TAXONOMY AND BIOLOGY OF SIPUNCULANS, WITH EMPHASIS ON THE MORPHOLOGY OF PHASCOLION STROMBUS...

Taxonomy and Biology of Sipunculans,with Emphasis on the Morphology ofPhascolion strombus (Montagu, 1804).

Annotated Bibliography 1555-1995Appended as Annex

Jorgen HyUeberg

Goteborgs' Universitet 1995

University of GiiteborgFaculty of Natural Sciences

Taxonomy and Biology of Sipunculans,with Emphasis on the Morphology ofPhascolion strombus (Montagu,1804)

Annotated Bibliography 1555-1995Appended as Annex

J0rgen Hyllebergcando scient.

Dissertation

Department of ZoologyUniversity of G5teborgMedicinaregatan 18S-413 90 G5teborgSweden

Pennanent address:Institute of Biological SciencesDepartment of Ecology and GeneticsAarhus UniversityDK-8000 Arhus CDenmark

Avhandlingen ilir filosofie doktorsexamen i zoologi (examinator professor Jarl Ove Str5mberg) som enligtbiologisk-geovetenskapliga sektionsnllmndens beslut kommer att offentligt ilirsvaras fredagen den 19 maj1995 kl. 13.15 i Kristinebergs marinbiologiska stations iliredragssal.

This thesis is dedicated to my wife

Karen

and our childrenTine, Kaare and Rune

In appreciation of their patience and encouragementwhilst this thesis was being written

Copyright J0rgen HyllebergTryckt och bunden:Matematisk Instituts trykkeriArhus Universitet 1995ISBN 91-628-1567-9

Cover photo: Section of basophilic cell receptaculum. Smooth epidermal organ of Phascolion strombus.Drawn from TEM photo. Scale bar I Ilm.

Table of contents

page

Abstract 5This thesis is based on the following papers 6The phylum Sipuncula. Introduction 7Form and function. Characters used in classification of the phylum Sipunl:ula 8Systematics of the phylum Sipuncula 14

Studies on Phaseolion strombus (Montagu, 1804) 16Introduction ... .. .. .. .. .. .... I7Materials and Methods 18Morphology and anatomy of body and introvert 19Morphology of the digestive system, with notes on food and feeding 28

Conclusions . .. . . .. .. ....... .. ..... ... 37Acknowledgements 38References .. ... . .. ... .. ..... ... ...... ... 39

ANNEX 1. Application of vital staining 41

ANNEX 2. Annotated bibliography. Systematics, ecology,physiology & biochemistry of the phylum Sipuncula 1555-1995 43

Irrigation in the sipunculid Phaseolion strombi (Mont.). 141

Fauna associated with the sipunculid Phaseo/ion strombi(Montagu), especially the parasitic gastropod Menestho diaphana (Jeffreys).

On the ecology of the sipunculan Phaseolion strombi (Montagu).

Phylum Sipuncula. - A detailed catalogue of valid genera,species, synonyms & erroneous interpretations of sipunculansfrom the world, with special reference to the Indian Ocean and. Thailand.

Phylum Sipuncula. - Cryptic fauna with emphasis onsipunculans in hump coral Porites lutea, the Andaman Sea, Thailand.

153

172

191

279

Taxonomy and biology of sipunculans

Hylleberg, Jorgen 1995. Taxonomy and Biology of Sipunculans, with Emphasis onthe Morphology of Phascolion strombus (Montagu, 1804). Annotated Bibliography1555-1995 Appended as Annex.

University of Goteborg, Department of Zoology, Medicinaregatan 18, S-413 90 Goteborg,Sweden

Permanent address:Institute of Biological Sciences, Department of Ecology and Genetics, Aarhus University,DK-8000 Aarhus C, Denmark

Key words: Sipuncula, Swedish west coast, Phascolion strombus, morphology, ultrastructure, epidermalorgans, intestinal system, vital staining, bibliography.

ABSTRACT

5

An overview of characters and taxonomy of thephylum is presented with emphasis on aspects ofvariability and the caution needed when sipunculansare to be classified. I describe epidermal organs andthe gut of Phascolion strombus with regard tomorphology, histochemistry, and biology. Newmorphological findings include (I) an expanded nuchal organ; (2) acidophilic gland cells of epidermalorgans opening to the surface via individual pores;(3) there is no evidence of an open pore for the nerveendings of epidermal organs. Nerves of the axial cellcolumn terminate in a cap; (4) the ultrastructure ofup to 2.5 /lm coarse granules in epidermal organsdisplays three parts with markedly different electrondensity; (5) a ca. 12 Jlm long structure with abundant tubules is present in epidermal organs of thesmooth region. - The morphology and histochemistry of the intestinal system'of sipuncuians is reviewed and compared to the gut of P. strombus. Thedistance from the mouth to the anus of this speciesis divided into II divisions. Based on methylene

ISBN 91-628-1567-9

blue stammg intra vitam, a pronounced circularinnervation is described on the oesophagus and theloop area of the posterior intestine. Histochemicalstaining showed abundant occurrence of lipofuscinin epidermal organs, particularly the thickened partofthe so-called holdfast papillae. The present studyagrees with the literature showing that the cuticle ofsipunculans is collagen-like, and that the thickenedpart of holdfast papillae contains iron. P. strombusdwells in discarded shells of various taxa, mainlygastropods. If the worm is pulled by the introvert,this is sacrificed. The zone of autotomy is located atthe transition of introvert and body. By use of abraded shells with attached glass windows, I found thatthe posterior body (but not the holdfast region)always maintains contact with the dwelling. P.strombus is a detritus feeder but the specific foodsource has not been identified. I append an annex onvital staining. This technique can give a quick impression of morphological structures. I append acommented bibliography clarifying some disturbingdisagreements in previous lists of references.

6 J0rgen Hylleberg

THIS THESIS IS BASED ON THE FOLLOWING PAPERS: .

I. Kristensen,1. Hylleberg, 1969. Irrigation in the sipunculid Phascolion strombi (Mont.). - Ophelia7(1): 101-112.

2. Kristensen, J. Hylleberg, 1970. Fauna associated with the sipunculid Phascolion strombi(Montagu), especially the parasitic gastropod Menestho diaphana (Jeffreys). - Ophelia 7(2): 257276.

3. Hylleberg, J. 1975. On the ecology of the sipunculan Phascolion strombi (Montagu). - In M. Rice(ed.): Proceedings of the International Symposium on the Biology of the Sipuncula and Echiura.Vol. 1: 241-250.

4. Hylleberg, J. 1994. Phylum Sipuncula. - Part 1. A detailed catalogue of valid genera, species, synonyms & erroneous interpretations of sipunculans from the world, with special reference to the Indian Ocean and Thailand. - Phuket Marine Biological Center Research Bulletin 58: 1-88.

5. Hylleberg, J. 1994. Phylum Sipuncula. - Part 2. Cryptic fauna with emphasis on sipunculans inhump coral Porites lutea, the Andaman Sea, Thailand. - Phuket Marine Biological Center ResearchBulletin 59: 33-41.


The phylum SipuDcula

INTRODUCTION

7

The phyletic status of Sipuncula is now generallyaccepted and the common name for a worm in thisphylum should be sipunculan (Stephen & Edmonds1972). The older literature refers to sipunculoids orsipunculids, depending on the name used for thehigher levels of classification at a given time (Sipunculoidea, Sipunculacea, Sipunculida). Assignment of sipunculans to genera has also changedconsiderably over the years (Hylleberg I994a). Inconsequence, the literature provides different spellings of specific names becaus~ the ending of specific names depends on the gender of a given genus. Ihave followed the classification and nomenclatureproposed in the latest revisions (listed in Hylleberg1994a) and I have changed my previous use of thename Phascolion strombi to Phascolion strombus.

Sipunculans are unsegmented coelomates characterized by a terminal mouth encircled by ciliated tentacles (rudimentary in a few species). The introvertis completely retractable. Anus is located middorsally on the anterior body, rarely on the introvert.One pair of metanephridia (rarely one nephridium)hangs freely in the coelomic cavity. The nephridiopores are located ventrolaterally in the anterior body.Most sipunculans are dioecious. Eggs or spermsdevelop in the coelom and exit through the nephridia. Development is of the spiral type, resulting in atrochophore larva (Akesson 1958; Rice 1975). Exceptions are the hermaphroditic Nephasoma minutum (Akesson 1958; Gibbs 1975), and the parthenogenetic Themiste lageniformis (Pilger 1987).Asexual reproduction has been found in Sipunculusrobustus (Rajulu & Krishnan 1969; Rajulu 1975)and Aspidosiphon elegans (Rice 1970; HyllebergI994b).

According to Hyman (1959) the first description ofrecognizable sipunculans was published in the formof two woodcuts in 1555. Subsequently, Linnaeus(1766) named the two species Sipunculus nudus andS. saccatus, but only S. nudus has survived as avalid species (Hylleberg I994a). After Linnaeusintroduced his Systema Naturae (1758, 10th ed.), anumber of well known sipunculans were described,e.g., Sipunculus phalloides (Pallas, 1774) and Phascolion strombus (Montagu, 1804). The first students of natural history assigned the sipunculans toVermes, Le., the old, heterogeneous pool of vermiform bilateral animals which differed morphological-

Iy from arthropods and molluscs. During the following 8 decades a better classification emerged.

Sipunculans were brought together and recognized asa separate group characterized by the absence of setaeand a total lack of segmentation. The first status wasmade by Selenka et af. (1883) who counted aworldwide total of 81 species. Hyman (1959) recognizing about 250 valid species, while Stephen &Edmonds (1972) listed 367 species. Recent revisions have reduced the number of valid species. Gibbs& Cutler (1987) counted 206 species while thenumber of species had reached the lowest number ofthis century when Hylleberg (1994a) counted 146revised, polytypic species and 17 subspecies. However, this number has slightly increased followingwork by Cutler (1995) who reviewed a total of 155species. The latter number is according to the publisher's announcement of Cutler's book. It was notreleased at the time of this writing.

The first attempt to evaluate the state of sipunculanaffairs was the International Symposium on Biologyof the Sipuncula and Echiura arranged by Mary Ricein Kotor, former Yugoslavia in 1970. At that timeabout 50 researchers worked with sipunculans, orsubjects related to sipunculans in the world. Thefollowing decades have only resulted in a moderateincrease of this number of students of the phylum.Most publications after the symposium deal withtaxonomy and morphology (Cutler & Cutler, Edmonds, Gibbs, Halder, Murina, Rice, Saiz Salinas,cf annex 2). Much work has also been done withfine structure, physiology and biochemistry(researchers from many countries, cf annex 2) whilelittle work has been carried out on the ecology(mainly by Gibbs, Hansen, Rice & collaborators, cfannex 2).

1started to study the ecology of Phascolion strombus (Hylleberg Kristensen 1969, 1970; Hylleberg1975) but have in recent years focused on morphology, and particularly taxonomy. I have collectedsipunculans from Thai waters for the last 15 years.This substantial collection, including quantitativesamples over 3 years, has now entered the stage offmal identification and description (Hylleberg 1994a,b). The task of identifying sipunculans is notstraightforward since the identification of mostspecies requires dissection followed by qualitativeand quantitative estimates of characters in order to

8 J0rgen Hylleberg

judge the taxonomic value. Varying emphasis hasbeen assigned to the amount of variation which hascaused a remarkable number of synonyms, considering the small number of researchers involved andthe low number of species in the world (Hylleberg1994a). Since the morphology of an organ can throwlight upon the function, especially such difficultaspects as food and feeding, I have studied selectedorgans of the cosmopolitan species Phaseolionstrombus in greater detail. Emphasis has been onepidermal organs and the intestinal system.

The present thesis comprises a brief overview ofcharacters and taxonomy of the phylum. Next, Iintroduce Phascolion strombus and the research Ihave carried out on this species. I describe epidermal

.organs and the gut with regard to morphology,histochemistry and innervation. I include a smallparagraph on food-feeding in spite of the ratherinconclusive findings. After all, this piece of work

highlights aspects of variability and cryptic behaviour of this species.! find it expedient to give remarks after each topic. These remarks replace a general discussion at the end of the paper. Finally thethesis is appended two annexes. The first one is onvital staining since this technique played an important role in the early descriptions of sipunculans.Second, I enclose a bibliography. It started as apersonal overview of publications, but soon developed into a major enterprise because I encountered anumber of disturbing disagreements between authors, casting doubt on correct spelling of names,year of publication, and details concerning cataloguing. After having ordered journals which subsequently turned out not to contain the requestedpaper, I decided to put my findings together in anannotated bibliography. It is unlikely to be withoutfault, but nevertheless I hope it can spare students ofsipunculans much of the frustration I have experienced during survey of the literature.

Form and functionCharacters used in classification of the phylum Sipuncula

Habitus of the body

Body shape. The approximately 150 species ofsipunculans (Hylleberg 1994a) display several characteristic forms of the body. Most sipunculans,such as species of Sipllnell/us, Xenosiphon, Goljingia. Nephasoma. and Aspidosiphon are cylindricalto subcylindrical. Aspidosiphon may have the shapeof a nearly perfect cylinder. Some species of Themiste, Antillesoma, and Thysanoeardia are pyriform,and some Onehnesoma are nearly globular. Theshape of the body is a character which is useful in ageneral sense; not as a key character. Pronouncedvariation may be caused by fixation, and it is obvious that species living in crevices and empty shellsof gastropods, polychaetes or foraminiferans, willvary in shape according to the shape of the dwelling,e.g., species of Phasea/ion, Aspidosiphon, andPhase%soma.

Body size. The length of sipunculans ranges fromthe large Sipuneu/us indieus measuring 550 mm(Kohn 1975) to 2 mm in Onehnesoma steenstrupiinudum as stated by Cutler & Cutler 1985. However,the majority of bodies are medium sized within therange of 20-60 mm in length. The size is not a verygood character except at higher taxonomic levelswhere the maximum size has been used (Gibbs &Cutler 1987). Pronounced variation in dimensions

of adults of a given species may be caused by environmental factors, sampling, or fixation.

Colour. Most sipunculans have uniformly white,grey or brownish bodies. However, black, brown, orred pigmented areas may be covering or dotting thebody to a variable degree, while blotches and bandsof pigment may occur on the introvert, especially inthe genus Phase%soma. A recent revision (Cutler& Cutler 1990) has shown that absence or presenceof pigment bands are consistent within a givenspecies of Phaseolosoma with the exception ofPhaseolosoma nigreseens.

Body wall. The body wall is so thin in some species that coelomic contents and the intestine can beseen through the skin. Females carrying eggs tend togive an orange or pinkish hue to the body of livespecimens, while males tend to become white because of the sperm. But body colour and presence ofa transparent body are only moderately good characters, because the intensity may depend on environmental factors and the size (age) of a given species.

Epidermal organs. All sipunculans have epidermalorgans which display a variety of shapes and sizes.The organs provide useful characters when treatedwith caution. This was not understood by someearly researchers who put much effort into detailed

Taxonomy and biology of sipunculans 9

description of the organs without realizing that size,shape and pigmentation are influenced by habitatand age. Hence, many species and subspecies havebeen erected on a wrong foundation. For example,Phymosoma asser and Phymosoma pe/ma weredescribed by Selenka & de Man (1883) on the basisof differences in skin bodies. However, these twospecies are identical, and furthermore identical withPhymosoma antil/arum Grube & Oersted, 1858(Cutler & Cutler 1983). Now, the correct designation is Antillesoma antillarum (el HyllebergI994a). However, epidermal organs can be a greathelp in identification of species, when used withcare. Cutler & Cutler (1990) analyzed the taxonomicvalue of pre-anal papillae in Phase%soma. Theyconcluded that the shape of these papillae is consistent and broadly useful to the taxonomist at thespecies level.Most epidermal organs have the same basic structure: glandular and neurosensory cells in a tissuecapsule lined with epidermal cells. Often one celltype dominates over the other one. Epidermal organswith only one of the two components are alsoknown, e.g., the unique bicellular glandular cells ofSipuneu/us nudus as shown by Akesson 1958, p.214. The structure, and presumably the ultrastructure, may provide additional good characters atspecific, as well as higher taxonomic levels. However, only few species have been studied, so it isimpossible to judge the overall value of epidermalorgans. Akesson (1958) suggested that the structureof epidermal organs might give valuable informationabout the grouping of the genera. I agree to thispoint of view.

Habitus of the introvert

Relative length of introvert. The length of theintrovert relative to the body length has taxonomicsignificance in a few cases, such as the generaSipuneulus and Xenosiphon, where the introvertalways is much shorter than the trunk. Otherwise,length of the introvert is a variable character and hasto be treated with caution. The introvert may be aslong as, or somewhat longer than the body, whilethe length may exceed trunk length considerably inthe genera Phaseolion, Onehnesoma, and Aspidosiphon. The anterior end of the introvert may be swollen to a variable degree (nearly globular in Phaseolion strombus, Figs. I & 3).

Mouth and oral disk. The mouth is located centrally on the ciliated oral disk. The shape and size of

the disk, together with the arrangement of tentaclesand the location of the nuchal organ, have significant diagnostic value at the levels of family andgenus.

Tentacles. The taxonomic importance of number,shape, and pattern of tentacles was first emphasizedby Selenka et al. (1883). Unfortunately, these characters are difficult to use. First, the introvert isusually withdrawn in preserved animals so detailshave to be studied in dissected specimens. Second,the number of tentacles of a given species is a function of size (age), and the number of tentacles maybe much smaller than is normal for the species as aresult of regeneration, following a predator's bite.The tentacles of most species are filiform or digitiform. They are reduced to small lobes in a few species (Nephasoma minutum), or may be broad andleaf-like (Sipuneu/us norvegieus), or branched structures (Themiste spp.). Most tentacles occur in pairs.Digitiform tentacles have a triangular cross section.The epithelium of a tentacle is constructed of multiciliated, pseudostratified, columnar cells on the oralsurface (Pilger 1987). The aboral surface is coveredwith microvilli, but not completely. I noticedpatches of ciliated cells on the aboral surface in P.strombus. There is no cuticle on the tentacles(Moritz & Storch 1970; Pilger 1982).

The nuchal organ. The nuchal organ is a lobed padof ciliated and vacuolized epithelium dorsally on theoral disk. It is innervated from the anterior brain.The nuchal organ can be found at the outer edge ofthe tentacles, usually in a dorsal inflection of thering of tentacles. Alternatively, the mouth may beenclosed by a ring of tentacles dorsal to the mouth.In the latter case, the oral disk has a cephalic collaron the ventral side. The position of the nuchal organhas high taxonomic importance. Gibbs & Cutler(1987) erected two classes of sipunculans based on 2patterns ofthe arrangements of mouth, tentacles, andnuchal organ as shown in the next paragraph.

Staining: an auxiliary technique. Most sipunculans have well defined nuchal organs, but the organmay be strongly reduced and difficult to visualize incertain species unless some kind of staining is applied. Staining works well, both vital stains (AnnexI) and methylene blue on fixed material studied inwater. Methylene blue is easily removed from thetissue with ethanol (Hylleberg & Nateewathana1991).

10 J0rgen Hylleberg

The nuchal organ of Phascolion strombus: Whenstained with methylene blue intra vitam, the nervesof the nuchal organ of P.strombus appear as straightblue fibres located in the space between the muscularlayer and the ciliated epithelium. The nerves continue from the organ into the subsequent section ofthe introvert. The organ is divided into an upperdiminutive lobe, and a large, ciliated lower lobeextending, in the shape of a hoof, towards the ventral side where there is a gap. The lower part of thisorgan has not previously been described (Fig. 1).The ciliated epithelium is strongly vacuolized. Thenuchal organ readily takes up neutral red and similarvital stains. When stained in vivo, the lower part ofthe organ appears reticulated in the dissecting microscope. Distal to the lower nuchal organ is a zonewithout papillae or hooks (Fig. I).

2

Figure 1. Anterior introvert of P. strombus from thedorsal side. (I) tentacles; (2) mouth; (3) nuchalorgan, upper part; (4) nuchal organ, lower part; (5)smooth zone: (6) bulb with hooks. Schematic drawing after live specimen stained with neutral red invivo.

Nuchal organ function. The function of the nuchalorgan is unknown. Akesson (1958; p.180), Hyman(1959, p.647) and Bullock & Horridge (1965, p.655) suggested that it may be of chemosensorynature as in polychaetes.

Introvert hooks. Sipunculans may be armed with avariety of hooks or spines on the distal half of the

introvert. The height and width of hooks have oftenbeen measured, and the degree of tip bending estimated together with the internal hook structure inorder to characterize species of sipunculans. Butrecently Cutler & Cutler (1990) showed that thediagnostic value of these hook characters has beenoverrated. The early researchers had simply overlooked the impact of environment and age on hookmorphology and described too many species basedon slight differences (Hylleberg 1994a). Cutler &Cutler (1990) showed thai all Phascolosoma carryhooks. They also found that the number of ringswith hooks displayed a pattern: half of the specieshad fewer than 50 rings (commonly 15-25), and theother half over 50 (often over 100). The height andother dimensions of hooks were not diagnostic formost species. In comparison, the internal structurewas potentially useful but had to be applied withcare. However, presence of a secondary tooth and ofposterior basal elaborations were taxonomicallyuseful. The findings from Phascolosoma may betaken to describe the general situation for sipunculans. Gibbs & Cutler (1987) expressed it this way:Morphological variation seems to be one of thehallmarks of the phylum, a feature that may accountfor the survival of this small group but one thatdoes not facilitate good taxonomy.

Internal characters

Retractor muscles. The introvert can be completelywithdrawn into the trunk by means of strong retractor muscles. Most sipunculans have one pair ofdorsal and one pair of ventral retractors, but thenumber of retractors may be reduced. Phascolion hasonly one or two retractors. The dorsal pair may becompletely fused and the ventral pair fused with thedorsal retractor over most of the length. Furtherfusion of dorsal and ventral retractors to one retractorcan be seen within the genera Phascolion andOnchnesoma (Cutler & Cutler 1985). The number ofretractors and the location of the attachment to thebody wall constitute an important taxonomic character although there is much variation, so the charactermust be applied with caution (Gibbs & Cutler1987).

The body wall. Musculature. The body wall ismade up of layers of tissue which I have numberedfrom 1 to 7 in this context. The outer surface of thebody is covered with a cuticle (1) secreted by theepidermal cells (2). The epidermis rests on a dermis(3) with numerous fine fibres of connective tissue,


pigment cells, and amoebocytes. If the dennis isweakly developed (e.g. in Phascolion strombus) theepidennis seems to rest directly on the outer layer ofthe circular muscle (4) which surrounds an innerlongitudinal muscle (5). Towards the coelomiccavity, the body wall is lined with a peritoneum (6)of flattened epithelial cells and scattered tufts ofciliated cells. Sandwiched between the circular andlongitudinal muscles, a thin layer of oblique musclestrands (7) may be more or less well developed.The structure of circular and longitudinal musclesconstitutes an important taxonomic character at thegeneric and subgeneric levels. In Sipunculus andXenosiphon both muscles are gathered into distinctcrossing bands while only the longitudinal musclegathers into bands in Phascolosoma and some Aspidosiphon spp. The longitudinal muscle occurs as asmooth unifonn sheet covered by the peritoneum ingenera such as Golfingia and Phascolion.

The coelom. The sipunculan coelom extends as asingle cavity from the beginning of oesophagus tothe end of the body without major interruption.Partitioning by mesenteria does not occur. Onlysome remnants of mesenteria connect oesophagus tothe retractors. Similarly, nephridia and parts of thedigestive system may be attached to the body wallby sheets of mesenteria.

Coelomic extensions. The coelom may continueinto coelomic extensions in the fonn of canals orsacs in the body wall. Coelomic extensions arecharacteristic of the order Sipunculifonnes, except inthe single species Phascolopsis gouldii (Gibbs &Cutler 1987).

Dissepiments. The ventral coelomic surface may beprovided with so-called dissepiments fonned bylayers of peritoneal tissue stretching transversallyacross the coelom in some species of Siphonosoma.Stephen & Edmonds (1972) placed those specieswith dissipiments in a separate subgenus Damosiphon (= Dasmosiphon, cf Hylleberg 1994a), butGibbs & Cutler (1987) did not agree. They foundthis character subject to great variation and of limited diagnostic value.

Keferstein bodies. The coelomic surface of thebody wall of some Siphonosoma also harbour socalled Keferstein bodies. These are small, but prominent 0.1-0.5 mm oval bodies of unknown function. They have not been assigned diagnostic value.

Vascular system: Sipunculans have a closed vascular system which is variously referred to in the literature as the polian vessel, polian vesicle, compensation sac, contractile vessel, or dorsal vessel. Itshould be noted that it does not meet the criteria ofa true invertebrate vascular system where blood iscirculated in vessels. The vessel of sipunculans is acoelomic structure where the content (referred to asblood) is moved by muscular contractions and cilia.The sequence of tissue layers is identical to thesequence in the body wall (Pilger 1982). There isnonnally only one dorsal vessel which is attachedalong its entire length to the wall of the oesophagus(see Fig. 16). Exceptions occur in Sipunculus andXenosiphon which have two vessels: a dorsal and aventral one. But, the vessels are long blind sacs asfound in other sipunculans (Hyman 1959). In somespecies the vessel is equipped with tubules(branched or unbranched), constituting an importanttaxonomic character (Gibbs & Cutler (987). Thegenus Thysanocardia is characterized by such villi(Gibbs et af. 1983). Anteriorly, the vessel is shiftedto a lateral position and connects to a circular vesselat the base of the tentacles. The circular vessel givesoff branches to the tentacles. Basically the bloodmoves towards the tip of each tentacle in the twoupper canals facing the ciliated oral surface. However, in P. strombus, I found that some blood wasshunted through lateral connections to the aboralchannel returning it to the circular vessel. althoughmost of the blood moved to the tip of the tentaclesbefore it returned through the aboral vessel (in accordance with Akesson 1958, p. 86). The blood isdominated by erythrocytes which are morphologically similar to those occurring in the coelomic fluid(Fig. 2).

Coelomic circulation. Ciliated cells, cushioned onthe peritoneum of the body wall and intestines,constantly move the coelomic fluid. Theel (1875)found that in Phascolion strombus, the main direction of movement is longitudinal from the beginning of oesophagus along the ventral side towardsthe posterior tip of the body, and in the oppositedirection along the dorsal side. I can confinn thisobservation. In addition to this main movement, Iobserved much movement following the body curvature, thereby effectively mixing the coelomic contents in live animals.

Coelomic fluid. Composed of inorganic ions anddissolved organic matter dominated by amino acids,

12 Jmgen Hylleberg

usually expressed as total ninhydrin positive substances (NPS). Foster (1970) extracted inorganicsolutes and amino acids from Themiste dyscritumexposed to various salinities. The coelomic fluidclosely matched the equivalents required to be isosmotic at all salinities. NPS appeared to be spared atlowered salinities, indicating that the intracellularisosmotic regulation was predominantly ionic.Oglesby (1968, 1969) has worked much withcoelomic fluid and osmoregulation.

Coelomic cell types. Include erythrocytes, leucDcytes, granulocytes which may be acidophilic, neutrophilic, and basophilic, swimming urn cells,morula cells, oval bodies, brown waste bodies, cellplates, multicellular bodies, multinucleate corpuscles, and germ cells. Fig. 2 shows cell types characteristic of the coelomic fluid of P. strombus infreshly made preparations or stained in vivo. Sipunculus nudus has special cells such as multinucleatecorpuscles and swimming urns. They have beenextensively studied by Bang and collaborators,Cuenot, Dybas, Herubel, Ohuye, Volkonsky andothers (cl annex 2).

Figure 2. Coelomic corpuscles of Phascolionstrombus. (A) erythrocyte showing vacuoles stainedin vivo with neutral red (black). (B) granular erythrocyte in coelomic fluid. (C) erythrocyte expellinggranular material when exposed to stress. (D) ovalbodies in coelomic fluid. (E) oval bodies stainedwith rose bengal to show vacuoles (black). (F) Largeamoeboid granulocyte containing vacuoles stainedwith neutral red in vivo (black and gray). (G) smallamoeboid granulocyte with neutral red vacuolesstained in vivo (black). (H, I, J) small amoeboidgranulocytes. (K) granular grey body, neutral redvacuoles stained in vivo (black).

Erythrocytes. Nucleated, circular, flexible diskswith a diameter of about 15-30 Ilm. They are whitein the reduced state and slightly red in the oxidizedstate. The pigment of erythrocytes is hemerythrin(also spelled haemerythrin), containing iron but notheme (Kennedy 1969). Erythrocytes burst if they dryout, get too warm, or are mixed with sea water. InPhaseolion strombus they will release granularmaterial, and one or more large vacuoles becomeprominent within the cells (Fig. 2). Ejection of thegranular content is very rapid.

Oval bodies. A unique feature of P. strombus according to Fange (1974). Oval bodies are normally4-12 Ilm long plates with rounded comers. Thethickness is 2-3 Ilm. Usually, they occur as singlebodies; occasionally as double bodies attached at thebroad surface. They have a central bulging vacuoleseen in phase contrast, or if stained with rose bengal, or nile blue sulphate. The oval body itself isnot stained. Franzen & Fange (1962) stated that thebodies are birefringent, unaffected by ethanol andether, but slowly dissolved in 10% HCI or concentrated nitric acid. Oval bodies contribute to theyellowish conglomerates referred to as brown bodiesand considered a waste product (Herubel 1907, p.348). Furthermore, oval bodies accumulate in fixedurns on the posterior gut wall.

Granular leucocytes (= granulocytes) are known tobe chiefly involved in defense mechanisms(Valembois & Boiledieu 1980). Dybas (1981) foundthat Phascolosoma agassizii granulocytes, regardless of variation in size and affinity to dyes, couldphagocytize latex beads and Staphylococcus aureusbacteria. I have observed that granulocytes of Phascolion strombus (Fig. 2, F-J) became amoebocytesafter a while and stretched out thin undulating pseudopodia.

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When observed in the microscope, the amoebocytesmoved with a speed of about 15 11m per minute on aglass surface.Theel (1875) and Towle (1975) distinguished between two types of granulocytes which they calledsmall and large phagocytic cells. At this level ofmorphological discrimination there is much similarity within the phylum. However, the ultrastructureof leucocytes revealed remarkable differences inlysosomal structure and glycogen content of 5 species studied by Ochi (1975). This indicates thatuseful discriminating characters may be discoveredin the TEM.

Reproductive system. Gonads are located at thebase of the retractors. They are shaped as indistinctribbons which normally are very difficult to observe,unless highlighted with methylene blue staining.There is no indication of their diagnostic value. Thegonads release immature eggs or sperms to thecoelomic fluid where the germ cells grow. Maturesex products are released through the nephridia. Fewstudies have been carried out on spermatogenesis(Franzen 1956) but there seems to be differences atthe generic level (Sawada 1980). More studies havebeen conducted on oogenesis (Sawada 1975). Riceas well as Akesson have provided much information(ef Annex 2). Without doubt, the morphology ofmature eggs can yield diagnostic characters at thegeneric level, and presumably also at the specificlevel, but only a limited number of species havebeen described so far (mainly by M. Rice, ef annex2). Very few taxonomic papers will include anythingon sex products in description of species.

Nervous system. Akesson (1958) has given anexceptionally elaborate description of the nervoussystem of 14 species in 8 genera. Otherwise, information about this system is limited to single species, and it has hardly been studied at all with special techniques for physiology of the nervous system(Bullock & Horridge 1965). Akesson (1958) suggested that the structure of the central nervous system might give valuable information about thegrouping ofgenera.

Cerebral ganglion and ventral nerve cord. Allsipunculans have an anterior, dorsal cerebral ganglion (also referred to as the brain). It is slightlybilobed in most species, but spherical in Nephasoma minutum as shown by Akesson 1958, p. 99.The brain is surrounded by a capsule of connectivetissue. Lateral branches from each side of the brain

form a circumenteric ring (also referred to as thecircumoesophageal connective) around the digestivetract and unite as the ventral nerve cord. Associatedwith the brain is the previously mentioned nuchalorgan. We also find neurosecretory cells which sendtheir axons to a neurohemal structure in front of thebrain.

Cerebral organ. A cerebral organ was prominent inlarvae studied by Akesson (1958) but may be reduced or absent in adults. The cerebral organ islocated at the anterior margin of the brain and moreor less separated from this by connective tissue. Itdevelops at the inner end of the cephalic tube infront of the ocular tubes and the nuchal organ. It issmall and can only be studied in sections. The cerebral organ has also been referred to as frons or frontal organ, but these terms are not recommended byAkesson (1958) and Hyman (1959) since the latternames cause confusion with another structure of thebrain which is termed digitate processes by Akesson(1958, p. 136).

Digitate processes. Hyman (1959) referred to abundle of outgrowths of the anterior part of the brainlocated above the cephalic tube and cerebral organ.The outgrowths project into the coelom, and theyare easily observed as papillae or lobes at the frontof the brain in dissected specimens. Akesson (1958,p.136) found that the cells are neurosecretory. Digitate processes are characteristic of Sipuneulus andXenosiphon.

Eyes. Two epidermal infoldings lying dorsally andventrally to the cerebral organ are called ocular tubes(Akesson 1958; Hermans & Eakin 1975). The tubespenetrate the cerebral capsule. Depending on thespecies, cerebral eyes may either be absent, or thetermination may be without pigment, or stronglypigmented, and at the highest level of organizationprovided with a refractory body. Well developedeyes can easily be observed in dissected specimensat the lateral margin of the brain. I noticed prominent eyes as a characteristic feature in the generaCloeosiphon, Golfingia, and notably Phaseolosoma, but the taxonomic significance of presenceor absence of eyes is unknown. Stephen & Edmonds(1972) did not give consistent reference to the eyes.

Peripheral nervous system. Two paraneural muscles (derived from the longitudinal muscle) run onthe sides of the ventral nerve cord (Akesson 1958, p.58). Nerves from this cord branches several times

14 J0rgen Hylleberg

and fonn networks in the body wall, intestinal system and nephridia. Three layers nervous tissue canbe distinguished in the body wall and the intestinalsystem of sipunculans, viz., a subperitoneal plexus,a system of ring nerves, and a subepidennal plexus.Further mention of the peripheral nerves are given insucceeding paragraphs on epidennal organs and thedigestive system in Phascolion strombus.

The excretory system. All sipunculan genera havetwo ventro-Iateral metanephridia with the exceptionof the genera Phascolion and Onchnesoma. Theyonly have one nephridium, nonnally the right one.The sipunculan nephridium is sac-shaped, more orless elongated and inflated distally (Fig. 3). There isan anterior ciliated nephrostome with a ciliated canalleading to the lumen of the nephridium. Anephridiopore in the anterior body connects thelumen with the surrounding medium. The coelomicside of a nephridium has a peritoneum on top of thecircular and longitudinal muscle fibres. The lumenis lined with an epithelium of large bulging cellswhich contain brown granules composed of neutralpolysaccharides, glucolipids, proteins, and lipofuscin (Serrano et ai. 1991). Storch & Welch (1972),Ocharan (1974), and Serrano et ai. (1989) have studied the fine structure of nephridia.A nephridium may hang freely in the coelom, or itmay be attached to the body wall with muscularstrands ·or sheets of mesenterium. The length ofnephridia relative to the length of the body, and thedistance over which a nephridium is attached to thebody wall, have previously been given high taxonomic significance (Stephen & Edmonds,1972), butthis has obviously been overrated (Cutler & Cutler1990). These characters are helpful only in a fewcases.

Staining: an auxiliary technique. Vital stammghas been extensively used to study nephridial fonnand function in sipunculans. Depending on thespecies, stains are taken up by the wholenephridium, cells of the nephridial wall, or certainareas of the nephridium, e.g. leaving the nephrostome and the canal to the nephridiopore unstained. (Baltzer 1934; Brumpt 1897; Moltchanoff1909; Herubel 1907; Tetry 1959). Indigo-carmineand acid fuchsin are selectively taken up bynephridia of sipunculans (in comparison, ammoniated carmine is not, cf Hyman 1959). 1 found thatthe brown nephridial granules take up neutral redintra vitam.

The intestinal system. The gut of most sipunculansappears as an intestinal coil comprising a singledescending and a single ascending branch. Exceptions to this general pattern are seen in Sipunculuswhere a further small coil is located in front of themain coil. This particular coil is referred to as thepost-oesophageal loop. Other deviations are theloose intestinal loops or slightly twisted guts asseen in the genera Phascolion and Onchnesoma. Thenonnal double coiled gut can be more or less tight.Cutler & Cutler (1986) found that some Nephasomahad this helix stretched out or loosely wound withspace between the individual coils. This type ofcoiling seemed to be constant and of taxonomicvalue. In comparison, the number of coils havepreviously been assumed to have diagnostic value,but as with all other characters of this phylum thereis pronounced variation, above all because the number of coils is correlated with size, so the conclusionis, that this is not a useful character (Cutler & Cutler 1988). In a succeeding paragraph on Phascolionstrombus, I discuss other problems associated withcomparative gut morphology. Researchers have useddifferent tenns to describe sections of the gut, so thefirst condition to use the intestinal system taxonomically must be to obtain a standardized tenninology. But guts have not received much attentionin the past. Hence, the conclusion to be drawn atpresent is that guts have limited diagnostic value.This includes rectal caeca (Gibbs & Cutler 1987;Hylleberg 1994a).

Spindle muscle. Many species have a spindle muscle which arises anteriorly in the body and supportsthe coils of the intestine. This muscle mayor maynot be well defined, and it mayor may not be fixedposteriorly to the body. These conditions have greattaxonomic significance at the levels of family andgenus.

Systematics of the phylumSipuncula

Various early attempts to divide sipunculans intogroups (or families) were not successful. The rightcombinations of distinguishing characters had notbeen identified. Stephen & Edmonds (1972) were


aware of the problems but nevertheless they attempted the first classification of the phylum intofour families by means of(1) the presence or absenceof coelomic sacs or integumental canals, (2) thestructure of the longitudinal musculature of thebody, (3) the tentacular patterns, and (4) the presenceof a cap or shield at the anterior body. Gibbs &Cutler (1987) revised the systematics of sipunculansand adopted a classification which comprised twoclasses, four orders, and six families. They redefmed17 genera and recognized 13 subgenera (Table 1).

Edward Cutler (1995) has provided the latest reviewof sipunculan classification. Unfortunately, Cutler'sbook was delayed by the printer so it has not beenpossible to include the results in this thesis. However, Cutler has informed me that he did not acceptthe new genus Cutlerensis, erected by Popkov(1991) for Aspidosiphon rutilofuscus. He foundCutlerensis acceptable at the subgeneric level only(pers. com. 1995). In consequence, I have added thischange to the classification.

Mouth, tentacles, and nuchal organ, located at theanterior end of the introvert, are characteristics ofsipunculans. Basically two patterns can be distinguished (Gibbs & Cutler 1987). First, the classSipunculidea with tentacles arranged in one orseveral, more or less complete circles around themouth. The dorsal nuchal organ (if present) is located in a crescent-shaped incision at the outer periphery of the tentacular ring. This location of tentacles and nuchal organs occurs in II genera. Second,the class Phascolosomatidea with tentacles restricted to an arch dorsal of the mouth, and enclosing the nuchal organ. This location of tentacles andnuchal organs occurs in 6 genera (Table I).

More detailed characterization ofthe pattern of tentacles has been applied at the family level. Stephen &Edmonds (1972) arranged the genera into 4 familieswhere Sipunculidae carried numerous tentacles or atentacular fold surrounding the mouth. Golfingiidaehad basically the same arrangement, but the tentaclesoccurred in groups, were dendritic, reduced to a fewlobes, or absent. Aspidosiphonidae had tentaclesarranged around the mouth, but never in a ring dorsal to the mouth. Finally, Phascolosomatidaealways had tentacles in a horseshoe-shaped ringdorsal to the mouth and encircling the nuchal organ.Gibbs & Cutler (1987) recognized 6 families.

Sipunculidae was maintained from Stephen & Edmonds (1972) while the other 3 families were rearranged and emended.New families were Phascolionidae and Themistidae. The latter family is characterized by tentaclescarrying 4-8 stem-like outgrowths of the oral disk.

Table I. Classification of the phylum Sipunculaaccording to Gibbs & Cutler (1987). Emendationmarked by asterix.

Class Sipunculidea Gibbs & Cutler, 1987

Order Sipunculiformes Gibbs & Cutler. 1987I. Family Sipunculidae Stephen & Edmonds. 1972

Genus Sipuneulus Linnaeus. 1766Subgenus (Sipuneulus) Gibbs & Cutler. 1987Subgenus (Auslrosiphon) Fisher. 1950

Genus Xenosiphon Fisher, 1947Genus Siphonosoma Spengel, 1912Genus Siphonomeeus Fisher. 1947Genus Phaseolopsis Fisher. 1950

Order Golfingiiformes Gibbs & Cutler. 19872. Family Golfingiidae Stephen & Edmonds. 1972

Genus Golfingia Lankester. 1885Genus Nephasoma Pergament, 1946Genus Thysanoeardia Fisher. 1950

3. Family Phascolionidae Cutler & Gibbs. 1985Genus Phaseolion Theel, 1875Subgenus (Phascolion) Gibbs 8:. Cutler. 1987Subgenus (Isomya) Cutler & Cutler. 1985Subgenus (,It!ontuga) Gibbs. 1985Subgenus (Lesenka) Gibbs. 1985Subgenus (Villiophora) Cutler & Cutler. 1985

Genus Onehnesoma Koren & Danielssen. 18754. Family Themistidae

Genus Themisle Gray, 1828Subgenus (Themisle) Gibbs & Cutler, 1987Subgenus(Lagenopsis) Edmonds. 1980

Class Phascolosomatidea Gibbs & Cutler, 1987

Order Phascolosomatiformes Gibbs & Cutler, 19875. Family Phascolosomatidae Stephen & Edmonds. 1972

Genus Phaseolasama Leuckart. 1828Subgenus (Phaseolosoma) Gibbs & Cutler. 1987Subgenus (Edmondsius) Gibbs & Cutler, 1987

Genus Apiansoma Sluiter, 1902Genus Antillesoma Stephen & Edmonds, 1972

Order Aspidosiphoniformes Gibbs & Cutler. 19876. Family Asphidosiphonidae Baird, 1868

Genus Aspidosiphon Diesing, 1851Subgenus (Aspidosiphan) Gibbs & Cutler, 1987Subgenus (Paraspidasiphan) Stephen, 1964·Subgenus (Cullerensis) Popkov, 1991

Genus Claeosiphon Grube, 1868Genus Lilhacrosiphan Shipley, 1902

16 J0rgen Hylleberg

Studies on Phascolion strombus (Montagu, 1804)

:.: ::'.:.-""':: '". --- ---- - ~ - -., ~ - '-- -- ---.. -- ---- -_ ...

"~~~:~;::':;I"'"•.~.-!:.---- ......:•._.,,--' . -; ."

9 ':"',',' "

Figure 3. External and internal characters of Phascolion strombus. (I) specimen dwelling in discarded TurrUella. (2) appearance of extracted specimen. (3) holdfast papillae. (4) smooth region with papillae. (5) hookon epidermal organ from bulb area, (6) dissected specimen. (7) metanephridium. (8) rectal diverticulum withciliated groove and fixed urns. (9) piece of intestine showing ciliated groove and appearance of the groovearea seen from the coelomic side. Compiled from plates in Theel (1875a & 1905).


INTRODUCTION

17

In the 1800's, before the Kristineberg Marine Biological Station was established, many Swedish reseachers visited the coast of Bohuslan to collect andstudy marine organisms. Hjalmar Theel was one ofthose pioneers. In 1874 he brought a microscope andother equipment to Kristineberg where he stayed inthe house of a private family for 3 months. On dayswhen sunlight was sufficient to permit microscopy,he used a razor to dissect and to cut slices of Phaseolion strombus so he could examine and drawanatomical details. He also set up a small aquariumto study behaviour of live individuals. Based ondetailed studies of sipunculans at Kristineberg, hefollowed a suggestion made by Keferstein (1865)and erected the new genus Phaseolion Theel, 1875.Furthermore, he was the first person to illustrate thenephrostome. He also showed the location of gonadsat the base of the retractors, thereby verifYing previous assumptions (Koren & Danielssen 1875), and hedescribed a third layer of oblique muscles in thebody wall which was a novel discovery. Strangeenough his discoveries were neglected by contemporary students of sipunculans. He felt hurt and used asubsequent paper to express his disappointment: "IfSelenka (1883) had not overlooked the papers referred to, his anatomical review would, it is fair toallege, have gained in value as regards, for instance,the segmental organs, the genital organs, etc."(Theel 1905, p. 9). 1sympathize with Theel becausemuch basic and correct information was provided byTheel as early as 1875. Invariably, the time and theway Theel made his discoveries must invite reflection for contemporary students of P. strombus.After Theel's pioneer work, a number of studies havefocused specifically on this species. Brumpt (1897)injected various into the body cavity and describedaccumulation dyes in vivo in cells of the nephridiumand intestine. Brumpt also described an associationbetween the sipunculan and a polychaete (Syllis),and he observed various types of shelter used by P.strombus.Moltchanoff (1909) described the nephridium of P.spitzbergense (= P. strombus, ef Hylleberg 1994a).Perez (1924, 1925) made ecological studies andfound a pyramidellid and a montacutid associatedwith the sipunculan. Wesenberg-Lund (1929) compared P. strombus from Greenland and the southernNorth Sea. She concluded that morphology andanatomy varied considerably, but the variations didnot imply presence of more than one species.Schleip (1934) studied regeneration of the introvert.After removal of the introvert, regeneration occurredfrom a strand of cells below the layer ofnerve cells

in the ventral nerve cord. Schleip (1935) noted thatposterior regeneration could not occur if part of theintestinal tract protruded through the wound. Arvy& Gabe (1952) studied the histology of the intestinal tract of P. strombus. They described distributionof alkaline phosphatase, lipase, glycogen, and othercompounds in the digestive tract. They suggestedthat cyclic variation in glycogen concentration wasrelated to the reproductive cycle. Arvy & Prenant(1952) studied occurrence of an entoproct associatedwith this sipunculan in France. Arvy (1952) found anew species of gregarine (Leeudina franeiana),parasitized by a sporozoan (Metehnikovella berliozi),in the gut of P. strombus. Gabe (1953) describedneurosecretion (function unknown) from this species. Bobin & Prenant (1953a,b) described entoproctsfrom the skin. Akesson (1958) described the nervoussystem, embryology and ecology of P. strombus.Nielsen (1964) identified two species of entoproctsattached to the body wall of this sipunculan. Mock(1965) noted range extension of this species to include the Gulf of Mexico. Tuzet & Ormieres (1965)continued studies on parasites and described newspecies of gregarines from the gut of this species.Nielsen (1967) stated that the two entoproct speciesLoxosomella atkinsae and L. murmaniea were regularly associated with P. strombus in Scandinavianwaters. Hylleberg Kristensen (1969) measured irrigation of habitations by P. strombus and described themechanism. Moritz & Storch (1970) investigatedthe fine structure of cuticle and epidermis. Storch &Moritz (1970) studied fine structure of cells involved in regeneration of the introvert. Hylleberg Kristensen (1970) described an ectoparasitic gastropodMenestho diaphana and a number of other organisms associated with the dwellings of P. strombus.Hylleberg (1975a) studied the ecology of P. strombus, mainly factors influencing the vertical distribution in GAso Ranna, the west coast of Sweden.Gibbs (1978) found that Menestho diaphana andMontaeuta phaseolionis also live in associationwith P. strombus in the vicinity of Plymouth. Gage(1979) studied the bivalve Montaeuta phaseolionis,and showed that six or more year classes of thiscommensal were present. Rice et al. (1983) compared the ecology of Phaseolion eryptus, in particularthe behaviour of animals in relation to shells andenvironmental factors, and compared it with P.strombus. Gibbs (1985) analyzed the north-eastAtlantic species of Phaseolion and concluded thatmuch of the variation in size, colour and skin papillae may be attributable to the size and type of shelterused by P. strombus.

18 J0rgen Hylleberg

Furthennore, infonnation about P. strombus can befound in publications concerning reviews of fauna,physiology, comparative anatomy, and geographicaldistribution of sipunculans, e.g., Cutler (1995),Cutler & Cutler (1985), Fischer (1914), Fischer(1922), Gerould (1913), Gibbs (1977), Hendrix(1975), Herubel (1907), Hyman (1959), Keferstein(1865), Theel (1905), Wesenberg-Lund (1935).

MATERIALS AND METHODS

Phascolion strombus was dredged in G<\so Rlinnanear Kristineberg Marine Biological Station, Sweden. Experimental work with live animals, and allhistochemical studies, were conducted at Kristineberg during 1967-75 (Hylleberg Kristensen 1969,1970). Map of the study area is found in Hylleberg(1975). Studies by transmission electron microscopy(TEM) were conducted in cooperation with JmgenMorup Jmgensen at the Institute of ComparativeAnatomy, Copenhagen University, Denmark in1968. Subsequent studies by scanning electronmicroscopy (SEM), and of thin sections, includingBodian-stained material were made in cooperationwith Jmgen Mmup Jmgensen at the Institute ofBiological Sciences, Department of Zoophysiology,Aarhus University, Denmark, during 1978-1995.

Fixed material: Specimens for histochemical staining were removed from the shelter and immersed in4 % fonnalin for a minimum of I hr. Specimens forparaffin, Epon® 2.5 /lm sections, or SEM studieswere fixed in Bouin or 4 % buffered fonnalin. ForTEM, pieces of tissue were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, postfixedin 1% buffered osmium tetroxide, dehydrated, embedded in Araldit®, sectioned with glass knives, andplaced on carbon coated grids stained with uranylacetate, followed by lead acetate. Series of 2 /lmsections were cut of tissue embedded in Araldit,using glass knives, stained with toluidine blue, andexamined by light microscopy. Unfixed material:Specimens were removed from the shelter andwashed in sea water. Complete individuals wereused for staining in vivo. Specimens were dissectedin sea water for supra vitam staining with methyleneblue. Specimens for histochemical staining werewashed in distilled water to remove surface salts.Death is not accomplished by this procedure, somost cells would still be alive when exposed to thestaining procedure. Complete individuals or dissected body walls were used. Unfixed mater.ial was cuton a freezing microtome and sections mounted inwater. Peroxidase. A solution of saturated benzidinewas prepared by addition of a little benzidine to 200

ml sea water. The solution was left for 24 hrs, andfiltered before use. Live animals without shelterwere immersed in this solution for 3 hrs (no signsof acute toxicity). Next, drops of 4% hydrogenperoxide were added until small bubbles appeared.Remarks: Dark blue colour develops in the epidermal organs. The colour will last for about I hr inanimals dissected in sea water. Thereafter, it changesto shades of brown and grey. Benzidine reacts withperoxidase (Goodpasture 1919) localized in granulesof phagocytizing cells (Dybas 1981). Hemoglobingives blue colour with benzidine in the presence ofhydrogen peroxide (pseudoperoxidase activity) butthe hemerythrine of sipunculans does not containhem (Kennedy 1969). Hence the reaction with sipunculan blood is negative. Inorganic phosphate.The molybdate method for inorganic phosphate(Pearse 1960, p. 945). Specimens were transferred tothe nitric acid and ammonium molybdate mixturefor a maximum of 5 min; subsequently immersed in35°C, freshly prepared benzidine and glacial aceticacid for I min; finally transferred to 30% sodiumacetate. Remarks: I found that the method workswell with unfixed, but not with fixed material.Sections cut on freezing microtome show blue c0

lour restricted to the cuticle, the cutis, and structuresof epidennal organs. Ferric iron. Perl's method(pearse 1960). Unfixed material. Remarks: A freshmixture of potassium or sodium ferrocyanide in HCIresults in deep prussian blue if ferric iron is present.I found that disturbing background colour can beeliminated with a green filter when sections areexamined by microscopy. Phenols. Million (Pearse1960, p. 791). Remarks: red colour develops withphenols. Carbohydrates & mucopolysaccharides.SCHIFF. (Baker 1960; Lison 1960). Unfixed material. Remarks: Red-purple colour develops with freealdehydes naturally present in the material, including aldehydes related to fatty acids. PAS (Baker1960). Unfixed material. Remarks: Red-purple c0

lour develops with carbohydrate such as glycogen,neutral mucopolysaccharides (chitin), mucoproteins,glycoproteins, and glycolipids. Acid mucopolysaccharides are negative. Deamination (Baker 1960, p.51). Unfixed material. Remarks: Destroys acidophilia of tissues, but does not nonnally affect hydroxylgroups, i.e. their PAS reactivity. Alcian blue(Clayden 1962, p. 96). Unfixed and fonnalin fixedmaterial. Remarks: The method is rather selective atlow pH and short time of staining. Mucopolysaccharides become deep blue. PFAS for lipids, fatty acids& Iipofuscins (Pearse 1960, p. 859}. Unfixed andfonnalin fixed material. Remarks: Unsaturated fattyacids give purple-red colour. Schmorl's method forlipofuscin (pearse 1960, p. 925) The mixture of


ferrichloride and potassium ferricyanide must beused within 30 min. Remarks: Substances whichreduce ferricyanide to ferrocyanide appear dark blue.These include argentaffm granules, melanin, lipofuscin, and components containing active sulfhydrylgroups. Long Ziehl-Neelsen's method for acid fastlipofuscin (pearse 1960, p. 926). UnfIxed material.Remarks: Acid fast lipofuscin is bright red, lipoprotein pink. Dam method for fat peroxidase (pearse1960, p. 927). Remarks: I did not get positive results with this test (n = 6). Reticulate fibres andcollagen: Gomori's trichrome staining. (Clayden1962, p. 74) Bouin's fIxation. Remarks: Nuclei arebrown-black (iron haematoxylin). Reticulate fIbresand collagen green (fast green). Cytoplasm, myofibrils and erythrocytes red (chromotrop 2R). Ferritinand apoferritin. The cadmium sulphate method(Pearse 1960). Unfixed material. Remarks: If ferritinis present, the typical yellow octohedral crystals ofthe cadmium-ferritin complex will be visible in themicroscope. I did not get positive results with thistest (n = 5). Osmium tetroxide (Cain 1950, p. 93)UnfIxed material. Remarks: It is a strong oxidizingagent which can be reduced by many easily oxidizable substances. Primary black staining of unfixedspecimens. Bodian protargol impregnation (Romeis§ 1816). Bouin fIxation. Remarks: Bodian (Romeis§ 1817) recommended a variety of fIxations depending upon the species and tissue. In general, Bouinshould be a good fixative for invertebrates. Bodianis a rather selective method for neurofibrils, butcollagen fibres and boundaries between cells mayalso be stained. In successful cases, the neurofibrilsbecome black against brownish, nonnervous material. SS and 8H groups: Alkaline tetrazolium reaction (Pearse 1960, p. 807). Fontanasch solution(Romeis § 1129) Unfixed material. Remarks: method for certain configurations of polyphenol,arninophenol and polyamine. Argentaffme granulesare stained brown-black. Lugol's solution (Romeis§ 705) Unfixed material. Remarks: nucleus andglycogen are stained brown. Vital staining. Allvital stains were dissolved in sea water at concentrations between 0.0001-0.005 % (in vivo) and 0.0020.05 (supra vitam): neutral red, vesuvian (also knowas bismarck brown), toluidine blue, methylene blue,trypan blue. Remarks: Vesuvian and neutral red canbe dissolved in oil, and certain cellular lipid globules may become stained by this mechanism (Baker1958, p. 290). Trypan blue gave best results atrelatively high concentrations: 0.005 % in vivo and0.05 %. supra vitam. The nervous systeIJ;! was studied in animals which were opened and incubated insea water with methylene blue (Hoechst aM, Methylenblau Medicin I, Chlorzinkdoppelsalz) dissolved

in a few drops of alcohol, diluted with fresh waterand then mixed with sea water. Nerves did not takethe stain following injection of dye, or when animals were incubated in vivo, as it has been usedwith Sipuneulus nudus (el Hyman 1959, p. 645).

Morphology and anatomyof body and introvert

INTRODUCTION

Akesson's (1958) comprehensive study of the nervous system of sipunculans constituted the onlyinformation available on this system in Phaseolionstrombus. I have supplemented Akesson's studywith studies by light microscopy, TEM, and SEM,but the interpretation of results turned out to bemore difficult than anticipated. Identification ofnervous tissue has a number of pitfalls as pointedout by Bullock & Horridge (1965). I have appliedthe classical methods of methylene blue intra vitamand reduced silver on paraffin sections. These methods have confirmed Akesson's results but they alsoresulted in staining of structures which have notpreviously been mentioned for sipunculans. Sinceall techniques have their shortcomings, I will brieflyrefer to comments by Bullock & Horridge (1965)before I continue with my own results.

Definition and the study of nerves.Methylene blue was considered capriciously selective by Bullock & Horridge (1965, p. 25). However,besides staining nerve cells and fibres, methyleneblue accumulates in granules of several types of cellsand stains fibres in connective tissue (Meyer 1955).The intensity of staining and the smoothness of thefibres could be criteria that separate nerves fromother cell types or elements. Nerve fibres often display dark blue swellings of variable size along thefIbres, so-called varicosities. Based on this criterion,I identified undescribed nerves of the peripheralsystem of P. strombus. I also found that pyriformbodies were stained with methylene blue. The ca. 2011m long, bodies were abundant (especially in thedigestive tract) and intimately connected with nervefibres. But I was not able to ascertain the exactnature of the polarity, or whether the bodies were infact part of the nervous system. The latter system isideally defined as an organized constellation ofneurons specialized for the repeated conduction of anexited state from receptor sites, or from otherneurons, to effectors or to other neurons (Bullock &Horridge 1965, p. 6). In practice, it is diffIcult to

20 J0rgen Hylleberg

use this ideal definition. A nerve cell may be hard torecognize since no specific criterion is certain. Nonnervous neuroglia, certain neurosecreting cells, andgland cells may look like nerves. In addition tomethylene blue, reduced silver methods, such asBodian, may provide evidence of elements of thenervous system. But findings have to be interpretedcautiously because of presence of non-nervous argentophilic cells and fibers (Bullock & Horridge1965, p. 25). This is the reason why I have used theterm black bodies for the about 5 !lm long, beanshaped, strongly argentaffine structures seen in epidermal organs, especially in the cutis. These bodiescould constitute varicosities on nerve fibres, butwithout further evidence it is impossible to specifYtheir relation to the nervous system.

THE BODY AND INTROVERT WALLS

CuticleThe cuticle (secreted by the underlying epidermis) isabsent on the tentacles. It increases in thicknesstowards the base of the introvert. Maximum thickness is on the anterior part of the trunk. The thickness is variable on the rest of the body (about 15!lm), but thin (about 2.5 !lm) on top of the epidermal organs.TheeI (1875) found that the outer surface of thecuticle had two systems of lines crossing each otherat right angles. The lines were diagonal to the bodyaxis. I can confirm Theel's observation; diagonalcrossing lines are separated by about 0.5 !lm. Thepattern was also confirmed by Moritz & Storch(1970). They found that the cuticle was made up offibrils orientated at right angles over each other,both on the introvert and the body. Goffinet et at.(1978) found the same pattern in Sipunculus nudusand Goljingia vulgaris. They concluded that thesipunculan cuticula is very similar in architecture tothe cuticle ofpolychaetes and pogonophorans.Ward (1891) found that the cuticle of Sipunculusnudus consisted of a substance optically like chitin,but it was shown not to contain chitin or cellulose,indicating that it is not identical with the cuticle ofarthropods. Carlisle (1959) found the cuticle ofSipuncllilis nudus to be entirely proteinaceous withno trace of chitin or mucopolysaccharides. Manavalaramanujam & Sundara (1982) recorded chitin in anIndian sipunculan. Otherwise chitin has been claimed absent from the phylum (Jeuniaux 1963; VossFoucart et at. 1977). Absence of chitin would constitute an important phylogenetic character (Florkin1975).

Voss-Foucartet at. (1977) found that the cuticle ofSipunculus nudus and Golfingia vulgare containscollagen, and that polysaccharides dominate overmucopolysaccharides. I studied Phascolion strombus with some of the methods used by Voss-Foucartet al. (1977). My conclusion is also similar. Thecuticle is basically proteinaceous but it also containscarbohydrate, mucopolysaccharide, pigment granules, and inorganic compounds. The cuticle of unfixed material is stained with trypan blue and rosebengal, both having protein affinity. It stains greenwith Gomori's trichrome, indicating reticular fibrilsor collagen (fast green), and it stains significantlywith PAS, indicating mucopolysaccharide. In comparison, the cuticle does not stain with SCHIFF(free aldehydes), iodine (glycogen), alcian blue(mucopolysaccharide), million (phenol), PFAS(unsaturated fatty acids), or sudan black (lipoid),indicating low content of carbohydrate and lipid. Itstains deep blue with Schmorl's method (lipofuscin)and reacts positively with the molybdate method forinorganic phosphate (blue colour).

Coating of the cuticleThe cuticle is covered with a mucoid coating on theintrovert and in the papillated region of the anteriortrunk, evidently consisting of a mixture of mucus,old exfoliated cuticle, and inorganic particles fromthe surroundings. Part of the coating is stained withtoluidine blue (metachromatic), Schmorl's method(lipofuscin), the molybdate method (inorganic phosphate), and benzidine which results in formation ofblue crystals, probably after having reacted withmanganese dioxide (Feigl 1960, p. 405).The cuticle of the smooth region is covered with athin layer of mucus taking toluidine blue in vivo.The coating is very adhesive. If placed in an aquarium with sediment, animals without shelter soonhave a muddy cuff covering the smooth and theholdfast regions. This is opposite to animals inshelters where the smooth zone always is free fromparticles while the posterior, and the anterior papillated zones, and to a smaller extent the holdfast region, are covered with particles among the papillae.

EpidermisThe epidermis of Phascolion strombus is characterized by many bundles of tonofilaments. Moritz &Storch (1970) found that the apical part of epidermalcells carried microvilli. The cytoplasm was poor inendoplasmatic reticulum; mitochondria were scattered, a golgi complex was found lateral or apical tothe nuclei which each carried one nucleolus, andlysosome-like granules were common.


The basal plasmalemma exhibited simple upfoldingswhich were rich in glycogen rosettes. The membranes between epithelial cells were strongly folded andcells were connected with desmosomes.Epidermis cells, which covered the smooth and theholdfast epidermal organs, were more flat, rich invacuoles, and small electron dense granules. Theywere also rich in Iysosomes.

EPIDERMAL ORGANS

Akesson (1958) found that epidermal organs are derived from the epidermal layer as elevations of thedermis. Epidermal organs of various sizes and shapes are present on the introvert and trunk of P.strombus. They show marked regional differencesand can be divided into 6 morphological types treated below: (I) hooked organs on the swollen regionof the anterior introvert; (2) organs of the introvert;(3) papillated organs of the base of the introvert andbeginning of body; (4) organs of the smooth regioncovering the anterior half of the body; (5) organs ofthe holdfast region succeeding the smooth region;(6) organs at the posterior end of the body.It has been discussed whether nerve endings are located in true pores in the cuticula, or whether thepores are corked with a plug, protecting the fibrils ofthe sensory cells from direct contact with the surrounding medium.If the cuticle is treated with nitric acid or KOH, itwill loosen from the rest of the body wall. The innersurface of the cuticle is provided with numerousexcavations where the epidermal were situated. Anarrow aperture is found in the center of each excavation (Theel 1875, p. 8; Akesson 1958, p.183). I canconfiml that there are holes in the cuticula at theposition of the nerve endings. But it is not easy todetermine whether the holes are artefacts created bythe method, or whether the holes originally havebeen closed with a cap. Therefore, a freeze dryedspecimen was studied in the SEM. This techniqueshowed that the holes are closed with a cap.

Hooked organs on the swollen region ofthe anterior introvert (1)

The hooks are slightly curved with the convex sidetowards the tentacles and the points directed posteriorly (Fig. 3). They are brown or colourless, triangular in lateral view, but the colour, form, abundance,and size may vary considerably. The length is about70 11m, the width at the base is about 45 !im, and atthe tip about 311m. The hooks contain a brownish,coarsely granular matrix which is a continuation of

the underlying epidermis. A narrow channel penetrates the hook to the tip. Histochemistry: The hooksstain metachromatically with extremely dilute toluidine blue in vivo, indicating secretion of mucusthrough the hooks. Function of the hooks: Theel(1875, p. 9) proposed that hooks function as barbson an anchor when the animal pulls itself forward.However, I suggest that the expanded bulb itself issufficient to give the necessary anchor effect. I foundthat the hooks also display a cleaning function.During protrusion of the tentacles, the hooks rollalong the aboral surfaces and cause adhering particlesto be removed. The hooks can be compared to arolling rasp. Remarks: The swollen region posteriorto the tentacles is usually armed with hooks, although they may be absent in adults (Akesson 1958,p. 186). In the juvenile stage, scattered hooks arealways present. According to Akesson (1958, p. 53)the course of hook development is not distinct inlarvae. Based on 2 11m sections, I found that theposition and formation of hooks resemble the horseshoe-shaped holdfast papillae. Histochemically,the hooks react like the yellow-brown, inner granules of holdfasts which contain significant amountsofferric iron (Peri's test). This is in agreement withGibbs (1985, p. 316) who demonstrated iron in theholdfast organs using a different technique.

Organs of the anterior part of theintrovert (2)

Small, only about 20 11m long epidermal organs arelocated on the bulb and the introvert. Akesson(1958, p. 186) noted that they often are aggregatedso close together that it seems as if they form asingle organ with several openings. Individual organs appear rounded in relaxed specimens. They areseparated by thin membranes of connective tissueallowing change of shape when the bulb is expanded. Akesson (1958, p. 187) noted three constituents of this type of epidermal organ: neurosensorycells, and two kinds of secretory cells. Histochemistry: I found that epidermal cells of these organsreadily take up neutral red in vivo indicating thatthey were rich in Iysosomes (see annex 1).

Organs of the base of introvert andanterior body (3)

The papillated epidermal organs are conspicuous atthe base of the introvert and on the anterior body.They differ from other epidermal organs in beingsurrounded by a thick cuticula (20 11m on the body,

22 J0rgen Hylleberg

5-1 0 ~m on the papillae). They connect with a massof cells under the epidermis on top of the circularmuscles (Fig. 4). Normally there are no open poresin papillated organs.. But Schmorl's method forlipofuscin showed presence of a maximum of 3pores, indicating that secretion may be given off bysome of the organs. Remarks: Akesson suggested(1958, p. 188) that a papillated organ ultimately isdetached from the cuticle and that a replacementorgan is formed from the cells in the underlyingtissue. Histochemistry: The cavity of a papilla islined with epidermal cells rich in lysosomes. Cellsof the central axis contain variable amounts of osmophilic substance (Fig. 4). Innervation: In 2 ~m

sections, stained with toluidine blue, I noticed clearnerve endings in the corking of the pore (Fig. 4).Otherwise, I did not study nerves in these organs,but Akesson (1958, p. 188) found that nerve cellsare centered in the proximal part of fully developedorgans.

Figure 4. Sagittal section of papillated epidermalorgan at the base of the introvert. (I) cuticula. (2)epidermis. (3) axial cells. (4) pile of cells. (5) circular muscle. Freezing microtome sectipn of Phascolion strombus, fixed and stained with osmium tetroxide. Dark shading of (3) indicates variable osmophilic staining. Schematic drawing.

Organs of the smooth region (4)

On an average the long dimension of contractedsmooth organs is about 100 ~m (fixed material).The cuticula above the organs is thin (about 2.5 ~m;

in agreement with Akesson, 1958). The surface issmooth and slightly convex (Fig. 5).

Axial cells: Akesson (1958) described the main axisof smooth organs as composed of a cylindrical aggregate of nerves and other cells (Fig. 5). In TEM, Ifound many electrondense granules and vacuoles.Even the nuclei appeared very black. Because of thisconfusing blacking of the cellular aggregate, it hasnot been possible to identifY nervous tissue withcertainty on the TEM pictures of this particularspecimen. Histochemistry: The axial material stainswith Million (phenol), Long Ziehl-Neelsen's method(acid lipofuscin), Osmium tetroxide (easily oxidizable substances), and PFAS (unsaturated fattyacids).

Figure 5. Sagittal section of smooth epidermalorgan at the anterior body. (1) cuticula. (2) epidermis. (3) basophilic cell with receptaculum and pore.(4) fine granular cell contents; one cell is shownwith a pore to the surface. (5) coarse granular cellcontents; two cells are shown with pores to thesurface. (6) axial cells. (7) presumed neurons at baseof organ (in cutis). (8) circular muscle. (9) longitudinal muscle. (10) peritoneum. (11) unidentified cellwith amorphous cell contents. Schematic drawing ofBodian-stained Phascolion strombus.


Acidophilic and basophilic cells: Further, Akesson (1958) distinguished between two types of cellsreferred to as acidophilic and basophilic cells ofsmooth organs. I found that·the proportions of thesecell types vary considerably along the body. Acidophilic cells dominate in the anterior part of thezone of smooth epidermal organs. The TEM showsthat basophilic and acidophilic material (sensu Akesson 1958) display a very complex structure of thegranules and other cellular contents, deserving amore detailed study. I have obtained the followingresults:

Non-granular cellular materialBasophilic cells sensu Akesson (1958). He found 5to 9 basophilic glandular cells located in the distalpart of the organ (Fig. 6).

I can confirm this finding. The basophilic cells havetrue pores as shown by Akesson (1958, p. 190).Each cell has an intracellular receptaculum with acanal to the surface. The TEM shows that numerousmicrovilli project into the receptaculum of thebasophilic cells (Fig. 7, and drawing on the presentcover).

, ,

•t3

I10

Figure 6. Smooth epidermal organ at the mid body.(I) termination area of axial cells. (2) pore area ofbasophilic cells. (3) pore area of eosinophilic, granular cells. Schmorl's staining for lipofuscin; unfixedPhascolion strombus. Inset: presumed innervationof axial and basophilic cell areas. Methylene bluesupra vitam. Scale bars: 10 11m.

Histochemistry: The cells contain basophilic mucopolysaccharide which stains deeply with alcian blueat pH 2.5. The neck of the receptaculum is stainedwith Schmorl (lipofuscin), Fontanasch solution(argentatfme reaction), and the inorganic phosphatemethod. The cells are not stained with Million(phenol), Long Ziehl-Neelsen's method (acid lipofuscin), PAS with and without deamination(carbohydrate), SCHIFF (free aldehydes) or PFAS(unsaturated fatty acids). There is no primary blacking with osmium tetroxide (easily oxidizable substances). The content is more or less amorphous inthe light microscope and TEM (Fig. 8, and drawingon the present cover).

Figure 7. Frontal section of receptaculum ofbasophilic cell in the upper part of smooth epidermal organ. Phascolion strombus. (I) cuticula.Drawn from TEM photo. Scale bar: 5 11m.

24 J0fgen Hylleberg

Fine granular cellular materialFine granular material is present in the cells referredto as acidophilic granular cells by Akesson (1958).Each cell connects with tQe surface through a canal.An intracellular receptaculum is absent (Fig. 5: No.4). The granules measure about 0.5-0.8 Ilm. Histochemistry: The material stains with Schmorl(lipofuscin), and the inorganic phosphate method. Itstains red with eosin, and purple with methyleneblue. There is dubious staining with PFAS andSchiff. Otherwise the material stains similar to thecoarse granular material mentioned below, but onlyweakly (Million, PAS, Long Ziehl-Neelsen's method, Osmium tetroxide, and PFAS).

Coarse granular cellular materialCoarse g~anular material is present in acidophilicgranular cells (Akesson 1958). He showed that thesecells lack receptacula and open directly to the surfacearound a central pore. I can confirm that all acido-

//i

I

Figure 8. Frontal section of a smooth epidermalorgan located at the anterior body of Phascolionstrombus. Section through the middle of the organ.(I) cuticula. (2) epidermis with only nuclei indicated. (3) basophilic cell. (4) unidentifil;d cell withamorphous cell contents. (5) coarse granular, e0

sinophilic cell. (6) tubular structure at edge of theorgan. Drawn from TEM photo. Scale bar: 10 Ilm.

philic cells lack receptacula. But they do not openaround a central pore: they have individual access tothe surface through 3-9 pores which measure about3-4 Ilm when fully open. (Fig. 5: No.5). The coarsegranules measure 1-2.5Ilm in diameter (Fig. 9). TheTEM shows that one half of each coarse granule islight, and the other half is medium electrondense.Often the surface appears crystalline, and each granule has a very electrondense cap. I have not seendescriptions of similar granules from other sipunculans.

Histochemistry: Schmorl's method stains lipofuscinpresent in the neck of the pores (Figs. 5 & 6). Similarly, the necks are stained deep blue with the inorganic phosphate method. The granular cell materialof unsectioned specimens strongly stains withmethylene blue (blue-green colour), Million (phenol,possibly reacting with tyrosine), Long ZiehlNeelsen's method (acid lipofuscin), osmium tetroxide (primary blacking), PAS (weakly after deamination), SCHIFF. (free aldehydes), and PFAS (unsaturated fatty acids). Benzidine stains part of thecoarse granular areas blue (oxidase). The histochemical tests indicate that this material is rich in proteins, carbohydrates, and lipids. Coarse granular material is always abundant in the anterior part of thesmooth region but the amounts are variable anddecrease in more distal organs of the smooth region(mid body). Remarks: Morphologically similargranules were described by Ochi (1969) in maleblood cells of the polychaete Travisia japonica.Lipid granules of uniformly high electron density

Figure 9. Coarse granules in smooth epidermalorgans at the mid body of Phascolion strombus.Granules in a web of tubules from the tubular structure, cf Fig. 8. Drawn from TEM photo. Scale bar:I Ilm.


were fused with 0.5-2 11m electron opaque granules(with an empty-like appearance). The boundary ofthe fused granules appeared in a marked straight orcurved line in sections. Highly electrondense granules were seen at the edge, but Ochi (1969) could notdecide whether they were getting in or out of thecomposite granule. Although morphologically similar, I find it unlikely that the composite granules inthe polychaete blood and the sipunculan epidermalorgan serve the same purpose. It is unknown howthe coarse granules are formed in the sipunculan, butthe source of granules may be golgi elements in thecells. The coarse granules are nested within an elaborate system of tubules projecting from an oblong ca.12 x 3 11m structure in the periphery of the organ. Itis difficult to identifY borderlines between cells onthe available pictures so the precise relationshipbetween the tubular structure and other cellular elements can not be ascertained (Fig. 8: No.6, Figs. 9& 10).I am not aware of descriptions of a similar structurein other invertebrates. The cells with coarse granuleshave a well developed endoplasmatic reticulum withribosomes indicating prominent synthesis of proteinand carbohydrate in agreement with the histochemical tests.

Figure 10. Frontal section of smooth epidermalorgan at the anterior body of Phaseo/ion strombus.Enlargement of tubular structure shown in Fig. 8(No.6). Drawn from TEM photo. Scale bar: 111m.

Innervation of smooth organsFig. 6 shows presumed innervation of the peripheryof basophilic cells (methylene blue staining supravitam). A marked blue streak developed at the baseof each pore, and more pronounced around the sensory buds of axial nerve cells (Fig. 6: inset). Inanterior smooth organs, methylene blue stained 6-10cells which stretched from the periphery of eachorgan towards the axial cells whose termination atthe surface of the cuticle also stained deep blue. Thepresumed neurons usually had a broadly lobed baseand a long slender neck, but ellipsoid shapes withlong necks were also common. The bottom ofsmooth organs contained a varicose central masswhich connected with a distinct nerve plexus linkingup all the epidermal organs of the body. From thecentral cell mass, a number of fine fibres stretched toepidermal cells underlying the cuticle. Attempts toidentifY the methylene blue cells on TEM picturesfailed because of the previously mentioned blackingof the presumed nerve cells. The thin sections stained with Bodian showed presence of an axial masscontaining nerve cells as found by Akesson (1958,p. 190). Furthermore, conspicuous 3-5 11m blackbodies were found at the bottom of cells and alsoimbedded in the cutis. The black bodies were connected with fibres to the epidermis, the axial cells,and the plexus of the body wall (Figs. 5 & II). Ihave not found mention of similar black bodies inthe literature so more studies are needed to show theaffiliation with nerves.

Figure 11. Sagittal section of epidermal organ inthe holdfast region at the mid body. The cuticularthickening (so-called horse shoe) not yet developed.(1) cuticula. (2) epidermis. (3) coarse granular cellwith pore to the surface. (4) axial cells. (7) presumedneurons at base of organ (in the cutis). - Drawn withthe aid of a camera lucida. Bodian-stained Phaseolion strombus. Scale bar: 10 11m.

26 J0rgen Hylleberg

Remarks: Akesson found that lateral nerves, givenoff from the ventral nerve cord, penetrate the longitudinal muscle and form a plexus between the circular and longitudinal muscles. The nerves divide andradiate into the cutis layer. The epidermal organs areinnervated from this plexus. My findings agree withAkesson (1958, p. 184). The net of fibres makingcontact with the glands are also in agreement withStehle (1953, p. 212) who found that a peripheralnerve entered the glandular epidermal organ throughthe cutis in Golfingia elongata. The nerve branchedand formed a plexus around each glandular cells.Akesson (1958, p. 195) agreed to this finding, butpointed out that some of the fibres were connectedto neurosensory cells, overlooked by Stehle.

Organs of the holdfast region (5)

A holdfast organ may measure 300 /lm across. Imeasured an average size of 140 /lm (range 89-178/lm; n = 37) on a specimen extracted from a Turritella shell. The crescentic anterior margin is thickened, usually pigmented and shaped like a pointedhorse-shoe. The rest of the surface is smooth. Thistype of organ forms a girdle posterior to the middleof the trunk.Akesson (1958, p. 192) found that basophilic cellspredominated and that acidophilic cells could beseen as strongly vacuolated rudiments in the proximal and peripheral parts. Most basophilic cells hadan elongated sac-like shape, and the granular contents tended to become amorphous distally. Smallreceptacles opened to the surface. I can confirm theseobservations. Schmorl's method for lipofuscin demonstrated 1-4 true pores connected with basophiliccells with a receptaculum and acidophilic cells without receptaculum (Fig. 12). The TEM showsbasophilic cells to be true mucus cells resemblinggoblet cells. The receptaculum of basophilic cellshas the same morphology as seen in smooth organs,viz., with numerous microvilli projecting into thelumen (Fig. 7). The acidophilic granular material isless abundant than in the smooth organs, and coarse,composite granules were not encountered in thestudied organ. Eosinophilic cells have an elaborateendoplasmatic reticulum (ER), and many golgielements, but both the ER and golgi appear differentwhen compared to the smooth organs. The ER iscurled and dense, and the golgi often appears asrings rather than stacks. In spite of the differentultrastructure in the TEM, the histochemical testswere basically identical regarding basophilic andacidophilic material of smooth and holdfast organs.The holdfast organs are also marked by prominentsynthesis ofprotein and carbohydrate.

The thick layer of material, characteristic of thehorse-shoe of these organs, has often been referred toas chitinous, but chitin is defmitely not involved.Three types of material can be distinguished in thecrescentic thickening of the anterior margin of aholdfast organ: The edge of the thickening close tothe cap ("central pore") is composed of yellowishgranules with a diameter of ca. I /lm. The yellowgranules gather into larger granules. There is a gradual transition to the next zone of granules. They aredarker brownish, somewhat oval granules with adiameter of about 5-8 /lm. I refer to those granulesas inner zone granules (Fig. 12: No 9). Finally thegranules form a compact dark brown-black tip. Irefer to this type of granule as the outer zone granules (Fig. 12: No 10). Histochemistry: Granules ofthe inner and outer zones stain red with Million(phenol) and Long Ziehl-Neelsen's method(lipofuscin). But the chemical nature of the granulesis uncertain.

Figure 12. Sagittal section of holdfast epidermalorgan at the mid body. (1) cuticula. (2) epidermis.(3) basophilic cell with receptaculum and pore. (4)mucus cell. (5) coarse granular cell with pore to thesurface. (6) axial cells. (7) presumed neurons at baseof organ (in cutis). (8) circular muscle. (9) innergranules of holdfast papilla. (10) outer granules ofholdfast papilla. - Schematic drawing of Bodianstained Phascolion strombus.


According to Kennedy (1969, p. 352) lipofuscin hasbeen found as brown, water insoluble and intracellular granules in nerve ganglia of sipunculans. Hereferred to lipofuscin as a compound derived fromfatty acid substances by oxidation. In accordancewith the Long Ziehl-Neelsen's method for lipofuscinthe reaction is positive in regard of a lipoid nature(Romeis § 1050, 1137). There is a strong xanthoproteic reaction (Romeis § 1247) of both innerand outer granules (dark yellow-orange). Trypan bluevacuoles intra vitam in the inner zone, also indicateoccurrence of proteinaceous substance (HyllebergKristensen, 1972). There is a positive Pearl's reaction for ferric iron of the inner zone granules in accordance with Gibb's (1985) demonstration of iron inthis zone. This is in concert with Romeis (§ 1137)who found that iron often is present in connectionwith lipofuscin (Abnutzungspigmente). However,the relationship between pigment and iron is notunderstood. Furthermore, I have applied histochemical spot tests which were negative in regard of camtinoids and melanin (Romeis § 1142, and § 1139).Schmorl's method (lipofuscin) was also negative.Remarks: The distribution, size, and colour ofholdfasts depend upon the size of the P. strombus,as well as the type of shelter (Hylleberg 1975a). Thevariable appearance of holdfasts has created muchtaxonomic confusion leading to the erection of several species and subspecies which are consideredsynonyms by contemporary taxonomists (HyllebergI994a).

Innervation of the holdfast organsWith the SEM and in thin sections stained withBodian, I found that the nerve endings terminate ina plug which usually projects into a nipple (Fig.12:on top of No 6). Methylene blue may stain 1-5 dotsin the nipple area intra vitam. Otherwise, I onlymanaged to stain the edge of the thickened horseshoe with this technique. The margin of the innergranular zone often turned deep blue and sent projections into the granular inner zone area as well astowards the base of the organ. This would agreewith Akesson (1958, p. 192) who found that thenerve cells usually are close to the armed side of theorgan. In Bodian-stained sections I found presumednerves in this position of the holdfast organ (Fig.13), indicating that the area may be equipped withreceptors. Nerves of the axial cells, which always aretilted compared to the sagittal plane (Figs. II &12), connect to the subepidermal plexus as found byAkesson (1958, p. 191).

Figure 13. Sagittal section of holdfast epidermalorgan at the mid body. (I) cuticula. (2) coarse granular, outer zone material. (3) epidermis. (4) axialcells with presumed neurons. (5) fibres presumedconnecting neurons with the plexus. Camera lucidadrawing of Bodian-stained Phascolion strombus.Scale bar: 10 Jlm.

Figure 14. Sagittal section of papillated epidermalorgan at the posterior body. (1) epidermal organwith pigment granules. (2) cuticula. (3) epidermis. Camera lucida drawing of P. strombus. Gomori'strichrome. Scale bar: 10 Jlm.

Organs of the posterior end (6)

Akesson (1958, p.186) found that papillated epidermal organs of the introvert and the anterior andposterior parts of the trunk are similar with respect

28 J0rgen Hylleberg

to general structure and cellular contents. I agree tothis finding. Schmorl's method did not show presence of pores. The cuticle contains many granulesor flakes which stain red .with Long Ziehl-Neelsen'smethod for acid lipofuscin (Fig. 14).

FUNCTION OF THE EPIDERMALORGANS

Empty Turritella shells were cut and abraded on oneside, creating 7 windows which were closed with acover slip tightly glued to the surface with Araldit.Phaseolion strombus were extracted from otherTurritella shells and placed in small aquaria with 3em sediment from the habitat and the windowshells. P. strombus established themselves insidethe shells and movements of the body were observedthrough the glass. A normal clay plug was formed atthe aperture (Hylleberg Kristensen 1969, 1970)In preserved specimens, the body is more or lesscylindrical, although twisted after life in Turritellashells. Epidermal organs of the smooth region areoval with the long axis following the body curvature. This shape is rarely seen in live specimens inwindow-shells. The smooth organs are round, ormore common: the long axis of an organ is in thelongitudinal direction.The holdfast papillae do not change shape when thebody is stretched, indicating that the function of theanterior thickening may be to secure form-stabilityof those organs. I have previously shown a cleaningfunction of the organs (Hylleberg 1975a). The concept of form-stability is in concert with the latterfunction, but form-stability may also be related tothe way of secretion or to the fact that receptor cellsare more numerous in this type than any other typeof epidermal organ. Akesson (1958, p. 192) recordedmore than 50 neurosensory cells in one organ.With window-shells, the smooth region is slender,freely moving in the shell lumen, rarely touchingthe glass window. The holdfast region can also bestretched, but is often inflated and firmly pressedagainst the glass. Only the posterior body is alwaysin contact with the shell, giving the impression ofan anchor. The anus is kept in a position outside theshelter, or at the outer edge of the clay plug.

AUTOTOMY

A zone with thin cuticula and muscle layers is located at the transition between introvert arid body, justin front of the anus (Hylleberg Kristensen 1969, p.109). Lateral branches of the ventral nerve cord are

fused and form a ring around the zone of autotomy(Fig. 15).Epidermal organs are located on either side of thiszone. Strands of oblique muscles are located between the circular and longitudinal muscles in thiszone. Oblique muscles pass the anus and extendposteriorly. If the introvert is seized, a P. strombuswill sacrifice the introvert by autotomy in this zone.(Hylleberg; in Kohn 1975, discussion p. 331)

Figure 15. Area between body and introvert ofPhaseolion strombus. Dissected specimen observedfrom the coelomic side. (I) ventral nerve cord. (2)zone of autotomy. (3) anus. (4) oblique musculaturein a layer between circular and longitudinal muscles.Schematic drawing.

Morphology of thedigestive system, with

notes on food and feeding

INTRODUCTION

Divisions of digestive tractsTheel (1875) gave the first description of the intestinal system of Phaseolion strombus, and Brumpt(1897) added information on the distribution ofchloragogue cells (Note: chloragogue cell is used byHyman (1959). Alternative spellings are chloragogene or chloragen cell). According to Stehle (1953),who made a very comprehensive study of the intestinal system of Golfingia e!ongata, the followinggut systems of other species were investigated indetail around the turn of this century: Andrews(1890) described the gut of Phase%psis gou/dii,


Wilcynski (1913) worked with Goljingia margaritaceum, Shipley (1890) with Phascolosoma varians,Metalnikoff (1900) with Sipunculus nudus. Cuenot(1900) made careful observations of Goljingia vulgare. In addition, Paul (1910) studied Nephasomaminutum, Arvy & Gabe (1952) described the histochemistry of the digestive tract of Phaseolionstrombus, and Michel & DeVillez (1984) investigated both histochemistry and fine structure of thedigestive tract of Phaseolopsis gouldii.Except for Phascolion strombus, all of the abovesipunculans have a straight oesophagus entering amore or less tightly coiled intestine comprised of adescending and an ascending part. A caecum marksthe end of the ascending intestine and the beginningof the rectum, which extends .across the body to thedorsal anus. In Phascolion, however, the intestine isbasically made up of 5-6 gently winding loops fixedto the body wall with a high number of musclesstrands (Figs. 3 & 16). Because of the high numberof winding loops it is not obvious how to comparethe gut of Phaseolion with other sipunculans, i.e.,to identify the parts corresponding to the descendingand ascending intestines of the genera with tightlycoiled intestines. Based on general morphology,methylene blue staining of the nervous system, andhistochemistry of the gut, I suggest that the descending homologue is about twice as long as the ascending part in Phaseolion. But comparisons withother species are difficult because of the limitednumber of studies. Only about 10 species have beenstudied in detail so far. Marked differences havebeen described, indicating that useful taxonomiccharacters can be found at the generic as welI as thespecific level. Unfortunately, some of the easy toobserve characters display much variation, e.g. thetotal number and the attachment area of individualmuscular strands (Figs. 3 & 16). Hence, the totalnumber of strands must be treated with caution as ataxonomic character.Stehle (1952, 1953) divided the intestinal system ofGoljingia elongata into 10 parts: pharynx, oesophagus, prestomach, stomach, anterior, middle andposterior intestines, rectum, and anus. BasicalIy, Iagree with this division, although the prestomachand stomach are not marked in P. strombus compared to G. elongata. However, Hyman (1959) questioned the general applicability of' the divisions proposed by Stehle (1953) as most of them were basedon small constrictions or slight differences in walIstructure. Working with Phaseolion strombus, Theel(1875) mentioned 3 divisions of the gut· of thisspecies, viz., oesophagus from the mouth to thebeginning of the ciliated groove, the intestine proper, and rectum from termination of the ciliary gro-

ove to the anus. Also working with P. strombus,Arvy & Gabe (1952) distinguished 7 divisions ofthe gut, viz., oesophagus, descending, ascending,and posterior intestines, rectal caecum, rectum, andanus. Arvy & Gabe (1952) also refer to the classicdivision into 4 divisions encompassing pharynx,mid & posterior intestines, and rectum.

MATERIALS AND METHODS

Figure 16. Divisions of intestinal system of dissected Phascolion strombus observed from the coelomic side. The introvert is cut and mouth (1) andpharynx (2) are excluded. The Fig. shows: (3) 0

esophagus. (4) anterior striped intestine. (5) posterior striped intestine. (6) yelIow intestine. (7) loop.(8) posterior intestine. (9) rectal caecum. (10) rectum. (11) anus. (T) arrows indicate transitions between divisions. (dr) base of dorsal retractor. (vr)ventral retractor. (n) nephridium. (c) ventral nervecord. (a) line of autotomy at transition of body andintrovert. Schematic drawing.

30 J0rgen Hylleberg

The gut of Phaseolion strombus was separated intoII divisions characterized in terms of morphology,histochemistry and function (Fig. 16). The peripheral nervous system was studied with methylene bluesupra vitam, and staining according to Bodian.Further mention of the applied techniques is givenin the general paragraph on Materials and Methods.

RESULTS

Digestive tract of Phascolion strombus

Mouth (1)

Food is collected on short tentacles and conveyed tothe centrally located mouth. The particles continueinto a buccal cavity which is lined with ciliated cellssimilar to those found on the oral side of the tentacles.

Figure 17. The first part of oesophagus of Phaseolion strombus is located in a muscular tube comprised of the fused retractors. The figure shows thepoint where oesophagus (I), with a~ached dorsalvessel (2), and ventral retractor (3), is separatedfrom the dorsal retractor (4). Schematic drawingbased on dissected live specimen.

Pharynx (2)

A short pharynx (stomodaeal invagination, Akesson1958) is surrounded by the fused dorsal and ventralretractors (Fig. 17).Innervation. As stated by Akesson (1958, p. 223).Nerve bundles in the pharynx have a position peripheral to the circular muscle layer (Fig. 18).

Oesophagus (3)

A straight oesophagus is attached to the ventralretractors for about half of the length by numerousmusculo-corumective strands (Fig. 19). A simplecontractile vessel is dorsally attached to the oesophagus for its entire length (Fig. 19).A sharp transition between oesophagus and theanterior descending intestine is formed by asphincter, a fastening muscle, and the blind termination of the contractile vessel (Fig. 16). The oesophagus epithelium is lined with abundant ciliatedcells.

Figure 18. Brain region of dissected Phaseo/ionstrombus seen from the coelomic side. Stainedsupra vitam with methylene blue. (I) left branch ofventral nerve cord entering the oesophageal ring. (2)circular nerves innervating the pharynx. (3) innervation of retractors. - Schematic drawing of live material.


Histochemistry. The oesophagus has many cellsrich in Iysosomes, especially in the peritoneum andcells lining the junction between oesophagus and thedorsal vessel. The present staining with neutral redin vivo, toluidine blue supra vitam, and histochemical tests by Arvy & Gabe (1952) emphasize thesharp borderline between the oesophagus and thenext section of the gut (Table 1).

Table 1. Vital staining of the intestinal system ofP. strombus. Cellular content of cells in (A) oesophagus, (B) striped intestine, (C) yellow intestine,(D) loop, (E) posterior intestine, (F) caecum, (G)rectum.

Section Neutral Toluidine Trypan Benzidineof gut red blue blue +H202

A ++ + + +B +++ +++ +++C ++ ++ +D ++ ++ +E ++ ++ +++F +G + + +++ +

Table 2. Methylene blue staining, supra vitam, ofthe intestinal system of P. strombus. Nervous structures in (1) oesophagus, (2) striped anterior intestine, (3) yellow intestine, (4) loop, (5) posterior intestine, (6) caecum, (7) rectum. A = Semicircularinnervation; B = Plexus; C = Pyriform bodies.

Section 2 3 4 5 6 7A +++ + + +++ + +B + ++ ++ ++ + ++ +++C++ +++ + +++ +++ ++

Innervation. Akesson (1958, p. 72) found that thenerve plexus in the oesophagus and the followingparts of the intestine originates from the oesophagusnerves but receives also fibres from the retractornerves in the anterior region. I have reached the sameconclusion as Akesson (op. cit). He also usedmethylene blue supra vitam technique. The areaanterior and posterior' to the transition between oesophagus and the striped anterior intestine hasnumerous semicircular nerves associated with thisepidermal plexus (Table 2).

descending1 2 3 4 5

loop6 7

ascending8 9 10 II

----------

: ~'... .....:...:..:...... ::.,

Figure 19. The transition between oesophagus andthe striped intestine of a dissected Phaseolionstrombus. Stained with methylene blue supra vitam.(1) dorsal vessel. (2) oesophagus with circular nervesand plexus. (3) ventral retractor. - Drawn with theaid of a camera lucida. Scale bar: 100 ~m.

ABCDEFG --(-) - (-H )......--

Figure 20. Divisions of the intestinal tract of Phaseolion strombus are indicated by horizontal barsand abbreviated as follows: (I) mouth, (2) pharynx,(3) oesophagus, (4) anterior striped intestine (5)posterior striped intestine, (6) yellow intestine, (7)"loop" where the intestine makes a sharp V-turn andthe basically descending intestine definitely becomesascending, (8) posterior intestine, (9) rectal caecum,(10) rectum, and (II) anus. The terminology variesamong students of sipunculan species. The suggested homologies are shown. Legend: A. = the present study of Phaseolion strombus; B = Theel(1875) for P. strombus, C = Arvy & Gabe (1952)for P. strombus; D = Paul (1910) for Nephasomaminutum; E = Stehle (1953) for Golfingia elongata;F = Michel & DeVillez (1984) for Phaseolopsisgouldii; and G = Hyman (1959) for sipunculans ingeneral.

32 J0rgen Hylleberg

The nerves extend from the ventral to the dorsal sidebut I could not ascertain whether they cross themidline. Occasionally they appeared to do so. Longitudinal nerves in the oesophagus continue in thefixing muscle. Remarks. Brumpt (1897) did notfind chloragogue cells in the oesophagus of P.strombus. I agree with this finding. Arvy & Gabe(1952) did not discover enzymatic activity but notedlimited storage of glycogen in the epithelium of P.strombus. The term oesophagus has been used bymost students of the digestive system (Fig. 20).Stehle (1953) stated that the oesophagus of Golfingia elongata is attached to the retractors for a distance by a mesenterium. Furthermore, the circularmuscles are weaker compared to pharynx, chloragogue cells are present, the cylindrical epithelium istall and ciliated, there are abundant mucus cells, anda constriction marks the transition to a prestomach.

Anterior striped intestine (4)

The anterior striped intestine has distinct longitudinal musculo-connective strands and granular, enzymatic cells of the lining epithelium. Brumpt (1897)noticed chloragogue cells which stained deep violetwith acid fuchsin R on the striped intestine of P.strombus. Histochemistry. Vacuoles in cells of themuscular strands of the striped intestine stain withtrypan blue in vivo. Enzymatic cells between thestrands do not accumulate trypan blue. In contrastthese enzymatic cells stain deeply with neutral redand toluidine while the muscle strands are unstained. A marked blue staining with benzidine startsshortly after the oesophagus and ends shortly beforethe striped intestine continues into the yellow intestine. Basic dyes stain numerous vacuoles along theperiphery of peritoneal cells in vivo and supra vitam.With methylene blue supra vitam, large cylindercells (referred to as cells of Leydig) contain a fewblue granules in the layer facing the ciliated groove.In comparison, all other cells of the epithelium,lateral to the cells of Leydig, are stuffed with deepblue granules. The ciliary groove is lined withmuscular fibres which do not take methylene blue.Apical parts of cells lining the ciliary groove containnumerous small methylene blue vacuoles. Cells ofLeydig, rich in glycogen, tum dark brown in Lugol's solution. Arvy & Gabe (1952) found lowalkaline phosphatase in the beginning of the stripedintestine of P. strombus. The enzymatic activitygradually increased from weak to medium and finally strong activity posteriorly. There was· no lipaseactivity in this first section of the striped intestine.The epithelium is lined with ciliated cells (Arvy &Gabe 1952). Innervation. The number of semicircu-

lar nerves is low in the striped intestine. By focusing through the tissues at high magnification, thefollowing layers can be distinguished: longitudinal,anastomosing fibres of a plexus, unipolar cells overthe ciliated groove, and some semicircular fibres(Table 2). The anastomosing fibres and the unipolarcells are part of the plexus first described by Akesson (1958, p.225). Remarks. The large, elongated,vesicular cells lateral to the ciliary groove, werereferred to as cells of Leydig by Cuenot (1900).According to Brumpt (1897), Leydig had previouslydescribed some large cells which he called"Fettkorper". I have not ben able to clarify if thosefat cells of Leydig are identical to the concept of thecells of Leydig applied by Cuenot. Anyhow, it hasbecome common practice to use the term Leydig'scells for the large cylindrical cells lining the ciliatedgroove (Fig. 3). Certainly, the cells do not containlipids. I found that they appear completely empty,except for a small nucleus, when stained with Bodian. This agrees with Arvy & Gabe (1952) whoshowed that the cells of Leydig are rich in glycogenwhich requires special staining to be seen in sections.I found a weak staining reaction with benzidine atthe start of the striped intestine indicating a differenthistochemistry of the epithelium (interpreted asabsence of oxidase activity). These differences maysignify an area corresponding to the zigzag stripedpre-stomach described by Stehle (1953) for Golfingia elongata. The prestomach has finely granulatedepithelial cells, and a valvula at the transition to thestomach. Two muscle strands are rooted at thistransition in G. elongata.Paul (1910) used the term "Mitteldarm" for thecombined descending-ascending parts of the intestine. He characterized the first part of the"Mitteldarm" as being striped, with granulatedepithelial cells. In the gut epithelium he saw some"Wimpersacke" termed "Krypten" by Stehle (1953),and "ciliated intestinal pits" by Hyman (1959).I interpret the striped intestine, part 4, of P. strombus, as corresponding with the stomach region ofGolfingia elongata (Stehle 1953), and the descending intestine, pars anterior, of P. strombus in thestudy of Arvy & Gabe (1952)

Posterior striped intestine (5)

A transition between the parts 4 and 5 of the stripedintestine is marked morphologically by a fasteningmuscle and the appearance of a clear ciliary groovestretching towards the caecum (Figs. 3 & 16). Theciliary groove is conspicuous on account of large,elongated, vesicular cells lateral to the groove (cells


of Leydig ef Cuenot (1900) and Hyman (1959).Histochemistry. The cells of Leydig take up verylittle neutral red in vivo. They appear as empty cellsunder the light microscope. In contrast, the enzymatic cells, bordering the cells of Leydig, are rich inIysosomes and appear as red bands when stainedvitally with neutral red. Histochemical tests (Arvy& Gabe 1952) did not indicate any marked changefrom section 4 to 5. Changes appeared to be gradualalong the entire anterior intestine. But, in section 5,that is the second half of the striped intestine, theynoted strong alkaline phosphatase and high lipaseactivity in apical part of cells, high concentrations ofcytoplasmic ribonuclein, and osmophile lipids.Remarks. The stomach of Golfingia elongata(Stehle 1953) is characterized by a loose laminapropria where the connective tissue contains individual muscle fibres and the bases of a tall, columnar,ciliated epithelium thrown into 6 longitudinal folds.Development of a so-called stomach (Hyman 1959)is marked in Golfingia but not in Phascolion. Isuggest that the posterior striped intestine of P.strombus corresponds to the stomach of Golfingiaelongata (Stehle 1953) and to the descending intestine, pars media & posterior, in the study by Arvy& Gabe (1952).

Yellow intestine (6)

The yellow intestine is sharply marked as a shift incolour caused by the colour of abundant chloragoguecells dominant on the surface of this section. Chloragogue cells are digitiform or pyriform. They contain yellow-brown granules imbedded in cytoplasmand bulge into the coelom. The lower part of thedescending intestine can easily be separated from thepreceding part by in vivo and histochemical stainingtechniques (Fig. 16). Histochemistry. Lipid droplets in the chloragogue cells stain with sudanblack. Supra vital staining with methylene blueshows a few blue granules in the cells of Leydigfacing the ciliated groove. With PAS (carbohydrates)the yellow intestine turns red while all other sections of the intestinal tract are purple. Innervation.Longitudinal, anastomosing fibres and unipolar cellsare found over the ciliated groove. Some semicircular fibres are stained in the yellow intestine, similarto findings in the striped intestine (Table 2). Abundant pyriform bodies have a more triangular outlinein the yellow intestine, and they point towards thelumen of the intestine. Remarks. Working with P.strombus, Brumpt (1897) noticed chloragogue cellswhich stained rose with acid fuchsin R on the firstpart of the yellow intestine (but not on the remaining part). Chloragogue cells may get loose, enter

the coelomic fluid, and eventually form largerclumps which ultimately pass through the nephridiato the outside (Baltzer 1934, p. 49).The "Zwischendarm" of Golfingia elongata (Stehle1953) is characterized by a weakly developed laminapropria with few muscle fibres, numerous folds ofthe epithelium, abundant secretory cells, includingmucus cells, ciliated intestinal pits, and gut papillaereferred to as "Darmpapillen" and posteriorly"Drusenanhang" (= external intestinal papillae andcoiled tubular gland, respectively in Hyman 1959). Ihave not observed such pits or papillae in Phaseolion strombus where the ciliary groove and cells ofLeydig are dominant features of the yellow intestine.The "Zwischendarm" of Golfingia elongata continues into the "Mitteldarm" (Stehle 1953). He characterized this part as having many secretory cells,gut papillae, and coiled tubular glands. The twoparts of the intestine are connected through a distinctvalvula provided with a muscularis. I have observedvalvula in the intestine of P. strombus, but not yetascertained how they correspond to Stehle's findings(1953).I suggest that "Zwischendarm-Mitteldarm" of Golfingia elongata (terminology of Stehle 1953), andthe ascending intestine of Phaseolion strombus(terminology of Arvy & Gabe, 1952) correspond tothe yellow intestine of this study.I confirm the finding of Arvy & Gabe (1952) thatcells of Leydig are rich in glycogen in this part ofthe gut. These cells are dominant in the descendingpart of the intestines of Golfingia vulgare and Phaseolopsis gouldii which I consider homologous tothe yellow intestine in Phaseolion strombus.I have found parasitic protozooans, in all dissectedP. strombus. The parasites are flat, ellipsoid, pointed at one end, and measure about 40 x 25 ~m whenfixed. They are prominent in the yellow intestine,where they concentrate in the glycogen rich cells ofLeydig, but they occur from the oesophagus torectum. I have identified them as Leeudina franciana Arvy, 1952. This gregarine was described fromintestinal cells of Phaseolion strombus collected atDinard, France (Arvy I952b).

The loop (7)

The "loop" may represent a rudiment of the coiledintestines characteristic of most sipunculan genera.In Phaseolion the last part of the yellow intestine isattached to the opposite part of the ascending intestine with about 10 fastening muscles (Fig. 16). Theloop formed by this process is usually somewhattwisted. Histochemistry. Neither vital staining norhistochemical tests showed anything special in the

34 J0rgen Hylleberg

base of an urn appears thickened, homogeneous, andparallel to the gut surface.

_10

_10

Figure 22. Fixed urns on the posterior intestine ofPhascolion strombus. Oval bodies are seen insidethe urns. Drawn with the aid of a camera lucida.Scale bar: 10 11m.

Figure 21. Varicose ring nerves from the intestinalloop of Phascolion strombus stained supra vitamwith methylene blue. - Drawn with the aid of acamera lucida. Scale bar: 10 11m.

loop. Reactions and staining patterns are similar tothe succeeding division of the posterior intestine.Innervation. Longitudinal, anastomosing fibres andunipolar cells are present'over the ciliated groove asin the preceding divisions. The sling contains moderate numbers of triangular pyriform bodies located intissues lateral to the ciliary groove, and pointingtowards the lumen of the intestine. Varicose semicircular fibres are well developed at the V-tum of theloop (Table 1 and Fig. 21). Remarks. Paul (1910)referred the loop to the second part of the"Mitteldarm" in Nephasoma minutum. The ciliatedgroove started in the "Mitteldarm", and the gut wasprovided with abundant ciliated intestinal pits(Wimpersacke) at the loop. Brumpt (1897) foundthat the chloragogue cells stained deep violet withacid fuchsin R compared to unstained cells in theadjoining part yellow intestine of Phascolionstrombus. Deep violet staining of chloragogue cellscontinued on the remaining part of the gut right tothe anus. The uniform staining pattern of chloragogenes on the loop suggests that the turning point ofthe loop does not signify a point of functional change. This would agree with the conclusion of Paul(1910). However, I have maintained the loop as aseparate division because the loop is easily identified on account of the muscle strands intimately connecting the anterior and posterior branches. In addition, the gut lumen is narrowed in the loop compared to the preceding and succeeding divisions, andthe abundant circular nerves (Fig. 21) indicate thatthe loop after all may play a specific role in thedigestive process.I suggest that the anterior section of the loop is partof the middle intestine of Golfingia elongata.(Stehle 1953), and that the anterior section corresponds to the last part of the ascending intestine ofP. strombus in the study by Arvy & Gabe (1952).The dorsal part of the loop should then correspondto the beginning of the posterior intestine. I suggestthat it corresponds to the second part of the"Mitteldarm", of Nephasoma minutum (Paul 1910).

Posterior intestine (8)

The posterior intestine is characterized by abundant,ciliated fixed urns from the sling towards the beginning of the rectum (Fig. 3). Chloragogue cells arealso common. Small muscular strands connect mostof the ascending intestine to the body wall (Fig.16). Fixed urns arise as elevations on the coelomicsurface (Fig. 3). Each urn is shaped like the hoof ofa horse. It has an interior lumen and sides coveredwith peritoneum. The crescentic opening is providedwith numerous cilia along the edge (Fig. 22). The


Histochemistry. The crescentic base of fixed urnsstains blue-green with Perl's method indicatingpresence of ferric iron. The crescentic bases are alsostained with trypan blue supra vitam. Staining withLugol's solution shows that cells of Leydig, containing glycogen granules, are more abundant in theposterior intestine than anywhere else in the digestive tract. Toluidine blue accumulates intra vitam ingranular cells (red-purple colour as in other parts ofthe gut system except caecum and rectum). Innervation. Methylene blue intra vitam shows a highnumber of pyriform bodies (Table 2). Otherwise thecircular innervation and plexus appear less prominent than in the preceding sections of the gut. Remarks. Fixed urns contain abundant oval bodiessimilar in size and shape to oval bodies in the c0,

elomic fluid (Fig. 22). However, urn-bodies do notcontain vacuoles, in contrast to oval bodies in thecoelomic fluid (el Fig. 2). The function of ovalbodies is unknown but I have regularly observedbodies excreted with faeces, or voided through thenephridium, indicating that they constitute wasteproducts. In Golfingia vulgaris, the roof of the urnis created by a vacuole (Hyman 1959, Fig. 2310).This construction has been observed in many speciesof sipunculans, including Phaseolion strombus.Herubel (1902) and Selensky (1908) mentioned thepresence of mixed cell debris in fixed urns of Goljingia vulgaris and Aspidosiphon respectively. Butoval bodies dominate in the fixed urns of P. strombus. Herubel (1902) remarked that when urns werepresent, chloragogue cells were always present. But,when urns were absent, chloragogue cells might, ormight not be present. It follows that when chloragogue cells are absent, then urns also are absent.Paul (1910) found that the ciliated groove started inthe "Mitteldarm", third part, of Nephasoma minutum. He did not describe urns or chloragoguecells, but found intestinal pits common in this partof the gut.The present usage of posterior intestine is identicalto the "Hinterdarm" in Golfingia elongata (Stehle1953) and the posterior intestine used by Arvy &Gabe (1952) for P. strombus. 1 suggest that it corresponds to the third part of the "Mitteldarm II ofNephasoma minutum (Paul 1910).

Rectal caecum (9)

The ciliary groove ends in a rectal caecum located atthe beginning of the rectum. By in vivo microscopyof dissected specimens, I noticed that the bottompart of the caecum has a delicate valve allowing thecaecum to be emptied into the rectum opposite tothe entrance from the ciliary groove. Rectal caeca

display numerous pyriform protuberances on theperitoneal surface when observed in vivo with phasecontrast at high magnification. Histochemistry.Arvy & Gabe (1952) noted mucous secretion in thediverticulum. There was no enzymatic activity.Stehle (1953) found secretory cells in the epitheliumbut nothing else that could indicate a specificfunction of the caecum. I found that toluidine blueaccumulates in scattered vacuoles (blue colour as inthe rectum, but not in other parts of the gut). Neutral red was only observed in vacuoles of the peritoneal cells. The significance of the toluidine bluecolour pattern is not understood. The pattern ofneutral red uptake is normal for the peritoneum..Innervation. Anastomosing longitudinal nervefibres branch off into the rectal caecum, but mostfibres continue laterally into the rectum. Remarks.Stehle (1953) described the caecum ("Divertikel") ofGolfingia elongata as the terminal part of the posterior intestine. It has many folds of the epithelium,ciliated cells and some secretion of mucus. I haveobserved that material enters the caecum of Phaseolion strombus together with mucus bands, about 1211m wide. The mucus bands disintegrate and arecompressed in the caecum. The nature of materialenclosed in the caecum is very variable as shown inthe following paragraph on food and feeding. Presence of motile granular cells (amoebocytes) indicates that intracellular digestion may occur.

Rectum (10)

The beginning of rectum is defined by the caecum, asphincter, and a fastening muscle (Fig. 16). Manysmall muscular strands connect the lower side of theposterior rectum to the body wall while a muscularmesenterial sheet attaches the rectum to the bodywall for the last ca. 30% of its length (Fig. 16).Shortly before the anus, the rectum is attached to thebody wall by a number of muscular strands referredto as the wing muscle. This muscle spreads like asheet to the left and right at the anus (Fig. 16).Histochemistry. Perl's method produces dark bluegranules, indicating presence of ferric iron in therectal epithelium. Toluidine blue supra vitam yieldsblue vacuoles, up to 10 11m in diameter. As with theposterior intestine, scattered cells may accumulatetrypan blue in large vacuoles, supra vitam. Innervation. Methylene blue staining shows a reducednumber of pyriform bodies compared to the posterior intestine, but a more well developed plexus. Thenumber of circular nerves is low, as found in thepreceding part of the gut. Remarks. The presentusage of rectum is identical to the terminology ofStehle (1953) for Golfingia elongata. and Arvy &

36 J0rgen Hylleberg

Gabe (1952) for P. strombus. The term is well defined and used by all students of gut systems (Fig.20). Stehle (1953) described the rectum of Go/fingiaelongata as being characterized by many folds of theepithelium which carried ciliated cells and had somemucus secretion at the beginning of its course. Paul(1910) found that the rectum of Nephasoma minutum had connective tissue with many musclefibres. He never observed faecal material in the rectum. I have made similar observations in Phaseolion strombus. The rectum was often empty, or nearlyso, in spite of an otherwise full intestine. The significance of this finding is not understood.

Anus (11)

The anus is a dorsal cylindrical tube which can beclosed by a terminal sphincter. This location andconstruction is a universal feature among sipunculans (Hyman 1959).

DIGESTIVE TRACT FUNCTIONALASPECTS

Total length of the digestive tractI measured the length of the intestine from mouth toanus to be about 4 times the body length. Thisagrees with Theel (1875). The oesophagus accountedfor about 18%, striped intestine 24%, yellow intestine 22%, "loop" 14%, posterior intestine I6 %, andrectum 6% of the gut length (n = 4).

Content and movement of the intestinal tractSelected particles wrapped in sticky mucus aremoved in the form of elongated boli from the mouthand pharynx to the oesophagus. Suspended in gutfluid, the boli move individually from the anteriorintestine to the sling. In animals with full intestines, the boli pile up into masses in the voluminousyellow intestine and can completely fill the slingand the ascending intestine right to the rectum. Thesticky membranes are more or less dissolved in theposterior part of the gut system. Animals with lowamounts of gut content keep boli suspended in fluidall along the digestive tract.There is a pronounced individual variation in termsof the degree of gut filling. All stages of fillingfrom nearly empty to completely full have beenobserved in animals collected at the same time (n =

15). Similar strong individual variation is characteristic of controlled feeding experiments in aquaria (n= 62).I observed action of the rectum during defaecation ina small semitransparent individual. Vigorous peri-

staltic movements took place in the rectum beforedefaecation, but then movements stopped, the rectalsphincter closed, and faeces slit slowly out of theanus. Subsequently the anus was closed, thesphincter relaxed, and a new portion of gut contententered rectum from the ascending intestine. Faeceshad a loose texture and fell apart in the water. Theirloose texture has been observed many times. I neversaw emptying of the rectal diverticulum which usually is full of fine grained particulate matter.Material enters the rectal diverticulum via the ciliated groove. In this groove, I have measured thespeed of particles to be 0.05 mm per second(dissected guts, n= 14). I calculated that on an average it would take ea. 20 min for a particle to travelfrom the beginning of the groove to the rectal diverticulum. In consequence, this route of particle transport is very fast compared to the movement of gutcontents in the gut lumen. The exact turnover timefor gut contents has not been measured in Phaseolion strombus but Hansen (1978) estimated the timeto be 19 ±1.5 hrs at 10 °C in Sipuneulus nudus.

Feeding behaviourPhaseolion strombus has a highly cryptic feedingbehaviour. Most of the time, the introvert is movedthrough the sediment a few mm below the surface.Phaseolion can probe the sediment 360 degreesaround itself without moving the shell used forshelter (Fig. 3). A worm can stay in the same location for a long time, or pull itself forwards after ashort time at a given location. The front part of ashelter is kept hidden in soft sediment. Only theapex of the shell will protrude into the water(Hylleberg Kristensen 1970).Analysis of tentacle response to offered food wasinconclusive. Animals in clean sea water withoutsubstratum were tested with surface sediment rich inbacteria and diatoms dropped near to, or on thetentacles. Mud adhered to the tentacles all over thesurfaces, but was not moved towards the mouth.Neither were particles on top of the ciliated surface.The tentacles were moved from side to side,touching each other for a while, or curling up fromthe tip towards the mouth, but none of these movements could be interpreted as active feeding.

Potential food sourcesUnfortunately, studies of the intestinal content of P.strombus did not provide much information towardsan understanding of the actual food source. Boliextracted from the oesophagus and the first descending part of the gut mostly contained a few smallzooflagellates (Bodo & Euglena) and small numbersof motile bacteria. Staining with rose bengal, accor-


ding to the method previously used with success instudies on polychaete feeding (Hylleberg 1975b),only revealed sessile bacteria in small numbers.Motile flagellates and bacteria were not observed insucceeding parts of the gut, indicating digestion,which may be intracellular to some extent. Myevidence is that I have observed many amoebocytes(about 9 /lm diameter when contracted) in boli ofthe oesophagus. At the end of the digestive tract, inthe rectal caecum, up to 6 live 6-48 /lm long benthicdiatoms were found in one specimen. Some specimens contained numerous live motile and sessilebacteria, and up to 150 live cylindrical flagellates,each measuring 5 x 2 /lm. On other occasions thecontent was nearly sterile. The function of the diverticulum is not known.Analyses of gut contents from other parts of thedigestive system were also inconclusive. Sometimesthe intestinal system contained many bacteria, flagellates and diatoms. On other occasions, nothing offood value could be identified. Sometimes thecounts of bacteria and flagellates showed a cleardecrease from the oesophagus to the rectum, indicating digestion, but in other specimens it turned outto be the other way round. Some individuals hadintestines full of ingested sediment while othersfrom the same collection had half full, or emptyguts. I am unable to explain these findings, but apossible explanation could be found in the conceptof gardening, which would result in individualfeeding rhythms (Hylleberg 1975b). If this were thecase, P. strombus should only ingest material whenit pays for the worm to eat. However, there is noevidence of such feeding behaviour in sipunculans.Comparison of gut contents with faeces has alsobeen non-conclusive. For the first it was very diffi-

cult to collect faeces from this species in situ. Faeces are voided at irregular intervals and the textureso loose that they easily mix with ambient sediment. 1 have only collected clean samples of faecesfrom live animals a few times. Each time, the faeceswas without remains of organism which could givea clue as to the digested food. I never saw meiofaunasuch as nematodes, harpacticoids, foraminiferans, orbivalves similar to the findings by Walter (1973)and Hansen (1978) in the much larger species Sipunculus nudus and Go/fingia vulgare. In experimentswhere Phascolion were offered mixed phytoplanktonand zooplankton, not a single cell could be found inthe guts after 6 hrs (n =2). A few boli were found,and they only contained mucus.

Gut filling time and defaecationStarved P. strombus kept in clean sea water withoutsediment were transferred to sediment and individuals dissected at intervals. It took 17 hrs before thefirst specimen was observed with a full gut, butanother worm contained only 2 boli after 18 hrs (n =13). In a similar experiment the first full intestinewas observed after 8 hrs (n = 8).Defaecation was measured by weighing worms without shelter at intervals. After 16 hrs in clean waterthe worms lost from 14.2-27.9% of the wet weight(n=4) In a similar 4 hrs experiment 2 worms gainedfrom to 1.3-3.9%, while one worm lost 15.5% inwet weight. I am unable to account for the variableresults, but they were not caused by error of themethod of wet weighing of cleaned worms. Repeated weighing of the same individual gave x = 58.64mg +/- s.d. 0.37 and 55.45 mg +/- s.d. 0.41 (n = 2x 10)

Conclusions

I have applied a number of vital stains during thepresent study of Phascolion strombus. My conclusion is that vital staining can give a quick impression of morphological structures, and not much more,with the exception of methylene blue applied supravitam in studies of the nervous system. Vital staining has been the basis for many findings by earlystudents of sipunculans, so with this in mind, Iconclude that it has been useful to try the techniqueon P. strombus. I have gained a better understanliingof the information in the old literature.The technique of vital staining can give a first impression of the disposition of certain. organs. For

example, it was vital staining which drew my attention to the expanded nuchal organ (Fig. I). Histological and histochemical techniques showed thatbasophilic and acidophilic gland cells of epidermalorgans open to the surface via individual pores (Fig.5) and that nerves ofthe axial cell column terminatein a cap. Subsequently it was possible to find thepores in thin sections. The trivial conclusion is thatif one knows what to look for, it is much easier tosee things and to interpret thin sections and photographs ofthe ultrastructure. Unfortunately, there is along way before this stage is reached with respect tosipunculans.

38 J0rgen Hylleberg

Little work has been carried out on comparativemorphology and ultrastructure of sipunculans.Hence, there is no basis for comparison of details inspite of the obvious implications for phylogeny andtaxonomy. This was also Akesson's (1958, p. 70)conclusion with respect to the nervous system.Before Akesson initiated his pioneer study, onlytwo writers had made comparative studies about thissubject.The present study has shown interesting ultrastructures in epidermal organs of Phascolionstrombus. The abundant occurrence of lipofuscin,the fine structure of coarse granules, and the tubular

structure present in eosinophilic cells of epidermalorgans constitute new findings. This indicates arewarding field of comparative studies withimplications for taxonomy as well as physiology.My studies on food and feeding were inconclusive. Iconclude that other techniques than direct counts ofbacteria and other potential food organisms must beapplied. Radioisotopes may help answeringunsolved question about the function of the ciliarygroove and the rectal caecum. Both structures arecharacteristic of sipunculaos, but their function inthe digestive process is still obscure.

AcknowledgementsI am grateful to many persons for help and advice since my first attempts to disclose some of the secrets ofsipunculan life. At the Kristineberg Marine Biological Station I received all possible support from theDirector Dr. B. Swedmark and other staff of the Station. I am grateful to laboratory engineer EyvindEmanuelsson and skipper Sylve Robertson. I also want to thank Dr. B. Akesson and Dr. R. Hinge for theirgreat encouragement and stimulation. At the Heisinger Marine Laboratory lowe gratitude to my supervisorDr. G. Thorson, and not least to Dr. K. W. Ockelmann and Dr. C. Nielsen for many good discussions, andfor letting me include their unpublished findings on the parasitic snail Menestho diaphana in my study fromKristineberg. At the Copenhagen University I received generous support from Dr. K. G. Wingstrand and Dr.K. J. Pedersen. At the University of Aarhus, I benefited from encouragement and very generous support byDr Tom Fenchel who, in capacity of being head of the institute, fulfilled all my whims and wishes. My timein Thailand has been supported by the directors of the Phuket Marine Biological Center. In this context I ampleased to acknowledge Mr. Urupun Boonprakob, Mr. Boonlert Phasuk, Mr. Komaet Charoenpanich, Mr.Udom Bhatia, Mr. Somsak Chullasom, and chief of the Reference Collection, Mr. Anuwat Nateewathana.Among representatives ofDanida and the Royal Danish Embassy in Bangkok, I am particularly grateful toChief ofthe Enreca Programme, Mr. Klaus Winkel and former ambassadors, Mr. McIlquham Schmidt andMr. F. Kirer for their support to science. Dr. lb Svane stimulated the idea of putting my findings onsipunculans together in the form of this thesis. I am grateful to my colleague Dr Tomas Cedhagen for helpwith all the practical matters and for valuable comments. lowe myoid friend and colleague Dr. JorgenMorup Jorgensen a very special thank. He has taken part in many discussions on sipunculans over the years,and he has encouraged the studies on ultrastructure.


ReferencesFurther reference is given to annex 2 (Bibliography of SipuncuIa)

39

Akesson, 1958. -see annex 2Allison, A. C. & M. R. Young. 1964. Uptake of

dyes and drugs by living cells in culture. - Life Sciences 3: 1407-1414.

Andrews, 1890. -see annex 2Arvy, 1952a. -see annex 2Arvy, 1952b. -see annex 2Arvy & Gabe, 1952. -see annex 2Arvy & Prenant, 1952. -see annex 2Baird, 1868. -see annex 2Baker, J. R. 1958. Principles of Biological Micro-

technique. London, Methuen & Co. Ltd. 357 pp.Baker, J. R. 1960. Cytological techniques. 4th ed.Baltzer, 1934. -see annex 2Bobin & Prenant, 1953a. -see annex 2Bobin & Prenant, 1953b. -see annex 2Brumpt, 1897. -see annex 2Brumpt, 1898. -see annex 2Bullock, T. H. & G. A. Horridge. 1965. Structure

and Function in the Nervous Systems of Invertebrates. Vols.1 & 2. W. H. Freeman and Co., San Fransisco. 1719 pp.

Cain, A. J. 1950. The histochemistry of Iipoids inanimals. - Biological Reviews 25: 73-111

Cappell, D. F. 1929. Intravitam and supravital staining.- Journal of Pathology and Bacteriology 32: 595.

Carlisle, 1959. -see annex 2Chlopin, N. G. 1927. Die Sekretorische Prozesse im

Cytoplasma. I. Uber die Reaktion der Gewebselemente auf intravitale Neutralrotfiirbung. - Archiv flirexperimentelle Zellforschung 4: 462.

Clayden, E. C. 1962. Practical section cutting andstaining. 4th edition. 198 pp.

Cohn, N. S. 1964. Elements of Cytology. - Burlingame Harcourt, Brace & World Inc., New York. 368pp.

Cooperstein, S. & A. Lazarow, 1953. Studies on themechanism of janus green B staining of mitochondria. lII. Reduction of janus green B by isolated enzyme systems. - Experimental Cell Research 5: 8297.

Cooperstein, S., A. Lazarow & Patterson. 1953.Studies on the mechanism of janus green B stainingof mitochondria. II. Reactions and properties ofjanus green B and its derivates. - Experimental CellResearch 5: 68-82.

Cuenot, 1900. -see annex 2Cutler, 1995. -see annex 2Cutler & Cutler, 1983. -see annex 2Cutler & Cutler, 1985. -see annex 2

Cutler & Cutler, 1986. -see annex 2Cutler & Cutler, 1988. -see annex 2Cutler & Cutler, 1990. -see annex 2Cutler & Gibbs, 1985. -see annex 2Dawson, 1933. -see annex 2Diesing, 1851. -see annex 2Dybas, 1981. -see annex 2Edmonds, 1980. -see annex 2Fange, 1974. -see annex 2Florkin, 1975. -see annex 2FeigI. 1960. Spot tests in organic analysis. Elsevier.Fischer, 1914. -see annex 2Fischer, 1922. -see annex 2Fisher, 1947. -see annex 2Fisher, 1950. -see annex 2Fontaine, A. R. 1965. The feeding mechanism of the

ophiuroid Ophiocornina nigra. - Journal of the Marine Biological Association of U.K. 45: 373- 385.

Foster, 1970. -see annex 2Franzen, A. 1956. On spermiogenesis, morphology of

the spermatozoon, and biology of fertilizationamong invertebrates. - Zoologiska Bidrag franUppsala 31: 355- 482.

Franzen & Fange, 1962. -see annex 2Gabe, 1953. -see annex 2Gage, 1979. -see annex 2Gerould, 1913. -see annex 2Gibbs, 1975. -see annex 2Gibbs, 1977. -see annex 2Gibbs, 1978. -see annex 2Gibbs, 1985. -see annex 2Gibbs & Cutler, 1987. -see annex 2Gibbs et al. , 1983. -see annex 2Goffinet et al. , 1978. -see annex 2Goodpasture, E. W. 1919. A peroxidase reaction with

sodium nitroprusside and benzidine in blood smearsand tissues. - Journal of Lab. Clinical Medicine 4:442-444

Gray, 1828. -see annex 2Grube, 1868. -see annex 2Grube & Oersted, 1858. -see annex 2Hansen, 1978. -see annex 2Hendrix, 1975. -see annex 2Hermans & Eakin, 1975. -see annex 2Herubel, 1902. -see annex 2Herubel, 1907. -see annex 2Hirsch, G. C. 1939. Form- und Stoffwechsel der

Golgikorper. - Borntraeger, Berlin. 394 pp.Hylleberg Kristensen, 1969. -see annex 2

40 kJrgen Hylleberg

Hylleberg Kristensen, 1970 . -see annex 2Hylleberg Kristensen, J. 1972. Structure and function

of crystalline styles of bivalves. - Ophelia 10:91108.

Hylleberg, 1975a. -see annex 2Hylleberg, J. 1975b. Selective feeding by Arenicola

pacifica with notes on Abarenicola vagabunda anda concept of gardening in lugworms. - Ophelia14:113-137.

Hylleberg, 1994a. -see annex 2Hylleberg, 1994b. -see annex 2Hylleberg, J. & A. Nateewathana. 1991. Polychaetes

of Thailand, Spionidae, (Part I): Prionospio of thesteenstrupi group with description of 8 new speciesfrom the Andaman Sea. - Phuket mar. bioI. Cent. Res.Bull. 55: 1- 32.

Hyman, 1959. -see annex 2Jeuniaux, 1963. -see annex 2Keferstein, 1865. -see annex 2Kennedy, 1969Koehring, V. 1930. The neutral red reaction. - Journal

of Morphology 49: 45- 137.Kohn, 1975. -see annex 2Koren & Danielssen, 1875. -see annex 2Lankester, 1885. -see annex 2Leuckart, 1828. -see annex 2Linnaeus, 1766. -see annex 2Lison, L. 1960. Histochimie et cytologie ani males.

3rd ed.Lowe, D. M., M.N. Moore & B.M. Evans. 1992.

Contaminant impact on interactions of molecularprobes with lysosomes in living hepatocytes fromdab Limanda limanda. - Marine Ecology ProgressSeries 91: 135- 140.

Manavalaramanujam & Sundara, 1982. -see annex 2Metalnikoff, 1900. -see annex 2Meyer, G. F. 1955. Vergleichende Untersuchungen

mit der supravitalen Methylenblaufarbung am Nervensystem wirbelloser Tiere (Mit besonderer Berlicksichtigung der Neurofibrillen bei Evertebraten undVertebraten, sowie des Bindegewebes der Insekten). Zoologische Jahrblicher Abteilung fUr Anatomie undOntogenie der Tiere 74 (3): 339-490.

Michel & DeViIlez, 1984. -see annex 2Mock, 1965. -see annex 2Moltchanoff, 1909. -see annex 2Montagu, 1804. -see annex 2Moritz & Storch, 1970. -see annex 2Nielsen, 1964. -see annex 2Nielsen, 1967. -see annex 2Ocharan, 1974. -see annex 2Ochi, 1969. -see annex 2Ochi, 1975. -see annex 2Oglesby, 1968. -see annex 2

Oglesby, 1969. -see annex 2Pallas, 1774. -see annex 2Paul, 1910. -see annex 2Pearse, A. G. E. 1960. Histochemistry. 2nd edition.

London, J. & A. Churchill Ltd. 998 pp.Pergament, 1946. -see annex 2Perez, 1924. -see annex 2Perez, 1925. -see annex 2Pilger, 1982. -see annex 2Pilger, 1987. -see annex 2Popkov, 1991. -see annex 2Rajulu, 1975. -see annex 2Rajulu & Krishnan, 1969. -see annex 2Rice, 1970. -see annex 2Rice, 1975. -see annex 2Rice et al. , 1983. -see annex 2Romeis, B. 1948. Mikroskopische Tcchnik. 15 Aufl.

Mlinchen. 695 pp.Sawada, 1975. -see annex 2Sawada, 1980. -see annex 2Schleip, 1934. -see annex 2Schleip, 1935. -see annex 2Selenka, 1883. -see annex 2Selenka et al. , 1883. -see annex 2Selenka & de Man, 1883. -see annex 2Selensky, 1908. -see annex 2Serrano et aI., 1989. -see annex 2Serrano et al., 1991. -see annex 2Shipley, 1890. -see annex 2Shipley, 1902. -see annex 2Sluiter, 1902. -see annex 2Spengel, 1912. -see annex 2Stehle, 1952. -see annex 2Stehle, 1953. -see annex 2Stephen, 1964. -see annex 2Stephen & Edmonds, 1972Storch & Moritz, 1970. -see annex 2Storch & Welch, 1972. -see annex 2Tetry, 1959. -see annex 2Theel, 1875. -see annex 2Theel, 1905. -see annex 2Thomas, 1932. -see annex 2Towle, 1975. -see annex 2Tuzet & Ormieres, 1965. -see annex 2Valembois & Boiledieu, 1980. -see annex 2Volkonsky, 1933. -see annex 2Voss-Foucart et al., 1977. -see annex 2Walter, 1973. -see annex 2Ward, 1891. -see annex 2Wesenberg-Lund, 1929. -see annex 2Wesenberg-Lund, 1935. -see annex 2Wilcynski, 1913. -see annex 2

ANNEXl


.Application of vital staining

41

From an ecological point of view, the application ofvital stains may give quick information aboutimportant secretory or excretory structures inanimals which have not been studied using ordinaryhistological techniques. For example, Fontaine(1965) used the metachromatic staining of mucuswith toluidine blue to study the feeding mechanismof brittle-star, Ophioeomina nigra. In this case a cellproduct was studied after secretion from the cells.Hylleberg Kristensen (1972) studied formation ofthe crystalline style in live Maeoma baltica byapplication of trypan blue in sea water. Hylleberg(1975b) also used neutral red to separate Iive fromdead diatoms since this stain is accumulated invacuoles (Iyso-somes) of live cells only. The use ofmethylene blue in supra vitam staining of nervoussystems is well known (Meyer 1955). Thistechnique is especially useful in studies on theperipheral nervous system of invertebrates wherenormal silver methods fail, e.g. Bodian. Nerves ofthe peripheral nervous system of invertebrates mayalso be difficult to identify in the TEM.The purpose of this section is to provide anoverview of application of vital stains which playeda very important role in the early studies onsipunculans (Thomas 1932; Dawson 1933; Volkonsky 1933).

The stains can be used intra vitam (staining ofelements in an otherwise undisturbed live animal),or supra vitam (staining of elements of the bodywhich are removed from an animal during life, butbefore cellular death has occurred). Collectively thetwo ways of staining are referred to as vital staining.Important vital stains are the acid dye trypan blueand the basic dyes neutral red, vesuvian (= Bismarckbrown), methylene blue, janus green B. Trypan blueand the first two of the above basic dyes are verylittle toxic. When stained in vivo with these dyes,Phaseolion strombus have lived for 4 months inaquaria, i.e., until the study was ended.The two main groups of vital stains are concentratedwithin the cells in a similar way, and certain cellstake up both acid and basic dyes. However, theuptake is normally selective. The basic dye neutralred is taken up by Iysosomes which fail to exhibitvital staining with the acid dye trypan blue. It is

characteristic that the neutral red vacuoles liberatethe stain as soon as the cell dies, and otherstructures, which never are stained intra vitam, arethen stained by the dye, e.g., the nucleus.Vital staining and post mortem staining aregenerally very different. The cells that actively takeup vital stains are generally those of the excretoryand phagocytic systems. Food vacuoles ofphagocytic cells have a particularly strong tendencyto take up vital stains. (Koehring 1930)

Neutral red as a vital stainThree types of neutral red vacuoles have beendescribed. Chlopin (1927) demonstrated that thestain binds to a protein component which becomescarrier of the dye. Chlopin termed these vacuoleskrinom. The krinom is not derived from the golgicomplex as far as it can be shown by lightmicroscopy. Hirsch (1939) surveyed the extensiveliterature on neutral red as vital stain and stated thatcertain golgi bodies have great affinity to neutral redin vital staining. These bodies are presumably theIysosomes which are derived from the golgicomplex, but other possibilities exist for the originof lysosomes (Cohn 1964). A simple chemicaldefinition of a lysosome is "a body rich in acidhydrolase" These bodies have great affmity toneutral red (Lowe et al. 1992). The red colour ofIysosomes stained in vivo indicates acid contentsbecause neutral red is an indicator: blue in stronglyacid solution, red in weak acid, and yellow in basicsolution. Lowe et at. (1992) used the selectiveuptake of neutral red by Iysosomes to study effectsof contaminants in living hepatocytes from dab,Limanda limanda. The neutral red assay makes useof the fact that only Iysosomes in healthy cells takeup and retain the supravital stain.Allison & Young (1964) concluded that one of thedistinctive properties of Iysosomes is their capacityto take take up and concentrate a wide range of basicand neutral substances at low concentrations. Thereis, however, some selectivity as shown by theirfailure to take up janus green B.

Janus green B as a vital stainThe enzymatic basis, the mechanism of the stainingreaction, and the properties of janus green B have

42 Jmgen Hylleberg

been studied by Cooperstein & Lazarow (1953) andCooperstein et al. (1953). The stain is selective formitochondria but the brand of stain is important forthe result (Romeis § 781): Franzen (1956) used it tostain mitochondria in developing spermatids. Hefound that mitochondria took up janus green B butnot neutral red. In my experience, janus green israther toxic when used in vivo with sipunculans.

Toluidine blue as a vital stainRomeis (§ 697) stated that nucleus, mucus, cartilageground substance, and granula of mast cells stainmetachromatically when the stain is used in ordinary

histological work. As a vital stain, toluidine bluehas high affinity for mucus.

Trypan blue as a vital stainCells of the reticulo-endothelial system take uptrypan blue in general, and several tissue elementsare stained diffusely, e.g., skin, mucous membranes,skeletal muscles, tendons, and connective tissue Thedye is readily accumulated in macrophages and to asmaller extent in fibroblasts (Cappell 1929).

REFERENCESare quoted in the preceding list of references.

TAXONOMY AND BIOLOGY OF SIPUNCULANS, WITH EMPHASIS ON THE MORPHOLOGY OF PHASCOLION STROMBUS...

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