DEVELOPMENT OF ATEIAL CHAMBER OF AMPHIOXUS. 445

The Development of the Atrial Chamber ofAmphioxus.

By

E. Ray Lanke»ter, M.A., L.L..D., F.R.S.,

and

Arthur Willey,Student of University College.

With Plates XXIX, XXX, XXXI, XXXII.

LAST year one of us (Mr. Willey) collected during the monthsof May, July, and August many hundreds of embryos andlarvae of Amphioxus in Sicily.

The material so obtained has been worked out in the labora-tory of University College. The period of the development,to which we determined first of all to give attention, was thatbefore which Hatschek's well-known work stops short. Seriesof sections were prepared in order to ascertain the mode inwhich the atrial chamber takes its origin and the subsequenthistory of the gill-slits, viz. as to how the slits on the left sideof the pharynx originate. The relation of the larval to theadult mouth and the details of the curious process of move-ment of the mouth from a unilateral to a median position wereincluded in the scope of our inquiries.

Amphioxus occurs in great numbers in a comparativelysmall lake, or pantano, which is situated behind, and sepa-rated from the sea by, the village of Faro, near Messina. It

TOL. XXXI, PAKT I I I . NEW SKR. G G

446 E. RAY LANKESTEE. AND ARTHUR WILLEV.

is connected with the Straits of Messina by a narrow canal,some two or three hundred yards in length.

The bottom of the pantano, in contrast to that of theStraits, consists of foul mud; and it may be mentioned in thisconnection, as stated by Professor Kleinenberg,1 that Amphi-oxus is only occasionally met with in the Straits, and isentirely absent from another larger pantano which liesbehind the neighbouring village of Ganzirri, and is joinedby a short canal to the one at Faro.

The embryos float on the surface, and are to be had bydredging on the surface at sunrise; but the readiest method ofobtaining them in quantity is to take the adults in glassesand allow them to spawn there, if they will. Spawning takesplace about an hour after sundown.

The ova, if fertilised, must be very carefully distributedamong several glasses containing clean, but unfiltered, waterfrom the pantauo. If the water is filtered, or if sea-water isemployed, or if too many ova are placed in one glass, they willcertainly either die or develop abnormally.

The first outward and visible sign of fertilisation is theseparation from the egg-cell of the yolk-membrane (Dotter-membran).

Most, if not all, of the ova obtained were discharged throughthe atriopore.

If Kowalevsky 2 had not seen them issuing from the mouth,it would not have been supposed that they could pass intothe pharynx in opposition to the constant outflow of waterbetween the gill-bars.

Segmentation always commences at dusk—between the

1 I desire to express my sincere thanks to Professor Kleinenberg, ofMessina, for his kindness and for the invaluable assistance which he gave toMr. Willey in accomplishing the object of the latter's visit to Sicily. I havealso to thank the Government Grant Committee of the Royal Society forthe funds which enabled me to obtain Mr. Willey's services in this inquiry.—E. R. L.

: "Entwick. des Am ph. lane." ('M6m. Acad. Imp6r. des Sciences de St.Petersbourg,' series vii, vol. xvi, 1866).

DEVELOPMENT OF ATBIAL CHAMBER OF AMPHIOXUS. 447

hours of seven and eight—and goes on very rapidly throughthe night.

The early stages have been so fully described by Hatschek1

that it will only be necessary to refer to them in the briefestmanner.

At 8 p.m. segmentation commences; at 11 p.m. invaginationcommences; at 1 a.m. the gastrula is complete; at 3 a.m. thegastrula begins to revolve by cilia within the yolk-membrane;and at 5 a.m. two pairs of myocoelomic pouches have beenformed, and the embryo ruptures the egg-membrane andbecomes free-swimming.

During the first day the embryo grows in length and addsseveral pairs of somites. By about eight o'clock on thesecond morning—that is,thirty-six hours after the commence-ment of segmentation—the embryo has acquired a mouth onthe left side of the body, and a gill-slit, which arises at first inthe median ventral line, and subsequently comes to lie on theright side of the body.

The anus is formed soon after the appearance of the mouthand first gill-slit.

The embryonic period is now at an end, and the larvalperiod begins. As Hatschek states, the only way of obtainingthe larval stages is by pelagic fishing. This consists indredging at depths varying from fifteen to twenty fathoms.At this depth the Amphioxus larvae float in the midst ofcountless thousands of Sagitta larvse.

A long, but not yet clearly ascertained interval (probablyabout a fortnight) elapses between the formation of the firstand second gill-clefts.

In the period during which it is free-swimming the larvaacquires from twelve to fifteen consecutive unpaired gill-slits,each one arising in the mid-ventral line, and then growing insuch a manner as to lie on the right side of the body. Thisapplies to the anterior two-thirds of the pharynx, but it is notquite clear yet as to whether the last two or three median slitsever move up to the l'ight side. Meanwhile, longitudinal

1 Claus's'Arbeiten/lSSl.

448 E. RAY LANKESTER AND ARTHUR WILLEY.

ridges, which are subsequently concerned in the formation ofthe atrium, have appeared (see Fig. 6). In this stage the larvarests habitually on one side at the bottom of the vessel in whiehit is kept, and does not bury itself in sand or mud.

At the time of the eompletion of the atrium, which occurs atthe close of the larval period, some remarkable changes in therelative position of parts of the body in the anterior regiontake place, by which the mouth becomes median, and the gill-slits are arranged in two series, a right and a left. The larvaemerges from this critical phase in its development as asymmetrical animal, but the details of the process of "symme-trisation"—the strongly marked character of which justifies theuse of an otherwise undesirable term—are still rather obscure.The larva, now really a young Amphioxus, with atrium andpaired gill-slits, ceases to lead a pelagic life, and takes to thesand, where it passes the rest of its life. In this condition itdoes not rest on one side on the sand, but buries itself uprighttail downwards with the oral hood alone projecting from thesand (Willey obs.). Hence in the adult condition there are notone-sided relations of the Amphioxus to its environment.

Spawning occurs at least from April to September inclusive.The best month, however, in which to obtain the embryos isJune, while all the larval stages, up to the passage into theadult form, are to be found during July and August.

Previous View as to the Format ion of the At r ium.

The.hitherto accepted method of formation of the atrialchamber- of Amphioxus is that described by Kowalevsky,1

and more fully by Rolph.2

Kowalevsky says that after a certain number of gill-slitshave been formed, two longitudinal folds appear on oppo-site sides of the body, which grow round and meet, andfinally fuse together in the median ventral line, leaving a wideaperture at one end—the atriopore. His figures, two of which

1 ' Archiv fur Mikrosk. Anat.,' vol. xiii, 1877.2 'Morphol. Jalnbuch,' vol. ii, 1S76.

DEVELOPMENT OP AT RIAL CHAMBER OP AMPHIOXUS. 449

are here reproduced (Figs. 1 and 2), bear this description out,

FIG. 1. FIG. 2.

—I7v. -

Copy of Kowalevsky's iigures of transverse sections through a larva of Am-phioxus with fully formed atrium. Tig. 1 represents a section takenbetween pharynx and atriopore; and Fig. 2, one taken just behind theatriopore of the same larva. Int. Intestine, at. Atrium, l.h. Coelom.

more or less, while .Rolph's schematic figures bear it outentirely. The latter are reproduced in Figs. 3, 4, and 5.

Fi&. 3.

Copy of Rolph's theoretical section through the pharyngeal region of a larvabefore the formation of the so-called epipleural folds. N. Nerve-cord.M. Muscles. D. Intestine, b. Epidermis. Ch. Notocbord. Lh. Ccelom.a. Intestinal epithelium.

The most serious error in Kowalevsky's view lies in the facttha t he makes the space in the lateral outgrowths coutinuous

450 E. RAY LANKBSTBR AND ARTHUR WILLET.

with the body-cavity, and consequently calls it " Leibeshohle,"or coelom.

FIG. 4.

X.%Copy of Eolph's theoretical section through au older larva, showing the com-

mencing longitudinal downgrowths. E. Epidermis. 2?,. Visceral epi-thelium of the (future) atrial cavity. Es. Parietal epithelium of same.V. Subcutaneous tissue. Other letters as in Fig. 3.

FIG. 5.

Copy of Rolph's theoretical section, showing the meeting together of the" e p i p l e u r a " in the ventral middle line. A. Alrium. R. Raphe.Other letters as in Figs. 3 and 4.

DEVELOPMENT OF ATRIAL CHAMBER OP AMPHIOXUS. 451

There is no evidence that this space is an offshoot of theoriginal myocoelomic pouches: it arises apparently as an inter-cellular space in the midst of the connective tissue; in fact, itwould seem to belong to that category of spaces to which theterm " pseudoccel" has been applied. If this should prove tobe its history it would stand in contrast to the spaces in con-nection with the dorsal and ventral fins, which have beenshown by Hatschek to be derived directly from the myo-coelomic pouches.

Rolph's figures (Figs. 3, 4, 5) do not profess to be more thandiagrams. They show the epipleur originating as a dependingridge on each side of the pharynx (Fig. 4). Into this ridge thecoelom is extended. The epipleura meet finally in the middleline below the pharynx according to this theory (Fig. 5). I t isno doubt true that the scheme of growth thus sketched byRolph, and based upon Kowalevsky's erroneous figures, wouldaccount satisfactorily for the condition of the atrial chamberand its epipleural walls, as observed in the adult. I t also givesa basis for the suggestion made by Kowalevsky that theepipleura are comparable to the opercula of Teleostean fish.

We shall now give an account of our recent observations.

F o r m a t i o n of t h e A t r i a l Chamber as nowdetermined.

The first indication of the commencing formation of theatrial chamber is to be found in larvee with nine or ten gill-slitson the right side. Behind the region of the pharynx we findthat the mid-line of the body has become marked with a narrowgroove, so that in section it is bifid (Fig. 6). The short up-standing ridges which limit the groove are the metapleura ofthe adult. Though at first solid, the connective tissue withinthe ridge soon becomes hollowed and forms a lymph-space,which we have not traced into connection with the coelom.These ridges can be traced from about the middle of the larva'sbody forward towards the pharyngeal region, where theydiverge considerably from one another (PI. XXX, figs. A, B, C).

452 E. BAT LANKESTElt AND ARTHUJi WILLEY.

That belonging to the animal's left side keeps a more or lessmedian position and can be traced (though but small in eleva-tion) when twelve gill-slits are present as a ridge situated atthe lower or ventral margin of the gill-slits, and dying out in

FIG. 6. PIG. 7.

FIG. 6.—Transverse section through a larva with eleven or twelve unpairedgill-slits, showing the minute sub-atrial ridges, d.m. Dorsal divisionof myocoel in which the fin-ray will lie when it is developed, nc.Nerve-cord, nch. Notochord. m. Muscle-plate, my. Cavity of myocoel.d.a. Dorsal aorta. Int. Intestine. d.l.m. Double-layered membraneseparating the myoccel from the splanchnocoel. sp. Primitive splanch-noccel. v.a. Ventral vessel, met. Metapleur. s.a.r. Sub- atrial ridges.

J?IG- 7_—Transverse section through a slightly older larva. The sub-atrialridges (s.a.r.) have fused for a short distance between atriopore andpharynx; but in the pharyngeal region the atrium is unclosed, and conse-quently the gill-slits still open directly to the exterior, at. Atrium.

the anterior region of the pharynx (PI. XXIX, fig. 6). Theright-hand ridge, or metapleur, takes a course to the right ofthe gill-slits (which, it will be remembered, are on the rightside of the body), and overhangs the upper limit of the slits to

DEVELOPMENT OF ATRIAL CHAMBER OF AMPHIOXUS. 453

a small extent. I t dies out in front of the first gill-slit, whereit bends towards the middle line.

The atrium is formed by a small horizontal growth (s.a.r. inFig. 6), which starts from the inner face of each metapleur andfloors in the deeper half of the groove or area between the twometapleura (Fig. 7, at.).

These horizontal growths may be called the sub-atrialfolds.

They are at first extremely small, and the atrial spacefloored in is a mere canal. Later the width of the atrial spaceincreases greatly, and the sub-atrial folds consequently widen

FIG. 8.

dim-

— met-

Transverse section through an advanced larva with fully-closed atrium. Thelatter has begun to encroach on the coelom (sphnchnoccei) (sp.). Lettersas in Figs. 6 and 7.

also, becoming that pleated expansible floor of the atrialchamber, with its transverse muscular layer, which all observersof Amphioxus know so well (Pig. 9, s.a.r.).

454 B. EAY LANK ESTER AND ARTHUR WTLLEY.

The atrial groove becomes floored in first in the region of theatriopore. The growth of the sub-atrial folds extends graduallyforwards, and the closure proceeds along one side (the right) of

r.-:,my;

sp~

Transverse section through an adult Amphioxus. The atrium has grownup so as to divide the primitive splanchnoccel into two portions—an inneror splanchnic, and an outer or pleural, portion {if!, and sp".). sp. Theportion of the primitive splanchnoccel which is not so affected by theatrium, and which persists as the dorsal coelom. sp". Pleural coelom.sp'". Its perigonadial dilatation, f.r. Pin-ray. Other letters as in pre-ceding figures.

the pharynx. The whole atrium thus formed is a very smalltube-like space. The closure by means of the small horizontalsub-atrial outgrowths in the region of the large gill-slits issomewhat difficult to explain. The small left metapleur

DEVELOPMENT OF ATRIAL CHAMBER OF AMPHIOXTJS. 455

actually moves in course of growth from the mid-line, andrises on to the right side somewhat (Pis. XXX and XXXI,figs. 6, 7, 14, and 14A, I. met.). At the same time the muchlarger right metapleur is deepened, and overhangs the slits.Then the little horizontal junction is effected, and we getactually a nearly tubular atrium receiving the openings of suc-cessive gill-slits. With subsequent growth the narrow atrialtube widens and pushes itself right and left, so as to encroachon the space hitherto occupied by the ccelom, and finally itextends so far dorsalwards as nearly to surround the alimentarycanal (see Figs. 8 and 9).

The evidence of this history, in the form of careful drawingsof various sections, at various stages in the closure of theatrium, together with drawings of whole larvae in two stagesof development, is given in the plates (Pis. XXIX, XXX,XXXI, and XXXII) accompanying this paper. It is importantto point out that the mode of formation of the atrium as anarrow groove, which closes and sinks (as it were) into thebody of the Amphioxus, is really different in importantrespects from the enclosure of a space by downgrowth of largefolds, though ultimately no doubt the two contrasted modes offormation come to the same thing so far as the more obviousmorphological relations are concerned. The mode of for-mation which really occurs in Amphioxus is readily har-monised with the existence of the post-atrioporal extension ofthe atrium which gradually tapers to a fine csecal canal. Italso gives us an essentially different view of the region called" epipleur " by Lankester, and generally so designated, fromthat which Rolph's theory necessitated. That portion of theepipleur into which the myotomes of the body-wall extend isseen now to be no downgrowth, no extension or fold. It isthe original unchanged body-wall which bounds the sides ofthe animal's body in front of the atriopore, just as much as itdoes behind. The only new growth in the atrial regionwhich takes part in the limitation of the surface is the sub-atrial growth formed by the two little horizontal folds whichfloor in the atrium when it is a mere canal. These in the

456 B. BAY LANKESTER AND ARTHUR WILLBT.

adult are represented by the region of longitudinally pleatedventral wall between the two metapleura.

The formation of the atrium as a narrow groove whichcloses, sinks into, and expands within the body of Amph i -oxus, is much more readily comparable to what is knownof the formation of the atrial chamber in the Ascidians than isthe Kowalevsky-Rolph scheme. In the Ascidian a pair of in-pushings are formed, each with a circular orifice of invagina-tion; they expand within the body, fuse with one another toform one cavity, and one of the circular orifices disappears.In Amph ioxus we have a single in-pushing with a longitu-dinal orifice of invagination, which closes as the invaginationforms, excepting at its hindermost border, and then expandsto a greatly increased volume.

The comparison of the so-called epipleura of A m p h i o x u swith the opercula of fishes has only a remote morphologicalbasis, and probably no genetic relationship exists betweenthese two structures. On the other hand, it is very probablethat whilst the median fin-rays and fin including the ventralfin with its double rays represent the median fins of fishes—themetapleura represent morphologically the primitively con-tinuous lateral fins. The duplication of the fin-rays in themedian ventral series of adult Amphioxus appears to be onlya complete carrying out of a tendency to bifid structurewhich is found in the young dorsal fin-ray (see Lankester—Amphioxus , 'Quart . Journ. Micr. Sci.,' vol. xxix, PI.XXXVI, B. fig. 11) ; and though in both dorsal and ventralmedian fins the fin-ray lymph-space is single, yet the floor ofthis space has a bilateral origin according to Hatschek.

The figures which are given in PL XXIX represent two stagesof the larvae of Amphioxus , an earlier with three gill-slitsand the rudiment of a fourth (figs. 1, 2, 3), and a later withtwelve gill-slits and the rudiments of two more (figs. 4, 5, 6).

Though the older larva is considerably larger than theyounger, the two are, for the sake of comparison, representedof the same size.

DEVELOPMENT OF ATR1AL CHAMJ3EB OP AMPHTOXUS. 457

Each larva is illustrated by three views: one a surface-view of the left side complete, one a surface-view of the rightside of the anterior end, and one a deep focus of the anteriorend.

The drawings were made from carefully preserved specimens(killed with corrosive sublimate), stained with carmine andmounted in balsam. They are diagrammatic in the sense thatthey represent the results of observation rather than an actualview as obtained by one focussing.

The most striking feature in both larvse is the large mouthon the left side. In the younger larva the form of the tail,with its peculiar larval fin-rays, is noteworthy. In manyrespects this larval tail-fin recalls that of young Teleosteanfishes. I t is also closely similar to that of some Ascidiantadpoles (e. g. Styela). The small number and large size ofthe myotomes (indicated by numbers in the drawings) in theanterior region of the body are also remarkable. No evidencecould be obtained by us of the intercalation of new myotomes,nor of the multiplication of anterior myotomes by division.

The new myotomes appear to form exclusively at the caudalextremity.

In the larger larva the full number of adult myotomes hasbeen attained, and the larval tail-fin has become greatly modi-fied, giving place to the mesoblastic expansion which forms thetail-fin of the adult.

When we remember that in the adult the oral sphincter liesin the vertical line of the apex of the tenth myotome, it is nota little astonishing to note the position of that myotome rela-tively to the alimentary canal in the younger larva, and even inthat which has attained the full complement of myotomes. Theindependence of the metamerism of the body-wall from thatof the gill-slits and alimentary canal is thus very sharplyindicated.

In the cephalic region of both the older and the youngerlarva we see two remarkable larval structures, which lie infront of the mouth—the one in front of the buccal cavity, andthe other within its area. These are the prseoral pit and the

458 B. RAY LANKESTER AND ARTHUR WILLEY.

club-shaped gland. They have been figured by Hatschek, whohas described the prseoral pit as consisting of a ciliated de-pression and a short glandular tube, and has traced to thisstructure the thickened ciliated epithelium which is found onthe inner face of the oral hood of the adult forming there—theso-called " Rader-organ."

Hatschek, in his important memoir in the 'Arbeiten a. d.Zool. Institute d. Univ. Wien,5 vol. iv, 1881, does not figureany larva later than one with a single gill-slit. In one of thewall-plates of Leuckart and Nitsche, however, received by usduring the progress of this work, there are a number of figuresof later stages, which have to some extent assisted us in arrivingat an understanding of the later unillustrated note by Hatschek(' Zoolog. Anzeiger/ 1884, p. 517). None of the publishedfigures exactly coincide with our younger larva as to age, andour later larva is even less closely represented in the diagramsabove mentioned, so that the figures we are able to publish arenew, and will probably be of service to naturalists. The club-shaped gland, though figured by Hatschek and earlier observers,has not been described. It is remarkable for its early develop-ment (observed by Hatschek), and for the fact that it seems toentirely disappear in the adult without leaving any trace. Thegland is a sac with a large lumen. It lies obliquely on theright wall of the buccal cavity, and, bending round below,tapers to a narrow canal as it rises on the left wall of thebuccal cavity, where it opens just below and external to themargin of the mouth. In the younger of the two larvaefigured the club-shaped gland has no internal opening; it endsblindly just below the notochord. But in the later stage(drawn in figs. 4>, 5, 6) the gland has acquired an opening intothe cavity of the mouth. This orifice is placed at the oppositeend of the glandular sac to its external opening (PL XXIX,fig. 5 ; and PL XXX, fig. 5, int. a., and fig. 2, exL a.).

By the side of and anterior to the club-shaped gland is atract of modified epithelium of the buccal cavity of about twicethe breadth of the gland itself, and divided by a median clearerspace into two parallel tracts. This strangely-placed group of

DEVELOPMENT OK ATEIAL CHAMBER OF AMPHIOXUS. 459

cells has sometimes the appearance in published drawings oflarvae—of being a shadow cast by the gland, or in some caseslooks like a duplication of it. It can be traced in the sectiondrawn in fig. 2, PL XXX, where it is marked me.

In the deep-focus drawings, PI. XXIX, figs. 2 and 5, anothertubular structure is figured, which is also seen in the transversesections (PI. XXX, figs. 2 and 3, neph., and fig. 4, neph. a.; andPI. XXXI, fig. 13, neph,). This, so far as we can judge fromthe dravvings given in Leuckart and Nitsche's diagram, is thestructure which Hatschek has described as a nephridium in thefZoolog. Anzeiger/ 1884, p. 517, without a figure. In thecondition in which we have observed this structure (viz. inlarvae ranging from the stage with three gill-slits up to closureof the atrial cavity) there does not seem to be any specialreason for regarding it as a nephridium. We should prefer tocall it the subchordal tube. It appears to end blindly ante-riorly, and to open into the buccal cavity near the recurvedextremity of the glandular tract which accompanies the club-shaped gland. The tube lies below, and to the left of, thenotochord.

The drawings of the larger larva (PL XXIX, figs. 4 and 6)show some interesting features as to the disposition of thegill-slits and the metapleura. In this larva the atrial tubehas formed from behind (the atriopore) forwards as far as thehindermost still very small gill-slits (gs. 9). It is a remark-able fact that all the gill-slits up to this stage originatein the median ventral line. This is true of the first and ofall that follow up to the fourteenth, and possibly some fewmore. It is, however, not true of the formation of newgill-slits after the right and the left lateral series of gill-slitshave become established. The figures in our plate show that,whilst gill-slit No. 1 occupies an entirely lateral area onthe animal's right side—not reaching below to the median line—this position is gradually receded from by the hinder slits,which from No. 6 onwards are seen to encroach more andmore on the left side. When we remember that gill-slitNo. 1 as well as all that follow it originated in the

460 E. BAY LANKESTER AND AETHUR WILLEY.

median line, it is clear that the anterior slits must undergo atranslation in growth which moves them up t h e r i g h t side.Now, if we look at the slits following No. 6, it appears asthough a translation of these hinder slits were in progress,tending to bring them into position on the left s ide whenfully formed. We do not, however, consider it likely thatsuch a movement of the hinder slits to the animal's left sidetakes place, but believe that they also in due time move upfirstly to the right side, alongside of those in front of them.We have found it impossible with our present material to tracethe immediately subsequent history (subsequent to the stagedrawn in figs. 4 and 6) of the gill-slits. We are of opinionthat Kowalevsky's very definite statement and figures given inthe ' Memoires de l'Acad. Imp. de St. Petersbourg,' 7th series,vol. xvi, No. 12, 1866, must be accepted. According to thataccount, after some dozen gill-slits have taken up their positionon the animal's right side—having moved into that positionfrom the median line—a new and startling change occurs.The whole series moves downwards across the median line andup the left side of the pharynx, so that the primitive right-sidegill-slits become the left-side series; and in the meanwhile anew series corresponding in number make their appearancenot one by one, but all together, in the right side of thepharynx, occupying, as it were, the position deserted by therotated primitive series. This movement of growth appears tobe a general one affecting the whole pharynx, for, simulta-neously with the translation of the primitive gill-slits fromright to left, the great larval mouth moves from its extra-ordinary position on the animal's left side, and, becomingrelatively very much smaller, takes up its permanent positionas an anterior median orifice whilst its hood and tentaclesappear. We have not, we regret to say, at present been ableto study any larvae in which these remarkable changes are inprogress. We have, however, many larvae in which they arecompleted. I t is noteworthy that these larvae are scarcely, ifat all, larger than that of PI. XXIX, fig. 6; and yet they havethe mouth reduced in size and nearly median in position, the

DEVELOPMENT OP ATRIAL CHAMBER OP AMPHIOXUS- 461

anterior closure of the atrium completed, and a symmetricallyplaced right and left series of gill-slits.

We have taken steps to obtain the critical stages in theliving condition during the present summer, and propose toascertain whether the second row of gill-slits originates by anykind of fission from the first. If not, it is a curious fact thatthe morphologically median plane of the pharynx of the younglarva becomes the left side of the adult, whilst the relations ofthe mouth to median plane, in adult and larva respectively, areeven more curiously divergent. It is probable enough that inthese differences the larva does not present the more archaiccondition, but an adaptational arrangement. We do not atpresent know what are the conditions of life which render itsexcessive asymmetry advantageous to the larva.

The closure of the atrium by the growth of the little hori-zontal sub-atrial ridges from the median face of each metapleuris shown in the sections of various larvae given in Pis. XXX,XXXI, XXXII.

In the drawings, figs. 4 and 6 of PI. XXIX, we can tracethe two metapleura in the still unenclosed region of thepharynx. The right-side metapleur is seen to have its freeedge somewhat high on the animal's side, whilst the leftmetapleur in the perforated pharyngeal region is almostcoincident with the median ventral line. (The reference linein fig. 4, PI. XXIX, lettered "edge of left metapleur," hasbeen by oversight carried up to the right metapleur. Itshould stop at the ventral line.) The right metapleur islarger and deeper than the left, which is barely traceable asa thickening of connective tissue, when its fellow of theopposite side is large and provided already with the character-istic lymph-space (see fig. 7, r. met. and I. met., PI. XXX).

The figures A, B, C, in PI. XXX, represent diagrammatic-ally three stages in the closure of the atrial tube, showing inA the metapleurs or metapleural ridges without any hori-zontal sub-atrial floor; in B the formation of this floor in thehinder region, where there are no gill-slits; and in C itscontinued formation so as to enclose the perforations of the

VOL. XXXI, PAET III . NEW SBB. H H

462 B. EAT LANKESTEIt AND ARTHUR WILLET.

pharynx. It must be pointed out that the sections are com-plicated and rendered a little difficult of interpretation at first,by the fact that the margins of the gill-slits are irregularlycurved and folded, so that they cross the plane of section, and(as in fig. 7, PI. XXX) the slit itself becomes divided in thesection by a part of the projecting margin. A further modi-fication in appearances is due to the greater or less opening ofthe gill-slits, which can be varied by muscular action duringthe life of the animal. The atrial tube or cavity is also foundto vary in size and dimensions as soon as it is formed, owingto the varying extension or contraction of its muscular floorformed by the union of the sub-atrial ridges (compare fig. 12,PL XXX, and figs. 18 and 20, PI. XXXII). As was pointedout by one of us in the case of adult Amphioxus distendedwith genital products (see Lankester, f Quart. Journ. Micr.Sci.,' vol. xxix, PI. XXXV, fig. 4), so here in the larva theatrium can be distended to such an extent as to practicallyobliterate the metapleural ridges and their lymphatic canals,which reappear when the distension ceases.

The description of the individual figures se r ia t im will befound, it is hoped, sufficiently explanatory of points whichhave not been specially mentioned in the general body of thememoir.

DEVELOPMENT OP ATRIAL CHAMBER OF AMPHIOXUS. 463

EXPLANATION OF PLATES XXIX—XXXII,

Illustrating Professor Lankester's and Mr. Willey's memoiron the " Development of the Atrial Chamber ofAmpMoxus."

PLATE XXIX.

PIGS. 1 and 2.—Eight and left surface-views of larva, with four gill-slits.Length T496 mm.

FIG. 3.—Head of latter, seen with a deeper focus. Club-shaped glandopen to exterior only.

FIGS. 4 and 5.—Right and left surface-views of larva, with fourteen gill-slits. Length 3'485 mm.

N.B.—In Fig. 4 the reference line belonging to the words " edge of leftmetapleur " has been carried too far, and touches the right metapleur. Itshould stop at the ventral line of the larva.

FIG. 6.—Head of latter, seen with a deeper focus. Club-shaped gland,open at both lower and upper extremities, externally and internally respec-tively.

PLATES XXX, XXXr, AND XXXII.

The italics in Plates XXX, XXXI, and XXXII have the significance givenbelow.

ant. al. Anterior opening of atrium, at. Atrium, at. p. Atriopore. hr. e.Modified intestinal epithelium bordering the gill-slits, d. a. Dorsal artery.d. I. m. Double-layered membrane, separating myoccel from splanchnoccel.d. m. Dorsal division of myoccel in connection with dorsal fin. d. w. Dorsalwall of atrium, ext. a. External aperture of club-shaped gland, g. s. Gill-slit. Int. Intestine. Int. a. Internal aperture of club-shaped gland, kd.Club-shaped gland. /. a. Left dorsal artery (unpaired). I. m. Lower lip ofmouth. I. met. Left metapleur. m. Mouth, m.e. Modified epithelium onwall of mouth-cavity, mm. Muscle-plates. my. Primary myoccel. my1.Secondary upgrowth of myocoel, between the muscle-plates and notochord andnerve-cord. n. c. Nerve-cord. nch. Notochord. neph. Nepliridiuin ofHatschek. neph. a. Opening of so-called • nephridium into mouth-cavity.o. h. Commencing oral hood. r. d. Right embryonic diverticulum from theintestine, r. c. So-called " renal " cells of W. Miiller. r. met. Right meta-

464 E. BAY LANKESTER AND ARTHUR WILLET.

pleur. s. a. r. Sub-atrial ridges or floor, s. o. Sense-organ (part of prseoralpit), som. Somatopleur. sp. Splanchnoooel. sp. p. Splanchnopleur. v. a.Ventral vessel, w. o. Ciliated organ (of prseoral pit).

GENERAL REMARKS.

The intestinal epithelium is ciliated throughout. The epithelium borderiugthe gill-slits is much modified, being divided up into innumerable small cells,the cell-divisions between which cannot be seen under ordinary circumstances.

The nerve-cord consists of a nucleated portion surrounding the centralcanal and a peripheral fibrous portion.

Nuclei are to be seen in the notochord, and in the superior and inferiorcanals of the notocliord.

There are nuclei in the muscle-plates, but, as Hatschek points out, there isno epithelium on the outer wall of the muscle-plates. The nuclei on theinner wall are sufficiently scanty.

The sense-organ and ciliated organ of the preeoral pit are derived togetherfrom the left anterior diverticulum of the arcbenteron of the embryo, whilethe right diverticulum becomes simply the space occupying the anterior endof the body. I t is included in Fig. 1, but not in Pig. 13.

A reference to the drawings of the whole animal in PI. XXIX will showapproximately through what regions the sections have been taken.

FIGS. A, B, C.—Three diagrams of larvse, seen from ventral aspect, toillustrate the origin and relation of the metaplenral ridges to one another,and the gradual closure of the atrium from behind forwards.

Fig. A. No atrium.Fig. B. Atrium behind pharynx.

Fig. C. First two gill-slits open to exterior, all the rest now open iutothe atrium.

3?IG. 1.—Transverse section through the region of the ciliated organ andsense-organ of the prseoral pit, just in front of the opening of the latter intothe former. The anterior commencement of the splanclmocoel, and the pos-terior portion of the right embryonic diverticulum are shown. The epitheliumof the preeoral pit is of hypoblastic origin (Hatschek). This larva had twelvegill-slits, and no closed atrium. Preparation : osmic acid, borax car., fol-lowed by Meyer's carmine.

FIG. 2.—Transverse section through the commencement of the mouth-opening, showing the external aperture of the club-shaped or tubular gland.It also passes through the tract of modified epithelium. The very thin pieceof epithelium, two thirds of the way up, is the cause of the clear space or linewhich gives a double appearance to the tract. The thickening of the rightmetapleur is tending to the right side. This larva had eleven slits. Prepara-tion : sublimate and acetic ; hsematoxylin.

DEVELOPMENT 01' ATEIAL CHAMBER OP AMPHIOXUS. 465

FIGS. 3 and 4.—Portions of sections through another larva of same age,and prepared in same way as the last, taken just posterior to the regionrepresented in Fig. 2, to show the opening of the nephridium of Hatschekinto the mouth-cavity. Notice that the nephridium lies immediately belowthe left dorsal artery.

N.B.—In the larva there are not two dorsal arteries—right and left—in thepharyngeal region, as there are in the adult; but only one, and that on theleft side of the notochord.

FIG. 5.—Section through first gill-slit of same larva, showing the club-sliaped gland opening into the mouth-cavity at its upper extremity. Theventral vessel lies on the right wall of the intestine in the pharyngeal region.No cavity yet in the right metapleur.

FIG. 6.—Section through same larva as Fig. 1 (twelve gill-slits), throughthe same region as preceding, to be compared with Fig. 5 (with elevengill-slits) where the mouth is half shut. In this case the mouth is wide open,and the appearance of the section is considerably altered owing to the expan-sion of the ventral portion of the ccelom. The right metapleur is moreadvanced, but still has no cavity in this region.

FIG. 7.—Section through the sixth gill-slit of the same larva. The doubleappearance of the slit is due to a fold in the wall of the slit. The right meta-pleur has a cavity here. The left metapleur has commenced as a thickening.

FIG. 8.—Section through twelfth and last gill-slit of same larva. Themetapleural folds are nearly equal. There is a very small cavity in the rightand none in the left fold.

FIG. 9.—Section through the post-pharyngeal region of a larva preservedwith osmic acid vapour, rather older than Fig. 8, but with no part of theatrium floored in. The various divisions of the myoccel will be understood bya reference to Hatschek's figures, reproduced in Professor Lankester's paperin this Journal, vol. xxix, PI. XXXVIA, figs. 6 and 7.

FIG. 10.—Section through the twelfth (last but one) slit of a larva of theage of that represented in PI. XXIX, fig. 4. Preparation : concentratedsublimate; borax carmine.

FIG. 11.—Section through the post-pharyngeal region of the same larva(cf. Fig. 9), showing the fusion of the sub-atrial ridges. The character ofthe latter as ridges on the inner faces of the metapleura is not so well seenhere as in other sections.

FIG. 12.—Section through the same region of another larva of the sameage, showing the method of fusion of the sub-atrial ridges as described in theletterpress. Preparation : osmic acid and picro-carmine.

FIG. 13.—Section through the compound sense-organ ( = prseoral pit) of alarva in which all the gill-slits, except the first two, opened into a floored-in

466 B. BAY LANKESTER AND ARTHUR WILLEY.

atrinm. It shows the sense-organ (s. o.) opening into the ciliated organ (to. o.),and the latter opening widely to the exterior. It also shows the independentorigin of the oral hood (o. A.). Preparation : concentrated sublimate ; boraxcarmine.

FIG. 14.—Section through the anterior opening of the atrium, in the samelarva. It shows very well the position of the atrium anteriorly on the rightside, also the sub-atrial ridges. Note the relatively huge size of the rightmetapleur, and the almost entire absence of any indication of the left meta-pleur; thus showing that the sub-atrial ridges are distinct structures from,and only secondarily dependent on, the metapleural folds ; and that the latterserve a function (probably vascular) other than that of merely contributingto the formation of the atrium. This section is between the second and thirdgill-slits. The second slit opens to the exterior, the third opens into theatrium. The large cells at the bottom of the right metapleur are still in theepidermis; whereas, in fig. 7, PI. XXX, they have migrated inwards.

FIG. 14 a.—Section through the same larva as the preceding, two or threesections farther back, showing a gill-slit (the third) opening into the laterallyplaced atrium.

FIG. 15.—Section between the tenth and eleventh slits of the same larva,showing an older condition of the atrium than that represented in Figs. 11and 12, with "renal" cells on the dorsal wall. Note also the large size of themetapleura.

FIG. 16.—Section through the post-pharyngeal region of the same larva,showing a still more advanced condition of the atrium. The gelatinous sub-cutaneous tissue has disappeared from the dorsal wall of the atrium, leavinga thin double membrane, consisting of coelomic and atrial epithelium( = somatopleur).

FIG. 17.—Section through the atriopore of same larva.FIG. 18.—Section through the last gill-slit but two of a larva wilh the

atrium floored in over three slits, showing expansion of atrium and temporaryobliteration of metapleural spaces. Preparation : osmic acid and picro-carmine.

FIGS. 19 and 20.—Sections through a larva in which the atrium had closedover two slits (the fourteenth and fifteenth), showing a narrow condition ofthe atrium in front (Fig. 19), followed by a more expanded condition behind(Fig. 20).

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