The Early Development ofAstropecten ir-regular is , with Remarks on Duplicity inEchinoderm Larvae.

By

H. G. Newth, A.R.C.Sc, D.I.C.,L e c t u r e r i n E m b r y o l o g y , U n i v e r s i t y of B i r m i n g h a m .

W i t h P l a t e s 4 0 a n d 4 1 a n d 2 T e x t - f i g u r e s .

C O N T E N T S .

P A G E

I N T R O D U C T I O N 5 1 9

M A T E R I A L A N D M E T H O D S . . . . . . . . 5 2 0

I . D E S C R I P T I O N . . . . . . . . . 5 2 1

1. F e r t i l i z a t i o n a n d C l e a v a g e . . . . . . 5 2 12 . G a s t r u l a t i o n . . . . . . . 5 2 33 . G a s t r u l a a n d B i p i n n a r i a 5 2 54 . A b n o r m a l L a r v a e . . . . . . . 5 2 7

I I . D I S C U S S I O N . . . . . . . . . 5 2 9

( a ) S y m m e t r i c a l L a r v a e . . . . . . . 5 3 0( l ) T w i n P o r e - C a n a l 5 3 0( 2 ) T w i n H y d r o c o e l a n d E n a n t i o m o r p h y . 5 3 3

(6) T w i n L a r v a e 5 3 7(c) A s y m m e t r y of t h e N o r m a l L a r v a . . . . . 5 3 9(d) A n H y p o t h e s i s t o A c c o u n t f o r L a r v a l D u p l i c i t y . . 5 4 1(e ) F a c t o r s i n G r o w t h 5 4 6( / ) C o n d i t i o n s o f E a r l y D e v e l o p m e n t . . . . . 5 4 8

S U M M A R Y . . . . . . . . . . . 5 5 1

L I T E R A T U R E R E F E R E N C E S 5 5 2

E X P L A N A T I O N o r P L A T E S . . . . . . . . 5 5 4

INTRODUCTION.

THE interesting, but fragmentary, accounts that we possessof the metamorphosis of starfishes belonging to the familiesAstropectinidae and Luidiidae (M. and C. Delap, 4 ; Mortensen,21), make it very desirable that the complete development

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of members of this group should be studied. Accordingly,during a two-months' stay at the Plymouth Laboratory in thesummer of 1921, and again in 1923, in the intervals of other,experimental work, I made repeated efforts to fertilize the eggsof A s t r o p e c t e n i r r e g u l a r i s . In each year only one ofthese attempts was successful. A plentiful supply of animalswas obtainable, but their gonads were small, and spermatozoaremoved from apparently ripe males were immobile whensuspended in sea-water—nor could they be made to functionby the usual device of altering the alkalinity of the water.Small, but apparently healthy, cultures of larvae resulted fromthe successful fertilizations. The larvae of 1923 perished fromunknown causes when only five days old, their early develop-ment having conformed exactly to that of the larvae of 1921,and enabled me to make a few additional observations. Thefollowing account is therefore based chiefly upon the earlierculture, which, though it disappointed my hopes of witnessinga complete development, furnished material of some generalmorphological interest, and made it possible for the first timeto identify the early Bipinnaria of the present species.

MATERIAL AND METHODS.

The parents of the successful culture were collected on July 21,1921, on the Eame-Eddystone trawling ground. Eggs wereobtained by gently teasing out the ovaries in filtered sea-water.Fertilizations were made in glass finger-bowls, and in these thelarvae were kept until they had assumed the typical Bipinnariaform and were feeding actively, a few drops of a culture of thediatom N i t z s c h i a being supplied as food. They were latertransferred to a large bell-jar with a mechanical plunger, andsimilarly fed. Freshly filtered water from outside the Plymouthbreakwater was used throughout, its alkalinity being foundto be practically unaffected by filtration. The temperaturewas kept almost constant at about 16° C. My friend, Mr. E.Ford, Naturalist at the Laboratory, very kindly took chargeof the culture when I left Plymouth, and the final sample waspreserved by him.

DEVELOPMENT OF ASTROPECTEN 521

The number of eggs fertilized was small, and fewer thanone hundred attained the early Bipinnaria stage—a numberwhich dwindled, owing to the usual causes of mortality, tillfinally, on August 15, there remained only two individuals,constituting the last stage here described.

The- figures are of animals preserved in Bouin's aqueouspicro-formol-acetic fixative, and in the case of whole larvae,drawn while in alcohol, in which they suffer no distortion,details being verified later in the stained and cleared prepara-tion. Sections were cut in collodion and wax.

I. DESCRIPTION.

1. F e r t i l i z a t i o n and C l e a v a g e .

F i r s t Day.—The eggs of the parent femalo, when removedfrom the ovary, still showed, for the most part, large, vesicularnuclei, indicating that the first maturation division had notyet begun, but in a few (less than 10 per cent.) this stage hadbeen passed. Only a very small proportion of the spermatozoataken from the parent male were active in ' outside ' sea-water.Eggs and sperm were set aside separately in a cool place, andafter about two hours it was estimated that between 10 percent, and 20 per cent, of the eggs had now undergone then-first maturation division. The average diameter of the matureegg is 0-21 mm.

No change in motility of the sperm had occurred during theripening of the eggs, and the addition of alkali was withouteffect. For insemination unmodified sperm-suspension wasused ; and at the end of five minutes' fertilization membraneshad been formed by practically all those eggs in which thegerminal vesicle had faded. The membrane, when fullyseparated, stood out from the surface of the egg at a distanceequal to about one-tenth of its diameter, and carried with itthe first polar body. The second polar body was seen later tobe formed within the membrane.

The following observation may be recorded here, in view ofits possible bearing upon the second subject of this paper.

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A single batch of eggs was left unfertilized overnight andinseminated next day. No further maturation took place, butthe ripe eggs segmented abnormally and none reached the blas-tula stage. Cleavage resulted in a wide separation of the blasto-meres, reminiscent of the effect of ' calcium-free' sea-water.1

Observations of the normal cleavage of the egg were not madein 1921, but in 1923 it was seen that after four or five regularand synchronous divisions segmentation became irregular andgave rise to an almost solid rnorula.

Nine hours after fertilization the blastula stage had beenreached. The embryo at this stage is of the wrinkled typealready described by other authors for various Asteroids,2 andby myself for two species of Cu cum a r i a (22).

Six hours later the blastulae were still in their membranes—which they now completely filled—and showed no sign ofciliation or of invagination. The mean diameter of ten of themwas 0-238 mm. The embryos being fairly transparent, it couldnow be seen that the blastula-wall was definitely epithelial,and that the sulci indenting its surface had become shortened,many of them appearing as pits rather than grooves. Fig. 1,PI. 40, exhibits the appearance of an embryo of this stage insection. It is noteworthy that the nuclei of its cells are—almost without exception—situated nearer to the morphologi-cally outer than to the inner surface of the blastula-wall—a fact demonstrable, of course, only by reading the whole seriesof sections.

'• I have since (1924) witnessed a similar spacing of the blastomeres incertain cultures of Aster ias rubens . The later development of thesewas perfectly normal, but the blastomeres were less widely separated thanin As t ropcc ten .

a It has several times been suggested to me that this type of blastulais due to the abnormal conditions of laboratory culture. Since writingthe above I have been able to induce its formation experimentally inEch inus and As te r ias , neither of which animals normally passesthrough such a stage; and in both cases the experimental conditionswere totally unlike those of the As t ropec ten culture. It is quitecertain that a wrinkled blastula is normal in the case of As te r inag i b b o s a—where it has never been described—for I have collectedembryos in this stage from the shore.

DEVELOPMENT OF ASTROPECTKN 523

2. G a s t r u l a t i o n .

Second Day.—Gastrulation was beginning at the end ofanother seven hours, and the embryos, now ciliated and freefrom their fertilization membranes, were rotating sluggishlynear the bottom of the bowl containing them.

Little or no smoothing-out of the folded blastula-wall precedesgastrulation in the majority of larvae, except over the smallcircular area that is actually invaginated. The folds are slowlyeffaced during the formation of the archenteron. In the earlieststages of this process the embryo is still approximately spherical,and the archenteron is easily to be distinguished from otherre-entrant parts of the blastula-wall as being roughly rect-angular in longitudinal section, while they are irregularlyrounded or pointed. Thus, the archenteron does not normallyarise in A s t r o p e c t e n as in S o l a s t e r endeca (Gemmill,6)—by the deepening of a pre-existing fold. Sections show thatthe cell-nuclei are now situated at the inner (or blastocoelic)ends of the cells, and that this is true of the whole integument.

Eapid general growth of the embryo, its elongation alongthe archenteric axis, and an increase in its transparency due toattenuation of the ectoderm, now begin, and continue through-out gastrulation. The egress of the infolded cells and theirincorporation into the general ectoderm go hand-in-hand withthese changes ; but this process is occasionally incompleteeven in the fully formed gastrula—which may thus retainvestiges of the sulci which look like supplementary archentera.These disappear later, leaving no trace.

The above observations have, in my opinion, their adverserelevance to certain attempted explanations of gastrulationthat have been put forward. In the first place, they makefinally untenable a view still occasionally advanced, in spiteof other evidence against i t : that reduced fluid pressure withinthe blastula determines the imagination of its wall. If thatwere true, we should be confronted in the case of As t ro -pec t en with a mechanical paradox: on the one hand,evagination of re-entrant folds, and increase in volume of the

M m 2

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embryo ; on the other hand, the concurrent ingrowth of thearchenteron. The first process, ex h y p o t h e s i , demandsan increase of internal pressure ; the second, a diminution.

Ehumbler's (26) ingenious interpretation makes invagina-tion depend upon (a) absorption of fluid by the inner endsof the cells concerned, (b) their consequent assumption ofa pyramidal shape, the base of the pyramid being inwards,and (c) the lateral pressure of neighbouring cells, due to inter-stitial multiplication. Here, again, the gastrulation of A s t r o -p e c t e n presents a difficulty. The invaginating area, it istrue, shows the usual pyramidal cells—but so, also, in manycases, do the zones of evagination (fig. 2, PI. 40). Assheton'scriticism that the observed cell-form should be regarded asa mechanical result—rather than a cause—of the infolding isthus strengthened.

Assheton's own theory of gastrulation (1) was developed inconnexion "with the regular and symmetrical process seen inA m p h i o x u s , and was supported by the behaviour of models.It is quite inapplicable to A s t r o p e c t e n . The theory,briefly stated, is that there exists between contiguous cellsa specific attraction acting from the neighbourhood of theirnuclei; and that a convex, free epithelium of such cells(e.g. a blastula), in which, over a small area, the cell-nucleihave migrated towards the convex outer surface, will tend toinvaginate the area in question. An obvious corollary—notstated by Asshefcon—is the evagination, or smoothing-out, ofany re-entrant portion of a curved epithelium in which theconverse arrangement of nuclei is found. The egg of A s t r o -p e c t e n furnishes, plainly, the ideal test of this hypothesis.It will be sufficient to repeat what has already been said :that in this animal the nuclei of the blastula are all nearer tothe outer than to the inner surface of the epithelial wall,whilst the nuclei of the gastrula—whatever the immediatepositions or movements of the cells to which they belong—are all near the inner, blastocoelic surface. The sections shownin figs. 1 and 2, PI. 40, though not specially chosen to illustratethe above points, exhibit sufficiently well the relations of the

DEVELOPMENT OF ASTROPECTEN 525

nuclei in blastula and gastrula ; and in each of these will heseen folds of ectoderm in the last stage of evagination, whosecells have the appearance—on Ehumbler's hypothesis—ofundergoing the diametrically opposite process.

3. G a s t r u l a and B i p i n n a r i a .

Th i rd Day.—There was great discrepancy in the rate ofearly development, so that the next sample preserved containedboth late gastrulae, in which the coelom was just appearing,and also larvae in which the Bipinnaria form was recognizable(figs. 3-5, PI. 40).

The gastrula differs from those of A s t e r i a s and P o r a n i a(Gemmill, 7 and 9) in that the archenteron, when fullyinvaginated, reaches appreciably less than half-way towardsthe anterior end, and in this way determines the characteristi-cally bulky preoral lobe of the young Bipinnaria. The primarycoelomic vesicle arises, as in A s t e r i a s, from the expandedtip of the archenteron, and in the living animal is a rather morevoluminous sac than is shown in fig. 3, PI. 40, where the resultof its partial collapse through fixation is seen. Wings of thisthin-walled expansion grow back on either side to form theenterocoel pouches, which are completely separate from oneanother save where they join the gut. The left-hand pouch is,from the first, larger than the right.

An extensive but shallow in-sinking of the future ventralsurface forms the circumoral field, and initiates the curve ofthe alimentary canal. In the middle of this depression thestomodaeum appears as a small and quite definitely circum-scribed invagination of the ectoderm, which comes into contactwith a blunt projection of the gut, and so remains till the endo-dermal oesophagus is constricted off from the stomach. Per-foration of the mouth then occurs, and about the same timethe enterocoels lose their connexion with the gut. Soon afterthis the left enterocoel acquires its communication with theexterior through pore-canal and hydropore (fig. 5, PI. 40),and faint indications of the ciliated band can be seen. Whether

526 H. 6. NEWTH

tan Auricularia stage occurs I cannot say with certainty. Thelength of the larva is now about 0-4 to 0-5 mm.

Six th and E i g h t h Days.—The larvae were nowhealthy, active Bipinnariae about 0-6 mm. long, with well-developed gut and ciliated bands. Fig. 7, PI. 41, makes furtherdescription unnecessary.

Twenty- f i f th Day.—The two larvae which alone reachedthis age were nearly identical in size and appearance, and thereis no reason to doubt that their external features representthose distinctive of the species (figs. 8 and 9, PI. 41).

Mortensen's illustrated descriptions of the two Japanesespecies, A s t r o p e c t e n scopar ius and A s t r o p e c t e np o l y a c a n t h u s , are the only accounts we possess of larvaeof the genus A s t r o p e c t e n ; and to the first of these thethree-weeks-old Bipinnaria of As t ropec t en i r r egu la r isbears a close resemblance. No appreciable increase in size,and no change in the coelom except its growth in length, hadoccurred since the eighth day. The boundaries of the entero-coels were not sufficiently plain in the whole preparations tobe included in my camera drawings, though it could be seenthat connexion between the anterior coeloms had not beenestablished in the preoral lobe, and that the hydrocoel hadnot yet appeared—facts that were confirmed by serial sections.

The ciliated processes are short and rounded. The preoralband is arched forward over the stomodaeum—though less sothan in As t ropec t en s c o p a r i u s . The median preoralprocess projects ventrally, and at its base the convex frontalarea is strongly narrowed without the formation of salientpreoral processes at that point. Of the remaining processesthe anterodorsals are the best developed ; the posterodorsals,posterolaterals, and postorals are small, though clearly defined.The length of the larva when preserved is now about 0-7 mm.

The Bipinnaria of As t ropec t en scopar ius , as figuredby Mortensen, shows practically no growth, and but littleincrease in the complexity of its processes, between the ninthday and a stage of apparently advanced metamorphosis. Itis probable, therefore, that though my latest stage is still far

DEVELOPMENT OF ASTROPECTBN 527

from metamorphosis the description here given will sufficefor the diagnosis of the larva of A s t r o p e c t e n i r r e g u -laris—at least among the Bipinnariae of British seas.

4. A b n o r m a l L a r v a e .

The commonest abnormality found in my cultures was thepresence of a hydropore and pore-canal on the right side aswell as on the left. This condition occurred in about one-third of the larvae examined, and is in no way exceptional,since it has been noticed in all Asteroids that have been experi-mentally reared in sufficient numbers. No observations weremade of the persistence of this character, but it was stillpresent in two otherwise normal Bipinnariae examined onthe eighth day.

A very different kind—or degree—of abnormality is illus-trated by the two individuals shown in fig. 6, PI. 40, and figs. 10,11, PI. 41.

Contemporary with the normal larvae shown in figs. 3-5,PI. 40, and discovered in the same fixed sample which con-tained them, were a number showing slight abnormalities ofdevelopment, mostly affecting the tip of the archenteron, and,with one exception, not of a sufficiently definite nature tojustify a description here. This exception was in the case ofthe animal depicted in fig. 6, PI. 40. The blind end of itsarchenteron is expanded in the usual way to form a flattenedsac, but from this sac two small oesophageal rudimentsproject, each met by a well-marked stomodaeum at the bottomof a corresponding circumoral field. The edge of the primarycoelomic vesicle is irregular, but two larger projections fromit probably represent the first appearance of a pair of entero-coels—both dorsolateral in position. There is no indication ofhydropore or pore-canal; but the circumoral ciliated band isvisible, faintly indicated on the ventral surface just in frontof the anus, and seen on either side in optical section as athickening of the ectoderm. The orientation of the animal asa whole is thus made certain.

The older abnormal larva (figs. 10 and 11, PI. 41)—a Bipin-

528 H. G. NEWTH

naria nine days old—was seen alive, but had unfortunatelyescaped notice until I made a final survey of my cultures beforeleaving Plymouth. When discovered it was active andapparently healthy. It possesses two fully formed gullets,a single stomach and intestine, but only one pair of enterocoelpouches, each with a pore-canal and hydropore. The ciliatedband is in four parts : a typical preoral band lies in front of

TEXT-FIG. 1.

Diagram of a twin Bipinnaria (hypothetical) in the Auricularia stage,to illustrate the probable homologies of the ciliated bands in theanimal shown in figs. 10 and 11, PL 41. P.O., postoral band,median loop completed by dotted lines.

each stomodaeum ; dorsal and posterior to these is the post-oral band, topographically normal; while, in addition, aciliated loop (m.l.) lies midway between the two frontal areas.This median loop appears at first to be a third preoral band,without a corresponding stomodaeum. I believe, on thecontrary, that it is a portion of the postoral band left isolatedby reason of the incompleteness of the twinning. Text-fig. 1illustrates this interpretation by showing a hypotheticalmonster in which the duplicity, both of ciliated bands and of

DEVELOPMENT OF ASTEOPBCTEN 529

alimentary canal, has been carried almost to the point ofseparating the constituent twins, A and B.

II. DISCUSSION.

In the absence of experimental analysis it would have beenpresumptuous to have offered any explanation of the twoabnormal larvae described above—had they stood alone.It happens, on the contrary, that they range themselves quitenaturally in series with a number of similar monstrositiesrecorded elsewhere. A consideration of certain properties ofthis series has led me to re-examine rather closely the currentexplanations of duplicity in Echinoderm larvae, and, as aresult, to adopt a view that differs, in some respects, fromthem all. Since my provisional hypothesis is one that mayprove fairly easy to test by experiment, I have thought itwould not be premature to set it forth here, together witha brief commentary on some of the relevant observations andopinions of previous workers.

Double monsters among Echinoderm larvae may be roughlydivided into two classes : one in which the duplicity affectslarval structures or the larva as a whole, and one in which itaffects the coelom and the rudiments of adult organs, leavingthe larval form apparently untouched. Examples of the firstclass, so far as I can find, have only once before been described(Gemmill, 8), and these I shall consider last.

Duplicity in the second class of larvae is of a very specialkind, and consists in the symmetrical repetition, on the rightside of the organism, of structures normally peculiar to theleft. It is not surprising, therefore, that those who haveimagined a bilaterally symmetrical ancestor for the groupshould have hailed these variations of development as aconfirmation of their views. With the characters of thisancestor I shall not be concerned here, except in so far as theyimply a fundamental—but ' latent'—bilateral symmetry inthe coelomic organs of normal Echinoderm larvae at thepresent day. It is a part of my thesis that this latent symmetrydoes not, in fact, exist.

530 H. G. NEWTH

(a) S y m m e t r i c a l L a r v a e .

(1) Twin Pore-canal.—The right enterocoel, as well asthe left, develops a pore-canal and hydropore. This conditionhas been observed in varying proportions of the larvae of manyAsteroids, and in certain Ophioplutei and Echinoplutei. Thefollowing are some examples of its incidence among experi-mentally reared Bipinnariae :

Species.Asterias rubensAsterias glacialis

Asterias glacialisAsterias vulgarisPorania pulvillus

Astropecten irregu-laris

Author.Gemmill (7)Gemraill (7)

Mortensen (20)Field (5)Gemmill (9)

Newth (presentt>at>er)

[iicidence.10 per cent.' At least 70 per cent, in cer-

tain cultures '.' About 50 per cent.'' Considerable numbers '.30-40 per cent, perfectly

bilateral, ' only about 25per cent, showed no trace '.

' About one-third of thelarvae '.

All these records are of larvae reared in the laboratory fromartificially fertilized eggs ; but it should be added that Gem-mill (7) reports an incidence of 5 per cent, among larvae ofA s t e r i a s r ubens ' spawned and fertilized naturally in thetanks ' (the conditions of cleavage and early development arenot stated). Apart from various less striking references to theoccurrence of this variation to be found in the literature ofexperimental zoology, there are two cases that have beenconsidered of special morphological or phylogenetic im-portance.

As t e r i a s vu lga r i s (Field, 5) calls for special mentionbecause it is quoted as a case in which the symmetrical develop-ment of two hydropores has been demonstrated to be a normal,though transitory, feature of development. This view of Field'sresults, though persistently repeated, is quite erroneous. Ithink that a careful collation of his statements should convinceany reader that only a comparatively small number of the larvaeobserved by him actually possessed two hydropores, and thathis conclusion regarding the ' normal' occurrence of the

DEVELOPMENT OF ASTKOPECTEN 531

character is nothing more than a rather hazardous inferencefrom the facts recorded. It is certainly of great interest, how-ever, that several larvae among the twins studied by Fieldwere obtained from the plankton, for this record—made morethan thirty years ago—still stands almost alone, in spite ofthe large number of pelagic Bipinnariae that have since beenexamined.

The second case is that of the larva of the Clypeastroid sea-urchin, M e 11 i t a p e n t a p o r a (M. t e s t u d i n a t a), studiedby Grave (11 and 12). Here the bilateral symmetry is saidto be completed in a different way : ' . . . two pore-canals areof constant occurrence. . . . Communicating with the singlemedian dorsal pore two well-developed canals are seen, onejoining the left, the other joining the right anterior enterocoel.The right canal is usually slightly smaller than the left butit is never entirely wanting. It persists in the adult as a smallclosed vesicle in the region of the ampulla which receives theinternal opening, or openings, of the madreporite and theterminal opening of the stone-canal' (Grave, 11). Unfor-tunately Grave does not describe the way in which this appar-ently symmetrical arrangement comes about. There wouldappear to be two possibilities : (a) the already separated entero-coels may each send out a canal which meets its fellow at thecommon hydropore ; or (b) the primary coelomic vesicle mayacquire its communication with the exterior while it is as yetundivided, in which case the right' pore-canal' would representthe remains of the original connexion between the enterocoels.Against the former alternative is a certain a p r i o r i im-probability, and also the fact that no comparable process hashitherto been observed elsewhere; while in favour of thelatter alternative are the observations mentioned in the nextparagraph. We have also Grave's own statement that theright ' pore-canal' persists as what we are fairly safe in callingthe madreporic vesicle of the adult—a closed vesicle in theregion of the ampulla of the stone-canal. Unless, then, we areprepared to draw the fantastic conclusion that the madreporicvesicle of Bchinoderms generally is a vestigial right poro-

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canal, the case for the complete bilateral symmetry of theMel l i t a Pluteus falls to the ground.

Gemmill (9) states of P o r a n i a that the coelom generallyseparates as a single dorsal sac which at once divides right andleft. It seems, further, that the appearance of the pore-canal, orpore-canals, normally follows immediately upon this separationof the enterocoels—so that there are three processes occurringin quick succession. Occasionally, however, the enterocoelsretained their dorsal connexion till even the third week (9,p. 34). The early stages of such larvae were not observed, butI have myself recently witnessed, as a rare abnormality inthe development of As t e r i a s r u b e n s , the acquisition ofa pore-canal by the undivided primary vesicle, the canal beinglater appropriated by the left enterocoel; and a very similarobservation is recorded by Gemmill (7) for the same species.There is in these cases what may be called a dislocation in thesequence of development, whereby the division of the primaryvesicle is delayed. It is not unreasonable to suppose that sucha dislocation may also occur in the reverse sense, the primaryvesicle dividing too soon instead of too late ; so that anyprocesses normally taking place in it as a prelude to itsdivision would be anticipated. The consequences and possiblecauses of such precocity will be dealt with below ; but itmay be recalled here as a significant fact that in Porania—•in which the events connected with the early differentiationof the eoelom occur so rapidly—there is an extraordinarily highpercentage of pore-canal twins. Certain abnormal E c h i n u slarvae that have been recorded as having dorsally confluentaxial sinuses sharing a single pore-canal may be mentionedin this connexion, though their early stages have not beenobserved (Ohshima, 24, cases 5 and 7).

No satisfactory explanation of twin pore-canal has beenoffered. Field saw in its occurrence the further expression ofan underlying, but much obscured, bilateral symmetry, due todescent from an ancestor in which that symmetry was com-plete. Other authors have since accepted the same view,MacBride asserting that it is ' the key to the understanding

DEVELOPMENT OF ASTROPECTEN 533

of Echinoderm development' (16, p. 466). G-emmill, however,on account of the widely varying frequency of the character,does not ' ascribe the incidence of double hydropore directlyto ancestral causes '—but to Homoeosis.

(2) Twin H y d r o c o e l and Bnan t iomorphy .—Theright side of the larva, as well as the left, develops—in varyingdegrees—a hydrocoel and its associated structures ; or theright side alone develops them, in which case the whole systembecomes a mirror-image, or enantiomorph, of the normal.The propriety of including the latter condition in the categoryof symmetrical larvae will appear later.

Among Asteroids, Ophiuroids, and Echinoids, individuallarvae with two symmetrical hydrocoels have been occasionallyrecorded for many years past; but only recently have theybeen obtained in large numbers, and studied in such detail asto warrant any conjecture with regard to their significanceor mode of origin. In only one case of this kind is a vestigialright hydrocoel described as being a constant feature of normaldevelopment ; and since great importance has been attachedto this case I shall consider it first and in some detail.

In his paper on O p h i o t h r i x f rag i l i s (14), MacBridedescribes and figures a single pluteus—taken from the plankton—in which there were two well-doveloped hydrocoels. Hedescribes also, as a normal feature of development both in thesea and in artificial culture, the separation of a vesicle ofvariable size and appearance from the right anterior coelom.This structure he considers, on account of its place of origin,to be the antimere of the hydrocoel. Such a view should,I think, be accepted with extreme caution ; because when theaccount of O p h i o t h r i x was published its author still believedthat a somewhat similar vesicle, formed from the right anteriorcoelom in A s t e r i n a and E c h i n u s , was a vestigial righthydrocoel. In Asteroids and Echinoids this structure has beenproved subsequently by Gemmill (7), and by MacBride himself(17), to be the madreporic vesicle, an organ quite distinct,which may be present in the same abnormal larva togetherwith a well-developed right hydrocoel. When it is added that

534 H. G. NEWTH

during the metamorphosis of O p h i o t h r i x the sac in questioncomes to lie close to the hydropore, and that ' sometimes aprojection of its inner wall is noticeable similar to that whichgives it acrescentic form in A s t e r i n a g i b b o s a ' (MacBride,14), the strong probability must be admitted that it is here,as in Asteroids and Echinoids, not the right hydrocoel but themadreporic vesicle. In a later publication (16) MacBride,after admitting the distinction between hydrocoel and madre-poric vesicle in Asteroids (p. 467), reiterates his opinion thatO p h i o t h r i x has a right hydrocoel (pp. 491 and 492), andthen states : ' A madreporic vesicle is formed, apparentlyin the same way as in Asteroidea.' This may mean that anadditional sac is formed in O p h i o t h r i x ; but I can find noreference to it elsewhere. If it does not exist there seems nopossibility of reconciling the three statements made, exceptby supposing the madreporic vesicle to possess different honio-logies in different groups of Echinoderms. I shall prefer toassume that an error of interpretation has been made andinadvertently perpetuated.

Excellent accounts of twin hydrocoel larvae in Asteroids haverecently been given by Gemmill (10), in Echinoids, by Mac-Bride (15 and 17), and Ohshima (23 and 24). For a full descrip-tion of cases and for references to previous observations thereader must consult these authors : I shall be able only tosummarize the large amount of material available.

(Temmill's account is based upon more than sixty twinhydrocoel larvae of A s t e r i a s r u b e n s found in his culturesduring 1912 and 1913. Many of these showed almost completesymmetry of coelomic organs up to an advanced stage. Thepresence of two hydrocoels and two hypogastric coelomsinvolved the absence of an epigastric coelom sensu s t r i c t o ,but a single, somewhat defective aboral complex of organsarose dorsally—in part over the dorsal wings of the hypo-gastric coeloms, in part over a pseudocoelomic space developedin the dorsal mesentery. Six or seven individuals actuallymetamorphosed and lived for a short time after. In thenormal development of A s t e r i a s asymmetry of the posterior

DEVELOPMENT OF ASTROPECTEN 585

coeloms declares itself, some clays before the appearance of thehydrocoel, by the outgrowth of a ventral horn of the leftposterior coelom and its fusion with the right middle coelom,a process with no counterpart on the right side ; and it isinteresting to find that in these twin hydrocoel larvae the firstsign of duplicity was the failure of this fusion to occur (10,p. 54). Gemmill ascribes this failure, in some cases, to mal-nutrition and a consequent inability of the ventral horn toextend; in other cases—the majority—he supposes it to be dueto excessive nutrition and a consequent enlargement of thestomach, which became a mechanical obstacle to the oxit-growing ventral horn. In either case the right middle eoelom,thus isolated, developed a hydrocoel if subsequent conditionswere favourable. One fact, recorded without comment, hereseems to me to be of capital importance : that the ventralhorn of the left posterior coelom, though it failed to reach itsnormal destination, united with a similar ventral horn of theright posterior coelom ; and apparently this occurred beforethe formation of the hydrocoels. In other words, the symmetryof the coelom was complete before the hydrocoels appeared.It is difficult to see how either excess or defect of food couldaccount for this.

In one respect all the twin hydrocoel larvae of A s t e r i a swere asymmetrical: not one had a right pore-canal or hydro-pore. These organs, however, when developed, usually atrophylong before a hydrocoel appears. It does not follow, therefore,that the two symmetrical variations are independent in origin.That conclusion would be valid only if it were proved that therewas no difference between the incidence of twin pore-canal in theearly stages of normal larvae and its incidence in similar stagesof twin hydrocoel larvae from the same batch of eggs.

Gemmill's double larvae were all developed from artificiallyfertilized eggs ; and among several hundred plankton larvaethat were surveyed not a single twin hydrocoel was found.From all these facts he concludes that laboratory conditionssuch as (a.) hurried maturation, &c, of the eggs, and (b) irregu-larities of larval nutrition, disturb the normal course of develop-

536 H. G. NBWTH

ment; but that such disturbance could not ' supply guidancein the production of double hydrocoel ' and serves only as anopening for the play of those agencies—atavism and homoeosis-which produce the specific effect.

MacBride (17, 18, 19), working with E c h i n u s m i l i a r i s ,claimed to have produced twin hydrocoel experimentally bythe transference of very young Plutei to sea-water of increasedsalinity. The incidence was 2 per cent, and ' at least 5 per cent.'in two ' treated ' cultures, while only one symmetrical indivi-dual was found among hundreds of larvae in the controls.MacBride explained his results by supposing that in thenormal right anterior coelom there resides a ' latent power 'to develop a hydrocoel, and that this was awakened by thestimulus of the hypertonic water. Other symmetricallydeveloped structures were due to the emanation of hormonesfrom the growing hydrocoel. Later, using the same methodsin the same laboratory, Ohshima (24) obtained more abnormallarvae in his controls than in his treated cultures ; but inthis case the commonest abnormalities were enantiomorphs,of which the incidence was more than 10 per cent, in about1,400 larvae examined.

Twin hydrocoel in Echinoids does not differ in essentialfeatures from the same variation in Asteroids : the right sidebecomes in many cases an almost exact, but reversed, copy ofthe left. Many differences occur, however, in the degree towhich the organs of the two sides are developed. A hydrocoelmay be absent altogether ; and in some cases where two werepresent MacBride found pedicellariae (aboral structures of theadult) also developed on both sides—a complication to whichI shall recur. Of twenty cases of twin hydrocoel reviewed byOhshima, two had pore-canal and hydropore on the right sideonly, and nine showed twin pore-canal. The numbers aretoo small to justify a positive inference, but the proportion oftwin pore-canal larvae is so large that correlation betweenthis condition and the presence of two hydrocoels is renderedvery probable.

The three main categories of abnormality just mentioned,

DEVELOPMENT OF ASTROPECTEN 537

(a) twin hydrocoel, (6) enantiomorphs, (c) absence of hydrocoel,are all, according to Ohshima, due to the same initial cause—arrest of development and consequent atrophy of the normalleft hydrocoel. This cause, acting alone, will produce larvaedevoid of hydrocoel (MacBride, 17), but in certain conditions,Ohshima maintains, the right anterior coelom, relieved of thenormal inhibitory presence of a left hydrocoel, may have its ownlatent capacity to form a hydrocoel aroused, and there willresult an enantiomorphic larva—or, if the left hydrocoel itselfrecovers, a twin hydrocoel larva. Prom the rarity of abnor-malities in the sea Ohshima concludes, with Gemmill, thatlaboratory conditions are responsible, accidental occlusion ofthe hydropore (e. g. by food-diatoms) being the main cause ofdegeneration of the hydrocoel. Like Gemmill, too, he relieson Homoeosis for the actual appearance of a right hydrocoel.But whether the two authors use this word in the same senseI am still unable to determine after repeatedly reading theirpapers with great attention ; and I can heartily endorse thecriticism of MacBride (18 and 19) that to invoke such a principleis to substitute a word for an explanation.

What is common to the interpretations of all three of theseworkers is this : apart from their admission of possibly con-tributory, but indefinite, disturbances of early development,they refer duplicity to agencies acting upon normal larvae ata stage just before hydrocoel formation. No attempt, however,has hitherto been made to eliminate conditions of early develop-ment that might be competent to cause duplicity ; and tillthat is done the assumption that the anterior coeloms are inany way normally equipotent cannot be said to rest on experi-mental evidence.

(b) Twin L a r v a e .

The larva as a whole is in some degree affected, as is evidencedby the duplication of axial structures such as the alimentarycanal. Apart from the present paper I know of only one recordof the occurrence of this form of abnormality.

Gemmill (8) found a number of such larvae in a culture ofNO. 275 N n

538 H. G. NEWTH

L u i d i a s a r s i 1 that had passed through their first stagesof development from the early blastula in transit from Plymouthto Glasgow in thermos flasks. Eleven gastrulae and nine youngBipinnariae are described. The gastrulae show various degreesof duplicity of the archenteron, ranging from one in which thatstructure is completely double from end to end, throughV-shaped, A-shaped, Y-shaped, A-shaped forms, to one,finally, in which the blind end of the archenteron is onlyslightly forked. The Bipinnariae correspond in their stage ofdevelopment to the A s t r o p e c t e n larva shown in figs. 10and 11, PI. 41. They exhibit degrees of duplicity and orienta-tions of twinning, similar to those met with in vertebrates, thegut and ciliated bands being taken as criteria. The conditionof the enterocoels is extremely interesting. It can be sum-marized by saying that where the endodermal oesophagus hasbeen doubled there is an attempt to form two pairs of entero-coels, that this has completely succeeded in all except twocases, and that, of the four sacs present in any larva, themorphologically left-hand one has a pore-canal. In the twoexceptional larvae—apparently owing to the exigencies ofspace—three instead of four sacs have been formed, and hereit is the left and the middle sacs which have pore-canals(8, figs. 19 and 21). The remaining double larva (fig. 16)is one in which the endodermal oesophagus is not obviouslyinvolved, though there are two stomodaea. It possesses a singlepair of sacs, apparently normal. In none of the larvae doesa free morphologically right enterocoel possess a pore-canal.

Gemmill attributes the formation of these monsters to theshaking sustained by the culture on its long journey, theconsequent ' early partial separation of cells or of cell masses 'having caused more or less doubling of the area of invagination.He definitely assimilates the appearances presented by theBipinnariae to those found in the twin monsters of Vertebrateteratology, which are due to the appearance of ' two foci ofembryo formation ' ; but he states, just as definitely, that

1 Gemmill speaks of L, s a r s i , but identifies it with the species bredby Mortensen (20), L. e i l i a r i s , which is much commoner at Plymouth.

DEVELOPMENT OF ASTROPECTEN 539

they are not comparable to ' double E c h i n u s rudiments . . .described in detail by MacBride '.

Ohshima (23 and 24) has cast doubt on this interpretation,and suggested that f u s i o n of individuals has occurred. Two orthree of the gastrulae and one of the Bipinnariae certainly lookin the figures as if they might have been formed in this way ;but as to the remaining sixteen or seventeen larvae, it is scarcelycredible that random approximation should have led to unionin pairs only, and moreover that this union should have beenof such a sort that the anterior and posterior ends of theconjugants always fused with their like.

The similarity of the L u i d i a twins to those of A s t r o -pec t e n , described on p. 527 of this paper, is obvious. Thefollowing facts may have a bearing on their occurrence. Thefamilies to which the two species belong are considered to benearly related ; the eggs of the two species are very similarin size, and somewhat large for animals with a pelagic larva( A s t r o p e c t e n , d = 021 mm.; L u i d i a , 1 d = 0-215-022 mm., i. e. they are about twice the volume of the egg ofA s t e r i a s r u b e n s , d = 0-16-019 mm.) ; both species passthrough a wrinkled blastula stage, the irregularity of whichmay facilitate—if not cause—irregularities of gastrulation.

(c) A s y m m e t r y of t h e N o r m a l L a r v a .

I have already reviewed several of the more striking andexceptional cases in which bilateral symmetry of coelomicorgans has been alleged to occur in normal development;and I have suggested that the evidence for such symmetryis insufficient. The evidence from more ordinary ontogenieswill be briefly examined.

In those Asteroids, Opbiuroids, and Echinoids which havea pelagic larva, four coelomic sacs are formed by the roughlysymmetrical growth backwards of two horns of a primaryvesicle, arising from the tip of the archenteron; by the separa-tion of these horns from their point of origin; and by their

1 Unpublished information for which I am indebted to ProfessorGemmilFs kindness.

540 H. G. NEWTH

subsequent division into two on either side. In those membersof the same groups which have more yolky eggs the processis often different, and may be manifestly asymmetrical.In the only other group in which a typical pelagic larva occurs—the Holothurioids—the process is always asymmetrical:the primary vesicle does not divide right and left, but eithermoves bodily to the left side or originally appears in thatposition, where it gives rise to the hydrocoel, after segmentingoff the single rudiment of the posterior coeloms. The view thatthe more symmetrical of these processes are also more funda-mental is based on a series of phylogenetic assumptions whichcannot be tested by existing methods of research, and whichtherefore need not be retailed here. There is, on the otherhand, some objective evidence for believing that even in themore symmetrical larvae the coelom is from the first in realityasymmetrical.

MacBride (14) early drew attention to a disparit}- in the rateof development of the two sides, at a time preceding theformation of the hydrocoel, in As te r in a, E c h i n u s , andO p h i o t h r i x , and stated that it foreshadowed ' that pre-dominance of the left side which plays such an important partin the metamorphosis '. I have already referred to the earlyasymmetry of the posterior coeloms of A s t e r i a s (p. 53o).As the result of experimental work, Eunnstrom (29) cameto the conclusion that asymmetry was determined much earlier,and observation of normal development bears this out. Inthe present paper I have stated that the left enterocoel ofA s t r o p e c t e n is initially larger than tho right ; Gemmillsays that in P o r a n i a (9) ' as a rule', in A s t e r i a s (7)' not infrequently at first', the left sac is slightly the larger ;and an examination of the figures of early stages given byvarious authors makes it probable that this is universal. Eunn-strom, indeed, claims in another paper (27) that the gastrulaof P a r e c h i n u s m.il iaris (Ech inus mi l i a r i s ) alreadyshows asymmetry of the anterior end of the archenteron ata stage when the coelom can hardly be said to be indicated :' Der vorderste Teil des Urdarms ist asymmetrisch gebaut.'

DEVELOPMENT OF ASTROPECTEN 541

When it is borne in mind that these early stages have generallybeen passed over with scant attention, such observationsacquire great significance.

To say that the asymmetry of a later stage is ' foreshadowed 'is surely to admit in metaphorical language that the organsupon which that asymmetry depends are already determined:that the dispositions which normally lead to their appearanceare present on one side and absent on the other. If it does notmean this, what does it mean ? If it does, then it is idle tospeak of characters of the left side being normally ' latent'on the right. For it is upon just such small adumbrations ashave been mentioned that—in the absence of experimentalanalysis—we usually rely for information on the processes ofdevelopment.

(d) An H y p o t h e s i s to Accoun t for L a r v a lD u p l i c i t y .

If the facts concerning abnormal Echinoderm larvae thathave now been reviewed are considered without phylogeneticpreconceptions, it will be almost impossible to avoid the con-clusion that they are of the same kind as the facts of vertebrateteratology. This similarity is, naturally, more striking in thecase of the twin Bipinnariae described by Gemmill and myself,where the duplicity involves chiefly larval organs which arebilaterally symmetrical; but the parallel is almost as completein cases of twin hydrocoel. If, for the sake of comparison, weregard the larval organs as simply a trophic appendage to thegrowing urchin or starfish, it is apparent at once that in normaldevelopment the rudiment of the adult bears much the sanierelation to the larva as—for instance—the embryo of a birdbears to its yolk-mass and embryonic membranes. The analogymight easily be elaborated. I need only point out here thatin both cases the system as a whole is asymmetrical with refer-ence to its original median plane, and that in both cases thedeveloping adult possesses its own asymmetry of organization.

Let us now consider a fowl embryo of the third day exhibitingthe kind of twinning known as sternopagus—that is to say,

542 H. G. NEWTH

an H-shaped double monster in which the anterior and posteriorends of each of the twins are free, and there is union in thethoracic region, with the two hearts—or a common heart—inthe isthmus (cf. Eabaud, 25). Abnormalities of this kind areby no means rare among vertebrates. The left-hand twinis normal in its relations, while the right-hand twin is enantio-morphic—its head being twisted so as to lie with the rightside instead of the left turned towards the yolk, and its heartshowing s i t u s i n v e r s u s . It will be seen that the systemas a whole is here symmetrical about a plane, the asymmetryof the one twin being balanced by the reversed asymmetry ofthe other. This is precisely the state of affairs in the twinhydrocoel larvae of Echinoderms.

Now there is good reason for believing that, in Vertebrates,such forms of duplicity as I have mentioned are due to theearlier partial fission of a region of active growth and differen-tiation—the edge of the blastopore or the primitive streak.We know also that mechanical and other interference with thesegmenting egg, or physiological inhibition of the closing blasto-pore, can cause a doubling of the ' foci of embryo formation '.Is there in Echinoderm development a comparable growth-centre to which subsequent duplicity of adult organs can bereferred ?

As regards the coelomic organs the answer, I think, must bethat there is such a region in the blind end of the archenteronat the time of the formation of the coelornic rudiment, and itis mainly in terms of the doubling of this region that I shallnow attempt to bring into line the several kinds of observedduplicity.

I. If my previous criticism has been just, Ave may say thatin the normal larva the characters of the enterocoels arealready determined at their first appearance ; and, further,we may assume that in the single vesicle from which theyspring, or in the tip of the archenteron, there occurs a segrega-tion of their potentialities right and left. Such a suppositionis quite in accord with what is believed to take place in otherembryonic processes. The undivided primary vesicle can be

DEVELOPMENT OF ASTROPECTEN 543

described, then, as b i v a l e n t with regard to the potentialcharacters of the right and left enterocoels (Text-fig. 2, A).

II. The presence of two or more less widely separated areasof invagination, during gastrulation, will lead to various degreesof duplicity of the archenteron (if Gemmill's interpretation iscorrect, the twin gastrulae observed by him are actual illustra-tions of this process). The extreme case is one where there aretwo complete archentera side by side as the result of widelyseparated invaginating areas ; and successive approximationsof these areas will lead to diminishing degrees of bifurcationof the anterior end of a single archenteron. But this bifurca-tion, whatever its degree, must necessarily involve the coelomicrudiment.

(1) Anterior doubling of the archenteron sufficient to affectthe fore end of the gut will at the same time lead to the forma-tion either of two completely separate primary vesicles, or oftwo which are in contact or joined side to side. Each will bebivalent, and each will attempt to produce two enterocoels—an attempt that would be expected to succeed in proportionto the divarication of the limbs of the forked gut:

(a) Complete success will result in the formation by thelarva of four enterocoels—two pairs of normal antimeres.

(b) Partial success will lead to the formation of three entero-coels, two lateral and one median, the latter equivalent to theright enterocoel of the left-hand pair p lu s the left enterocoelof the right-hand pair. This median sac will be bivalent,and will possess the capacity of forming a pore-canal.

These relations are exhibited in Text-fig. 2, rows C and D.The conditions there portrayed—excepting the middle termof each,sequence—are not suppositional, but are those presentin actual larvae described by Gemmill (see p. 538). Now, thetwin Bipinnaria of A s t r o p e c t e n (figs. 10 and 11, PI. 41)shows, besides anterior duplicity, a certain degree of back-to-back twinning, which is revealed by the facing outwards of thestomodaea and the appearance between them of part of thepostoral ciliated band. In other words, the dorsal region isnarrowed ; and this, on the assumptions made, will have the

544 H. G. NEWTH

same effect on the twin primary vesicles as that of a lesserdegree of bifurcation of the archenteron, i.e. the medianenterocoel will be suppressed, and the two sacs formed will be

TEXT-FIO. 2.

Diagram illustrating the relations of the coelom and gut in normaland double Echinoderm larvae. Larvae shown in dorsal aspect;stippling indicates normal potentialities of left enterocoel;vertical rows are individual sequences; horizontal rows, equivalentstages. A, Normal L a r v a e : segregation of potentialitiesundisturbed, single hydro pore. B, S y m m e t r i c a l L a r v a e :segregation anticipated, precocious division of coelomic rudiment,two hydropores. C and D, Twin L a r v a e . C, anteriorduplicity; archenteron forked; larval gut affected; threeenterocoels—middle one bivalent, with pore. D, parallel duplicity,archenteron and all derived organs completely duplicated. (Let-tering as in Pis. 40 and 41.)

bivalent as in the next case to be considered. The A s t r o -pec t e n larva is, thus, one term in a series of forms showingdiminishing duplicity of the alimentary canal and ciliated

DEVELOPMENT OF ASTEOPECTEN 545

bands, and at the same time—quite consistently—of thecoelom also. A final term in this series remains to be discussed.

(2) Anterior doubling of the archenteron so slight as not toreach the oesophageal region will affect the coelomic rudimentonly ; and, on analogy with the appearances in twin L u i d i aand A s t r o p e c t e n larvae, we may expect the external formof the larva not to show—at first—any duplicity at all. Thedoubling, in fact, may now be supposed to constitute, orproduce, the precocious division of an apparently singleprimary coelomic vesicle whose lateral halves become theenterocoels, as in a normal larva. Each enterocoel, however,will be bivalent and capable of forming a pore-canal and, insuitable conditions, a hydrocoel (Text-fig. 2, B). The particularmanifestations of this degree of duplicity will depend uponlater conditions. The pore-canal is an organ formed soonafter the separation of the enterocoels, and since it is of smallsize its appearance is independent of the food-supply—in factit is formed even by unfed larvae. A right pore-canal wouldthus be the first structural indication of duplicity in an out-wardly normal larva. The hydrocoel, on the other hand, hasrightly been called by MacBride an ' expensive' structureformed after the initial impetus of development has beenexhausted. The appearance of even one hydrocoel is dependenton abundant food ; the development of two requires a super-abundance. This is a partial explanation of the greaterfrequency of twin pore-canal.

Duplicity of the archenteron has been assumed throughoutto be traceable to an early stage of invagination, and we maytherefore describe the duplicity of the coelomic organs, &c.which arises in this way as being o b l i g a t o r y .

If, however, in the above scheme I have dealt with duplicityparticularly in terms of the archenteron and the coelom, ithas been for the sake of simplicity of exposition, and withoutany intention to claim autonomy for these parts. In the caseof twin larvae the external structure is no less affected thanthe internal; and there are indications that this is true oftwin hydrocoel larvae. It was urged by Grave (IS), as an argu-

546 H. G. NEWTH

ment against invoking atavism to account for a right hydrocoel,that a right amniotic invagination was not possessed by theancestor, but nevertheless occurred in symmetrical larvae.MacBride explained its presence by supposing that a stimulusfrom the underlying hydrocoel activated the ectoderm. Butthe same author has homologized the amnion of Echinoidswith a part of the larval stomodaeum delayed in its appear-ance—a view the correctness of which has since been madealmost certain by Mortensen's observations on P e r on el la(21). If it is indeed correct, the appearance of an amnion onthe right side of a twin hydrocoel larva is not remarkable,and no causes more recondite than those here suggested seemto be called for to explain it. Both right hydrocoel and rightamnion can be referred to the twinning of median structures—archenteron and stomodaeum—of which they are respectivelythe derivatives.

(e) F a c t o r s in Growth .

Hitherto I have designedly spoken of the ' potentialities' oforgan-rudiments because the term is non-committal with regardto the nature of the processes of growth that are involved.Both the facts and their attempted explanation may, however,be expressed in terms of the metabolic gradients that are—some of them certainly, others by inference—concerned indevelopment, without any modification of the fundamentalconception set forth.

Child (2) demonstrated the presence of a well-marked axialsusceptibility gradient in the unfertilized eggs, cleavage stages,blastulae, gastrulae, and young Bipinnariae of A s t e r i a sf o r b e s i i . The gradient axis coincided in all cases with themorphological axis, the apical region being at the animal poleof the egg, and at the anterior end of the gastrula and youngBipinnaria. Unfortunately Child was unable to obtain laterlarvae, but it is extremely significant to find that this initialgradient in the Bipinnaria became less and less distinct asdevelopment proceeded, and—at least as regards the ectoderm—that it was finally reversed in a certain percentage of larvae,

DEVELOPMENT OP ASTROPEOTEN 547

the posterior part of the body showing a slightly higher sus-ceptibility. An outline sketch of a larva in this stage showsa young Bipinnaria in which the enterocoels and pore-canal—though they are not figured—would be expected to bewell established.

Throughout gastrulation there is a centre of growth anddifferentiation at the tip of the archenteron, which gives riseultimately to the coelom. Soon after this the essentially larvalcharacters are nearly all present as rudiments, the animalbegins to feed, and the susceptibility gradient disappears.Henceforward the new growth is, in the main, associated withthe extension of the coelom backwards along the gut on eitherside; and I suggest that the appearance of the reversed gradientnoted by Child is a manifestation of the fact that the advancingtip of the enterocoel is a metabolic apex. It is at, or near to,this apical region that the pore-canal is formed ; later thewhole apical region is cut off as the posterior coelom on eitherside ; whereupon, on the left side, the physiological isolationthus conferred upon the anterior coelom enables it to form anew apical organ—the hydrocoel. Eunnstrbm (28) concludesfrom his experiments on E c h i n u s that the difference betweenthe two asymmetrical sides of the larva is quantitative, andconsists in a difference of metabolic rate which appears earlyin development. Indeed, accepting the evidence of normal,left-sided development alone, we should suppose that from thefirst separation of the enterocoels the gradient on the leftside was more strongly marked than that on the right; or,in other words, that there was a secondary gradient from left toright across the larval body. This gradient—ultimately theaxial gradient of the adult, with its dominant region on theoral surface—probably originates in the undifferentiatedprimary coelomic vesicle, and what I have called a segregationof potentialities is here, as in other cases, the establishment ofa metabolic axis. If this is the case we have a possible secondarycause of duplicity suggested to us. In seedling plants theremoval or inhibition of a growing tip will, in some cases, causethe formation of paired axillary shoots. If these appear

548 H. G. NBWTH

simultaneously and grow initially at the same rate they mayboth persist, but if one appears earlier or grows more rapidlyit inhibits the growth of the other (cf. Child, 3, p. 152). Thesimilarity of these relations to those between the two hydrocoelsof double Echinoid larvae (Ohshima, 24) is very striking, andpoints to an essential identity of the growth-processes concerned.

In the next section I shall attempt to show that there are,in fact, conditions in laboratory cultures which may mechani-cally damage or physiologically inhibit the tip of the archen-teron, and so produce in a previously normal larva what maybe called Facultative Duplicity in contrast with the ObligatoryDuplicity already noticed. It is only necessary to add here that,given the presence of either of these conditions in the earlylarva, the relations of dominance and subordination betweenregions of higher and lower metabolic rate will account formany, if not all, of the discrepant facts mentioned in theearlier part of this discussion. Among these are the transitorynature of the right pore-canal in Asteroids, the production ofenantiomorphs in Echinoids (dominance of the right-handmember of a pair of bivalent enterocoels), the development ofpedicellariae on both sides of twin hydrocoel Echinoplutei, andfinally the occurrence of an enantiomorphic Anrieularia—whichotherwise seems quite inexplicable (Ohshima, 24).

(/) Cond i t i ons of E a r l y D e v e l o p m e n t .The unnatural conditions present in ordinary laboratory

cultures have not been left out of account by other authors,but their importance has, I think, been greatly under-estimated.To obtain eggs for fertilization it is often necessary to detachthem more or less forcibly from the ovary—by ' shredding out '(Geinmill), shaking the ovary in a bag of bolting-silk in sea-water, or some such process. Thus obtained, the eggs aresometimes mature, and presumably ready for fertilization,or they may mature after standing for an hour or two, duringwhich time they are exposed to influences that may affecttheir polarity in a manner to which I shall refer below. Apartfrom the chance of gross mechanical injury there is thus a

DEVELOPMENT OF ASTROPECTEN 549

possibility that, with reference to the establishment of theaxial gradient, eggs may be matured and fertilized pre-cociously.

The effect of raised temperature upon development has, ofcourse, been studied in detail in a number of animals, butgenerally as regards greater departures from the normal thanthose occurring in carefully conducted fertilization experi-ments. Heat has a marked effect in producing abnormalitiesof segmentation and gastrulation (including duplicity) in bothvertebrates and invertebrates, and it must be remembered thateven in the favourable conditions of the Plymouth Laboratoryit is impossible to maintain cultures at a temperature as lowby several degrees as that of the sea. Des Arts in the case ofC u c u m a r i a f rondosa showed that small increases intemperature can seriously interfere with the normal develop-ment of that animal.

The conditions of the eggs during cleavage are exceedinglyunnatural, and are such, moreover, as may easily entail drasticinternal changes. It is safe to say that in the majority ofEchinoderms, in which the eggs are shed freely in the sea andremain unattached, they are subjected in a state of nature tono external influence that could be d i r e c t i v e . Since theyare in suspension they are equally oxygenated on all sides,and their orientation with reference to gravity is either con-stantly changing, or can attain its optimum in the case of eggswith more marked polarity. Now, in artificial cultures theeggs, after a few initial changes of water, lie upon a glasssurface undisturbed throughout their cleavage—in many casesclosely packed side by side, even when in a single layer. It iswell known how the egg of the frog is affected in its develop-ment by inversion and the consequent redistribution of yolkin the two-cell stage ; and although the polarity of the oligo-lecithal Echinoderm egg is generally so little marked that itwill lie in any position in which it happens to fall, we are notjustified in assuming that, in the unnatural stillness of a culture-bowl, gravity can have no effect. These conditions of immo-bility will also modifjr respiration. The oxygen concentration

550 H. G. NEWTH

of the water immediately adjacent to the eggs will be quicklyreduced. Oxygen reaches such eggs by diffusion downwardsfrom the surface of the water, possibly also by feeble convec-tion currents ; in either case oxygenation will be greater onthat side of an egg which happens to be uppermost. Similarly,the waste products of metabolism, escaping upwards only,will be in greatest concentration nearest the glass surface.Since respiration is active, there will thus be imposed on an eggduring cleavage, if not previously during maturation, a gradientin oxygen consumption that may, or may not, coincide withits axis. It is at least conceivable that some modification ofthis original axis, or the permanent establishment of a sub-sidiary axis, may be the result.

If it be objected that in spite of these conditions the vastmajority of the larvae of certain species show no duplicity atall, I can only say that, for the production of a viable doublemonster in this way, optimum direction and intensity of themodifying influence would be necessary, and only a small per-centage of eggs could be expected to find this optimum bychance orientation.

"When cleavage is completed, and the embryos escape from theirmembranes, there are still external influences that may well beteratogenetic. There are, in crowded cultures, unwontedimpacts of the blastulae upon one another and upon the wallsof their aquarium, which may cause displacement of cells orgive an unnatural stimulus to invagination ; and decantationfrom bowl to bowl is probably a more violent shock to earlylarvae than any they would normally sustain in the sea, involv-ing often brusque changes of temperature, alkalinity, and evensalinity. If the view that I have put forward is correct, thereis, however, one period during which the young larva mustbe peculiarly susceptable to such external influences—the periodat which the coelom appears. MacBride, in his paper on theexperimental production of twin hydrocoel in E c h i n u s ,attributes the observed duplicity to the transference of thePlutei to sea-water of increased salinity at a particular, criticalstage of their development. It is most significant to find that

DEVELOPMENT OP ASTROPECTEN 551

he expressly describes this stage as t h a t at which s epa ra -t ion of t he en t e rocoe l s o c c u r s . Not only is thisapproximately the stage at which I have supposed a segregationof potentialities to take place, but the agent employed—hyper-tonic sea-water—is one that, by causing a slight shrinkage ofthe larva as a whole, might be expected to produce partialcollapse or other deformation of the thin-walled primarycoelomic rudiment, such as occurs in the case of the third-daylarva of A s t r o p e c t e n under the action of fixatives. It istrue that, in the larva described by MacBride as typical of hiscultures at the time of transference, the enterocoels are justlosing their communication with the gut; but it must beremembered that in any culture there is considerable variationin the rate of development of individuals, and that only asmall percentage of double larvae was obtained. In view of thevery different results obtained by Ohshima, who used the samemethod, great importance must not perhaps be attached tothe use of hypertonic water. Nevertheless, in all Ohshima'sexperiments the cultures—including controls—were transferredfrom finger-bowls to Breffitt jars when they contained ' one-day-old larvae with pyramidal body and a pair of rudimentarypostoral arms ' (Ohshima, 24). Now this is a stage at whichthe coelomic rudiment is still in connexion with the gut, andis even nearer to what, on my assumptions, would be anoptimum moment for inhibition than that at which MacBride'shypertonic water began to act. It will be recalled that Ohshimaobtained many more abnormalities in his cultures than Mac-Bride.

SUMMARY.

I. (1) The normal development of A s t r o p e c t e n i r r egu -lar i s is described up to the twenty-fifth day.

(2) About a third of the larvae possessed two pore-canals,and larval twinning was observed in two cases.

II. There is insufficient evidence for believing that normalEchinoderm larvae possess a ' latent' bilateral symmetry.

552 H. G. NEWTH

III. The following provisional conclusions are reached regard-

ing duplicity in Echinoderm larvae :

(1) The various kinds of duplicity form a series.

(2) They are of the same nature as those found in vertebrate

embryos, and are probably due to similar causes.

(3) They may be determined by

(o) Alteration of the polarity of the egg;

(b) Interference with processes of early development affect-

ing gastrulation;(c) Physiological inhibition or mechanical deformation of

the tip of the archenteron.

(4) Their ultimate facies, in the case of (c), is determined

largely by excess or defect of nutrition.

LITERATURE REFERENCES.

(Further references may be found in the papers of Gemmill (7) andOhshima (24).)

1. Assheton, R. (1910).—" The geometrical relation of the Nuclei in anInvaginating Gastrula (e.g. Amphioxus) considered in connexionwith Cell Rhythm and Driesch's conception of Entelechy ", ' Arch. f.Entw.-Mech.', vol. 29. Leipzig.

2. Child, C. M. (1915).—" Axial gradients in the early development of theStarfish ", ' Ainer. Journ. Physiol.', vol. 37, p. 203.

3. (1915).—' Individuality in Organisms'. Chicago: the Universityof Chicago Press.

4. Delap, M. andC. (1906).—'Notes on the Plankton of Valencia Harbour.'Fisheries, Ireland, Sci. Invest. 1905, VII.

5. Field, G. W. (1892).—"The larva of Asterias vulgaris", 'Quart.Journ. Micr. Sci.', vol. 34. London.

6. Gommill, J. F. (1912).—" The Development of the Starfish Solasterendeca, Forbes " , ' Trans. Zool. Soc '. London.

7. (1914).—" The Development and Certain Points in the AdultStructure of the Starfish, Asterias rubens, L.", ' Phil. Trans. Roy.Soc.', B, vol. 205. London.

8. (1915).—" Twin Gastrulae and Bipinnariae of Luidia sarsi,Diiben and Koren ", ' Journ. Mar. Biol. Assoc.', vol. 10, no. 4.Plymouth.

9. (1916).—" The Larva of the Starfish Porania pulvillus (O.F.M.) ",' Quart. Journ. Micr. Sci.', vol. 61. London.

•DEVELOPMENT OF ASTROPECTEN 553

10. Gemmill, J. E. (1916).—" Double Hydrocoel in the Development andMetamorphosis of the Larva of Asterias rubens " , ibid. London.

11. Grave, C. (1902).—" Some Points in the Structure and Developmentof Mellita testudinata " , ' Johns Hopkins Univ. Circ.', v. 21.

12. (1911).—" Metamerism of the Echinoid Pluteus ", ibid., no. 231.13. MacBride, E. W. (1903).—" The Development of Echinus esculentus

together with Some Points in the Development of E. miliaris andE. aeutus ", ' Phil. Trans. Roy. Soc.', B, vol. 195. London.

14. (1907).—"The Development of Ophiothrix fragilis " , 'Quart.Journ. Micr. Sci.', vol. 51, pt. ii. London.

15. (1911).—"Two Abnormal Plutei of Echinus, and the lightwhich they throw on the Factors in the normal development ofEchinus " , ibid., vol. 57, pt. ii. London.

16. (1914).—' Text Book of Embryology '. London.17. (1918).—" The Artificial Production of Echinoderm Larvae with

Two Water-Vascular Systems, and also of Larvae Devoid of aWater-Vascular System ", ' Proc. Roy. Soc.', B, vol. 90. London.

18. (1921).—Note appended to Professor Ohshima's paper (23).Ibid., vol. 92, p. 175. London.

19. (1922).—Note appended to Professor Ohshima's paper (24).' Quart. Journ. Micr. Sci.', vol. 66, p. 149. London.

20. Mortensen, Th. (1913).—" On the Development of some BritishEchinoderms " , ' Journ. Mar. Biol. Assoc.', vol. 10, pt. i. Plymouth.

21. (1921).—'Studies of the Development and Larval Forms ofEchinoderms '. Copenhagen.

22. Newth, H. G. (1916).—" The Early Development of Cucumaria:Preliminary Account", ' Proc. Zool. Soc.', 1916. London.

23. Ohshima, H. (1921).—" Reversal of Asymmetry in the Plutei ofEchinus miliaris " , ' Proc. Roy. Soc.', B, vol. 92. London.

24. (1922).—" The occurrence of Situs inversus among artificially-reared Echinoid Larvae", ' Quart. Journ. Micr. Sci.', vol. 66.London.

25. Rabaud, E. (1914).—" La Teratogenese", ' Encyclope'die Scienti-fique '. Paris.

26. Rhumbler, L. (1902).—" Zur Mechanik des Gastrulationsvorganges,inbesondere der Invagination ", ' Arch. f. Entw.-Mech.', vol. 14,pts. iii and iv. Leipzig.

27. Runnstrom, J. (1917-18).—" Zur Entwicklungsmechanik der Larvevon Parechinus miliaris " , ' Bergens Mus. Aarb.' Bergen.

28. (1918).—"Analytische Studien iiber die Seeigelentwicklung " ,IV. Mitteilung, ' Arch. f. Entw.-Mech.', vol. 43, pt. iv. Leipzig.

29. (1918).—Ditto. V. Mitteilung, ibid.30. (1920).—" Entwicklungsmechanischo Studien an Henricia san-

guinolenta Forbes und Solaster spec", ibid., vol. 46, pts. ii and iii.Leipzig.

NO. 275 o O

554 H. G. NEWTH

EXPLANATION OF PLATES 40 AND 41.

LETTERING EMPLOYED.

u.d., anterodorsal process ; an., anus ; or., archenteron ; c.b., circum-oral ciliated band; Co./., circumoral field; ex., evaginating cells;

f.a., frontal area; f.m., fertilization membrane ; H., hydropore ; Hr.,right hydropore ; IN., intestine; L.G., left enterocoel; m.l., medianloop of the postoral band; m.p.p., median preoral process ; Oes., oeso-phagus ; P.C., primary coelomic ve3icle ; P.O., postoral ciliated band ;S.G., right enterocoel; S., stomach ; St., stomodaeum ; su., cavity ofsulcus.

PLATE 40.

All drawings were made with the camera lucida.Fig. 1.—Blastula of A s t r o p e c t e n , fifteen hours old. Section showing

evagination in progress. The cells in the blastocoel are parts of the blastula-wall. Magnification 330—in figures. Drawn under Zeiss 3 mm. apochr.,Leitz comp. oe. 6.

Fig. 2.—Longitudinal section of early gastrula, showing both invagina-tion and evagination in progress. Magnification, &c, as in fig. 1.

Figs. 3-6.—Third-day larvae all taken from the same preserved sampleand illustrating early stages in the development of Bipinnaria. Drawnfrom stained and cleared specimens under Leitz 8 mm. apochr., comp. oc. 6.Magnification 136—in figures. Ectoderm shown in optical section (black),its thickness accurately indicated.

Fig. 3.—Completed gastrula. Primary vesicle formed.Fig. 4.—Young post-gastrula stage, seen from the left side. Wings

of the primary vesicle have grown out right and left, the left being thelarger. The endodermal oesophagus touches the stomodaeum.

Fig. 5.—Post-gastrula stage slightly later than that shown in fig. 4 andseen from the dorsal surface. Oesophagus constricted off from stomach ;hydropore just appearing and enterocoels losing connexion with gut;ciliated band faintly indicated above stomodaeum.

Fig. 6.—Abnormal post-gastrula at the same stage as the normal larvashown in fig. 4. Ventral aspect. For description see text, p. 527.

PLATE 41.

Figs. 7-11 are all camera lucida drawings made from animals still in70 per cent, alcohol. Magnification 100—in figures. Leitz obj. 3, oc. 15.

Fig. 7.—Bipinnaria nearly six days old ; ventral aspect.Fig. 8.—Bipinnaria twenty-four days old ; ventral aspect.Fig. 9.—Bipinnaria twenty-four days old ; ventro-lateral aspect. This

is not the same individual as that shown in fig. 8, though almost identicalin size and shape.

Fig. 10.—Abnormal Bipinnaria, eight days old ; right dorso-lateralaspect. For description see text, p. 527.

Fig. 11.—The larva shown in fig. 10 ; ventral aspect.

Quart. Journ. Micr. Sci. Vol. 69, N. S., PI 40

FIG. 5 FIG. 6

Newtk, del.

Quart. Journ. Mkr. Sci Vol. 69, N. S., PL 41

FIG. II