Morphology, Behavior, and

Histogenesis of the Pelagosphera

Larva of Phascolosoma agassizii

(Sipuncula)

MARY E. RICE

m

SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY • NUMBER 132
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SMITHSONIA N CONTRIBUTION S T O ZOOLOG Y • NUMBE R 13 2

Morphology, Behavior, and

Histogenesis of the Pelagosphera

Larva of Phascolosoma agassizii

(Sipuncula)

Mary E. Rice

SMITHSONIAN INSTITUTION PRESS

City of Washington

1973
ABSTRACT

Rice, Mary E. Morphology, Behavior, and Histogenesis of the Pelagosphera

Larva of Phascolosoma agassizii (Sipuncula). Smithsonian Contributions to

Zoology, number 132, 51 pages, 14 plates, 1973.—Morphology, behavior, and

histology are described for the pelagosphera larva of Phascolosoma agassizii

Keferstein. Observations are reported on histology, origin, and development of

the following larval structures: body wall, coelom and coelomic elements, retractor muscles, digestive tract, buccal organ, lip gland, nervous system, nephridia,

larval glands and terminal organ. Functional significance of certain structures,

particularly organs associated with the mouth, is considered. Moreover, available

information on developmental histology of other sipunculans is reviewed and

intra- as well as inter-phyletic comparisons are discussed. An account is given

of the general morphology of the larva and a few observations are noted on

larval locomotion and feeding behavior in the laboratory.

Official publication date is handstamped in a limited number of initial copies

and is recorded in the Institution's annual report, Smithsonian Year.

SERIES COVER DESIGN: The coral Montastrea cavernosa (Linnaeus) .

Library of Congress Cataloging in Publication Data

Rice, Mary E.

Morphology, behavior, and histogenesis of the pelagosphera larva of Phascolosoma agassizii

(Sipuncula).

(Smithsonian contributions to zoology, no. 132).

Bibliography: p.

1. Phascolosoma agassizii. 2. Larvae—Worms. I. Title. II. Series: Smithsonian Institution.

Smithsonian contributions to zoology, no. 132.

QL1.S54 no. 132 [QL391.G5] 591'.O8s [595M7] 72-8959

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C., 20402 - Price $1.25 (paper cover)
Contents

Page

Introduction 1

Acknowledgments 2

Materials and Methods 2

Review of Early Development 3

Morphology and Behavior of the Larva 3

Larval Histogenesis 5

Body Wall . 5

Coelom and Coelomic Elements 7

Retractor Muscles 7

Digestive Tract 9

Mouth and Associated Structures . 11

Nervous System 14

Nephridia 16

Larval Glands 17

Terminal Organ . 20

Comparisons with Other Phyla 21

Summary 22

Literature Cited 22

in

Morphology, Behavior, and

Histogenesis of the Pelagosphera

Larva of Phascolosoma agassizii

(Sipuncula)

Mary E. Rice

Introduction

As reported in an earlier paper (Rice, 1967), the

development of Phascolosoma agassizii Keferstein is

indirect with a pelagic, lecithotrophic trochophore

followed by a prolonged pelagic, planktotrophic

pelagosphera stage.1 The trochophore is characterized by an apical tuft, a prominent equatorial band

of ciliated prototroch cells, mesodermal bands on

either side of a closed gut and a stomodaeum closed

to the exterior by an overlying egg envelope. Metamorphosis of the trochophore results in a second

larval form, designated as a pelagosphera, in which

the metatrochal ciliary band has replaced the prototroch as the chief locomotory organ, the mouth

and anus have opened to complete the gut, the coelom has expanded into a spacious cavity and a

terminal attachment organ has formed. In this

paper observations are reported on the morphology

and behavior of the pelagosphera larva of Phascolosoma agassizii, bred from known adults and

reared in the laboratory. The histology and deMary E. Rice, Department of Invertebrate Zoology, National

Museum of Natural History, Smithsonian Institution,

Washington, D.C. 20560.

1A pelagosphera has been defined as a sipunculan larva,

resulting from the metamorphosis of a trochophore, which

swims by means of a prominent ciliated metatroch and in

which the prototroch either has been lost or has undergone

a marked reduction (Rice, 1967:164).

velopment of the major organs and tissues of the

larva are described, and intra- and interphyletic

comparisons are discussed.

Sipunculan larvae were recognized as early as

1850 when Max Mueller, working at Trieste, described and illustrated a larva which he presumed

to belong to the genus Phascolosoma. A year later,

in 1851, A. Krohn criticized this work, asserting that

the larva depicted was not that of Phascolosoma,

but rather belonged to Sipunculus nudus. In 1883,

Hatschek published a treatise on the development

of Sipunculus n.udus, describing in detail early development as well as the morphology and metamorphosis of the larval stages. His material was

collected in the plankton at Messina, Sicily. Interest in larvae from oceanic plankton was elicited by

Mingazinni's (1905) description of a form which

he erroneously identified as an adult, creating a

new genus and species, Pelagosphaera aloysii. The

description was made from only one preserved and

contracted specimen which had been collected in

the Pacific between New Zealand and New Caledonia at a depth of approximately 500 meters by

the planktonic expedition of the Italian Naval ship,

Liguria. Working with material from the same expedition, but from waters off India and Ceylon,

Senna, in 1906, described similar forms. He concluded that they were larval forms and thus corrected Mingazinni's interpretation. Spengel (1907),

in an independent investigation, also exposed

1
SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

Mingazinni's error. Comparing the larvae with

those of Sipunculus nudus in the Bay of Naples,

he suggested that the structures which Mingazinni

claimed to be gonads might be instead glandular

appendages of the esophagus. Thus Mingazinni's

mistake was soon realized, but the name that he

created, pelagosphera, remains well entrenched in

the literature and is today used to designate the

larval form of sipunculans which succeeds the stage

of trochophore (Rice, 1967).

Other reports of planktonic larvae have been

made by Heath (1910), Dawydoff (1930), Stephen

(1941), Fisher (1947), Akesson (1961a), Damas

(1962), Jagersten (1963), Murina (1965), Scheltema

and Hall (1965), and Hall and Scheltema (1966).

Only two of these authors have attempted to assign

adult specific affinities to the planktonic larvae.

Fisher (1947) identified a larva collected off Cape

Hatteras as Sipunculus polymyotus on the basis of

the number of longitudinal muscle bands. Murina

(1965) designated larvae from the Gulf of Aden

as Sipunculus aequabliblis and from the Northwest

Pacific as Sipunculus norvegicus. Most of the earlier reports were based on preserved and contracted

specimens and it is only in some of the more recent

studies on living planktonic larvae (Jagersten,

1963; Murina, 1965; Hall and Scheltema, 1966)

that larval morphology, particularly of the head

region, has been correctly interpreted.

Four developmental patterns have been recognized within the phylum Sipuncula, only one of

which includes the long-lived planktotrophic pelagosphera larvae of oceanic waters (Rice, 1967). The

four categories of development are defined as follows: (1) direct development with no pelagic stages,

(2) indirect development with one pelagic larval

stage, the lecithotrophic trochophore, (3) indirect

development with two pelagic larval stages, a lecithotrophic trochophore and a short-lived lecithotrophic pelagosphera, (4) indirect development

with a lecithotrophic trochophore and a long-lived

planktotrophic pelagosphera larva. Included in this

last category are the development of Phascolosoma

agassizii, Sipunculus nudus, and unidentified species which have oceanic planktonic larvae.

Previous studies of sipunculan development

which include significant accounts of histogenesis

have been reported for 6 species, representing all 4

developmental categories. The species are Golfingia

minuta, category 1, with direct development (Akesson, 1958), Phascolopsis gouldi and Phascolion

strombi in category 2 (Gerould, 1907; Akesson,

1958), and Golfingia elongata and G. vulgaris in

category 3 (Akesson, 1961b; Gerould, 1907). In

category 4 histogenesis has been described for only

one species, Sipunculus nudus; developmental features of this planktotrophic species have been found

to differ markedly in several respects from the species in the first three categories with lecithotrophic

development. The present account of histogenesis

of the pelagosphera larva of Phascolosoma agassizii

provides additional information on planktotrophic

development in sipunculans and serves to broaden

the basis for comparative evaluation of developmental features within the phylum and for interpretation of affinities with other groups.

ACKNOWLEDGMENTS

This study represents part of a dissertation submitted to the University of Washington in partial

fulfillment of the requirements for the degree of

Doctor of Philosophy. The author wishes to express her appreciation to Dr. Robert L. Fernald,

Director of the Friday Harbor Laboratories, for his

advice and encouragement and to thank him for

use of the facilities at the Friday Harpor Laboratories. I am also deeply grateful to Dr. Paul Illg

of the University of Washington for his many helpful and valuable suggestions throughout the course

of this investigation. Support was provided, in

part, by a Public Health Service fellowship 5—Fl—

GM-16, 344-03 from the National Institute of General Medical Science.

Materials and Methods

Adult specimens of Phascolosoma agassizii were

collected from the San Juan Archipelago on the

northwestern coast of the state of Washington and

from Vancouver Island, British Columbia. They

were found in intertidal habitats either under rocks

or in a coarse gravel mixed with sand. Collections

were made just previous to or during the breeding

season which lasts from the middle of June to late

August or early September (Rice, 1967). Animals

were maintained at a temperature of 10° to 12° C

in glass dishes in which the sea water was changed

once or twice daily. When spawning occurred, the

fertilized eggs were removed and placed in tall
NUMBER 132

covered glass petri dishes in which the embryos and

larvae were reared. Dishes with developmental

stages were maintained at 10° to 12° C on the sea

water table or in a refrigerated incubator; water

was changed daily for early embryonic stages, but

less frequently for later larval stages. Food for the

larvae was supplied by additions of unfiltered sea

water, supplemented by mixed diatoms and dinoflagellates collected from tidal pools.

For histological studies, unless noted otherwise

in the text, larvae were fixed in ice-cold 2 percent

osmium tetroxide, buffered with 0.2 M S-collidine

at a pH of 7.5 (Bennett and Luft, 1959) and embedded in Epon according to the method of Luft

(1961). One or two micron sections were cut with

glass knives on a Porter-Blum ultramicrotome, affixed to glass slides and stained with Richardson's

stain (equal parts of 1 percent azure II and 1 percent methylene blue made up in 1 percent sodium

borate) for examination with the light microscope

(Richardson et al., 1960). Additional stains for

specific structures are mentioned in the text. Before

fixation the larvae were relaxed in a mixture of

saturated aqueous chloretone and sea water (1:2)

(Clement and Cather, 1957).

Review of Early Development

The egg of Phascolosoma agassizii, ovoid in

shape, but somewhat flattened sagittally, is relatively low in yolk content and undergoes spiral,

unequal cleavage to give rise to a freely swimming

blastula with modified blastocoel (Rice, 1967). Gastrulation occurs chiefly by epiboly, although invagination plays a minor role, forming a narrow

archenteron (Plate la,b). Mesodermal bands, derived from two large mesodermal teloblasts, are

situated on either side of the endoderm cells which

are distinguished by their small green cytoplasmic

granules. At the site of the closed blastopore, the

stomodaeum is formed by an invagination of rapidly dividing ectodermal stomatoblasts. From the

rosette cells in the center of the apical plate, the

cilia of the apical tuft extend through the pores

of the overlying egg envelope and the embryo, at

its equator, is encircled by two equatorial rows of

ciliated prototroch cells (Plate \a-d). A remnant of

the blastocoel persists on the inner side of the prototroch cells as the "prototrochal cavity" into which

yolk granules from the prototroch are discharged

during early development (Plates \c,d, 2a-d).

The trochopore stage extends from 2i/£ to 9 or

10 days of age (Plates lc,d, 2a,b). In the early trochophore the essential size and shape of the egg

envelope are retained, but at approximately 5 days

of age gradual changes begin to take place. This

period of gradual change in the trochophore, extending over 4 or 5 days and leading up to the

more rapid changes of trochophoral metamorphosis, is referred to here as premetamorphosis (Plates

2a-d, 3a). During premetamorphosis alterations occur in size and shape of the trochophore, a narrow

coelomic cavity is formed by the splitting of mesodermal bands, and the differentiation of larval organs such as gut and retractor muscles is initiated.

Metamorphosis of the trochophore into the pelagosphera larva occurs at the age of 9 or 10 days

and lasts for a period of 12 hours or less at 10° C

(8 days at 12° C). It is marked by a rapid increase

in length (30 percent), expansion of the coelom,

opening of mouth and anus, reduction of the prototroch, formation of the metatroch, and emergence

of the terminal organ (Plate Sac).

In the following report on morphology, behavior,

and histogenesis of the pelagosphera larva of Phascolosoma agassizii, descriptions, unless otherwise indicated, will refer to the larva as it appears at 20

days of age. Larvae have been maintained in the

laboratory up to an age of 7 months, with relatively

few observed changes other than an increase in

size, a change in relative proportions, and a modification of cuticular patterns. Duration of larval

life under natural conditions is unknown. Transformation to the juvenile worm has not been observed and all efforts to collect larvae from the

plankton for comparative studies have been unsuccessful.

Morphology and Behavior of the Larva

The body of the pelagosphera larva of Phascolosoma agassizii is marked by four distinct regions:

head, thorax, elongated trunk, and terminal organ

(Plates 5c, 4a-f, 5). The average length of the larval body at 20 days of age is between 400 and 500

microns; by stretching or contracting, a given larva

may increase or decrease this length as much as 50

percent. The average maximum width is approximately 100 microns.
SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

The head of the larva is covered by cilia on the

ventral surface and divided into two ventral lobes

by a median ciliated groove which leads into the

mouth. The groove begins as a wide channel at

the tip of the head and becomes narrower as it approaches the mouth. Below the mouth is a ciliated

lobe (Plates 4c,d, 5, 8b) which will be referred to

as the "lip" in conformity with Jagersten's (1963)

terminology for pelagosphera larvae. The lip usually projects out at right angles from the ventral

surface of the head, but when the head is withdrawn into the trunk, the lip is pulled in parallel

to the head. When the larva is feeding on the substratum this lip may be pushed back so that it is

flattened out against the substratum in the same

plane as the ventral surface of the head. The dorsal

surface of the head lacks ciliation with the exception of the prototrochal cilia which are now reduced in size and number. Located in a dorsolateral position about half of the distance down

the length of the head are a pair of eyespots, hemispherical in shape and of a deep orange pigmentation (Plates 4b, \\b,d). The entire head may be

withdrawn into the trunk when the larva is inactive or when it is startled by some stimulus such

as a movement of the surrounding medium.

Not only may the head be withdrawn into the

trunk, but also the region immediately posterior to

the head, known as the "thorax," may be withdrawn when introversion is complete. In the terminology of Jagersten (1963) the head plus the

thorax make up the introvert. Hence the thorax

may be defined as that part of the larval body adjoining the head which is retractable into the

posterior portion of the larva. It includes the metatroch and is characterized by a thinner cuticle than

that of the trunk.

The trunk includes that portion of the larval

body posterior to the thorax with the exception of

the terminal organ which is considered to be a distinct region of the body. When extended the trunk

is wedge-shaped, being broadest anteriorly and becoming increasingly narrow toward the posterior

end. It is at the same time curved so that the

dorsal surface is slightly convex and the ventral surface concave (Plates 3c, 4a,c,f, 5). The anus is

clearly visible on the dorsal surface of the trunk,

usually about halfway along its length, but its relative position varies according to the degree of body

extension (Plates 4a,c, 5). Other openings, not so

easily observed, occur in the proximity of the «mal

opening. Slightly anterior to the anus in ventrolateral positions are the two nephridiopores. Dorsolaterally, on either side of the anus, are the

openings to the pair of large sacciform glands

(Plates 4b, 14a-c). Three pairs of dorsolateral papillae, each papilla bearing a single bristle, appear

between 10 and 20 days. The most anterior pair

is in the midtrunk region at the level of the stomach

and the other two pairs are located in the posterior

third of the trunk (Plate \4d). When the larva

has reached the age of 28 days the cuticle of the

trunk is covered over its entire surface with numerous small papillae (Plates 4c,e, 5, 7b-d, IQd,

13d). These cuticular papillae must not be confused with the epidermal papillae described above.

The cuticular papillae are responsible, in Jagersten's (1963) terms, for the "rough" appearance of

the body surface. On the basis of the appearance

of body surface, Jagersten divided the pelagosphera

which he examined into two species: "rough" and

"smooth."

From the posterior end of the trunk there extends a narrow appendage, usually 50 microns in

length, but extensible to a length of 170 microns.

This appendage is known as the terminal organ

(Plates 3r, 4a,c,e,f, I4e,f). Such an organ has been

described by Hatschek (1883) in the larva of Sipunculus nudus and has been noted by Jagersten (1963)

in some unidentified pelagosphera. The terminal

organ can be retracted into the posterior end of the

trunk and this is the usual position when the larva

is swimming or when it is quiescent. The organ

manifests adhesive properties and its primary function seems to be the attachment of the larva to the

substratum.

The larva of Phascolosoma agassizii begins to

feed as soon as it completes metamorphosis. At this

stage it is chiefly a bottom feeder, although it is

also able to feed on small planktonic organisms.

Most commonly the larva attaches by the terminal

organ to some debris on the bottom of the container and from this point of attachment it may

stretch out great distances, either parallel to the

bottom or upward at any angle. One frequent pattern of feeding is for the larva to stretch out along

the bottom and then, with mouth applied to the

substratum, to pull the head backward toward the

point of attachment; the body is arched as the head

progresses toward the terminal organ and the
NUMBER 132

mouth takes in diatoms and various sorts of debris

as it moves along the bottom. The larva may also

assume an arched position as it sweeps around the

attached terminal organ in a complete circle. The

ciliated ventral surface of the head is flattened upon

the bottom, the lip bent backward, while the larva

grazes over the substratum and clears the debris

from the area surrounding it. From time to time

the larva may release itself from its posterior attachment and swim with the ventral surface of the

head applied to the substratum and the terminal

organ pointing upward. In this manner it can cover

more territory than would be possible in a fixed

position, or this may be only a means of finding a

new location for settling. The larva may also move

about by swimming along the bottom in an arched

position with the terminal organ dragging along

behind, unattached; or it may crawl along in the

manner of an inch worm. At other times it may

swim freely through the water, propelled by the

activity of the metatrochal cilia, or it may remain

quiescent on the bottom, unattached with the introvert retracted. The larva appears to feed for the

most part when it is attached with the introvert

extended. Food intake is apparently accomplished

by ciliary activity on the ventral surface of the

head and by the occasional eversion of the buccal

organ. Possible functions of the buccal organ will

be discussed in more detail in the treatment of the

histogenesis of this organ (pp. 11-13). Occasionally

the larva bends both the head and the posterior end

in a ventral direction so that the lip and terminal

organ touch, or else the terminal organ is pushed

past the lip to one side of the head. Whatever may

be accomplished by this activity is not obvious; in

fact, the activity may be abnormal since it is most

likely to occur when there is no substratum on

which the larva may settle. It is possible also that

a secretion is transferred from the posterior sacciform glands or from the terminal organ to the lip,

or from the lip to the terminal organ. Jagersten

(1963) reported that the terminal organ was pushed

into the mouth, but in Phascolosoma agassizii this

was not observed.

The metatrochal cilia function mainly in locomotion. When the larva is attached, these cilia aid in

the movement of the anterior end around the point

of attachment, but they do not beat continuously.

When the introvert is retracted the metatrochal

cilia usually cease to beat.

Larval Histogenesis

The description of histogenesis in each organ or

structure in the larva of Phascolosoma agassizii will

be preceded by an account of its histology and followed by a comparative review of the information

available on the histology and development of the

structure in other sipunculans. The larval structures to be described are the following: body wall,

coelom and coelomic elements, retractor muscles,

digestive tract, buccal organ, lip gland, nervous

system, nephridia, larval glands, and terminal organ. In addition to the descriptive considerations,

functional significance of many of the organs will

be discussed.

BODY WALL

Cuticle.—The cuticle of the larva is distinctive

in each region of the body; hence the four regions

will be discussed separately.

In the head there are two distinct types of cuticle: the dorsal cuticle differs from the ventral and

has a separate origin from it (Plates 5b,c, lid). The

dorsal cuticle of the head is very thin and no structure can be resolved within it. It is derived from

the inner layer of the vitelline envelope, the outer

layers being sloughed off soon after metamorphosis.

The dorsal cuticle stains very lightly with Richardson's stain as does the ventral cuticle and both react positively to the periodic acid-Schiff stain. The

ventral cuticle, penetrated by numerous cilia, is

slightly thicker than the dorsal and is divided into

two layers by a darkly staining central striation. It

originates from the lining of the stomodaeum and

bears no affinity to the original vitelline envelope.

The cuticle of the thorax is structurally the same

as that of the trunk, but it is much thinner (Plate

5). There is no sharp demarcation between the

two, but rather a gradual transition. This cuticle,

composed of two layers, is derived from the vitelline

envelope (Plate 13b). The inner layer, wider and

more lightly staining, represents the combined inner and middle layers of the vitelline envelope and

the outer layer of the cuticle is derived from the

outer layer of the envelope. The inner layer gives

a faint positive reaction to the periodic acid-Schiff

stain and the outer layer is characterized by metachromasia.

Between 3 and 4 weeks of age small papillae form
SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

in the cuticle of the trunk (Plates \c,e, 5, Ib-d,

13d, He). These are filled at their base by the substance of the inner layer, but within the apex,

staining bright blue with Richardson's stain, a new

substance makes its appearance. The papillae are

covered externally by the outer cuticular layer.

The cuticle of the terminal organ is identical to

that of the trunk and continuous with it. It differs

only in that it is much thinner.

The fate of the egg envelope and the formation

of the cuticle are not uniform in all species of

sipunculans. Gerould (1907) observed the shedding

of the egg envelope in Phascolopsis gouldi and

Golfingia vulgaris at the time of metamorphosis.

The envelope ruptured at the postoral circlet of

cilia and the entire posterior portion was immediately lost. Anteriorly the envelope remained attached longer but was eventually sloughed off by

the repeated introversions of the head. A fine granular cuticle beneath the egg envelope became the

larval cuticle after the shedding of the envelope.

Selenka (1875) described a transformation of the

egg envelope into the larval cuticle in Golfingia

elongata. In the light of his own observations

Gerould doubted the validity of Selenka's description, but Akesson in 1961(b) confirmed Selenka's

original report. Akesson (1958) also had reported

that the egg envelope in Phascolion strombi and

Golfingia minuta was distended to form the larval

cuticle. Beneath the larval cuticle he observed a

second narrow cuticle, presumably the definitive

cuticle of the juvenile form. The egg envelope, although distended, did not become elastic and when

the larva contracted the envelope bulged out from

the body. Bulges in the larval cuticle of Phascolosoma agassizii have been seen only in abnormal

larvae or in larvae that have been subjected to

relaxants and fixatives. The egg envelope in P.

agassizii is transformed into an elastic larval cuticle

and no second cuticle has been observed.

In Sipunculus nudus the egg envelope is cast off

at the initial metamorphosis along with the remnants of the prototroch cells (Hatschek, 1883). In

this species the relationship of the egg envelope to

the embryo is quite different from that in other

sipunculans (Gerould, 1903). The trochoblasts

spread around the inside of the egg envelope forming a complete envelope or "serosa" surrounding

the developing embryo. The cells degenerate and

are shed with the egg envelope at the beginning of

the pelagic larval stage.

Epidermis.—In the larva the epidermal cells are

marked by their vacuolar cytoplasm and large nuclei with homogeneous, darkly staining nucleoplasm

and prominent nucleoli (Plates 3c, 5,7c). Very fine

cytoplasmic extensions of the epidermal cells penetrate into the cuticle.

The epidermal cells of the trunk are derived

largely from the descendants of the second cell. In

the early stages these cells are filled with yolk, but

by the end of the trochophore stage much of the

yolk has disappeared. After the formation of the

coelom, large intercellular spaces appear in the epidermis between the points of the epidermal attachment to the peritoneum (Plate 3a). As the cells

expand into these spaces, their cytoplasm becomes

intensively vacuolated (Plate 36). At the metamorphosis of the trochophore to the pelagosphera the

epidermis is stretched out into a much thinner

layer (Plate 3c).

The epidermal glands will be considered in a

later section on larval glands (pp. 18-19).

Muscles of the Body Wall.—The muscles of the

body wall are small and inconspicuous and are not

easily identified in any stage that has been studied

(Plate lb,c). In the larva the longitudinal muscles

appear in sagittal section as long, very fine, darkly

staining fibers on the outer side of the peritoneum

and the circular muscles are represented by small

darkly staining dots, apparently attached to the

outer side of the longitudinal muscles. The adult

arrangement of longitudinal muscles into bundles

of fibers is not discernible in the larva.

Immediately posterior to the metatroch there is

a broad circular sphincter which increases in

breadth with age (Plate 76). In a larva of 2 months

this sphincter may reach from the metatroch to the

level of the stomach. At fixation it often contracts,

pushing the stomach into an abnormal anterior

position. The normal function of the sphincter is

to close the anterior opening of the body after the

head has been retracted. In sagittal section the

sphincter is seen as a number of relatively large

spherical fibers well separated from each other and

located to the inside of the epidermal cells.

Nothing can be said concerning the origin of

the muscles of the body wall. In prelarval development small nuclei, resembling those of muscle, are

seen scattered among the epidermal cells, but it has
NUMBER 152

not been possible to demonstrate whether these

give rise to the circular and longitudinal muscles.

There is little agreement among other authors

as to the derivation of the muscles of the body

wall. Gerould (1907) reported the origin of the

circular muscles in Golfingia vulgaris and Phascolopsis gouldi to be ectomesodermal, but he does not

mention the source of the longitudinal muscles.

Hatschek (1883), on the other hand, believed that

all of the body wall musculature in Sipunculus

nudus was derived from endomesoderm. In his

study of Golfingia minuta and Phascolion strombi

Akesson (1958) found that the circular muscles

were formed from ectomesoderm and the longitudinal muscles from endomesoderm.

Peritoneum.—The peritoneum lines the coelom

of the larva, covering the inner side of the body

wall and continuing around the esophagus and

rectum to surround the gut. The cytoplasm of each

peritoneal cell extends out from the nucleus as a

thin membrane which joins that of neighboring

cells to form the peritoneal lining. The nuclei

of the peritoneum are distinguished from those of

the epidermis by their pronounced chromatin granules and their lightly staining nucleoplasm.

The peritoneum is derived from the lateral

bands of mesoderm which in turn arise from the

early mesodermal teloblasts (Plate 16). The lateral

bands spread around the embryo and split into

inner and outer layers, splanchnic and somatic,

respectively, to form the coelom (Plates 2b-d, la).

The splanchnic mesoderm surrounds the gut and

the somatic forms the inner lining of the body wall.

At first the nuclei of the peritoneum are very close

together (Plate 3a), but with the elongation of

the body they are pulled apart and the cytoplasm

is stretched out into a thin mesothelium (Plate 3c).

The ciliated peritoneal cells of the adult have

not been identified in the larva.

coelomic cells which are moved about by the nearly

continuous contraction and expansion of the larval

body. There are two types of coelomocytes (Plates

la-d, 14c). One is spindle-shaped with occasional

variations in form that suggest amoeboid activity.

The cytoplasm of this cell type stains darkly and

in older larval stages it is packed with basophilic

granules. The second cell type is round and flat

and the cytoplasm in younger larvae stains lightly

with relatively few inclusions. At two months the

cytoplasm of the second cell type is characterized

by clusters of granules and vacuoles and the formerly central nucleus is in an eccentric position

(Plate 7c).

In the premetamorphic larva, cells resembling

the second cell type appear to be budding off from

the splanchnic peritoneum into the newly formed

coelom (Plate 7a). These coelomocytes increase in

number with age, but there is no evidence for their

later derivation or for the derivation of the spindleshaped cells. The round, flat cells of the larva are

similar to and may be identical with the red cells

of the adult, whereas the spindle cells may represent

the small amoebocyte of the adult. There are 3

types of adult coelomocytes, however, that have

not been recognized in the larva. They are the

large amoebocytes with large granules, the vesicles,

and the urns.

The coelomocytes described by Gerould (1907,

pi. 9: fig. 80b, c) in Golfingia vulgaris and Phascolopsis gouldi are very similar to the round flat cells

of Phascolosoma agassizii. Although he presented

no evidence for their origin, Gerould suggested

that they were probably derived from the peritoneum in the posterior coelom. In addition to

these coelomocytes, Gerould also found within the

coelom 19 cells with large nuclei and scant cytoplasm, which he believed to be the remains of the

discarded prototroch cells.

COELOM AND COELOMIC ELEMENTS

The expansive coelom of the larva extends without interruption from the most anterior part of the

head into the terminal organ and it is open to the

exterior only by way of the nephridial organs.

The coelom is formed between 6 and 7 days by a

splitting of the splanchnic and somatic layers of

mesoderm.

Within the coelom there are freely suspended

RETRACTOR MUSCLES

Spanning the coelom lengthwise are four retractor muscles, two dorsal and two ventral, which

function to withdraw the head into the trunk. In

the early larva up until the age of one month each

muscle is composed of eight fibers and each fiber

is a single cell with only one nucleus. A cross-section

of the muscle reveals a circular arrangement of

fibers joined together by a thin peritoneal mesen-
8 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

tery enclosing a central cavity (Plates 12a, 13b).

The fibers of each of the dorsal retractors begin

in a dorsolateral position near the base of the

brain, attached in a straight row to the peritoneal

lining (Plate 13a). The most median fibers arise

more anteriorly than the outermost which arise

slightly posterior to the level of the mouth. At

the level of the ventral lip the fibers and their

surrounding peritoneum pinch off from the peritoneal lining forming a circle with a central space

(Plate 13fr). The muscle then extends posteriorly

through the coelom to the first posterior bristle

where it again attaches to the peritoneal lining of

the dorsal body wall, fanning out into a row of

fibers. Just below the bristle the two outermost

fibers insert into the body wall (Plate 14d). Two

more fibers are inserted at the level of the posterior

glands and the last four fibers in the region of the

anus.

The fibers of the ventral retractors arise from

the body wall on either side of the mouth near the

emergence of the circumesophageal connectives

from the brain (Plate 13a). The four most anterior

fibers lie between the epidermis and the connectives

and it is only posterior to the connectives that the

fibers come into contact with the peritoneum. The

four anterior fibers are joined by four additional

fibers in the region of the buccal organ and the

eight fibers plus the interconnecting peritoneum

separate from the peritoneal lining, forming a

circular muscle similar to that of the dorsal retractor (Plate 136). The ventral retractors pass

posteriorly between the buccal organ and the lip

glands to the level of the first bristle where each of

the ventral retractors gives off two fibers, one close

to the ventral nerve cord and another more laterally. The circle of fibers then flattens out and

becomes continuous with the ventral peritoneal

lining. At the level of the nephridia two more fibers

are inserted (Plate 13c) and the four remaining

fibers in each of the ventral retractors insert far

posteriorly at the level of the second bristle, thus

exceeding by far the dorsal retractors in length.

The nucleus of each muscle fiber is located anteriorly along the length of the fiber and all of the

nuclei of a muscle are relatively close together

(Plate 6rf). In both dorsal and ventral retractors

the nuclei are found at the level of the lower

esophagus or upper stomach, although the ventral

nuclei may extend slightly farther posteriorly.

The retractor muscles of Phascolosoma agassizii

appear to be derived from ectoderm, but it has not

been possible in this study to identify with certainty

the rudiments in very early stages. At the time of

coelom formation groups of cells with nuclei which

resemble those of the later muscles are found

bulging into the coelom in the position of the future

ventral retractors (Plate 6a, b). These cells are

located at the anterior end of the newly forming

posttrochal coelom and appear to be connected by

cytoplasmic extensions to the ectodermal cells of

the stomodaeum and the ventrolateral apical plate,

just above the prototroch. In the position of the

future dorsal retractors the early coelom is narrow

and a cluster of cells corresponding to that in the

ventral region has not been identified; however,

a few cells of similar cytology have been recognized

just anterior to the coelom. Within a period of 24

hours after the ventral cell clusters first are seen,

both ventral and dorsal retractors make their appearance as long fibers with central nuclei which

extend from the lateral ectoderm of the apical

plate through the coelom to the posterior somatic

mesoderm (Plate 6c). The ventral retractors with

their very thick fibers are much larger when first

formed than the dorsal retractors with their relatively thin and tenuous fibers. Both ventral and

dorsal retractors are intimately associated with

peritoneal cells, the nuclei of which, although

usually smaller and rounder, are not always easily

distinguished from those of the muscle.

The above observations are interpreted to signify

that the retractor muscles originate from four areas

of ectomesoderm located in dorsolateral and ventrolateral positions in the apical plate. These

rudiments grow downward, retaining their cytoplasmic connections in the head, and push into the

coelom taking with them an investing layer of

peritoneum. Long cytoplasmic processes or fibers are

extended through the coelom and attach posteriorly

to the outer layer of coelomic mesoderm.

If the observations of this study are complete and

if the interpretations are correct, then the formation of retractor muscles in Phascolosoma agassizii

differs from that observed in studies of other

sipunculans. Gerould (1907) in his study of Golfingia vulgaris and Phascolopsis gouldi stated that

the muscles were apparent in the early trochophore

prior to the division of the coelomomesoblast, extending from the sides of the apical plate, from
NUMBER 152

which they were derived, posteriorly to the region

of the postoral circlet. He suggested that the muscles

passed through the loose layer of mesoderm at the

time of metamorphosis.

Hatschek (1883) proposed that in Sipunculus

nudus the retractors were derived from somatic

mesoderm. He was unable, however, to trace their

early development and recognized them only after

they were already formed within the coelom.

Confirming Gerould's observations, Akesson

(1958, 1961a) found in three species of sipunculans,

Phascolion stro?nbi, Golfingia minuta, and Golfingia elongata, that the retractor muscles were derived from ectomesoderm and that they were

formed outside of the mesoderm in very early stages

before the formation of the coelom. In Phascolion

strombi and Golfingia minuta the development of

the muscles was reported in detail and it was

observed that each retractor had two rudiments,

an anterior and a posterior, which grew toward

each other and fused to form the completed muscle.

This manner of development could not occur in

Phascolosoma agassizii where each fiber of the larval

muscle is a single cell which extends the entire

length of the muscle.

The reported incongruities demand further

studies on Phascolosoma agassizii and a reinvestigation of Sipunculus nudus before conclusions can

be drawn as to the mode of development of the

retractor muscles of sipunculans and the specific

variations.

The larval retractor muscles are retained as the

retractor muscles of the adult sipunculan. In Phascolosoma agassizii it has been demonstrated that

the muscle fibers increase in number with the age

of the larva. In the larva of 28 days each muscle is

composed of only eight fibers arranged as a tube

with a central cavity, whereas at 2 months the

number of fibers has greatly increased, completely

filling the central cavity (Plate 13d).

DIGESTIVE TRACT

The gut of the larva is regionalized into four distinct morphological and cytological portions: the

long, broad esophagus with posterior sphincter, the

expanded and bulbous stomach, the thin, looped

intestine, and the short rectum.

Esophagus.—The epithelial lining of the esophagus is composed of cuboidal cells with dense, long

cilia which form a central swirl in the lumen

(Plates 1b, 13d). The epithelium is lined with an

inner cuticle which is continuous with that of the

ventral head and stains brightly with periodic

acid-Schiff. The nuclei are relatively large and the

cytoplasm appears to be homogeneous with small

lipid droplets scattered through it. On the outer

side of the epithelium is a thin layer of muscle

fibers covered by peritoneum. The muscle layer

increases in thickness at the junction with the

stomach, forming a tight sphincter (Plate 76).

When the head is retracted into the body the

esophagus is pushed into a lateral position.

The esophageal lining is formed from the stomodaeal invagination and is therefore ectodermal in

derivation. The invagination begins at approximately 60 hours and by the end of the trochophore

stage it has elongated in an anterodorsal direction

and the cells have developed cilia (Plate Id). As the

stomodaeal invagination sinks inward the anterior

end of the stomach moves into a more dorsal position. At the beginning of the premetamorphosis

stage (6i/2 days) the invagination opens into the

cavity of the stomach and, as elongation proceeds,

the stomach is moved into a position directly

posterior to the esophagus (Plate 2b).

Stomach.—The stomach is very large and bulbous

in shape and its maximum width is nearly equal

to that of the coelom (Plates 36, 5). Its diameter

may exceed that of the mid-esophagus two or three

times and that of the esophageal sphincter over

lour times. The lining of the stomach is constructed

of a single layer of cuboidal epithelium enclosing

an expansive central cavity and, in contrast to the

esophagus and intestine, there is no obvious intervening muscle between the epithelium and the

surrounding peritoneum (Plates Ib-d, 10c, 126).

The apical border of the epithelial cells is striated

as well as weakly ciliated, the cilia being much less

concentrated and shorter than those of the esophagus (Plates 76, 126, c). Within the cytoplasm of

the epithelial cells there are numerous lipid droplets of varying sizes, all larger than those of the

esophagus. In larvae older than one month, the

lipid droplets coalesce to form one huge sphere

of lipid in each cell (Plate 7c, d). The remainder

of the cytoplasm is highly vacuolated, giving the

appearance of a reticulum. The epithelial nuclei

have a large eccentric nucleolus and the nucleoplasm, with prominent chromatin granules, stains
10 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

more lightly than that of the esophageal cells. Although the cuboidal epithelial cell is by far the

most common, there is a second cell type that is

rather sparsely scattered throughout the epithelium

and is most readily observed in the older stages.

This is a smaller cell, pyramidal in shape and with

homogeneously staining basophilic cytoplasm.

The larval stomach is derived from endoderm

and is the first portion of the gut to be differentiated. It can be distinguished at two and one-half

days when characteristically small, green cytoplasmic

granules appear in the endoderm and a cavity, the

future lumen of the stomach, appears in the midst

of the endoderm cells. The cavity elongates as the

cells of the posterior portion of the stomach undergo division. In the premetamorphosis stage the cells

of the posterior stomach give rise to the rudiment

of the intestine which makes its appearance as a

small round knob at the end of the stomach (Plate

2b).

In the early trochophore the green granules increase slightly in size and, along with the relatively

densely concentrated yolk granules, mark the cells

of the stomach as distinct from all others (Plate

la). In the late trochophore some of the yolk

granules coalesce and, when the coelom is formed,

these large clusters of yolk are cast off into it.

During premetamorphosis the remainder of the

yolk disappears, its fate undetermined. The green

granules now vary in size; some are quite large

and are recognizable as lipid droplets. After metamorphosis the small granules disappear and the

lipid droplets increase in size and decrease in

number until there is in each cell only one very

large sphere (Plate 7c, d). From this evidence it is

assumed that the green granules described in the

early stages were in actuality small lipid inclusions.

Whether these lipid inclusions are responsible for

the green coloration in the living larva or whether

there is some other constituent that has not been

defined cytologically is not known. The two are,

however, linked together by circumstantial evidence in that they both appear at approximately

the same stage of development. The esophagus, although not colored in the living larva, does contain

some lipid, but it is not present in the same concentration as in the stomach and intestine, both

of which are marked by green coloration.

At the time of metamorphosis the cavity of the

stomach increases in size and the bulbous shape is

assumed. In older larvae (2 months or more) the

stomach enlarges ami elongates to form a long

cylinder which may extend more than half the

length of the body. The fate of the larval stomach

is unknown. Although it occupies a place of great

prominence in the larva, it has no morphological

counterpart in the adult. Since only those sipunculan larvae with a prolonged pelagic life, such as

Sipitnculiis nudus (Hatschek, 1883) and other unidentified pelagosphera (Akesson, 1961a; Damas,

1962), possess a well-defined stomach, it is likely that

the stomach is of functional significance to a pelagic

mode of existence.

Intestine.—The stomach narrows abruptly into

the thin intestine which in the larva up to age of

60 days is looped but not coiled. There is a descending portion which extends anteriorly toward the

dorsal anus. As it leaves the stomach the intestinal

lining is composed of a relatively thick, ciliated

cuboidal epithelium with a densely basophilic cytoplasm. Within a short distance the wall becomes

exceedingly thin, the cells are flattened and the

cytoplasm vacuolated (Plates 12rf, 13c). The intestinal epithelium is ciliated and there are small

lipid granules scattered throughout its length. The

wall of the intestine is completed by a thin muscle

layer and a peritoneal covering.

The intestine originates from endoderm which

grows out as an extension from the posterior end

of the stomach in the late trochophore stage (Plate

2b). Growing dorsally it attaches to the dorsoposterior ectoderm in the position where the future

anus will form (Plate 106). At metamorphosis there

is an elongation of the body posterior to this point

of attachment, extending the coelom far below

the anal region and as the intestine continues to

grow it loops downward into the posterior coelom.

From the time of its formation there are lipid

droplets dispersed throughout the cytoplasm of the

intestine, but they never become as large or as

concentrated as those of the stomach.

Rectum.—The larval rectum is a short tube

surrounded by a thin layer of muscle and an outer

covering of peritoneum (Plates 5, 10c). The epithelium of the rectum is very much thicker than

that of the preceding intestine and the cells are

heavily ciliated. The cytoplasm is basophilic with

no visible inclusions other than yolk granules in

the early stages, and the inner border of the cells

stains strongly with periodic acid-Schiff, as does
NUMBER 1S2 II

that of the esophageal cells. The rectum continues

into the anus and at the anal opening the larval

cuticle and its associated epidermis turn inward to

meet the rectal epithelium (Plates 5, 10b, c). Surrounding the anus there is a thick sphincter muscle.

The rectum is formed during premetamorphosis

from a dorsoposterior ectodermal proliferation.

The rudiment of the rectum is composed of only a

few cells which are soon joined by the growing

intestine (Plate 10b). A ciliated cavity forms among

the ectodermal cells and before metamorphosis this

opens into the intestinal lumen. A slight depression

appears in the cuticle opposite the rectal rudiment

and at this point the cuticle ruptures during metamorphosis, forming the anus and thus opening the

rectum to the exterior.

In all species of sipunculans that have been

studied the esophagus is formed from a stomodaeal

invagination, the rectum from a cluster of ectodermal cells referred to as the proctodaeum, and

the remainder of the gut from the entomeres of

the blastoporal region. In the different species of

sipunculans there is a great difference in the time

at which a lumen appears within the gut and the

time at which the gut is completed. Golfingia minuta, with a large egg rich in yolk, remains lecithotrophic for 2 months and Phascolion strombi for

one month (Akesson, 1958). In Golfingia vulgaris

and Phascolopsis gonldi a lumen appears within

the archenteron at 5 or 6 days, but they do not

begin to feed until the second or third week of

age (Gerould, 1907). The gut of Sipunculus nudus

is functional at 3 days (Hatschek, 1883) and that of

Phascolosoma agassizii at 9 or 10 days.

MOUTH AND ASSOCIATED STRUCTURES

The term mouth will be used in a broad sense

to include the anterior ciliated channel of the

ventral head, the ventral, posterior lip, and the

buccal groove through which the buccal organ

may be protruded.

The ciliated channel divides the ventral surface

of the head into two lobes and continues into the

esophagus, forming the dorsal and lateral boundaries of the esophageal opening. The ventral

boundary of the opening is formed by the union

of two nonciliated folds. The channel of the ventral

surface of the head is derived from the roof of the

stomodaeum at metamorphosis.

Posteriorly the mouth is expanded into the ventral lip (Plates 5, 8b), a lobe which usually extends

out perpendicularly from the head, but which may

be withdrawn along with the head into the trunk

or may, when the larva is feeding, be flattened out

against the substratum in a 180° angle from the

head. The lip is partially bifurcated by a deep

ciliated groove which begins near the distal end

of the lip and extends to a ciliated pore near the

proximal end of the lip. The lip glands, to be

described later, open into this pore. Distally the

ciliation of the lip is limited to the central groove

and a few lateral cilia, whereas that portion of the

lip proximal to the pore is evenly ciliated. When

the mouth opens at metamorphosis, the floor of the

stomodaeum is extended outward to form the surface of the lip (Plates 3b,c, 5, 9d). The cuticle surrounding the lower portion of the mouth is

stretched out with the stomodaeal extension to form

the sides of the ventral lip.

Buccal Organ.—At the base of the lip is a long,

transverse slit, the width of the mouth, which extends downward as a nonciliated infolding for

some distance along the ventral side of the esophagus (Plates 8«, b, We). The slit, which will be

termed the buccal groove is expanded in the center

of its posterior extremity and ventrally it may show

one or more folds. The entire invagination is lined

by a very thick cuticle which is stained by periodic

acid-Schiff. On the dorsal side, the cells of the

epithelium are very thin and lie in close association

with the esophagus along much of its length. On

the ventral side of the infolding and extending

around under the posterior end is the buccal organ;

when the infolding is everted the buccal organ protrudes to the exterior.

The buccal organ hangs down into the coelom

of the larva as a large muscular sac within which

there are two main cavities: posterior-dorsal and

anterior-ventral; the latter may be divided again,

making a small third cavity. The organ is circumscribed by a sheet of muscle which attaches ventrally to the proximal portion of the lip and, looping down around the posterior extremity of the

buccal groove, attaches dorsally to the muscle layer

of the esophagus (Plates 3f, 5, 8a, b, \\b, c). The

epithelium of the organ lines the ventral side of the

buccal groove and, with its overlying cuticle, is the

first portion to reach the exterior when the organ

is protruded. The epithelial cells are very elongate
12 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

with thin necks leading to rounded tips which

bulge out into the cavity of the organ. The partition that divides the cavity into chambers is formed

by an extension of epithelial cells across the cavity,

connecting to the outer muscle wall. The cytoplasm

of these cells stains lightly and appears to be

fibrous (Plate 86).

The formation of the buccal organ is not entirely

understood. The outer muscular layer is formed

from cells which grow downward from the sides of

the anterior esophagus and posterior stomodaeum

and join beneath in a solid mass (Plate 9c,d). The

cells are first recognized at the beginning of the premetamorphosis stage and by the end of metamorphosis they have spread out as a single layer to

form a muscular sac which hangs down from the

esophagus. It has not been possible to discern

whether the cells originate from the ectoderm of

the apical plate above the esophagus or whether

they are derived from the mesoderm along the

sides of the esophagus. The buccal groove has not

been observed until the day preceding metamorphosis. At this time it extends downward as an invagination from the floor of the stomodaeum, but in

contrast to the stomodaeal lining, it has a heavy

cuticle and lacks ciliation (Plate 9d).

The buccal organ was first described in 1883 by

Hatschek in the larva of Sipunculus nudus. Hatschek referred to this organ as the "Schlundkopf," a

word which can be translated as the upper pharynx,

esophagus, or buccal mass. The structure was

described as a fold in the esophageal wall which

extended posteriorly as a groove, then curved

upward with the two layers of the fold tightly apposed to surround a round vesicle (Hatschek, 1883,

pi. 5: fig. 57a). The lumen of the vesicle was traversed by spindle-shaped cells. The vesicle was

reported to arise from mesoderm and the fold from

an ectodermal evagination of the esophagus. In

Phascolosoma agassizii the fold does not continue

around the organ, but instead the organ is enclosed

by a muscular sac.

In a study of pelagosphera collected by the Dana

Expedition, Damas (1962:13—14) again described

this organ and called it the "machoire." Damas'

representation of the organ agrees in its essential

aspects with the findings in P. agassizii. He described a transverse groove in the posterior mouth

with a thick cuticle, covered ventrally by a unique

epithelium of tall, cylindrical cells. Beneath the

epithelium was an oblong transverse mass of turgid

tissue composed of muscle fibers and within this

mass there were numerous cavities. The histological

details of the turgid tissue as presented by Damas

may not be entirely reliable since his specimens

were fixed aboard ship in diluted formalin 30 years

prior to his investigation.

Jagersten (1963) observed the organ in living

pelagosphera and referred to it as the "pharyngeal

bulb." Although he noted that it was in a position

at the base of the lip, he gave no information on

the structure of the organ.

The term buccal organ has been used in the

present study because it does not imply a molluscan

homology as does "buccal mass" (Hatschek, 1883),

nor does it attribute a speculative function as is

implicit in the term "machoire" (Damas, 1962).

Jagersten's term, "pharyngeal bulb," is not appropriate, since there is no differentiated pharynx in

the larva and, even if there were, this term would

connote a muscular expansion in the pharyngeal

tube, rather than a saccular evagination.

Investigators have pondered the possible functions of the buccal organ, but thus far no one has

provided a conclusive demonstration of its significance in larval life. Hatschek's (1883:39) only

comment on the function was "Die Function dieses

sogenannten Schlundkopfes ist mir ziemlich rathselhaft geblieben." Although he did not observe the

eversion of the organ, Hatschek noted that pressure on an overlying coverslip resulted in the extrusion of both the esophagus and the "Schlundkopf." Damas (1962) did not examine living larvae,

but he proposed that the organ might act as a

rocker, pushing food accumulated in front of it

back into the esophagus, Jagersten (1963) observed

in the living pelagosphera a "kneading" action of

the buccal organ and he supposed that such an action might serve to break up large pieces of food

into smaller pieces; however, he did not give evidence to support this supposition.

In the present study a cinematographic record of

a larva of Phascolosoma agassizii was made as it

moved along a filamentous diatom, continually

everting the buccal organ. The diatom was of too

great a length to be ingested intact by the larva.

The mouth of the larva was applied to the diatom,

the body arched, and the terminal organ attached

to the substratum. At each eversion of the buccal

organ the head of the larva moved forward. After
NUMBER 1S2 13

a series of eversions the diatom strand was bent

inward, appearing to be directed into the esophagus. In a final eversion the strand was pushed out

of the mouth and the larva abandoned the diatom.

From these observations it would appear that the

buccal organ is not only able to push food into the

esophagus as proposed by Damas (1962), but also

it can reject objects, pushing them away from the

esophagus. The latter action might be of value if

large and extraneous objects adhere to the presumably sticky secretion of the lip gland. The extrusion of the buccal organ, moreover, blocks the

esophageal opening and, if living organisms were

being ingested, this motion might prevent their

escape. Another function, possibly performed by

the continual eversion of the buccal organ against

a substratum, could be the loosening of small

diatoms and debris which would then be swept

into the digestive tract by the directed ciliary movement. Although a function in the feeding process

seems to- be the primary significance of the buccal

organ, it may also play some role in locomotion.

The fact that the head moves forward when the

buccal organ is applied to the substratum suggests

this possibility. On the other hand, the head can

also move forward by means of ciliary activity

alone. A possible sensory function cannot be eliminated even though the heavy cuticle of the organ

makes such a function seem improbable. An innervation has not been demonstrated, but the connectives from the brain are very closely associated

with the organ at either end of the buccal groove

(Plate 12a).

Lip Gland.—In the larva of Phascolosoma agassizii the lip gland, composed of four pendulous

lobes, hangs down from the inner surface of the lip

into the anterior coelom (Plates 36, c, 5, 8a, 6).

The two median lobes of the gland are approximately 50 microns in length and are slightly longer

than the two lateral lobes. Each lobe, suspended

from the lip by a long neck, ends in a bulbous

expansion. At their point of attachment to the lip,

the necks of the lobes surround the lip pore (Plate

8/;). Because of this arrangement it is inferred that

the glands discharge a secretion into the pore.

In larvae up to an age of 20 days each lobe of

the lip gland is composed of a single large cell, but

in later larvae the number of cells increases so that

a single lobe may consist of several cells. Each cell

is characterized by a large nucleus with an exceptionally large nucleolus and a nucleoplasm that

stains deep blue with Richardson's stain. The

cytoplasm is highly vacuolated and penetrated by

a fine reticular network (Plate 86). Large patches

of darkly staining material are scattered through

the cell, sometimes concentrated at the periphery

or around the nucleus, and from these patches

thick strands of similar material radiate into the

surrounding cytoplasm. In larvae that have been

embedded in methacrylate the gland gives a negative reaction to periodic acid-Schiff and alcian blue,

but with safranin O small areas within the gland

stain metachromatically.

The lip gland is derived from ectoderm. At the

beginning of the premetamorphosis stage, four

large cells are distinguishable among the cells of

the ventral wall of the stomodaeum by their large

size, basophilic cytoplasm, and large nuclei each

with a single prominent nucleolus (Plate 9a). As

the cells increase in size the cytoplasm becomes

progressively vacuolated; by the time of metamorphosis the characteristic cytology of the larval gland

has been assumed (Plates 36, c, 9b). At their first

appearance the cells are widely scattered and at

this time the lip pore cannot be identified. The

process by which contact is established between the

gland cells and the lip pore is not understood.

The lip glands were first described by Max Mueller (1850) in a larva which he presumed to belong

to the genus Phascolosoma. The glands were designated as the unpaired organ and described as a

bilobed body opening into a ciliated excretory duct

beneath the introvert. The significance of the organ

was not clear to Mueller, but he suggested that it

was either a secretory gland or a gonad. Later, in

1883, Hatschek depicted the gland as the "Anhangsdruse" in the larva of Sipunculus nudus and reported it to be an esophageal invagination, the cells

at the blind end of which grew toward the coelom

to form the glandular masses. The gland was found

to be composed of two glandular appendages, each

with tall prismatic cells that ended at the lumen

of a ciliated excretory duct. The duct opened near

the posterior wall of the mouth. Mingazinni (1905)

figured the glands in a pelagosphera larva that had

been collected at a depth of 500 meters in the South

Pacific between New Caledonia and New Zealand.

Unaware of Hatschek's work, he believed the glands

to be gonads and therefore considered the animal

to be an adult sipunculan, creating a new genus
14 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

and species, Pelagosphaera aloysii. Senna (1906) in

a study of other planktonic sipunculan larvae,

recognized the glands to be the esophageal evaginations described by Hatschek. In pelagosphera larvae from Monterey Bay, California, Heath (1910)

found similar structures and identified them as

larval glands, rather than gonads. In his examination of 1,900 pelagosphera from the Dana Expedition, Damas (1962) reported a typical bilobed gland

suspended in the coelomic cavity from the end of

a ciliated canal. The canal opened to the exterior

on the ventral surface of the anterior introvert. The

cells of the gland were arranged in the shape of

a fan around a central cavity, a continuation of the

ciliated canal. Akesson's (1961a) description of the

glandular organ of four pelagosphera collected by

the Galathea Expedition agrees essentially with that

of Damas. The term lip gland was proposed by

Jagersten (1963) who was the first to observe that

the duct of the gland opened onto a ventral lobe

of the mouth which he designated as the lip. Jagersten's observations have been confirmed here in the

larva of Phascolosoma agassizii; thus his term, lip

gland, has been adopted in this report.

The structure of the lip gland of P. agassizii

differs from that described in the literature in that

it is divided into four appendages, all of which

converge at a pore on the base of the lip. There is

thus no long "excretory duct" as found in other

pelagosphera and there is no central cavity within

the gland. Rather than numerous cells around a

central cavity, in the early larva of P. agassizii there

are only four cells in the gland, each lobe consisting of a single cell suspended from the region

of the pore.

There is very little information available on the

nature of the glandular secretion. Akesson (1961a)

reported a basophilic staining with the Azan stain

and Damas (1962) demonstrated a weak reaction

with periodic acid-Schiff. In the gland of Phascolosoma agassizii there was no reaction with periodic

acid-Schiff, but scattered patches of cytoplasm manifested an intense basophilia with Richardson's stain.

Areas of the gland were stained a bright orange

with safranin 0, a reaction characteristic of mucus

(Li I lie, 1951:285). The results are confusing, however, in that there was no manifestation of metachromasia with Richardson's stain and there was

no reaction to alcian blue. Further work is necessary before the nature of the secretory product can

be denned. If it should be mucus, then such a

secretion might function in a ciliary-mucus pattern

of feeding or in adhering the ventral surface of the

head to the substratum as the larva moves along in

search of food.

Both the buccal organ and the lip gland are

larval organs with no known fate in the adult. Akesson (1961a) hypothesized that the lip gland is

homologous to the ventral sensory organ of the

adult in the genera Phascolosoma, Siphonosoma,

and Aspidosiphon. The ventral sensory organ has

been described as a tubular organ which opens on

the ventral midline beneath the collar and terminates at the anterior end of the ventral nerve cord.

In some species the termination is expanded into a

cavity with a central statocyst (Akesson, 1958). The

ventral sensory organ in the adult of Phascolosoma

agassizii lias been found in the present study to be

a simple, narrow, nonciliated tube ending blindly

at the anterior end of the ventral nerve cord at the

point of junction of the circumesophageal connectives. In the larva the position of the lip pore is

similar: the connectives from the brain approximate

each other on either side of the lip pore and continue along the sides of the lip groove. The transformation of the larva to the adult form has not

been observed in this study; hence it has not been

demonstrated whether the lip pore and lip groove

of the larva are in any way related to the sensory

organ of the adult.

NERVOUS SYSTEM

The central nervous system of the larva of Phascolosoma agassizii consists of a cerebral ganglion,

circumesophageal connectives, and an unsegmented

ventral nerve cord.

The cerebral ganglion or brain is located dorsally

in the head in close association with the overlying

epidermis (Plate 1 la, b). Localized in the most

dorsal portion of the brain are small nuclei with

dense chromatin granules and a lightly staining

nucleoplasm. Ventral to the nuclei is a mass of

cytoplasmic processes, the neuropile of the brain

(Plate Mb). Two pairs of nerves originating from

the neuropile have been identified: one pair extends to the dorsoanterior surface of the head and

another pair to the esophagus. Embedded laterally

near the surface of the head in the posterior portion

of the brain is a pair of eyes, each composed of
NUMBER 132 15

a hemispherical pigment cup which opens in an

anterolateral direction (Plate lib, d). The optic

nerve has not been identified.

The circumesophageal connectives leave the

brain near the level of the eyes and extend ventrally

to either side of the ciliated channel of the mouth

(Plates 12a, 13a). They pass posteriorly along the

channel, then laterally around the buccal groove

and, finally, ventrally on either side of the lip pore

and lip groove. Beyond this point they become very

thin and their union has not been observed in

younger larvae. The connectives are characterized

by a few fibers and many small nuclei, similar to

those of the brain. Usually intimately associated

with the epidermis or esophagus, the circumesophageal connectives are not well-defined structures.

The circumesophageal connectives are joined at

the distal end of the lip by the ventral nerve cord

(Plates 3b, c, 5, 8b). Up until the age of one month

the nerve cord is double throughout most of its

length. In the region of the thorax it is very slender,

composed of two longitudinal rows of nuclei joined

by a transverse, slightly thickened mesentery which

probably represents the neuropile. The two rows

of nuclei are attached by mesenteries to the peritoneum of the body wall and the lip glands. The

nerve cord is thickened in the trunk region where

it lies within the epidermal layer of the body wall

as two longitudinal cords (Plates \2b-d, 13c). The

neuropile of each cord is dorsal or dorsomedial to

the nuclei or in some regions the fibers of the

neuropile may be scattered among the nuclei. The

separation between the two neuropiles is not always

distinct in the most anterior regions, but posteriorly

the neuropiles are clearly separate.

At the age of two months in most larvae the

nerve cord has united. In the thorax it is bilobed,

the two lobes joined at their bases with nuclei in

a lateral position on the outer side of each lobe

(Plate 13<i). In the trunk the two longitudinal

cords have fused to form a single cord, suspended

by a thickened peritoneum. The nuclei of the cord

are ventral and the neuropile dorsal as in the adult.

Posteriorly the nerve cord becomes extremely thin

and it has not been possible to trace it to the posterior end of the trunk.

The central nervous system is ectodermal in

origin. The rudiments of the brain are formed

from the lateral cells of the apical plate by an

inward proliferation in the early trochophore and

by the end of the trochophore stage the brain rudiments are well developed (Plate lla). The cells of

the brain surround the rosette cells and extend

downward to the prototroch, forming the sides of

the apical cavity. Beneath the cavity the lateral

brain rudiments are united by a central commissure, the future neuropile. The nuclei of the brain

rudiments are small and ellipsoidal, measuring approximately 4x5 microns, with prominent chromatin granules and very little surrounding

cytoplasm. When the mouth opens at metamorphosis the ventral portion of the brain is pushed

into a more dorsal position, the apical cavity is

obliterated, the rosette cells degenerate, and the

two lateral lobes of the brain are united.

The pigment granules of the eye appear late in

the trochophore stage in a dorsolateral cell on

either side of the apical plate. The two cells are

located directly above the prototroch at the external surface beneath the egg membrane. During

the premetamorphosis stage the granules increase

in number and assume the form of a cup with the

opening directed posterolaterally. At metamorphosis the opening of the cup is turned in an

anterolateral direction, retaining its position near

the surface.

The circumesophageal connectives appear to

originate from the brain, growing down on either

side of the esophagus. By the time of metamorphosis

the connectives are complete.

The nerve cord is proliferated inward from the

ventral ectoderm posterior to the stomodaeum. At

the end of the trochophore stage a few scattered

cells, very similar in cytology to those of the brain,

make their appearance in this position. In the premetamorphosis stage at the time of post-trochal

elongation the ventral nerve cord is clearly visible

as two loosely arranged rows of small nuclei near

the ventral surface of the developing embryo. The

two rows are separated posteriorly by epidermal

cells, but in the region beneath the stomodaeum the

cells are more scattered and there is no distinct

separation. The neuropile has not been identified in

prelarval stages.

In all other studies on sipunculans the ventral

nerve cord has been reported to arise from a median

unpaired thickening of the ectoderm of the trunk,

beginning in the anterior trunk region and progressing posteriorly (Hatschek, 1883; Gerould,

1907; Akesson, 1958, 1961a). In Phascolosoma agas-
16 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

sizii the development of the ventral nerve cord

begins as a ventral ectodermal proliferation of

scattered cells in the anterior trunk region, but it

differs from other sipunculans in that it progresses

posteriorly as two separate longitudinal cords. By

the time the larva has reached the age of 2 months,

the two cords have united.

Gerould (1907) reported a transitory metamerism

of the nerve cord and the mesoblasts into two to

four segments. Later in letters to W. K. Fisher and

G. Pickford, he repudiated this report, explaining

the apparent metamerism to be the result of larval

contraction (Hyman, 1959:657). Neither Hatschek

nor Akesson found any evidence for segmentation

in the ventral nerve cord, nor was any such evidence found in Phascolosoma agassizii.

Both Gerould (1907) and Hatschek (1883)

asserted that the circumesophageal connectives were

formed by a bifurcation and forward growth of

the anterior end of the ventral nerve cord. Akesson

(1958) was unable to determine whether the connectives originate from the brain or the ventral

nerve cord. Because in Phascolosoma agassizii the

connectives appear before the nerve cord is well

developed, it is suggested that they arise from the

brain.

Eye spots similar to those of Phascolosoma agassizii

have been described in the embryos of Golfingia

vulgaris, Phascolopsis gouldi (Gerould, 1907), and

Golfingia elongata (Selenka, 1875). The eye spots

as figured by Gerould are of the inverse type, unlike

the everse adult eye which is embedded within the

brain at the inner end of an ocular tube. In the

larva of Phascolosoma agassizii it has not been

possible to demonstrate whether the larval eyes are

inverse or whether they are rudiments of the adult

eyes. Akesson (1958) observed rudimentary ocular

tubes in the larvae of Phascolion strombi and

Golfingia minuta, but he did not find pigmented

eye spots in these species. Two pairs of eye spots,

one large and one small, have been reported in the

larva of Sipunculus nudus (Hatschek, 1883) and in

unidentified pelagosphera (Senna, 1906; Jagersten,

1963). In Sipunculus nudus one pair of eye spots

appeared in the early gastrula and a second in the

older larva. Akesson (1958) hypothesized that the

first pair might be the inverse type and the second

the everse eye characteristic of the adult.

NEPHRIDIA

In the larva of Phascolosoma agassizii there is a

single pair of nephridia, each member of the pair

with a U-shaped ciliated canal opening at one end

to the exterior by way of an epidermal pore and at

the other end to the coelom through a ciliated

funnel (Plate lOd). The external pores are situated

in a ventrolateral position on either side of the

ventral nerve cord, slightly anterior to the level of

the anus. From their point of attachment in the

region of the pore the nephridia are suspended

freely within the coelom, usually extending posteriorly, although they may be pushed anteriorly

by the movement of the gut or the introversion of

the head (Plate 4b). At the age of one month the

nephridia measure approximately 40 microns in

length.

The arm ol the nephridium that is associated

with the funnel is narrower in diameter than the

arm that terminates at the external pore, the

difference being more marked in the region of the

external openings (Plate Id). The nephridium is

four cells in width, with two cells surrounding the

funnel canal and two cells around the pore canal.

The number of cells along the length of the

nephridium increases with its growth. The cells of

the pore arm are characterized by green granules,

often enclosed within large vacuoles. The cytoplasm

of both the pore and funnel arms is densely concentrated with mitochondria and the nuclei are

large with finely dispersed chromatin and prominent

nucleoli (Plate Id). The nuclei of the cells of the

funnel are small and resemble those of the

peritoneum that surrounds the remainder of the

nephridium.

The rudiments of the nephridia have been

identified in the premetamorphosis stage at the

time of coelom formation (Plates Id, 10a). A

nephridial rudiment consists of two enlarged cells

located ventrally within the mesoderm bands in

the middle of the post-trochal region of the developing embryo. The distinctive nuclei are large

and lobate with a lightly staining nucleoplasm and

one or two prominent nucleoli. In the premetamorphosis stage the somatic and splanchnic bands

of mesoderm split anteriorly and posteriorly to the

nephridial rudiment to form the coelom, leaving

the nephridial cells as a bridge connecting

endoderm and ectoderm (Plates 2d, 3b, 10a, b). At
NUMBER 132 17

the time of metamorphosis the nephridial cells are

separated from the gut, but retain their connection

with the body wall as they are overgrown by cells

of the somatic peritoneum (Plate 10c). It appears

probable that the more interior nephridial cell

gives rise to the funnel arm of the nephridium and

the more exterior cell to the pore arm. A proliferation of small cells within the epidermis joins the

outer nephridial cell to form the external pore,

and cells resembling those of the peritoneum surround the nephridium to form the funnel. The

pores and the funnels were not observed until

several days after metamorphosis.

There is no protonephridium apparent at any

time during the development of Phascolosoma

agassizii and the larval nephridium, as described

above, undoubtedly becomes the definitive nephridium of the adult. The exact origin of the larval

nephridium has not been conclusively determined.

The position of the nephridial cells in the mesoderm bands and the relationship of the developing

nephridium to the coelom suggest that the nephridium of Phascolosoma agassizii is mesodermal in

origin, growing out from the coelom toward the

periphery. The source of the nephridial cells,

however, has not been traced and the possibility of

their migration inward from the ectoderm cannot

be excluded. Moreover, the exact derivation of the

cells of the coelomic funnel is not known, and it is

possible that the funnel is formed from coelomic

epithelium as a separate structure and at a later

stage than the tubular portion of the nephridium.

In the literature on sipunculan development

there are discrepancies concerning the mode of

formation of the nephridium. Hatschek (1883)

reported a purely mesodermal origin of the

nephridium of Sipunculus nudus. He was able to

recognize two nephroblasts within the mesoderm

at a very early stage by their distinctive yellow

pigmentation. The division of each yellow cell

was found to give rise to a U-shaped chain of cells

with a central lumen opening at one end through

the epidermis and at the other into the coelom. The

anlage of the coelomic funnel he reported to be

composed of cells of the peritoneal lining. The

reported origin of the nephridia of Sipunculus

nudus from cells in the mesoderm bands is thus

similar to that observed for Phascolosoma agassizii.

In the species studied by Akesson (1958) and

Gerould (1907), the nephridium was not observed

at its inception because of large amounts of coelomic

yolk. Both authors assumed, nevertheless, that the

nephridium proper was formed as an ectodermal

invagination and that the funnel arose from peritoneum. Akesson'-s assumption of an ectodermal

participation in nephridial formation was based on

the morphology of the larval nephridium in

Golfingia minuta. The central cavity of the

nephridium in this larva is lined with a cuticle that

is continuous with that covering the body wall.

Akesson supposes, therefore, that the cells surrounding the cavity are ectodermal and that the

more peripheral cells and the cells of the funnel

are derived from mesoderm.

The observations of Akesson and Gerould, although incomplete, suggest that the nephridia of

the species they studied are composite structures,

conforming to the concept of mixonephridia by

Goodrich (1945). Observations on Phascolosoma

agassizii and Sipunculus nudus, on the other hand,

do not rule out the possibility that the nephridial

structure may be a coelomoduct, derived entirely

from entomesoderm. In view of the information

available to him from the studies of Hatschek

(1883) and Gerould (1907), Goodrich (1945)

assumed the sipunculan nephridium to be a

mixonephridium. Further evidence on the exact

source of the "nephroblasts," the derivation of the

coelomic funnels, and the comparative cytology of

the cellular components is now necessary to clarify

the nature of the nephridia of the Sipuncula.

LARVAL GLANDS

The glands in the larva of Phascolosoma agassizii

are designated as the lip glands, the posterior

sacciform glands, the epidermal glands, and the

glands of the head. The lip glands have been

discussed previously as an associated structure of

the mouth.

Posterior Sacciform Glands.—The posterior sacciform glands are thus termed because of their

shape and their position in the posterior half of

the larva. There are two glands, one on either side

of the anus in a dorsolateral position (Plate 46).

The glands are cellular sacs, composed of a single

layer of cells surrounding a central cavity that opens

to the exterior through a wide aperture in the body

wall (Plate 146, c). In the larva of one month of
18 SMITHSONIAN CONTRIBUTIONS TO ZOOLOCY

age each gland projects into the coelom to a length

of approximately SO microns.

The cells of the gland are of two different types:

one presumed to be secretory and the other supportive. The nucleus of the secretory type is round,

approximately 4.5 microns in diameter, with a

homogeneous, darkly staining nucleoplasm and a

very large, eccentric nucleous (Plate 146). There

is only one such cell in each sacciform gland and

it is located at the rounded extremity of the gland.

The cytoplasm of this cell is thicker than that of

the other cells of the gland and it is intensely

basophilic. The nucleus of the second cell type,

the supportive cell, is smaller, measuring 2x3

microns, with fine chroma tin granules. Many cells

of this type form the wall of the gland surrounding

the central cavity. The cytoplasm of the cells, also

basophilic, becomes increasingly thin as the central

cavity enlarges and the gland increases in size. The

central cavity is filled with tightly packed globules,

the secretory product of the gland. These globules

are stained a light blue with Richardson's stain, but

they are not stained by alcian blue or periodic acidSchiff. Hence, the chemical nature of the secretion

has not been determined and the possible function

of the sacciform glands in larval life remains

enigmatic.

The posterior sacciform glands develop from

ectoderm late in the premetamorphosis stage. An

ectodermal cell on either side of the rectal rudiment

increases in size; the cytoplasm of the cell becomes

basophilic and the nucleus moves into a position

toward the coelom (Plate 14a). In that part of the

cytoplasm nearest the body wall, small globules,

similar to those later found in the central cavity

of the gland, make their appearance. This cell is

identical to the cell at the extremity of the mature

gland, and it is presumed to be the secretory element of the gland. Smaller ectodermal cells are

proliferated around the base of the larger cell, and

as the gland grows these cells are extended to. form

the walls of the gland, surrounding the central

cavity. The aperture through the body wall is

completed a few days after metamorphosis.

Glands such as the posterior sacciform glands in

the larva of Phascolosoma agassizzi have not been

described in any other species of sipunculans.

Epidermal Glands.—There are three pairs of

epidermal glands in the larva at 20 days of age

(Plate I4d). Located in dorsolateral positions on

the trunk, one pair is near the middle of the body

and two additional pairs are relatively close

together in a more posterior region. In living

larvae each gland appears as a small epidermal

papilla from which a very fine bristle, 5 to 10

microns in length, protrudes to the exterior. In

sectioned material the glands are found to be

pyriform in shape, the more pointed end leading to

a narrow canal which passes through the larval

cuticle. The rounded base of the gland, covered by

peritoneum, projects for a distance of approximately 10 microns into the coelom. The gland is

comprised of a cluster of small epidermal cells surrounding a diminutive cavity which opens into the

cuticular canal. Within the cavity is a homogeneous

secretory material, staining light blue with Richardson's stain, and from the base of the cavity the

bristle extends outward through the canal. In

contrast to the other epidermal cells, the cytoplasm

of the gland cells is scant and the nuclei are

characterized by fine chromatin granules and an

absence of nucleoli.

The epidermal glands are derived from ectodermal

proliferations, first evident late in the premetamorphosis stage. The most anterior pair of glands, the

earliest to appear, is fully developed with extended

bristles at the end of metamorphosis. By 17 days

bristles are also apparent on the two posterior pairs

of glands. All of the bristles have vanished in larvae

older than one month and many additional

epidermal glands have formed without bristles

(Plate 13d).

Epidermal glands similar to those in Phascolosoma

agassizii have been described in all studies of

sipunculan larvae; however, associated bristles have

been reported only in Golfingia elongata (Selenka,

1875; Akesson, 1961b) and Phascolion strombi

(Akesson, 1958). In Phascolion strombi, studied by

Akesson (1958) at Kristineberg, the bristles were

not arranged in pairs, but rather in six circlets,

each circlet with four bristles. Golfingia elongata,

also from Kristineberg, showed the same arrangement (Akesson, 1961b), but larvae of the same

species at Roscoff entirely lacked bristles (Gerould,

1907; Akesson, 1961b) and those at Nice possessed

only three pairs (Selenka, 1875). In an effort to

explain this variation among different populations

of the same species, Akesson (1961b) pointed out

that Golfingia elongata at Kristineberg lived at

depths of 30 meters, whereas those at Roscoff in-
NUMBER 132 19

habited the intertidal zone. Such a correlation

between depth of habitat and presence of bristles

was not found in the present study in which larvae

from an intertidal population of Phascolosoma

agassizzi were observed to possess three pairs of

bristles.

Akesson (1958) speculated on the functional

significance of the glands and bristles, suggesting

that the secretion of the gland in Phascolion strombi

might aid in dissolving the holes which the larvae

inhabited in gastropod or scaphopod shells, or that

it might have a protective effect against enemies.

Although he was unable to demonstrate a sensory

element in the gland, Akesson proposed that the

bristles might be tactile organs. In the study of

Phascolosoma agassizii neither sensory cells nor

sensory nerves could be demonstrated in the glands;

however, larval nerves are very difficult to trace

and sensory cells might not have been recognized,

therefore it cannot be concluded that a sensory

element does not exist. Epidermal organs in the

adult of this species have a sensory function as well

as secretory, and it is not unlikely that the organs

of the larva might also have a dual function.

Glands of the Head.—There are many different

glands in the head of the larva of Phascolosoma

agassizzi. Some of the glands secrete a material

that stains with periodic acid-Schiff, in others

neurosecretory granules can be demonstrated, and

in still others the secretion has not been characterized.

Opening on the ventrolateral surface of the head

are two pairs of long, thin glands, approximately

7 microns in width, packed with small globules that

stain very intensely with periodic acid-Schiff (Plate

llrf [g1, g-]). One pair opens directly above the

lateral extremities of the buccal groove, passing

posteriorly along either side of the esophagus for a

distance of 40 microns. A second pair, somewhat

shorter, opens more anteriorly and extends into the

head coelom. Also staining with periodic acid-Schiff

is a third pair of smaller, club-shaped glands, approximately 15 microns in length, that opens

farther anteriorly on the ventral surface of the

head (Plate \\d [g3]). These glands are filled with

loosely packed globules, smaller than those of the

long, thin glands. The secretion of these three

pairs of glands is discharged onto the ventral surface of the head and may aid in the trapping of

food particles which would then be swept by ciliary

action into the esophagus.

Neurosecretory granules have been demonstrated

in cells in the brain and in cells along the sides of

the esophagus. After fixation in osmium tetroxide

and embedding in methacrylate the larvae were

sectioned at 1 micron and stained in paraldehyde

fuchsin by the method of Clark (1955). Prominent

neurosecretory granules were stained a very deep

red. In larvae embedded in Epon and stained with

Richardson's stain, very large cells in the brain and

along the esophagus were assumed, because of their

position and cytology, to be the neurosecretory

cells of the methacrylate-embedded material. The

cytoplasm of these large cells was intensely

basophilic with vacuolar inclusions and the nucleus

contained a prominent nucleolus Plates 8b, lid

[g5])-

There are other glands with an unidentified

secretion that are apparently unicellular and open

to the exterior. The secretion, foamy in appearance, does not stain with Richardson's stain,

periodic acid-Schiff, or paraldehyde fuchsin. One

pair opens on the ventral surface of the head between the apertures of the long, thin glands described above, and the others open on the anterior

head along the ventral boundary of the brain (Plate

llr, d[g*]).

The origin and fate of the glands of the larval

head in Phascolosoma agassizii are not known. In

the larvae of Phascolion strombi, Golfingia minuta,

and Golfingia elongata, Akesson (1958, 1961b)

described a larval cerebral organ, derived from the

rosette cells, with a secretion characterized in the

first two species by basophilic, argentaffine staining

properties. The more proximal cells of the organ

are eventually incorporated into the brain, whereas

the remainder form the cerebral organ of the adult.

The cells that are incorporated into the brain are

not secretorily active in the adults of these species;

however, Akesson (1961b) points out that cells in

the same position in the brain of the adult

Sipunculus nudus are neurosecretory and he assumes, because of their identical position, that they

are homologous to the incorporated cells of other

species. From these observations and assumptions,

Akesson concludes that primitive epidermal secretory cells transform into neurosecretory cells within

the brain. Since Akesson did not examine the larva

of Sipunculus nudus and Hatschek (1883), in his
20 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

study of the larva of this species, did not describe

a larval cerebral organ, further observations on the

larva are necessary before Akesson's conclusion can

be confirmed. In the larva of Phascolosoma agassizii

no distinct cerebral organ was found, but neurosecretory cells, of uncertain origin, were already

present within the brain of the larva. Whether

other glandular cells later transform into additional

neurosecretory cells has not been determined in the

present study.

TERMINAL ORGAN

The terminal organ is a retractable appendage

located at the posterior end of the larva (Plates

5c, 4a,c,e,f, \-ie,f). Usually a bulb-shaped organ, 25

microns at its widest dimension and 50 microns in

length, the terminal organ may be elongated to a

length of 170 microns. The function of the organ

is the attachment of the larva to the substratum.

When the larva is swimming or lying unattached

on the substratum the organ may be retracted

within the body.

Withdrawal of the terminal organ into the

posterior portion of the trunk is accomplished by

the contraction of two retractor muscles. These

muscles originate within the terminal organ and

insert dorsally on either side of the dorsal midline

at the level of the middle pair of epidermal glands

(Plate 14/). Each retractor muscle is composed of

six fibers and differs from the retractor muscles of

the head, each of which has eight fibers.

The terminal organ is covered by a thin cuticle,

a continuation of the cuticle of the trunk. The

cuticle at the posterior tip of the organ is interrupted by an aperture into which several gland

cells open (Plate 14e,/). Gland cells, vacuolar in

appearance, are not stained by Richardson's stain,

periodic acid-Schiff, or alcian blue. Even though

the secretion has not been characterized by its

staining properties, it is assumed that it is adhesive

in nature because of the attachment of the larva

to the substratum by means of the terminal organ.

The terminal organ and its retractor muscles

form from an ectodermal proliferation, first observed during the premetamorphosis stage in a

ventral position near the posterior tip of the body.

An invagination appears in the egg membrane

opposite the ectodermal proliferation and at the

time of metamorphosis the ectodermal thickening

is pushed out through the invagination, taking

with it the surrounding cuticle, to form the terminal organ (Plate 14e). The cuticle at the posterior

tip of the organ is ruptured, thus creating the

aperture through which the secretion is discharged.

At the end of metamorphosis the larvae settle on

the substratum, an indication that the gland cells

have become active in secretion.

A terminal organ has been reported in other

benthopelagic or pelagic sipunculan larvae. In

the larva of Sipunculus nudus Hatschek (1883)

described an ectodermal thickening to which bristles

were attached. The thickening was connected internally to the ventral nerve cord by thin fibers and

he supposed it to be a tactile organ. Usually retracted, the organ was only rarely observed to be

extended. Akesson (1961a) described a similar

organ in unidentified, preserved pelagosphera

larvae and noted the similarity to the terminal

organ of adult species of Sipunculus. In the larvae

of an intertidal population of Golfingia elongata,

Akesson (1961b) found a terminal glandular organ,

but in a deep-water population of the same species

at Kristineberg he did not find such an organ.

Larvae in the former population were observed to

adhere to the substratum by means of the organ,

but no mention was made of retractility. In more

than half of the species of pelagosphera that

Jagersten (1963) examined, he observed a retractile

"tail," considered to be identical with the terminal

organ described by Akesson (1961a). Jagersten

reported an odd behavior in which the "tail" was

placed into the mouth possibly, he speculated, to

discharge a secretion. In Phascolosoma agassizii a

similar behavior was observed whereby the body

was bent so that the terminal organ touched the lip

or else was pushed past the head (p. 5). This

action was most frequently noted when there was

no substratum on which the larva could settle, and

what role, if any, it served in normal behavior was

not determined. The primary function of the

terminal organ in this species is attachment of the

larva to the substratum. It would be of interest to

ascertain whether the pelagosphera studied by

Jagersten ever use the terminal organ under natural

conditions to attach to a substratum, or to some

debris floating among the plankton. If not, then

the behavior that Jagersten reports might be explained as an adaptation to planktonic life and the
NUMBER 132 21

terminal organ in such forms may serve an entirely

different function from that of attachment.

Comparisons with Other Phyla

The phylum Sipuncula with spiral cleavage and

a trochophore larva is, by definition, a member of

the Protostomia. Close affinities with both annelids

and mollusks are apparent from developmental

studies and much of this evidence has been reviewed in detail by Hatschek (1883), Gerould

(1907), Akesson (1958), and Clark (1969).

Comparisons of cuticle formation reveal that in

many polychaetous annelids, as in sipunculans, the

egg envelope may undergo a transformation into

the larval cuticle. Such a transformation is found

in the sipunculan species Golfingia elongata,

Phascolion strombi, Golfingia minuta (Akesson,

1958; 1961b), Phascolosoma agassizii, Golfingia

pugettensis, and Themiste pyroides (Rice, 1967)

and in numerous polychaetes, some of which are

Clymenella torquata, Arenicola cristata (Wilson,

1892), Pomatoceros triqueter (Segrove, 1941), Nereis

diversicolor (Dales, 1950), Diopatra cuprea and

Autolytus fasciatus (Allen, 1959; 1964).

A larval structure, the metatrochal band of cilia,

common to all pelagosphera larvae of sipunculans,

is homologous in sipunculans and polychaetes, as

indicated by the cell lineage studies of Gerould

(1907). In contrast to that of most polychaetes,

however, the metatroch of the pelagosphera is the

primary locomotory organ and at the time of its

formation, or soon thereafter, the prototroch is

lost or diminished. In Sipunculus nudus the prototroch is unique in that it completely envelops the

embryo. This structure has been compared by

Gerould (1907) to a similarly modified prototroch

that constitutes the velum of some aplacophorans

and primitive lamellibranchs.

A protonephridium, found in the trochophores

of the many polychaetes and some mollusks, is

lacking in sipunculans. The larval nephridium of

the sipunculan, a U-shaped tubular structure opening at one end to the exterior and at the other to

the coelom, becomes the nephridium of the adult

with no essential change. Its precise derivation has

yet to be determined, but studies of Phascolopsis

gouldi, Golfingia vulgaris (Gerould, 1907) and

Golfingia minuta (Akesson, 1958) suggest that it is

a composite structure, formed from ectoderm

and coelomic epithelium. In Sipunculus nudus

(Hatschek, 1883), however, and in Phascolosoma

agassizii a purely mesodermal origin is suggested,

but not proved. Goodrich (1945) considered the

sipunculan nephridium to be a mixonephridium, a

structure characteristic of most polychaetes but not

of mollusks.

The nervous system develops similarly in

sipunculans and annelids and is comprised in both

of a cerebral ganglion, circumesophageal connectives, and a ventral nerve cord. The nerve cord

develops as a ventral ectodermal thickening which

is double in annelids, but in most sipunculans is

median and single. An exception is the species

Phascolosoma agassizii in which the nerve cord is

paired at its inception, although later it is united

into a single cord. Gerould (1907) considered the

unpaired ventral nerve cord to be a primitive

feature, relating the sipunculans to primitive

annelids such as Polygordius. Akesson (1958) took

an opposite view, pointing out that among the

turbellarians, annelids, and mollusks the orthogonal

nervous system of turbellarians should be considered the primitive one and that the nerve cord of

sipunculans therefore was not a primitive but

rather a derived feature. The rudimentary, paired

nerve cord in Phascolosoma agassizii is interpreted

in this study as a reminiscence of a double nerve

cord that probably appeared in the development of

the ancestors of extant sipunculans, relating the

sipunculans more closely to the annelidan stem.

The anus of the sipunculan larva does not arise

at the posterior region of the blastopore as in most

polychaetes, but rather it arises at the site of a

dorsal ectodermal proliferation, the rectal rudiment.

In a few polychaetes, such as Hydroides (Shearer,

1911), the anus develops similarly.

A striking and important difference from the

annelids is the complete lack of segmentation of the

nerve cord and the mesoderm. Although Gerould

in 1907 described a temporary division of the nerve

cord and mesoderm of Phascolopsis gouldi into two

to four segments he later retracted this report

(Hyman, 1959:657). No other investigator has noted

any indication of mesodermal or nervous segmentation in sipunculan development. The arrangement

of epidermal bristles into several circlets in the

larvae of Golfingia elongata and Phascolion strombi

is regarded by Akesson (1958, 1961a) as evidence of

a transitory metamerism. The papillae from which
22 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

the bristles arise, however, are ectodermal and there

is no involvement of the mesoderm. The bristles, a

secretion of the papillae, are readily dissolved by

fixatives and are in no way homologous to the

chaetae of polychaetes.

Jagersten (1963) noted the likeness of the lip of

the pelagosphera to the foot of a gastropod larva.

Previously Gerould (1907) had suggested a possible

homology of the lip gland (glandular organ) and

the buccal organ ("Schlundkopf") to two similarly

placed invaginations in the veligers of chitons: the

pedal gland and radular sac. These organs, however, have not been observed in the lecithotrophic

pelagosphera which are assumed to represent a more

primitive development (Rice, 1967); hence, it

seems probable that they are adaptations of the

planktotrophic larva for feeding activity.

Summary

The pelagosphera larva of Phascolosoma agassizii,

as observed from living specimens reared in the

laboratory, is characterized by a ventrally ciliated

and bilobed head, a thorax with prominent metatroch which, along with the head can be withdrawn

into the body, an elongated, wedge-shaped trunk

which in older larvae is marked by characteristically

papillated cuticle, and a posterior terminal organ.

The larva attaches to the substratum by the terminal organ and in this position it feeds on bottomdwelling diatoms and debris. It is able to release

itself from the substratum and swim freely through

the water by means of the metatrochal circlet of

cilia. Larvae were not observed to transform into

the adult form.

In a histological study of the structure, origin,

and development of larval organs, the following

observations were made: The stomodaeum is

formed at the site of the closed blastopore and the

proctodaeum arises later as a new formation in a

dorsal position. The coelom results from the

splitting of the mesoderm layers and the retractor

muscles, originating from ectomesoderm, make their

appearance soon alter the formation of the coelom.

At the time of metamorphosis of the trochophore

into the pelagosphera larva the egg envelope is

transformed into the elastic, extensible cuticle of the

larva. The nephridium arises from two conspicuous

cells within the mesoderm bands. The ventral

nerve cord, derived from trunk ectoderm, is double

at its inception, but by the age of 2 months the two

longitudinal cords have united. There are three

pairs of epidermal glands with bristles and, in

addition, there is one pair of posterior sacciform

glands opening to the exterior on either side of the

anus. Two organs of the mouth, the buccal organ

and the lip gland, previously described only in the

larva of Sipunculus nudus and in unidentified

pelagosphera larvae, are present in the pelagosphera

larva of Phascolosoma agassizii.

Finally, the histogenesis of selected structures in

sipunculans shows many similarities to the polychaetous annelids and, to a lesser extent, to the

mollusks.

Literature Cited

Akesson, B.

1958. A study of the Nervous System of the Sipunculoideae with Some Remarks on the Development of

the Two Species Phascolion strombi Montagu and

Colfingia ininuta Keferstein. Undersdkningar over

Oresund, 38:1-249. 105 figures. Lund: C. W. K.

Gleerup.

1961a. Some Observations on Pelagosphaera Larva. Galathea Report, 5:7-17, figures 1-11.

1961b. The Development of Golfingia elongata Keferstein

(Sipunculidea) with Some Remarks on the Development of Neurosecretory Cells in Sipunculids.

Arkiv for Zoologi, series 2, 13 (2) :511-531, figures

1-11.

Allen, M. J.

1959. Embryological Development of the Polychaetous

Annelid, Diopatra cuprea (Bosc). Biological Bulletin, 116:339-361, figures 1-30.

1964. Embryological Development of the Syllid, Autolytus fasciatus (Bosc) (Class Polychaeta) . Biological

Bulletin, 127:187-205, figures 1-45.

Bennett, H. S., and J. Luft

1959. S-collidine as a Basis for Buffering Fixatives.

Journal of Cell Biology, 6:113-117.

Clark, R. B.

1955. The Posterior Lobes of the Brain of Nephthys and

the Mucus Glands of the Prostomium. Quarterly

Journal of Microscopical Science, 96:545-565.

1969. Systematics and Phytogeny: Annelida, Echiura,

Sipuncula. Chapter 1, pages 1-68, in Marcel Florkin

and Bradley T. Scheer, editors, Chemical Zoology,

Volume IV. New York and London: Academic Press.

Clement. A. C, and J. N. Cather

1957. A Tcchnic for Preparing Whole Mounts of Veliger

Larvae, Biological Bulletin, 113:340.

Dales, R. P.

1950. The Reproduction and Larval Development of

Xereis diversicolor (O. F. Miiller). Journal of the
NUMBER 132 23

Marine Biological Association of the United Kingdom, 29:321-360.

Damas, H.

1962. La collection de Pelagosphaera du "Dana." Dana

Report, 59:1-22, 10 figures.

Dawydoff, C. N.

1930. Quelques observations sur Pelagosphaera, larve de

sipunculide des cotes d'Annam. Bulletin de la

Socie'te de Zoologique de France, 55:88-90.

Fisher, W. K.

1947. New Genera and Species of Echiuroid and Sipunculoid Worms. Proceedings of the United States

National Museum, 97 (3218):351-372.

Gerould, J. H.

1903. Studies on the Embryology of the Sipunculidae, I:

The Embryonal Envelope and its Homologue.

Mark Anniversary Volume, pages 439-452, 1 plate.

New York.

1907. Studies on the Embryology of the Sipunculidae, II:

The Development of Phascolosoma. Zoologische

Jahrbu'cher, Abteilung t'iir Anatomie und Ontogonie der Thiere, 23:77-162, plates 1-11.

Goodrich, E. S.

1945. The Study of the Nephridia and Genital Ducts since

1895. Quarterly Journal of Microscopical Science,

86 (342-344): 113-392, figures 1-100.

Hall, J. R., and R. S. Scheltema

1966. Morphology of North Atlantic Sipunculid Larvae,

(Abstract). American Zoologist, 6(3):338.

Hatschek, B.

1883. Ueber Entwicklung von Sipunculus nudus. Arbeiten

aus dem Zoologischen Institute der UniversitatWien und der Zoologischen Station in Triest, 5:61-

140, plates 1-6.

Heath, H.

1910. Pelagosphaera, a Larval Gephyrean. Biological Bulletin, 18:281-284, figure 1.

Hyman, L. H.

1959. The Invertebrates: Smaller Coelomate Groups.

Volume 5. New York: McGraw-Hill.

Jagersten, G.

1963. On the Morphology and Behaviour of Pelagosphaera Larvae (Sipunculoidea). Zoologiska Bidrag

Fran Uppsala, 36:27-35, figures 1-2.

Krohn, A.

1851. Ueber die Larve des Sipunculus nudus, nebst vorausgeschickten Bemerkungen iiber die Sexualverhaltnisse der Sipunculiden. Archiv fur Anatomie,

Physiologie, und wissenschlaftliche Medizin, 368-

379. 1 plate.

Lillie, R. D.

1954. HistopatUologic Technic and Practical Histochemistry. 501 pages. New York: The Blakiston Company, Inc.

Luft, L. H.

1961. Improvement in Epoxy Resin Embedding Methods.

Journal oj Biophysical and Biochemical Cytology,

9:409-414.

Mingazinni, P.

1905. Un Gefireo pelagico: Pelagosphaera Aloysii n. gen.,

n. sp. Rendiconti delle sedute solemn delta R. Accademia Nacionale dei Lincei, 14:713-720, figures

1-2.

Mueller, Max

1850. Ueber eine den Sipunculiden verwandte Wurmlarve.

Archiv fur Anatomie, Physiologie, und wissenschlaftliche Medizin, 438-451, 1 plate.

Murina, V. V.

1965. Some Data on the Structure of Pelagospheres—

Sipunculid Larvae. Zoologicheskii Zhurnal [in

Russian], 44:1610-1619, figures 1-8.

Rice, M. E.

1967. A Comparative Study of the Development of

Phascolosoma agassizii, Golfingia pugettensis, and

Themiste pyroides with a Discussion of Developmental Patterns in the Sipuncula. Ophelia, 4:143-

171, figures 1-4.

Richardson, K. C, L. Jarett, and E. H. Finke

1960. Embedding in Epoxy Resins for Ultrathin Sectioning in Electron Microscopy. Stain Technology, 35:

313-323.

Scheltema, R. S., and J. R. Hall

1965. Trans-oceanic Transport of Sipunculid Larvae Belonging to the Genus Phascolosoma. (Abstract).

American Zoologist, 5 (2) :216.

Scgrove, F.

1941. The Development of the Serpulid Pomatoceros

triqueter L. Quarterly Journal of Microscopical

Science, 56:543-590.

Selenka, E.

1875. Eifurchung und Larvenbildung von Phascolosoma

elongatum (Kef.) Zeitschrift fsir wissenschlaftliche

Zoologie, 25:442-450, 2 plates.

Senna, A.

1906. Sulla struttura di alcune larve (Pelagosphaera) di

Sipunculidi. Pubblicazioni del (R.) Instituto di

Studio superiori practici e di Perfezionamento in

Firenze; Sezione de Scienze Fisichee Naturale, 50-78.

Shearer, C.

1911. Development and Structure of Trochophore of

Hydroides uncinarus (Eupomatus). Quarterly

Journal of Microscopical Science, 56:543-590.

.Spcngel, J. W.

1907. Eine verkannte Sipunculus-I^T\e. Zoologischen Anzeiger, 31:97-99.

Stephen, A. C.

1941. The Echiuridae, Sipunculidae and Priapulidae Collected by the Ships of the "Discovery" Committee

during the Years 1926 to 1937. Discovery Report,

21:237-260, 2 plates.

Wilson, E. B.

1892. The Cell Lineage of Sereis. Journal of Morphology,

6:361-480.
2 4 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 1

Early developmental stages of Phascolosoma agassizii: blastula, gastrula, trochophore. Epon, one

micron, Richardson's stain.

a. Sagittal section of a 24-hour embryo showing rosette cells (r) with apical cilia, modified

blastocoel (b), prototroch cells (p) and three-layered egg envelope (en) perforated by fine

pore canals.

ft. Sagittal section of a 48-hour embryo showing rosette cells (r) , blastocoel (b), blastopore

(bp), mesentoblast (ms), and archenteron (ar).

c. Sagittal section of trochophore showing gut cavity (gc) , prototrochal cavity (pc), prototroch

cells (p) and posterior cavity (psc). Note yolk granules in the prototrochal cavity. 4 days

(10-C).

d. Oblique sagittal section of trochophore. 51/2 days (10°C). Note apical cavity (ac), prototrochal cavity (pc) and stomodaeum (st). The prototroch cells (p) have lost most of their

yolk granules at this stage. Prototrochal cilia are clearly shown in this photograph.

[ac —apical cavity, ar=archenteron, b=blastocoel, bp = blastopore, en —egg envelope, gc=gut

cavity, ms^mesentoblasts, p = prototroch cell, pc = prototrochal cavity, psc=posterior cavity,

r—rosette cell, st = stomodaeum.]
NUMBER 132 25
2 6 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 2

Late trochophorc and piemetamorphosis stages of Phascolosoma agassizii. Epon, one micron,

Richardson's stain.

a. Frontal section of late trochophoie showing rosette cells (r) with apical cilia, apical cavity

(ac), prototrochal cavity (pc), and protonoch cells (p). The prototroch cells and rosette

cells have lost their yolk granules. 6 days (10°C).

b. Frontal section of prcmetamorphosis stage. 7 days (10°C). Note apical cavity (ac), prototrochal cavity (pc) , coelom (cm) , and the peritoneal cells lining the coeloni. Three regions

of the gut can be identified: esophagus (es), stomach (stm) and intestine (in).

r. Frontal section of premetamorphosis stage showing cells of the ventral retractor muscles

(vrm) traversing the coelom (cm) . 7i/2 days (10°C).

d. Frontal section of premetamorphosis stage through nephridial rudiment (ne). Posterior to

the nephridial rudiment the coelom (cm) is apparent as a narrow slit between splanchnic

and somatic mesodermal layers. ~\/2 days (IO°C) .

[ac—apical cavity, cm —coelom, es=z esophagus, in = intestine, ne —nephridial rudiment, p =

prototroch cells, pc = prototrochal cavity, pe=peritoneum, r —rosette cells, stm = stomach, vrm

— ventral retractor muscles.]
NUMBER 132 27
2 8 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 3

Metamorphosis from late trochophore stage to pelagosphera larva of Phascolosoma agassizii.

Epon. One micron. Richardson's stain.

a. Sagittal section of premetamorphosis stage. 6 days (12°C). Note the intercellular spaces in the

epidermis. The egg envelope (en) posterior to the prototroch (p) has begun to lose its

porosity and lamellation.

b. Parasagittal section of the beginning metamorphosis stage. 8 days (12°C). The egg envelope

(en) has ruptured in the region of the mouth and the stomodaeal lining has everted to form

the ciliated ventral surface of the head (vh).

c Sagittal section of recently metamorphosed larva. 9 days (12°C). The coelom (cm) has

expanded, the metatroch (mt) has become the functional locomotory organ, the terminal

attachment organ (to) has emerged, the gut (evident here only in part) has been completed,

and the egg envelope (en) has undergone a transformation into the larval cuticle (cu).

[bo—buccal organ, br—brain, cm = coelom, cu—cuticle, en —egg envelope, ep=epidermis,

es=esophagus, in=intestine, lg=lip gland, lprrlip pore, mo=;mouth, mt = metatroch, ne

— nephridium, np=neuropile, p = prototroch, pc = prototrochal cavity, pel = prototrochal cilia,

pe = peritoneum, psg=posterior sacciform gland, stm = stomach, to = terminal organ, vh=ventral ciliated head, vnc=ventral nerve cord, vnn —ventral retractor muscle.]
NUMBER 132 29

pctcu

to-
30 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 4

Photographs of pelagosphera larvae of Phascolosoma agassizii: living larvae and whole mounts.

a. Eleven days. Whole mount, fixed in 10 percent buffered formalin, mounted in euparol.

No stain. Lateral view.

b. Four weeks. Fixed in osmium vapor (2 minutes) after relaxation in a 1:2 solution of saturated chloretone in sea water (3 minutes). Dorsal view.

c. Four weeks. Living larvae. Lateral view. Metatroch is retracted.

d. Ten weeks. Living larva. Lateral view of head. Metatroch is retracted.

e. Ten weeks. Living larva. Lateral view of posterior terminal organ (to). Note papillated

cuticle (cu).

/. Ten weeks. Living larva. Lateral view of entire larva with retracted lip and metatroch.

Note change from 4-week stage (Ac) in relative proportions of body.

[a —anus, cu = cuticle, e —eye, in—intestine, l = lip, mt — metatroch, ne=nephridium, psg=posterior sacciform gland, stm—stomach, tozrterminal organ, vh —ventral ciliated head.]
NUMBER 132 31
32 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 5

Sagittal section of larva of Phascolosoma agassizii. 28 days. The terminal organ, present at this

stage, is not shown in this section. Epon. One micron. Richardson's stain.

[a = anus, bo=:buccal organ, co=coelomocytes, cm = coelom, cu = cuticle, es=esophagus, ep

r=epidermis, in = intestine, lg=lip gland, lp^lip pore, mo=mouth, mt = metatroch, ne

=:nephridium, re—rectum, stm —stomach, vhzrventral ciliated head, vnc=ventral nerve

cord.]
NUMBER 132 S3

vh

mt

—mo

-ep
3 4 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 6

Sections showing formation of ventral retractor muscles and coelom in Phascolosoma agassizii.

Epon. One micron. Richardson's stain.

a. Frontal section of an early premetamorphosis stage showing the cells of the ventral retractor

muscle (vrm) growing into the newly formed coelom (cm). 6i/2 days (10°C).

b. Frontal section of premetamorphosis stage, slightly later than above. 7 days (10°C). Cells

of the ventral retractor muscle (vrm) have invaded the coelom (cm).

c. Frontal section of premetamorphosis stage showing completely formed ventral retractor

muscle (vrm) traversing the coelom (cm). 7i/fe days (10°C).

d. Oblique frontal section of metamorphosed larva. 11 days (10°C). The coelom (cm) has

expanded and the cells of the ventral retractor muscles (vrm) have elongated.

[cm —coelom, es = esophagus, mt = metatroch, pc=prototrochal cavity, vrm = ventral retractor

muscle.]
NUMBER 132 S5
3 6 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 7

Sections showing gut, coelomocytes and cuticle in prcmetamorphosis and larval stages of Phascolosoma agassizii. Epon. One micron. Richardson's stain.

a. Frontal section of premetamorphosis stage. 7i/2 days (10°C). Note coelomocyte (co) which

appears to be attached to the splanchnic peritoneum. The stomach (stm), in contrast to the

esophagus, is marked by a dense concentration of darkly staining granules of varying sizes.

b. Sagittal section of 28-day larva showing spindle-shaped coelomocytes (co), heavily ciliated

esophagus (es), weakly ciliated stomach (stm), and circular muscles of the postmetatrochal

sphincter (crm).

c. Sagittal section of 43-day larva. Both round and spindle-shaped coelomocytes (co) are

evident as well as the longitudinal musculature of the body wall and the papillated cuticle.

d. Cross-section of 57-day larva through the stomach (stm) and nephridium (ne). Each cell

of the wall of the stomach contains one large darkly staining granule or globule. In the

nephridium the ciliated canals of the two arms are apparent: the upper is associated with

the coelomic funnel and the lower terminates at the external pore.

[cm—coelom, co=coelomocyte, crm=circular muscle, cu = cuticle, en=egg envelope, ep=epidermis, es=;esophagus, in = intestine, lm = longitudinal muscle, mt = metatroch, ne = nephridium, pc=prototrochal cavity, pe = peritoneum, stm = stomach.]
NUMBER 152 37
38 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 8

Sections of larval organs of the mouth. Larvae of Phascolosotna agassizii. Epon. One micron.

Richardson's stain.

a. Frontal section of 60-day larva showing buccal organ (bo) with buccal groove (bg) and lip

glands (lg).

b. Sagittal section through lip and lip pore (lp) and associated organs of the lip of a 28-day

larva. The arrow points to a neurosecretory cell overlying the esophagus.

[bg=buccal groove, bo —buccal organ, es=esophagus, lg —lip gland, lp = lip pore, mt = metatroch, vnc=ventral nerve cord.]
NUMBER 132 39
40 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 9

Sections showing formation of organs of the mouth. Phascolosoma agassizii. Epon. One micron.

Richardson's stain.

a. Frontal section of premetamorphosis stage. 7 days (10°C). Note the two large cells of the

future lip gland (lg) and the stomodaeum (st).

b. Frontal section of premetamorphosis stage showing lip gland (lg) with cytoplasmic vacuoles

and inclusions and stomodaeum (st). 9i/2 days (10°C).

c. Frontal section of premetamorphosis stage showing buccal organ (bo) and stomodaeum

(st). 9 days (10°C).

d. Sagittal section of premetamorphosis stage. 9i/2 days (10°C). Note stomodaeum (st), buccal

groove (bg), buccal organ (bo), lip gland (lg), and stomach (stm).

[bg—buccal gland, bo z= buccal organ, lg=lip gland, st=stomodaeum, stm —stomach.]
NUMBER 132 41
42 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 10

Sections showing formation and structure of nephridium and anus of Phascolosoma agassizii.

Epon. One micron. Richardson's stain.

a. Frontal section of premetamorphosis stage. 7i/2 days (10°C). The nephridial rudiment

(ne), consisting of two enlarged cells, is located within the mesodermal band which has

split anterior and posterior to the developing nephridium to form the coelom (cm).

b. Sagittal section of premetamorphosis stage showing rectal rudiment (re), intestine (in),

stomach (stm) and nephridial rudiment (ne). Note a slight invagination of the egg envelope

indicated by the arrow opposite the rectal rudiment; the envelope later will rupture at the

site of the invagination to form the anus. 8i/2 days (I0°C).

c. Sagittal section of recently metamorphosed larva. The anal opening (a) has been formed,

completing the intestine (in), and the nephridial rudiment (ne) has enlarged. 10 days

(10'C).

d. Sagittal section of 28-day larva showing the well-developed nephridium (ne). The arrow

points to the ciliated funnel opening into the coelom.

[a—anus, cm=coelom, cu=cuticle, in=intestine, ne=nephridium, pc—prototrochal cavity, re

=rectum, stm—stomach.]
NUMBER 132 43
4 4 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 11

Sections of premetamorphosis stage and pelagosphera larvae of Phascolosoma agassizii showing

development of the brain and the structure of the buccal organ and glands of the head. Epon.

One micron. Richardson's stain.

a. Frontal section of premetamorphosis stage through the region of the developing brain (br)

and neuropile (np). 7i/2 days (10°C).

b. Frontal section of 60-day larva showing brain (br), neuropile (np), eye (e), esophagus

(es), and buccal organ (bo).

c. Frontal section of 60-day larva. Head glands, designated by the arrows, open to the exterior.

They are the same as that labeled g4 in the sagittal section pictured in lid.

d. Sagittal section of 28-day larva. Five different glands of the head are apparent in this

section: gl, g2, and g3 are PAS-positive; g4 are the "foamy" glands with unidentified secretion; g5 have been characterized as neurosecretory cells.

[bg=buccal groove, bo=buccal organ, br=brain, drm=dorsal retractor muscle, e=eye, es=

esophagus, gl-5=glands of the head, np=neuropile, vrm=ventral retractor muscle.]
NUMBER 138 45
4 6 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 12

Cross-sections of a recently metamorphosed pelagosphera larva of Phascolosoma agassizii. 10

days (10°C). Epon. One micron. Richardsons' stain.

a. Cross-section through region of the lip.

b. Cross-section through region of the mid-stomach. Double nature of the ventral nerve cord

(vnc) is clearly apparent.

c. Cross-section through anus and nephridium.

d. Cross-section of posterior body through intestine.

[a = anus, bg=buccal groove, cec=circumesophageal connective, drm=: dorsal retractor muscle,

es=esophagus, in = intestine, ne=nephridium, stm —stomach, vnc —ventral nerve cord.]
NUMBER 132 47
48 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 13

Cross-sections of pelagosphera larvae of Phascolosoma agassizii. Epon. One micron. Richardson's stain.

a. Cross-section of a 22-day larva through the region of the head. Note that in this region the

dorsal retractor muscles (drm) are attached to the peritoneum of the body wall.

b. Cross-section of a 22-day larva through the metatroch (mt) and bilobed lip (1). Dorsal

and ventral retractor muscles (drm, vrm) are seen suspended in the coelom as circles of

fibers.

c. Cross-section of a 22-day larva through the region of the nephridia (ne). Some of the

fibers of the ventral retractor muscles (vrm) insert into the body wall in this section.

d. Cross-section of a 57-day larva through the region of the contracted postmetatrochal sphincter. At this age the epidermal glands (epg) are well developed, the retractor muscles (drm,

vrm) have increased in thickness and number of fibers and the nerve cord is bilobed.

[cec=zcircumesophageal connectives, cu=cuticle, drm = dorsal retractor muscle, epg=epidermal

gland, es=:esophagus, in = intestine, l = lip, lgmlip gland, mtr=metatroch, ne —nephridium,

vnc=ventral nerve cord, vrm —ventral retractor muscle.]
NUMBE

R 132

49

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50 SMITHSONIAN CONTRIBUTIONS TO ZOOLOGY

PLATE 14

Sections of premetamorphosis and larval stages of Phascolosoma agassizii. Stages in the formation of the posterior sacciform glands and the terminal organ are illustrated. Epon. One

micron. Richardson's stain.

a. Sagittal section of beginning metamorphosis stage showing posterior sacciform gland (psg).

The nucleus of the gland cell is distinguished by the large eccentric nucleolus.

b. Oblique cross-section of the distal end of the posterior sacciform gland (psg) of a 28-day

larva through the secretory cell with its characteristic nucleus. The large, unstained granules of the central cavity of the gland are of unidentified composition.

c. Sagittal section of posterior sacciform gland (psg) in a 43-day larva. Filled with amorphous

secretory material, the central cavity of the gland is surrounded laterally by supportive cells

and distally by the secretory cell. The cavity is continuous with the exterior by way of the

proximal canal which penetrates the body wall.

d. Sagittal section of 22-day larva showing anterior epidermal gland (epg) with bristle (brs).

e. Sagittal section of beginning metamorphosis stage showing the terminal organ (to) in an

early stage of formation. Note the gland cells (gl) which open to the exterior through the

aperture at the distal end of the terminal organ.

/. Sagittal section of the terminal organ (to) of a 22-day larva. This section shows the aperture at the end of the terminal organ and the retractor muscles of the terminal organ (trm).

[brs = bristle, co—coelomocyte, cu=cuticle, epg = epidermal gland, gl —gland of terminal organ,

ne=znephridium, pe= peritoneum, psg=posterior sacciform gland, stm=stomach, to = terminal

organ, trm —retractor muscle of terminal organ, vrm — ventral retractor muscle.]
NUMBER 132 51

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