors to be closely con- nected with the development of the pericardimi. It arises as an evagination of the posterior wall of the branchial sac, and a little further back divides into two blind pouches, which remain separate in the buds, but in the embryo unite to form the "cul de sac epicardique" of Van Beneden and Julin; the latter is continued into the stolon to form the double- walled partition. The development of the epicardium will be again referred to in connection with the pericardimn, with which it stands in very close relation in some Ascidians.
In Distaplia Salensky (27) has described the epi- cardial sacs as arising in the buds at an early stage by eva-
-24-
gination from the posterior end of the inner vesicle; the two sacs are not formed at the same tine, and the left one is always larger that the right.
In the buds of the Polyclinidae the epicardium is formed in the same 7.'ay; two small diverticx^la, a right and a left one, are given off from the posterior end of the bran- chial sac, from which they afterv/ards become detached. They soon, however, unite to form a single tube, which is contin- ued out into the postabdomen, where it is destined to fur- nish the inner vesicles of the buds produced by transverse constriction of that region of the body.
The existence of an epicardium in Botryllus is de- nied by Hjort (8), but maintained by Pizon (22). According to the latter the inner vesicle at a very early stage gives off two anterior lateral diverticula, one on each side, which later form the peribranchial cavity, and also two posterior lateral diverticula. These four pouches are at first sepa- rate, but soon the two on each side fise in the middle region of the bud. V^hen the peribranchial cavity is separated from the inner vesicle, the posterior diverticula are cut off at the sane time, and now appear as posterior prolongations of the cloacal cavity, with which they always remain in free communication. They are what Pizon calls the "diverticules perivisceral^;:" , and in later stages completely envelop the digestive tract. From the fact that these cavities arise as two diverticula from the posterior end of the inner vesi- cle Pizon regards them as homologous with the epicardial
-25-
tubes of other Ascidians, and states (I.e., page 29), that "la formation de cette cavite peri viscerale n 'est pas secon- daire ©t qu'elle s'est annoncee, des le debut, par deux pe- tits diverticules posterieurs de la vesicule primitive, en niemetenps que les diverticules anterieurs correspondants qui engendreront la cavite peribranchiale . "
These perivisceral diverticula, however, differ from the epicardial tubes of Clavelina, Distaplia and the Polyclinidae in that they communicate with the cloacal cavi- ty.
Hjort (I.e., page 594) states, that the "einheit- liche Peribranchialblase sich nun derart weiter entwickelt, das sie nicht nur den Abscnnitt des Kiemendarnies , sondern den ganzen Darmtractus unwachst", and Salensky (27), who ac- cepts the conclusion of Pizon as to the homology of the peri- visceral diverticula, thinks that Hjort evidently saw the 'epicardial sacs' in Bo try 11 us, but failed to recognize them as such. Salensky believes that the connection of the 'epi- cardial sacs' with the cloaca in Botryllus, must be regarded as a result of the early separation of the peribranchial ca- vity from the inner vesicle.
In the light of these considerations it is possible that the posterior extensions of the peribranchial sacs, which I have described as arising in the buds of Perophora viridis,are likev/ise homologous with the epicardial tubes of other Ascidians. It is to be remembered, however, that if such be the ease, which I think very doubtful, their direct
origin from the inner vesicle has been completely lost, as they do not appear until quite a late stage, and then merely as prolongations backi^^ard of the lateral portions of the pe- ribranchial cavity, after the latter have been entirely cut off from the inner vesicle. This would, therefore, be a still more modified condition than that which is found in Bo- tryllus .
Pizon (I.e., page 105) makes the statement, which is not, however, illustrated by figures, that he has confirm- ed on the buds of Perophora Listeri the results of Kowalewsky (13: Amaroucium proliferum) and of Van Beneden and Julin (33: Glavelina Rissoan-t) in regard to the origin of the epicardial tube. "Ce tube", he says, "resulte bien de la reunion de deux petits diverticules qui naissent a droite et a gauche du sac branchial et qui s'isolent conpletement de celui-ci a un moment donne . " Such a description is not in the slight est accord with my obser-'ations, and if an epicardial tube arises in this manner in the buds of the European Perophora, it certainly does not in Perophora viridi s.
The Branchial Sac or Pharynx. That portion of the original inner vesicle, which is left after the separation of the peribranchial cavity, becomes the pharynx. At its ante- rior end it finally opens to the exterior through the bran- chial orifice, and after the appearance of the gill-slits it comjnunicates with the peribranchial cavity, w..ile posteriorly it leads off into the digestive tract.
-27-
The formation of the branchial sac in the buds of Perophora viridis is complicated by reason of the peculiar shifting of the inner vesicle, which has been described a- bove . The whole vesicle, with tne exception of tne anterior end, Y/hich, as already stated, is but slightly involved in the process, becornes shifted or revolved through about 90 , in such a way that the original right wall of the vesicle comes to lie ultimately on the ventral side. This right wall, as has been shown, is early found to be much thicker than the rpst of tne vesicle, the difference being due, not to an increase in thickness of this region, but to the flat- tening of the cells composing the remaining portion of tne vesicle. It is this thickened v/all, originally on the right side, which forms the floor of the pharynx in that part of the vesicle which is concerned in the displacement. Very soon after tne beginning of the change in position, a shallow longitudinal groove is found on the inner surface of the ves- icle in the middle region of the bud, lying on tne rignt side on a level with the lov;er border of the pericardial rudiment. This is the first appearance of the endos tyle ; its position is shown at end in Fig. 10, but at a slightly earlier stage it is found even higher up. In this figure and the next two it is seen that tne groove runs through about the middle of the thickened area, that is to say, above and belov; it there are equal portions of tne thick wall, which will lie to its right and left when the change in position of the vesicle is fully accomplished.
• 28-
The groove rapidly extends anteriorly and posteri- orly, and at the same time becomes deeper and broader. When it reaches its definitive position in the mid-ventral line, it stretches throughout the entire length of the branchial sac.
It v/ill not be necessary to speak of the differen- tiation of the endostyle into the various zones of cells which go to make it up, as these have been described by nu- merous authors: Delia Valle (3), Herdmann (7), Lahille (17), and others.
It is to be especially noticed in Figs. 10, 11, 17 and 21, that the rositions of tne pericardial rudiment and endostylar groove in reference to each other remain the same during the displacement of tne vesicle. Prom this fact it is evident that the thick portion of tiie vesicle is carried down bodily, and that no interstitial growth takes place in this region during the process, else the distance betv/een the pericardium and endostyle would not remain the same. It cannot be said that the pericardial rudiment might compensate by its own growth for any increase in extent of that part of the wall against which it lies, for it covers practically about the same area as long as it adheres to the vesicle. It would seem, therefore, that the change in position of the inner vesicle is brought about by the stretching out and flattening of the cells in all but the thick area, and that the latter is borne or rather pushed dov/n tov/ards the ventral side .
-29-
The Gill-Slits . The gill-slits are not formed un- til after the peribranchial cavity has been completely sepa- rated from the branchial sac. The first to appear lie far back towards the posterior ' end, but very soon they begin to break out in spots all over the sides of the branchial sac.
The tendency to arise in vertical rov/s becones ap- parent, when only very fev/ are present, but eacii slit is a separate and independent formation. I have never observed the origin of one slit from another, such as occurs in the larvae of Ascidians.
Pig. 5, represents a stage when about eight slits have been formed on each side; as the anterior pouches of the peribranchial sacs grow further and further fon.vard, new rov/s of slits are laid down along their free margins.
The first indication of a gil]-slit is a small cir- cular, thickened area of the branchial wall, which at this spot becomes slightly evaginated until it touches the visce- ral wall of the peribranchial cavity^ ^ig. 22, a. The cells of the latter at this point become thickened somewhat, and now a fusion takes place between the two v;alls; this is seen in the lower portion of Pig. 22, b. The opening, which breaks through the centre of the fused patch of cells, is drawn out later in the long axis of the bud into a narrow slit, which is provided v/ith cilia in the usual way. The upper part of Pig. 22, b^, shows a slit just after the opening has been formed.
The Branchial and Cloacal Orifices . The branchial orifice arises at a tolerably late stage, and is first indi-
-30-
cated by a great increase in thickness of the ectoderm at a point opposite the extreme anterior end of the branchial sac. This thickened area becomes invaginated until the bottom of the pit touches the endodermal wall, and a complete fiision of the two soon takes place: Pigs. 23, a and h, and Fig. 5. The cells in the centre break down, and the cavity of the pharynx is put into conmunication with the outside. As is shown in Pigs. 23, a_ and h, many mesodermal cells attach themselves to the inner surface of the ectodermal depression, become greatly elongated, and are eventually transformed into muscle fibres .
In Botryllus, according to Pizon (22), it is the branchial wall which thickens and evaginates to fuse with the ectoderm, while the latter plays but a small part in the pro- duction of the orifice. The irocess, as it occurs in P e r o - phora virldis, is quite similar to that described by Kov/alev/- sky (13) for Phallusia, and by Van Beneden and Julin (33) for Clavelina .
The cloaca 1 orifice is formed in exactly the same manner by the union of an ectodermal invagination v;ith the wall of the cloaca at the anterior end of the latter, cl .o, Fig. 5, and Pig. 20. It will not be necessary to speak here of the various appendages and ciliated grov/ths of the pharynx which arise later, namely, the tentacles, papillae, languets, dorsal lamina, and peripharyngeal bands, as these are merely differentiations of the pharyngeal epithelium and have been sufficiently described by numerous authors.
THE DIGEST I VE TRACT.
Some time before the displacement of the inner ves- icle is completed, and when the folds which wil] cut off the peribranchial cavity are not very deep, the wall of the inner vesicle high up on the left at the extreme posterior end, be- comes much thickened, and soon evaginates to produce a little blind pouch, the rudiment of the digestive tract. Fig. 24, d_. tr. This lateral diverticulum grows out as a tube, which at once bends sharply downwards and for.vards, v/hile, as the shifting of the vesicle continues, its opening into the lat- ter is carried up nearer and nearer the mid-dorsal line, where it will ultimately come to lie. The tube soon turns abruptly on itself to form a close U, and nov; grov/ing up- wards along the outer wall of the left peribranchial sac un- til it reaches the dorsal surface, ^i^t- finally bends directly forward and stops short at the posterior v/all of the cloacal cavity. At this point the distal extremity of thetube fuses with the cloacal wall, an opening breaks through, and the anus is established. The differentiation into oesophagus, stomach and intestine takes place very early, and is apparent at a stage considerably younger than that shown in Fig. 4. The course and development of tne tube are sufficiently il- lustrated by Pigs. 4 and 5.
As the bud grov;s and increases in length, the di- gestive tract enlarges enormously, the U becomes opened more and more, and the intestine describes a v;ide curve which lies
• 32-
well forward against the outer wall of tne left peribranchial sac, Fig. 5. 'with tne anterior extension of the digestive tract and the posterior prolongation of tne left peribran- chial sac, the whole tract, which lies entirely on the left side of the bud, comes eventually to be closely enveloped by the outer wall of tne peribranchial cavity.
The "pyloric gland" or "organe refringent " of Giard arises as a tubular diverticulum from the lower ante- rior face of tne enlarged portion of the digestive tract which will become the stomach. Before reaching the intes- tine the tube bifurcates, and each branch in its turn gives off two others, which also divide, the whole system of dich- otomously branching tubules finally forming a lace-work sur- rounding the whole intestine.
The development of this problematical organ is al- ready well advanced at the stigo shown in Pig. 5, o .r . This figure, together with Pig. 25, which shows a portion of the stomach wall, leaves no doubt that the tube is directly deri- ved from the digestive tract. This origin was maintained by Delia "(''alle (3), but denied by Roule (25), who stated that the "organe refring'nt" is not a part of the digestive tract, but communicates v;ith the heart, and therefore belongs to the vascular system, an opinion already held by Kuppfer. (16)
Delia Valle's vievf is also supported by Pizon (22), whose description of the development of the organ in Botryl- lus , agrees minutely v;ith ray observations on Perophora.
■53-
The terminal branches of tne system of tubules which ramify over the surface of the intestine, end in little enlargements or ampullae, the -.vails of v/hich are very thin, and lie closely pressed against the intestinal' wall . The cells of the duct are cylindricil, and gradually pass over into the flat cells of the ampullae. I have failed to find any cilia on the latter, as Chandelon (2) has described in Perophora. Fig. 26, shoves a cros:---3ection of the intestine surrounded by the thin-v;alled tubules and ampullae, tne flat cells of which contain very deeply stained nuclei. On one side of the figure one of the ducts is cut longitudinally, just where it forks near the surface of the intestine.
Different viev/s have been held concerning the func- tion of the "organe refringont". Krohn /S^ , Kuppfer (16) and Giard (G) have regarded it as a renal organ, but as the ampullae always contain a clear, unstainable fluid, and never concretions or epithelial debris, this view has been discard- ed. A second hypothesis, that it is a digestive gland v/hich gives its secretion to the intestine, has been held by Chan- delon (2) and Delia Valle (3), the latter attributing to the organ an hepa to-pancreatic function. Pizon (22), however, believes that the flat cells of the ampullae possess no glan- dular characters, and cannot be reconciled v;itn a secretory function, but he is inclined to regard the organ as a chy- lif erous apparatus . He says (I.e., page 95), "Je suis plutot porte a croire que 1 '^pi theli^jm des ampoules ne se- crete rien, et qu'il se charge simplement d'absorber les pro-
■34.
duits de la digestion qui sont assirnilables et qui n'ont pas ete pris par les parois de I'intestin. Ces produits quit- teraient ensuite la cellule pour aller se melanger au sang, dont les corpuscules sont precis^ment extrernernent norabreux autour des anpoules ter.-ninales . " Although Pizon's hypoth- esis would seem the most probable one, as the histological structure of the organ is not such as to suggest a glandular function, still the role played by the "organe r^fringent"' must remain uncertain, until the nature of the liquid con- tained in the tubules is determined.
THE PERICARDUTI AND HEART.
Concerning the origin of the common rudiment of the pericardium and heart investigators have given wiaely diver- gent accounts, some deriving it from endoderm, others from mesoderm. Although it is very certain that this structure arises diferently in different Ascidians, still in the buds of one and the same form statements of authors are at vari- ance. Seeliger (29) describes the pericardium as arising in the buds of Glavelina from an enormously large evagination of the ventral portion of the branchial sac, which later be- comes separated as an independent vesicle. He did not, how- ever, distinguish the epicardial sacs, and mistook a part of the latter for the pericardium. Van Beneden and Julin (33) shov,red conclusively that the diverticulixm of the branchial sac observed by Seeliger and called by him the pericardium.
-35-
is merely a part of the stolonic partition-wall and is not concerned in the formation of the heart. According to the Belgian authors, who described in detail the development of the pericardi;ini in the buds of Clavelina, the pericardium and epicardium at first form a common cavity with the inner ves- icle. A separation takes place later in such a way that the epicardiiim remains in communication '.vith the inner vesicle, while the pericardium becomes entirely cut off from the lat- ter, but retains its connection with the stolonic partition. Van Beneden and Julin maintain that the union of the epicar- dium with the branchial sac is never lost in the bud-develop- ment of Clavelina, and therefore do not agree with Seeliger's statement that the diverticulum, which he observed and er- roneously regarded as the pericardium, beco?'ies separated from the branchial sac. Seeliger's description of the early con- striction of the inner vesicle from the stolonic partition is not confirmed by the Belgians, who showed that the pericar- diiim, originally a part of the inner vesicle preserves its connection with the partition wall, as explained above.
A somewhat similar origin of the pericardium to- gether v/ith the epicardium from the inner vesicle is stated by Pizon (22) to occur in the Polyclinidae, for example in Circinalium and Amarouoium.
Our knowledge of the derivation of this structure in the buds of Botryllus is very much less certain. Pizon (22) declares that the pericardium arises as a little diver- ticulum from the lov/er wall of the inner vesicle, which be-
comes completely constricted off as an elongated tube. His conclusion as to the endodermal origin of the peribranchial rudiment cannot, however, be unhesitatingly accepted, since his figures do not satisfactorily establish the correctness of his description, while the supposition that he has not followed the development with sufficient care, is very strong, Salensky (27: page 527) calls attention to the fact that the little circle of epithelial cells which Pizon marks with the letters Per in Fig. 7, Pi.T., "wohl auch einen Ouerschnitt der unteren Wand des Kleimendarmes darstellen kann", and that
naf a.ta.72 t>/~o t-e n /Aa.fi'/'cs
it is the same structure as the pericardii™ figured in later stages .
The first appearance of the pericardial rudiment observed by Hjort (8) in Botryllus, was a small clump of cells lying against the ventral wall of the inner vesicle in the posterior part of the bud to the right of the middle line. As to the derivation of these cells, Hjort was unable to say whether they were mesodermal cells or cells which had wandered out from the endoderm, but he distinctly states that an evagination of the inner vesicle does not occur at this point.
In the buds of Dlstaplia Salensky (27) observed a similar collection of cells lying against the lower wall of the branchial sac and surrounded by mesodermal cells. He maintains that there is no ground for attributing an endo- dermal origin to the rudiment, which is from the beginning sharply marked off from the v/all of the branchial sac, and
• 57-
he therefore concludes that the pericardium is derived from the mesoderm. The result to which my observations on the bud-developnent of Perophcra viridis have led me, in regard to the origin of t.ie peribranchial rudiment, is in accord with that of Salensky.
At about the stage represented in Fig. 9, a very loose patch of cells is found applied to the outer surface of the inner vesicle higi: up on the right side in the posterior end of the bud. Before this time many isolated cells are seen adhering to the wall of the vesicle at numerous points, Figs. 7 and 6, but when the difference in thickness between the right side and the r.- st of the vesicle is just becoming apparent, a marked tendency in the scattered cells to acciimu- late in one spot is noticed. At first there is but a single layer of cells joined loosely toget/ier end to end, and forming a somewhat elongated patch; this is the rudiment of the peri- cardium., v^nich is the first organ to make its appearance. In Fig. 27, u, v/hich is drawn from a frontal section, an extreme- ly early stage is sho'.vn. That the rudiment is formed by the coming together of free mesodermal cells , I believe there is no reason for doubting. At the stage represented in this figure, the similarity between many of the cells scattered freely about in the space between the ectoderm and endoderm, and those which form this eel] -mass, is perfectly apparent. There is certainly not the slightest evidence that the wall of the inner vesicle evaginates, or its cells proliferate at this point; the line of demarcation between the two struc-
• 38-
tures is distinct throughout and shov/s no interruption in its continuity.
The rudiment does not long remain of one layer, but by the addition of other cells and by active cell-division it soon becomes thicker and more compact. Fig. 27, _b. The cell- boundaries are gradually lost, and the solid mass \s now firmly attached to the wall of the vesicle. Pig. 27, _c. The rudiment, which has nov/ an elongated form, does not lie hori- zontal, but posteriorly is at a higher level than at its an- terior 3nd .
When the shifting of the inner vesicle begins, the clump of cells is borne pasi.ively dov/n tov/ards the ventral side, but long before it has reached its definitive position, a cavity has appeared in its centre, around v/hich the cells become arranged in an epitheliiMi to form an elongated closed sac. Pigs. 27, _d and e_.
The position of the rudiment at various stages du- ring its descent has already been observed while considering the displacement of the inner vesicle from Pigs. 9, 10, 11, 17 and 21.
About the time that this change in position is ac- complished, the pericardial sac loses its attachment to the branchial wall, and grows considerably larger and wider. The cells composing the sac become very much flattened and atten- uated, except in the dorsal wal] , v/hich is soon folded in longitudinally to form the heart in the usual way. Fig. 28. The pericardium, in its definitive position, is placed under
the posterior floor of the pharynx just to the right of the median line. It does not lie horizontal, i.e. parallel with the surface of the stolon, but the posterior is higher than the anterior end. This inclination is seen in Figs. 4 and 5, in v/hich the pericardii;ra is indicated at pc . In the latter figure, which shows about its final position, the peri- cardium is seen to extend from a point at a level nearly as high as the upper end of the stomach straight down to the stalk which connects the bud with the stolon.
THE DORSAL TUBE AND GANGLION.
Of all the organs of the Ascidian bud, that which has given rise to the greatest amount of discussion is the nervous system. Its origin and development have been mat- ters of much dispute, and so wide is the difference of opin- ion concerning points of fundamental importance, that there is little hope at present of harmonising the conflicting statements of the various authors.
A close relation between the dorsal tube and gan- glion has been affirmed by many v/ho hold to a common origin of the two, but is strenuously denied by others who assert that the dorsal tube arises independently, and has nothing whatever to do with the nervous system. Different authors have ascribed to these striictures an ectodermal, a mesodermal and an endodermal origin, and have thereby exhausted the en- tire series of possibilities.
•40-
Kowalev/sky (12), for the buds of Perophora, was the first to describe an endoder;r,al origin of thp nervous-system.
According to him, the dorsal v/all of the branchial sac evaginates to form a tube, v/hich retains its connection v;ith the branchial cavity and v/hich he calls the "llervenrohr . " In his later work on the budding of Ascidians (13) he des- cribes the rudiment of the nervous-system in Amaroucium and Dideninum as "ein sehr langes, an vordern Ende zeiralich brei- tes Rohr, dessen Lumen mit der Hohle des Kiemanseokes zu com- municiren scheint" (I.e., page 455). He did not follov/ the development of this tube, which he held to be derived from the endodermal vesicle, and was ignorant of its relation to the hyphophysis and nervous-system of the adult animal. It is probable, however, that he sav/ the ganglion in Amaroucium at least, but failed to recognize it, for he says (I.e., page 465), "Bemerkenswerth ist noch, dass iiber dem Nervenrohr sich eine Anhaufung von sehr blassen Zellen befindet, welche bei '•."•eiterer Entwicklung zu verschwinden scheinen." Amaroucium is one of those Ascidians, in v/hich the ganglion lies above the hypophyseal tube.
Ganin (5), v;ho studied the bud-development in Pi- demnum and Botryllus, derived the nervous-system from a ves- icle which he described as being cut off from the inner ves- icle of the bud, and converted into a long cylindrical tube lying over the dorsal wall of the branchial sac. The gan- glion, according to Ganin, becomes differentiated from a part of this tube, the remainder of v/hich forms a ciliated organ
-41-
communicating with the branchial cavity. 'His description is very obscure, however, and the only points to be noticed are, that the dorsal tube, accordinr: to this author, is derived from the endodermal vesicle, and that it gives rise to the ganglion.
Giard (6) and Delia 7a lie (3 and 4) v/ho studied the bud-development in different species of Ascidians, contribu- ted nothing of value concerning the nervous-system, but both ascribe a common origin to the dorsal tube and ganglion.
The viev/s of Seeliger (29) are very different from the foregoing. According to hi:., the dorsal tube and gan- glion in the buds of Clave lina arise from a common rudiment, which is derived from mesodermal cells. This belief was not based on direct observation, since he did not examine suffi- ciently young stages, but was arrived at through theoretical considerations. The great similarity between the individual cells of the nerve-rudiment and the free mesodermal cells in the body-cavity of the bud, Seeliger holds in good evidence for the mesodermal origin of this structure. He furthermore points out that the cells composing the ganglion of the lar- va, would be carried off in the blood after the disintegra- tion of that organ, ana give rise in the bud to some of the mesodermal cells. The latter would therefore be "directe Abkcmmlinge eines friihere gangliosen Organs", and it would be but natural for them to resume the function which they had once possessed. Van Beneden and Julin {33) in their work on the development of the buds of Clavelina, state, that the
• 42-
nervous-system is derived from the ectoderm, and first ap- pears as a cord of cells lying close against the ectodermal wall. Their description, however, is very incomplete and unsatisfactory.
Our more recent knov^rledge of the subject is due to the researches of Pizon (22), Oka (20), and Hjort (8) on Bo- tryllus, of Salensky (27) on Distaplia, and of Gaullery (1) on Glosophorim and Diplosona. The first tnree authors, al- though they are in agreement concerning the origin of the dorsal tube, differ widely in respect to the derivation of the ganglion. According to all tnree, the dorsal tube in the Botryllus bud arises as an anteriorly directed evagina- tion of the dorsal wall of the branchial sac, ending blindly in front, but freely opening into the branchial cavity at its posterior extremity. This tube grov/s forward, and its ante- rior end fuses with the wall of the branchial sac, whose cavity is then put into communication with the lamen of the tube, while the posterior connection becomes obliterated. The definitive opening of the hypophyseal tube is therefore secondary. So far v/e find these authors agree, but it is quite otherwise when we come to consider the origin of tne ganglion.
Pizon maintains that the ganglion of the bud is de- rived directly from a fine nerve-string, which grov/s out from the ganglion of the parent-bud, or in the first from tnat of the larva, into the young bud. His view is not based on ac-
43-
tual observation, and his arguments, which are far from sat- isfactory, fail to convince. He avers that a constriction of the ganglion from the wall of tne dorsal tube does not take place, but, on the contrary, the figures of Kjort (8: Botryllus) prove fairly conclusively that such a constriction does actually occur. Kjort 's contention that the ganglion is formed from the thickened ventral wall of tne hypophyseal tube, is based on a study of an unbroken series of stages and is clearly borne out by his figures. In a short note""" on the budding of Botryllus, v/hich was published recently, I added additional evidence in support of Hjort's view, and re- produced a drav/ing v/hich showed beyond a doubt that the thickened ventral v/all of the dorsal tube is pinched off to form the ganglion. The account given by Oka is entirely different. According to this observer, cells wander out from the ectoderm, fasten themselves to the ventral wall of the dorsal tube, and there form tne ganglion. These wander- ing ectodermal cells were also observed by Pizon, who descri- bed them as giving rise to a portion of the genital gland, to muscular fibres, and to certain cells of the blood. The principal difference, then, bet'.veen the three authors is, that whereas Pizon and Oka hold to an independent origin of hypophysis and ganglion, Hjort maintains that there is a com- mon rudiment for the tv/o structures.
The results of Salensky (27) on the bud-development
Johns Hopkins University Circulars, No. 119, June, 1895. -44-
of Distaplia do not stand in the slightest agreement with any of those obtained for Bo_tryllus. The nervous-syste.'n of the Distaplia-bud is of ectodermal origin, according to him. Cells sink down at a very early period from the ectodermal wall, and form a solid mass, which later acquires a cavity, increases in length, and produces a tube. The latter be- comes differentiated into three parts; the anterior gives rise to the hypophysis, the middle to the ganglion, and the posterior portion to the visceral nerve. The hypophysis and ganglion have, therefore, a common origin. This mode of formation, however, is only true of the primordial bud; for all the other buds, which are produced from it by fission, de- rive their nervous-system by division directly from that of the parent along with the rtst of their organs.
Tt might be mentioned that in the buds of Pyrosoma, Salensky (26) has described a similar ectodermal origin of the nervous-system, although Seoliger (30) in the same form aerives the common rudiment of ganglion and hypophysis from mesodermal cells.
Salensky's results on Distaplia are directly con- tradicted by Hjort and Frl . Bonnevie (10). The latter find no trace of the nervous-system in the early stage at which Salensky describes its first appearance, but maintain, on the contrary, that a forwardly directed diverticulum is later formed from the dorsal wall of tne inner vesicle, just as in Botryllus, and that the ganglion is differentiated from the wall of the dorsal tube.
-45-
Pizon (22), in his v/ork on Botryllus , states that he has made observatio s on the develop'nent of the dorsal tube in the buds of a number of other Ascidians. In Perq- phoraand Clave lina he observed tne tube over the dorsal v^all of the branchial sac, but did not obtain stages which were young enough to enable him to determine its origin. He con- cludes, however, on the insufficient evidence of Kovmlev;sky 's observations on Perophora, that the dorsal tube arises as a diverticuluiTi of the endoderrnal wall. Since in Glavelina the later stages in the development of the dorsal tube are simi- . lar to those of Perophora, he holds that in this Ascidian al- so the origin is the sane. In two of tne Polyclinidae, viz: Amaroucium proliferurn and Circinalium, in in Didemnurn niveun; and in Astellium sponglf orrne, he has observed the dorsal tube arising as an endodermal diverticulaTi, v/hich acquires a sec- ondary opening into the branchial sac at its anterior extrem- ity, just as in Botryllus .
In none of tnese forms did he determine the origin of the ganglion, but he comes to the unwarranted conclusion that this structure is derived, independently of the dorsal tube, in the sane way as he has described for the Botrylli- dae .
Hjort (9) has recently studied the development of the neuro-hypophyseal system in the buds of glossophormi sa- bulosum, one of the Polyclinidae , and Caullery ( 1) in Glos- sophorum l\iteurn, Gircinali;m concrescens and Diplosoma gela- tinosum, and although both of these authors find that the
-46-
dorsal tube arises in the manner described by Pizon, that is, as an anteriorly directed endoder.-aal diverticulum, they give a different account of the origin of tne ganglion. . In all the species studied, tne ganglion is formed as a differen- tiation of tne dorsal v/all of the hypophyseal tube, and has therefore a comnon rudiment v/ith the latter. Their results are in agreement v/ith vj-hat Hjort has found in Botryllus, ex- cept that in all of the above-mentioned Ascidians the hypo- physis lies belov; the ganglion, v/hereas in Botryllus it is above .
Finally, Ritter (24) who has recently described the bud-development of Goodseria, a genus in which budding had not been observed before, finds a complete agreement, concern- ing the origin of the neuro-hypophyseal system, with Hjort 's work on Botryllus .
In the same paper Ritter gives a preliminary ac- count of some observations on the development of the buds of Perophora anne^ctens (Ritter) and P. Iiis.tejM^- ^^^ both spe- cies he derives the common rudiment of the hypophysis and ganglion from cells which wander out from the dorsal wall of the inner vesicle.
After this short review of the state of our know- ledge regarding this much confused subject , I shall now give an account of my ov/n observations on the development of the dorsal tube and ganglion in the buds of Perophora viridis_.
My results, which are based on a study of an unin- terrupted series of stages, have led me to believe that the
•47-
conclusion :';hich Seeliger drev/, from purely theoretical con- siderations, concerning tne origin of tnese structures in Clavelina is also true of Perophora viridis. I shall try to show tnat tnere is every reason to believe that tne dorsal tube and ganglion are derived from .'7iesoder:nal cells .
The dorsal tube is formed long before the ganglion , and the rudiment from which it v/ill arise is first indicated just after the collection of cells v/hich is to produce the pericardiam makes its appearance. 'Vhen the difference in thickness between the right side and the rest of the vesicle is becoming apparent, hence at a time --.vhen tne rotation is about beginning, in the anterior portion of the bud, a little to the left of the median dorsal line, there is seen an ir- regular elongated patch of cells very loosely grouped togeth- er and lying on the outer surface of the inner vesicle, Pig. 28, d^.t. The free mesodermal cells in the space between the ectoderm and endoderm are especially numerous in this region, and are closely associated with the collection of cells ad- hering to the vesicle. Although I have examined my sections with the greatest care under a ■'"/12 oil-i.'nmersion lens, from the very first appearance of the rudiment, I have failed to find any indication of cell-migration from the endodermal wall, and therefore cannot confirm Hitter's statement (I.e. page 368) that an "indistinguishable transition from the cells of the 'endoderm' to those of the neuro-hypophyseal anlage is to be traced", and also that cells can be found "in the act of migrating from the "endoderm" into the anlage."
-48-
The line of separation between the rudiment and the wall of tne vesicle is seen to be perfectly distinct and clearly marked, and there is no evidence whatever of prolif- eration of endodermal cells at any point; Fig. 29, a. Du- ring the early stages of development the cells which are to form the dorsal tube, and many of the mesodermal cells, are absolutely identical in appearance, and exui.At the same amoeboid character. So gradual is the transition from the free mesodermal cells to the cells of the rudiment, that it is at first impossible to say where the former end, and the latter begin, Pig. 29, b.
I believe, therefore, that there is no evidence whatevejr for holding that the dorsal t_ube does not aris_e frojn free mesodermal cells of t^he blood.
By further additions from outside and by active cell-multiplication -.vithin the mass, the rudiment gradually increases in size; its cells become more closely packed to- gether, and soon form an elongated solid cord lying close against the dorsal wall of the vesicle in the anterior end of the bud. Fig. 29, c^. Fig. 29, a_, _b and _c illustrate the development up to this point; a is drawn from the same sec- tion as Fig. 28, h and _g from the series to which Figs. 10 and 11 respectively belong. It has been stated above that the anterior portion of the inner vesicle is only slightly involved in the displacement already described, but that it
is to a certain extent, is proved by the fact that the rudi-
first appears ment of the dorsal tube not exactly in the mid-dorsal line,
A
■49-
but a little to the left of this, Pig. 23. 3y the time the shifting of the vesicle has proceeded soraev/hat further than is shown in Fig. 11, the string of cells, which is now solid, has been carried up to the median plane.
Very shortly after it has reached its definitive position, a lumen appears in the centre of the rudiment throughout its entire length, and around this the cells be- come arranged into a one-layered epitheliiim. Pig. 27, d_ and
By following the course of development up to this point, it is seen hov/ an epithelial tube does actually arise from free mesodermal cells, a thing which Hjort has charac- terized as most improbable. In criticising Seeliger's view of the origin of the neuro-hypophyseal system in Clave lina, this author says, (8: page 602) "die Wahrscheinlichheit da- fur, dass ein Ganglion und ein epitheliales Rohr sich aus zusammengehauf ten Mesodermzellen bilden sollte, scheint mir so gering zu sein, wie fur die Auffassung Herdman's dass die Innere Blase der Knospenanlage einen solchen Ursprung habe . "
About the time that the peribranchial cavity is completely cut off from the inner vesicle, the anterior ex- tremity of the dorsal tube fuses with the dorsal wall of the branchial sac, an opening breaks through, and the lumen of the tube is put into communication with the branchial cavity. The posterior end of the tube abuts against the anterior wall of the cloaca, but neve-.r opens into the latter, in con- trast with the condition found in Botryllus and many other
-50-
Ascidians .
Fig. 50, represents a median sagittal section of a bud before the complete separation of the peribranchial cav- ity, and therefore before the dorsal tube has acquired an opening into the branchial sac. The section passes through the entire length of the tube, d . t_, v/hich is seen to be clo- sed at both ends and made up of an epithelium of one layer.
In my preliminary work on the budding of P.erophora, already referred to, I made the statement that, "the ganglion is formed by a thickening of the d_orsal wall of the tube, which eventually becomes constricted off in the manner des- cribed by Kjort for Botryllus, although in the latter it is the _ventral v/all of the tube which gives rise to the gan- glion." More careful study of very young stages, however, has convinced me that the above is not an accurate descrip- tion of the formation of the ganglion.
After the communication between the dorsal tube and branchial sac has been established, a few cells, identical in appearance with mesodermal cells, are found adhering to the dorsal surface of the tube throughout the greater part of its length; this elongated, loose patch of cells constitutes the rudiment of the prur-licn, £l. Fig. 31, a_.
It is a difficult question to decide whether these cells are entirely mesodermal, as their appearance indicates, or whether they are derived from proliferation oi' tne wall of the tube, fot- in many places the outline of the latter is broken, and there is no sharp demarcation between the cells
-51-
of the rudinent and those of the tube, as seen in Pig. 51, a.
]"[any sections, however, such as the one shov/n in Fig. 31, _b, v/hich represents a slightly older stage, leave little room for doubt that nuclei do wander out into the ru- diment. But, on the other hand, I think that this figure shows equally well that mesodermal cells are added to the mass from the outside.
I have, therefore, come to the conclusion that the ganglion has a double origin, and that both the wall of the tube and free mesodermal cells co-operate in forming it. It is to be remembered, however-, that ultimately it is entire ly a mesodermal structure, as we have seen hov/ the dorsal tube is constructed out of cells of the blood.
The ganglionic- rudiment is at first a very irreg- ular heap of cells, and is clearly associated at the periphe- ry with surrounding mesoderm.al cells. The cell-boundaries are completely lost very early, and the mass rapidly increases in size by multiplication of nuclei within, by further ac- quisition of cells from without, and by continued migration of nuclei from the wall of the tube. Pig. 31, c. The nuclei now arrange themselves in a couple of layers around a central core, in which fine fibrils are laid dov/n, and the ganglion becomes completely marked off from tne v/all of the tube; the definitive cti'ucture is nov/ attained.
'"ig. 31, d and _e, illustrates the latter course of development .
?;Iy observations, therefore, have led me to believe
that the hypophyseal tube and the ganglion are formed only in part from a common rudiment, and in this respect to take a middle ground between Kjort, Salensky, and Caullery, on the one hand, who have described a common origin for these struc- tures in the Ascidians studied by them, and Oka and Pizon on the other, who maintain that they arise independently. In deriving the hypophysis and ganglion, hov/ever, from tne mes- oderm, I differ widely from all previous observers with the exception of Seeliger. Concerning the origin of the dorsal tube in the buds of Perophora my results are totally opposed to the conclusion of Pizon, which as stated above, is not based on sufficient evidence, namely, that "le tube dorsal des Perophores a la meme evolution que le tube dorsal des Bo- tryllides", (I.e., page 130).
A study of the younger stages would have convinced him of his error.
Perophora viridis, at all events, presents an ex- ception to the general rule laid down by Pizon, that "Chez toutes ces families d'Ascidies composees (Clavelinidae, Pero- phoridae, Botryllidae, Polyclinidae, Distomidae, Didemnidae, et Diplosomidae ) I'organe vibratile debute par un tube aveu- gle, forme par un diverticule de la vesicule endodermique primitive." (I.e., page 131.)
THE SEXUAL ORGANS.
:.!y observations on the development of the sexual
organs have not been carried beyond quite an early stage, but so far as they go, they clearly agree with the description
given by Van Beneden and Julin (33) for the buds of Peropho- ra Listeri .
Shortly after the peribranchial cavity has been completely divided off from the branchial sac, a small col- lection of cells appears between the two arms of the U-shaped digestive tract and at the level of the duct of the "organe refringent", almost at the point where the latter is connect- ed with the stomach. The sexual organs arise from this lit- tle sp?ierical mass of cells, which are at first but loosely held together and identical in appearance with the mesodermal cells of the blood. ?ig. 32, a, ./hich is precisely similar to Van Beneden and Julin 's Pig. 5, b^, PI. XVI, shows the in- timate relation between the cells of the blood and those of the rudiment, so that there can be no doubt that the latter are of mesodermal origin. The connection, already described by the Belgian authors, of some of the peripheral cells of the mass with surrounding mesodermal cells by protoplasmic processes, is distinctly seen in the figure. A small ir;reg- ular cavity is also shown in the centre of the clump of cells'-
Some of the free mesodermal cells lying above the rudiment, g . c . Pig. 32, a, are seen to be spindle-shaped; these soon become joined, end to end, to form a solid cord united at one end to the spherical mass of cells, and taking a course parallel to that of the intestine, g.c . 32, b. This figure represents a later stage, in which the cavity is considerably
-54-
enlarged and the genital cord, g^.c, is present as a solid single rov; of cells, (Compare Fig. 2, Pl.XI-I, of /an 13ene(3en and Julin. )
A furrow, which appears opposite to tne attachment of the cord, novr divides the hollow sphere into two lobes, the cavities of which are not completely separated, but re- main in communication. In Fig. 32, c^, the division of the originally simple sphere is seen, but the section is not in the proper plane to show the connection between the two cavi- ties .
One of these lobes gives rise to the testis, the . other to the ovary, according to Van Beneden and Julin, who have described in detail how, from the primitive, simple sphere and the single cord of cells, testis, ovary, vas def- erens and oviduct are all differentiated.
Ily observations would therefore seem to support the view of the Belgian authors that the male and female sexual organs do not arise from separate vesicles, as described by Kov/alewsky (12) in the buds of Perophqra, but are formed from one and tne same rudiment.
SUr.CvIARY OF RESULTS.
1. The ruciiment of the Perophora bud, like that of all other Ascidians, consists of two vesicles, an outer and an inner one. The former is derived from the ectoderm of tne stolon, tne latter from the thickened evaginated wall
■55-
of tne stolonic partition.
2. At an early stage the right side of the inner vesicle is found to be much thicker t/ian the remaining por- tion, and by a peculiar process of rotatory growth or dis- placement of the vpsicle, is carried dovm to the ventral side of the bud, where it forms the floor of the pharynx. This process seems to be due to tne growth and flattening out of the cells composing the whole wall of the vesicle except in the thickened region. I am at a loss to explain why the dis- placement of the vesicle should occur, and to discover the phylogenetic significance of it, if it have any.
3. The peribranchial sacs arise asymaetrically . As tne displacement proceeds, the wall of the inner vesicle is folded in, at the point where the right side of the vesi- cle joins the stolonic partition, to form the le_ft peilV°^?.5." chial sac. The connection of the latter with the partition of the stolon is retained until a much later stage. A lon- gitudinal furrow appearing high up on the right side of the inner vesicle, separates off tne right peribranchial sac, and is gradually borne ventrally, as the shifting of the vesicle continues. The constriction of the whole peribranchial cav- ity eventually takes place in such a way as to cut off from tne peribranchial sac a :)-shaped bag, composed of the median dorsal connecting piece or cloaca, and the two lateral por- tions of the cavity. Anterior and posterior extensions of. the latter gro\7 out and surround respectively the anterior and posterior ends of the pharynx. The posterior prolonga-
tions are possibly to be regarded as ho.'nologous \7ith the epi- cardial sacs of some other Ascidians.
4. The endostyle appears early as a longitudinal groove in the middle of the thickened portion of the vesicle; from its primitive position on the ri^ht side it is moved dov/n to tne ventral mid-line by thp displacement of the ves- icle.
5. The digestive tract grows out laterally as a blind tube from the posterior end of the inner vesicle high up on the left side. During the change in position of the vesicle, its opening into the latter is carried up into the median plane.
The "organe refringent" arises as a tubular diver- ticulim from the anterior face of the stomach, and produces a dichotomously branched system of tubules, which surround the intestine and terminate in little dilated vesicles or am- pullae .
6. All the evidence goes to show that the peri- cardium is formed from free meso.iernal cells. It first ap- pears as a clump of cells adhering to the outer surface of the inner vesicle far wp on the right side, and through the shifting of the vesicle is brought down to the ventral side.
7. The dorsal tube and ganglion are formed only in part from a common rudiment, and there is every reason to believe tnat both are derived solely from tne mesoderm. The former appears as an elongated, solid mass of cells, which lies close against the outer surface of the inner vesicle,
a little to the left of the median dorsal line. '■:rnen the displacement of the vesicle is completed, the rudiment lies in the Tiedian plane; it then acquires a lumen, which is put into communication anteriorly v/ith the cavity of the pharynx. The ganglion has a double origin, and is formed by prolifer- ation of the upper wall of the dorsal tube and also from free mesodermal cells.
8. The sexual organs have a comiaon mesodermal or- igin, both testis and ovary arising from one and the same spherical rudiment.
C ONCLUD I NG REMARKS .
The results which I have obtained from the study of the budding of P_erophora viridis furnish additional evi- dence in support of the viev/ that the development of the bud and that of the larva do not proceed along parallel lines. The attempts which have been made to harmof||inise the facts of budding with the germ layer theory, have benn totally fu- tile in the cuse of Ascidians, and ariy hypofiasis "Jhi':'\ ex- plains budding in the Ascidians as a procesr. of regeneration , by which the organs of the parent, or their-layers , give rise to sim.ilar organs in the bud, must, in the light of known fc.'Cts, be ruled out.
The rudiment of the bud in all groups of Compound Ascidians is composed of two vesicles, one wi*:hin the other, enclosing between them free mesodermal cells. The outer
• 58-
vesicle Is always derived from the ectoderm of the parent, and gives rise to the ectodermal covering of the bud. The origin of the inner vesicle, however, is not the sane in all Ascidians. In the Botryllidae it arises, in both larva and bud, from the perlbranchial wall, which is formed in the first place from, the ectoderm of the embryo. This vesicle is, therefore, ultimately of ectodermal ori^^in in Botryll us , whereas in al] other Ascidians it comes from an endcdermial structure of the parent.
Although derived in the two cases from different germ-layers, the inner ve'sicle may go to form the same organs in the bud, but organs which are of widely different origin in the larva; for example, the digestive tract and nervous system. (Pljort, Botryl] us (8), Distaplia (10), Glcssophorum (9).
The fate of the inner vesicle is consequently di- rectly opposed to the application of the germ-layer theory in its ordinary meaning to the Ascidian bud.
In Perophora viridis the important part played by cells, which there is every reason to regard as mesodermal cells, in the bud-development, only increases the disagree- ment. Organs which in other Ascidians are formed from ec- toderm or endoderm, are here of mesodermal origin. The ner- vous-system and pericardium, v/hich are respectively ectodermal and endodermal productions in the larva, develop out of free mesodermal cells.
Since in the development of the bud and larva the
the sane end is reached by entirely different roads, and in the foi-mer organs do not proceed from corresponding organs, or even their germ-layers, in the latter, and moreover, since in the bud a rudiment derived from one and the sam.e larval germ-layer may give rise to structures of widely different nature, one is compelled to believe witn Hjort, that "die Knospen der zusanmengesetzte Ascidien ein Entv/icklungsprocess ist, in v/elchem sam.tliche Organe durch 'Neubildung' aus einer
sehr primitive Anlage entstehen." "Die Knospe muss
ihre eigenen Gesetze haben und muss, da sie aus einer wesent- lich anderen Anlage hervorgeht als die Larve, auf andere Weise gebildet werden. Ebenso wie dass Ei, m.uss das :iate- rial, die Anlage, welche den Ausgangspunkt fur die Entwick- lung bildet, als ganz undifferenzirt gedacht werden und muss alles enthalten, was zur Bildung eines ganzen Individuimis no- tig ist, ebenso wie die Blastula des Eies," (9:, page 225).
The behaviour of the 'm.esooerm' in the bud-devel- opment of Perophora viridis is of much interest, ov/ing to the number and variety of organs in whose formation nesodermal cells are concerned. As we have seen these cells give rise to the pericardium, the dorsal tube, the ganglion, and the sexual organs; they also produce the musculature, and, as Kowalewsky (14) and Seeliger (32) have shown in other Asci- dians, in Perophora also they doubtless become the cells of the cellulose test.
I have spoken of the cells which go to form the pericardium, nervous-system, etc., as 'mesoderm.al • , because
-60-
they are identical in appearance v/ith the amoeboid cells which are scattered about abundantly in the body-cavities of tne buds and the sinuses of the stolon. As I have emphasis- ed repeatedly in describing the formation of tne various or- gans in question, there is not the slightest discoverable ev- idence that cells are given off from the wall of the inner vesicle at the points where such organs arise, or at any oth- er place, for that matter, and this fact, together with the similarity betv/een the free mesodermal cells and the cells which go to make up these rudiments, is strong proof that the latter are genuine mesodermal cells.
The origin of these cells is a question which is
practically undeterminable, and direct proof that they are
of tne mesoderm all descendants of the larva, is of course impossible, al-
though the assumption that they are, seems to me to be a jus- tifiable one. The statement that they do not arise from cells which wander out from the endoderm or ectoderm, is ba- sed, it is true, on negative evidence, since after the most diligent search it cannot be discovered that any are derived in this manner.
It is, perhaps, going a little far to absolutely deny such an origin, for it would be impossible from the na- ture of the case to say that at no time does the endoderm or ectoderm give off cells to the blood, even although it cannot be shown that such is the fact.
The positive evidence furnished by their mesodermal appearance, however, together with the negative evider. ce de-
rived fron the entire absence of any indication of another origin, almost amounts to a certainty that these cells are truly mesodermal.
If then this assumption be admitted, a discussion of the nature of these cells and the part played by them in the developing bud is not out of place.
In the case of a fixed organ, like the ganglion or pericardii.m, which has a definite and determined position, the cells destined to form it, must by virtue of their motile power come together at the right time and place.
Weismann (35; pages 161-52) in discussing the pro- cess of gemmation in Glavelina, as described by Seeliger (29), supposes that t.-ie nescdermal cells "contain very different kinds of idioplasm: ox\p , for instance, might contain 'muscle determinants', and another, 'nerve-determinants', and a third •blood-corpuscle-determinants'." He further adds, that "until we knov/ more of the actual facts concerned, we can on-' ly -- ho\7ever unsatisfactory such an assumption may be -- at- tribute to the cells a tendency to become attached at defi- nite points according to the manner in which they have pre- viously been determined."
It seems to me, however, that the opposite assump- tion, which Weismann regards as less likely, namely, "that these cells develop into muscle, nerve, or sexual cells according to their point of attachment," is more in accor- dance with the facts presented by the bud-development of Pe- rophora viridis.
We have already seen in the very young bud, when it consists nerely of two sinple layers, and before there is the slightest indication of the appearance of any organ, that mesodermal cells are attached in many places to the inner surface of the ectoderm, and outer surface of the endoderm, but that they are nowhere more numerous at any one spot than another, Pigs. 7 and 8. Tneir poY,rer of amoeboid movement over any surface v/ith v/hic;T they come in contact, would ac- count for their presence on the walls of the vesicles.
It would seen more probable that these cells are all alike and undifferentiated, and that the nature of the organs to which they give rise is determined, not by any pre- arranged condition of their idioplasm, but by the particular point to which they happen to become attached. I regard it as a significant fact that mesodermal cells are found, not only at the places v/here organs will arise, but also at many other points. Those of the cells which chance to fall, as it were, on fertile soil, will undergo further development, and under the formative influence exerted upon them by that portion of the wall to which they adhere, will be utilised in building up a definite structure.
All parts of the walls cannot possess a specific determining po'.ver, and such cells as lodge on barren ground, are not further modified, and do not furnish material for the formation of organs.
According to this viev/, one mesodermal cell is the equivalent of any otner, and it is only a few that find fa-
vorable positicns, and have their latent possibilities called forth.
Those which become attached at a point high up on the right side of the inner vesicle in the posterior region of the bud, v.'ill forin the pericardiim; others on the dorsal side, at the anterior end, v/ill give rise to the dorsal tube, and still others, v/hich lodge on the upper wall of the latter, will help to construct the ganglion; sorrie adhere to the in- ner surface of the ectoderm, lengthen out and become muscle- fibres, some wander through the ectoderm, and on the external surface are transformed into the cells of the cellulose test, while others find a definite place in the posterior region of the bud and develop into the sexual organs.
This viev/ is opposed to the supposition of Seeliger spoken of above, that in Clavelina the ganglion of the bud is formed from free cells of the blood, v/hich had earlier com- posed the larval ganglion and been liberated on the dissolu- tion of that organ; these cells would, therefore, have al- ready possessed a ganglionic nature, and would merely resume in the bud their former function.
On any such assumption, it is almost impossible to imagine how isolated specific cells, moving freely about in the blood, could reach their proper destination. On the re- verse assumption, hov/ever, the presence of rnesociermal cells at any particular point is accidental, but once there, their potentialities are called out under the specific formative influence of the place of attachment.
Baltimore, Id., April 18, 1696.
LIT'^RATURE CITKD.
1. Caullery, Li.
2. Chandelon, T. --
3. Delia Valle, A,
4. Delia Valle, A. --
5. Ganin, M. -■
6. aiard, A. --
7. Herdaan, Y/.A.
8. Hjort, J.
Contributions a I'etiide des Ascidies composees. -- Thesis, Paris, 1895. Recherches sur line annexe du t;ibe di- gestif aes Tuniciers. -- Bulletin Acad. Royale de Belgique, T. XXXIX, 1875.
Recherches sur I'anatomie des Ascidies composees. --Arch. Ital. Biol. T. II, 1882.
Sur le bourgeonnement des Didernnides et des Botryllides. -- Arch. Ital. Bi- ol. T. TI, 1882.
ITeue Thatsachen au.s der Rntwicklungs- gGschichte der Ascidien. -- Zeitsch. wiss. Zool. XX Bd., 1870. Recherches s-ar les Ascidies composees ou Synascidies. -- Arch. Zool. Rxper., T. I, 1872.
Report on the Tuniccita. -- Challenger Reports, Vol. VI, 1882. Uber den Entwicklungscyclus der zu- samrnengesetzten Ascidien. --- i.Iittu. zool. station Neapel, X, 3d., 1893.
9. Hjort, J. -- Beitrag zur Keirnblatterlehre und Ent-
wicklungsinechanik der Ascidienknospung -- Anat. Anz., X Bd . , No. 7, 1894.
10. Hjort, J. und Prl . Bonnevie. — Uber die Knospung von
Distaplia jaagnilarva. -- Anat. Anz., X Bd. , 'lo. 12, 1895.
11. Kovmlewsky, A. -- V»'eitere Studien u?jer die Entwicklung
der einfachen Ascidien. -- Arch. nikr. Anat., VTT 3d., 1871.
12. Kowalewsky, A. -- Sur le bourgeonne?rient du Perophora
Listeri. -- (Trans, from the Russian, by A. Giard) -- Rsv. sc . nat. "lont- pellier, T. I[[, 1874.
13. Kov7alev/sky, A. -- Uber die Knospung der Ascidien. - =
Arch. mikr. Anat., X Bd . , 1874.
14. Kowalev/sky, A. -- Einige Beitrage zur Bildung des Man-
tels der Ascidien. -- 'Tem. kc . Sc . 3t. Petersb. T. 28, 1892.
15. Krohn, A. -- Uber die Fortpf lanzungsverhaltnisse
bei den Botrylliden. -- Arch. Naturg., XXXV Bd., 1859. -- Uber die fruheste Bilding der Botryllusstocke . -- Ibi- d era .
16. Kuppfer, C. -- Zur Entwicklung der einfachen Ascidien
-- Arch. mikr. Anat., VIII Bd . , 1872.
17. Lahille, P. -- P.echerches sur les Tuniciers des cotes
de Prance. -- Tnesis, Toulouse, 1890.
-55-
18. Iietschnikoff , E. --Uber die Laven und Knospen von Botryl-
lus . — Bull. Ac. f;C. St. Petorsb., T. 1:3, 18o9.
19. Metschnikoff , E. --Enbryorialentwicklung der einfachen As-
cidien. -- Ibidem.
20. Oka, A. -- Uber die Kncspimg der Botrylliden.-
Zeitsch. wiss. Zool., LIVBd., 1892.
21. Patten, V/. -- Orienting small objects for sectioning^
and fixing tnem when moiinted in cells. -- Zeitsch. wiss. Mikr., XI Bd . , 1894.
22. Pi7.on, A, -- Histoire de la blastogenese chez les
Botryllidps. -- Ann. Sc . Nat., Zool (7), T. XIV, 1893.
23. Ritter^ W.PJ. -- Tunicata of the Pacific Coast of North
America. 1. Perophora annectens, n.sp. -- Proc. California Ac. Sc . , Vol. IV, 1894.
24. Ritter, -".E. -- On biidding in Goodsiria and Perophora.
-- Anat. Anz., X Bd . , No. 11, 1895.
25. Roule, L. -- Recherches sur les Ascidies simples
des cStes de Provoiice (Phallusiadees ) . Ann. 'ius. Nat. Hist. Marseille, T. II, 1884.
26. Salensky, V/. -- Beitrage zur Srnbryonalentwicklung der
Pyrosomen. -- Zool. Jahrb. Abtn. f. Anat., IV, VKd., 1891, 1892.
27. Salensky, W. -- Morphologische Studien an Tunicaten,
-57-
II. -- :iorph. Jahrb. XX Bd . , 1893.
28. Salensky, v/. -- Beitrage zur Entwicklungsgeschichte
der Synajcidien T, IT. -- Mitth. zool. Stat. TTeapel, XI Sd . , 1894, 1895.
29. Seeligftr, 0. -- Eiblldung und Knospung von Clavelina
lepadif orinis . -- Sitz. Ber. Akad , Wien, 'latn.-- "Taturwiss. Classe, LXXXV 3d. , T Abth. , 1882.
30. Seeliger, 0. -- Zur Entwicklungsgescaichte derPyroso-
men. -- Jena. Zeitsch. TTaturwiss . , XXI I [ Bd. , 1889.
31. Seoliger, 0. -- Uber die Kntsteh' ng des peribranchia-
len Raunen bei den Ernbryonen der Asci- dien. -- Zeitsch. wiss. Zool., Il/I, Bd., 1893.
32. Seeliger, 0. -- Einige Beobachtungen uber die Bildung
des ausseren Mantels der Tunicaten. - Zeitsch. v/iss. Zool., LVI Bd . , 1893.
33. Van Beneden et Julin. -- Recherches sur la morphologie
des Tuniciers. -- Arch, de Biologie, T. VI, 1887.
34. verrill, A.E. -- Brief Contributions from tiie Museum of
Yale College. No. XVI. On the Distribution of Marine Animals on the Southern Coast of ITe\7 England. Amer. Journ. Sc . and Arts, (ser.3) Vol. IT, p. 359, 1871.
35. v/eismann, A. -- The. Crerm-plasm. (Trans.) wew York,
1893.
36. '.Villey, A. -- Studies en the Pro tochordata, I, II.
Quart. Journ. micr. Sc, Vols. XXXIV, XXXV, 1893.
-69-
EXPLANATIOK OF THE FIGURES.
All the drawings have been made with a camera luci' da. The lenses used viere those of Zeiss, and are referred to in the description of each figure.
Reference Letters .
a_. Anus.
amp . Ampulla of the "organe refringent."
bl . s. Blood-sinus of stolon.
br . Branchial sac.
br . 0^. Branchial oi'ifice.
_c. g. Cavity of rudiment of sexual organs.
c 1 . Cloaca.
c3^._^. Cloacal orifice.
£. p_c. Cavity of pericardial sac.
jc. t. Cells of test.
d . t . Dorsal tube.
a_. jtr. Digestive tract.
d.w.p£. Thickened dorsal wall of pericardiT-ra, which becomes
invaginated to form the heart.
£.br._o. Ectodermal invagination to form the brancnial orific(
— ■Sl'S-' Ectodermal invagination to form the cloacal orifice.
ect . Ectoderm,
en. Endoderm.
end. Endostyle.
-70
£._l.p_b._c. Fold to form left peribranchial cavity.
£.r.pb._c. Fold to form right peribranchial cavity.
g._c. Gienital cord.
gl . Ganglion.
g. _s. Crill-slit.
int. Intestine.
l.a.ex. Left anterior extension or pouch of peribranchial cavi ty .
1. d. t. Lumen of dorsal tube.
l.pbr.c. Left peribranchial cavity.
l.p.ex. Left posterior extension or pouch (left epicardial sac?) of peribranchial cavity.
oe . Oesophagus.
0. r. "Organe refringent."
pc. Pericardium.
pt. Double-v/alled partition of stolon.
r.a.ex. Right anterior extension or pouch of peribranchial cavi ty .
r .pbr . c . Right peribranchial cavity.
r.p.ex. Right posterior extension or pouch (right epicar- dial sac?) of peribranchial cavity.
r.v/.i.v. Thickened wall of inner vesicle on right side.
rd.d.t. Rudiment of dorsal tube.
rd.g. Rudiment of sexual organs.
r d . gl . Rudiment of ganglion.
rd . pc . Rudiment of pericardium.
'1-
St. stomach.
_stol. Stolon.
t. Cellulose test.
•72-
Fig. 1. Total preparaticn of very young bud, shov;ing evagi- nation of ectodern and partition of stolon. B. 4.
Fig. 2. Slightly older bud, seen from right side. 3. 4.
Fig. 3. Still older bud, seen from right side, and shov/ing anterior elongation and constriction of the stolon- ic connection. B. 4.
Fig. 4. Bud, seen from right side, at a stage when devel- opment of peribranchial cavity, pericardium and di- gestive tract is well advanced. B. 4.
Fig. 5. Bud, considerably older than last, shov/ing exten- sions of peribranchial cavity, formation of gill- slits, and further development of digestive tract. B. 4.
Fig. 5. Transverse section of stolen, showing double-v-'alled partition. F. 2.
Fig. 7. Transverse section of stolon at point v:here a bud is beginning to form. The ectoderm on one side is thickened, and the double-walled partition is much thickened and dilated. F. 2.
Fig. 8. Transverse section through very young bud and sto- lon, at about the stage of Fig. 1. The' walls of the partition are coming togetner to close off the inner vesicle. "'esodermal cells are found lying against the outer surface of the latter. ?. 2.
Fig. 9. Transverse section through posterior end of bud at about the stage of Fig. 3, showing thicke^ned v;all of inner vesicle on right side, and beginning of
pericardial rudiiBent . P. 2.
Pig. 10. Transverse section through posterior end of bud,
showing an early stage in the displacernent or shift- ing of the inner vesicle. The wall of the latter is being bent in at the point indicated by tne line a. D. 2.
Pig. 11. Transverse section through posterior end of bud
slightly older than last, showing further progress of the displacement of the vesicle. The peribran- chial rudiment is at a much lower level than in the last figure, and the wall of inner vesicle is being bent in further at a_. D. 2.
Pig. 12. Transverse section through the middle region of
same bud as the one shov/n in Pig. 11. The section lies in front of the connection with the stolonic partition, and shov/s the beginning of the fold which will form the right peribranchial cavity. D. 2.
Pigs. 13-18. Series of transverse sections of a bud at
about the same stage as that shown in Pig. 4. "^he sections are taken respectively at the levels in- dicated by the lines, a, _b, £, d, _e and f, in Pig. 4. Pig. 1-', line a, the most anterior secticn, shows undivided portion of branchial sac, and dor- sal tube. "Pig. 14, line b, is a little furtner back and shov/s anterior extension of right peri- branchial cavity. Pig. 15, line _c, is taken imme-
•74-
dieitely in front of the clcacal cavity, and snows the extensions of tne peribranchial cavity on both sides. The section passes through the extreme posterior end of the dorsal tube. Fig. 16, line
d, passes through the anterior ena of the cloacal cavity, '/nich is seen to con?;oct the lateral por- tions of the peribrancnial cavity. Fi^:. l^, line
e, is taken from the posterior end of the bud, and shows the connection of the left peribranchial cav- ity v^ith the stolonic partition. The pericardial- sac is also shown in this region. Fig. lo, line
f, snows the undivided extreme posterior end of the branchial sac. The section passes througn tno ai- lated portion of the digestivF tract, the stomach, and behind the connection '-rith the stolonic parti- tion. All are drawn with D, 2.
Figs. 19-21. Series of transverse sections of a bud of
about the same age as the one represented in Fig. 5. Fig. 19, is a section through the anterior re- gion, and shov/s the extensions of the peribranchial cavity. Fig. 20, is dravm from a section .vhich is taken from the middle region of the bud, and shows the median portion or cloaca connecting the lateral sacs of the peribranchial cavity, which is nov/ en- tirely cut off from, the branchial sac. Fig. 21, sho\'/s the posterior extensions (epicardial sacs?) of the peribranchial cavity, and the remnant of the
■75-
connection v/ith the stolcnic partition. D. 2.
Pigs. 22, a and b. These sections illustrate the develop- ment of the .^ill-slits . Tn a, tiie thickened bran- chial wall is slightly cvaginated and is in contact with the thickened patch of cells in the visceral wall of the peribranchial cavity. The lov/er por- tion of h^ shov/s the fusion between the two walls, and the upper portion, a stage after the formation of the opening. P, 2.
Pigs. 23, a and b^. Stages in the formation of the branchial orifice. In a, the much-thickened invaginated ec- toderm is seen nearly touching the branchial v/all. The fusion of the two walls is shov/n in _b. P. 2.
Pig. 24. Transverse section through the extreme posterior end of a bud, showing the origin of the gut, as a diverticulum on the left side of the branchial sac. D. 2.
Pig. 25. Section through the wall of the stomach and a por- tion of the duct of the "organe refringent", snov/- ing clearly the connection of the latter with the digestive tract. ?. 2.
Pig. 26. Transverse section of the terminal portion of the
. intestine, soov/ing the surrounding ducts and ampul- lae of the "organe refringent" with tneir deeply stained nuclei. 7. 2.
Pig. 27, a, b, c^, d, and e_^ Sections illustrating the devel- opment of the pericardium. i is from a frontal
section of a very young bud, ind sho'-7s tne rudiment wrien it consists merely of a few cells loosely at- tached to the inner vesicle. The remaining fig- ures are drav/n from transverse sections. In h and c, the rudiment has increased in size, cells are being added from without, and nuclei v/ithin the mass are seen to be dividing. Tn d and e^, a cav- ity has appeared in the centre of t}ie rudiment, which is now much larger, and the cells are becom- ing arranged into a one-layered epithelium. .■' . 2.
Fig. 28. Transverse section through tne anterior end of a biid slightly older than Pig. 9. The rudiment of the dorsal tube is shown, and consists at this stage of a few scattered cells, adhering to the wall of the inner vesicle a little to the left of the median dorsal line. Tne difference in thick- ness between the right side and the rest of tne ves- icle is seen to be but slight in t.iis region. D.2.
Fig. 29, a, b, c, d, and_e . Transverse sections representing stages in the development of the dorsal tube. In a a fevf cells are seen loosely grouped together and lying on trie wall of the inner vesicle. In b^ the association witn surrounding mesodermal cells is apparent. In c and d the mass has become compact, and cell-boundaries have entirely disappeared; in the latter figure the I'omen in the centre has begun to form. In e tne tube is completely formed, and
-77-
the cells are arranged around the lumen in a one- layered epithelium. i'^ . 2.
Fig. 30. Median sagittal section of a bud at about the stage of Pig. 4, shov/in;':^ the position and extent of tne dorsal tub«^ . ""ne peribranchial cavity is not yet completely constricted off from tne branchial sac. j;. 2. 7ii~. ^1 -_'^i_b,_c,_d , and _e . Transverse section shov/ing the devrlopment of the ganglion. Tn a and _b a few cells resembling . lesodermal cells are seen lying * on the dorsal side of tne dorsal tube. The wall of the latter is broken at this point, and nuclei appear to be wandering out into the rudiment . In £ and _d the rudiment is much enlarged, and in tne latter trie nuclei are beginning to arrange tnera- selves in peripheral region vf tne rudiment. In _e the wall of the dorsal tube under the ganglion has been reformed, and the central portion of tne latter is now free from nuclei and occupied by fine fibrils. P. 2.
Figs. 32, £, b, and c_. Sections illustrating the early de- velopment of tne sexual organs. Tn a an early stage is shovm, when the rudiment is merely a loose mass of cells, surrounding a slight cavity. A couple of tne elongated cells v/hich will form the genital cord are also ser-n. In b the cavity of the rudiment is much enlarged, and the genital cord
is prominent, and attiched to tne sphere; in both these figures the genital rudiment is seen to lie close to the wall of t;ie digestive tract. In c an older stage is shov/n, in which the cavity of the sphere has become divided into two; tne genital cord, although only represented in part, now ex- tends nearly to the posterior wall of tne cloaca, and lies close to, and parallel with, the intes- tine. 7, 2.
-79-
VI T A.
The writer of tnis paper, George Lefevre, v/as born in the city of Balti'nore, "Id., on Septe:nber 16th, 1869.
He received his early education in private, schools of Balti-nore, and in the fall of 1888, entered the Johns Hop- kins University as an undergraduate student in the Chemical- Biological Course.
After taking the degree of Bachelor of Arts in the same University, he began grad.iate studies in tne Biological Department, making Zoology his principal subject, and Animal Physiology and Botany his first and second subordinate sub- jects, respectively.
In 1892-93, he was assistant in Histology, and the same year held the University Scholarsnip in Biology.
In 1893-4, he v/as again assistant in Histology; ir June, 1894, he v/as appointed Fellow in Zoology, and in June, 1895, received the Bruce Fellowship.
■80-
^i^»3fe..Ji^
'4>t rf :h
■««*.«il.«.M.'»l.
li t^ if*
-p -i^ %^-' i
I p t
Off • I ^ ^
^ ^. II it. i. i >, - -
.-. .. .- Jfe >•■ » t' *i k? !• ^. ... , ^4 |i yi. M i it i ii' M ^ it I
^^ 1^ *: ?|t t It^ * ■*> ilf^ * 1*^ *^ *^' i
!jsr
r; ^' h
I t i * ^'
: I «■ > ?. .» >
<: * # »
S 4. .1, J * t Hi *. V * »
■f .1 > i
> 0 i *
.^ »■ It: *= '^ W- 0 n ^ :^ - .'^ T .-/"-. ~ -^-. . ]!;■ i » * ji * ^ a' i ^ t i. II .1 .» tj**^*
i ,1- # i • ,#. .ft 0 * i i jR ,* .* » * • «•*
>■ W # H" % * U >: f ft t: ^ > * I ^^J» iL» * * ♦' * ^ i^- ^ Ji » *■ ^ » > *^ it. -9- >' •• * , . V Jl^*^r>Ml'>ii r i^ ^\^ji» >':>■>'. ^^V**. • *. '4 1^ it i it * *■ * M * ^ * * * * « ;^ ^.f-.V 'H^ II. * .»>^ r?; »► ^^ -^■■. f>:. *. ife. *^'. "^.^^J^J'J^^ *^j\^\.v ' ■* * * ^' •« i» i*. «^ :jf ^ .« * ,i .ill ^JIr*Mv;'-
,' 1:^ It i^iiillli ^ % 1^ ii iji pi n j» i^ # !• ^ p» If . *-r^^^-'
u t^t^tmMmmmm ' #' # > »»'»(*#> ^' i» ii^ <• a p n » t. r.
i 1. ir If ■« ^"^ «* -i^ i' 11^' p. >'• ;• ,4.; %■ m ^: ^ ^' Ik 0 jif j^ t '' ^ •
- - - -, ~ ... ..- ..' , • . ^ p —-'-'•■ ^
'''<^.^'^''*^*mmi^&%
;>T<:.X«..
-I M » *
;V A; . . .
( -.A; ji
■4 '4 ^n
'^ '^ ,^« : - . ^
s
'*>.«.*.'«-;^^--.^