Body structure of marine sponges

Summary Each choanocyte chamber of Petrosia ficiformis is formed by a slightly outpocked choanocyte epithelium and by a ring of three or four uniflagellated cone cells surrounding the apopyle. The apopyle opens into a small aphodus, which leads the water flow to larger excurrent canals. Pinacocytes of the incurrent canal system cover the basal surface of the choanocytes and separate them from the incurrent canals and the mesenchyme. The water flows into the chambers by pores in the pinacocyte cover and then through gaps between adjacent choanocytes. To our knowledge this is the first report of a leuconoid canal system in which choanocyte chambers are covered by a pinacocyte epithelium of the incurrent canal system that isolates the chambers from the mesenchyme. A future comprehensive revision of the types of canal systems in sponges seems to be necessary. Permanent affiliation: Department of Biology and Health Sciences, University of Hartford, West Hartford, CT 06117, USA     

Body structure of marine sponges

Summary Specimens of Haliclona elegans (Bowerbank, 1866) are covered by a thin, double layered dermal membrane extending over large subdermal spaces. The pores in the dermal membrane are formed by single porocytes with one or sometimes several pores in the center of the cell. The subjacent tissue shows a faintly developed mesenchyme and numerous big choanocyte chambers projecting into lacunar spaces of the incurrent canal system. The outer surface of the chambers is directly covered by the pinacocyte epithelium of the incurrent canal wall, which also separates them completely from the mesenchyme. Water influx into the chambers is guaranteed by prosopylar openings in the pinacocyte cover at the outer chamber surface. The chambers are connected to the excurrent canal system in the eurypylous way by wide apopyles, each of which is surrounded by a small ring of flagellated cone cells. About 15% of the choanocyte chambers in H. elegans contain central cells, which are thought to derive from migrating pinacocytes of the canal systems.     

Die Aufnahme partikulärer Nahrung beiReniera sp. (Porifera)

The choanocyte chambers of the marine spongeReniera sp. protrude with their curved outer surface free into the incurrent canals. The water is sucked into the chambers by cavities between the choanocytes. Particles up to 1 µm in diameter may enter the chambers with the water current. These particles are trapped on the outer surface of the choanocyte collars and are ingested by the choanocytes and processes of the pinacocyte epithelium of the incurrent canal system, which project into the chambers. Bigger particles are retained in the incurrent canals mainly on the outer surface of the choanocyte chambers. They are ingested by pinacocytes of the canal wall and transported to cells of the mesenchyme. The present investigation shows the great importance of the pinacocyte epithelium of the incurrent canal system for suspension feeding inReniera sp.     

Comparative study of the choanosome of Porifera: 1. The Homoscleromorpha

The choanoderm and pinacoderm of representatives of the two families of Homoscleromorpha sponges, the Oscarellidae and Plakinidae, have been examined by transmission and scanning electron microscopy. Different fixative procedures have shown the dramatic influence of fixation conditions on the morphology of choanocytes. These two families of sponges have the following morphological features in common: flagellated endopinacocytes with short apical microvilli and basal pseudopods; the presence of a very thin and dense sheet of matrix material which limits the mesohyl. There are, however, only minor differences in the flagellar morphology, granule content, and anchoring system of their choanocytes. Two findings are of particular interest: (1) the presence of glycocalyx bridges between the microvilli of the choanocyte collar; and (2) the discovery of a new cell type, the apopylar cell, which has a morphology intermediate between that of pinacocytes and choanocytes. The apopylar cells limit the apopylar opening of the choanocyte chamber and indicate the transition between choanoderm and pinacoderm.     

A new type of central cell in the choanocyte chambers of Pellina fistulosa (Porifera,Demospongiae)

Central cells of a hitherto unknown type, forming a continuous, perforated layer at the level of the distal collar ends in each choanocyte chamber, have been found in the choanocyte chambers of Pellina fistulosa. The collars project through the pores of the perforated central cell layer. The spaces between the collar ends and between the collars and the cone cell ring in the apopyle region are sealed by the central cell cytoplasm. The latter represents an impermeable barrier for particulate material as well as for water and thus enhances the filtration efficiency by preventing a bypass of water and particles between the collar apices.     

Ultrastructure and embryonic development of a syconoid calcareous sponge

Abstract. Recent molecular data suggest that the Porifera is paraphyletic (Calcarea+Silicea) and that the Calcarea is more closely related to the Metazoa than to other sponge groups, thereby implying that a sponge‐like animal gave rise to other metazoans. One ramification of these data is that calcareous sponges could provide clues as to what features are shared among this ancestral metazoan and higher animals. Recent studies describing detailed morphology in the Calcarea are lacking. We have used a combination of microscopy techniques to study the fine structure of Syconcoactum Urban 1905, a cosmopolitan calcareous sponge. The sponge has a distinct polarity, consisting of a single tube with an apically opening osculum. Finger‐like chambers, several hundred micrometers in length, form the sides of the tube. The inner and outer layers of the chamber wall are formed by epithelia characterized by apical–basal polarity and occluding junctions between cells. The outer layer—the pinacoderm—and atrial cavity are lined by plate‐like cells (pinacocytes), and the inner choanoderm is lined by a continuous sheet of choanocytes. Incurrent openings of the sponge are formed by porocytes, tubular cells that join the pinacoderm to the choanoderm. Between these two layers lies a collagenous mesohyl that houses sclerocytes, spicules, amoeboid cells, and a progression of embryonic stages. The morphology of choanocytes and porocytes is plastic. Ostia were closed in sponges that were vigorously shaken and in sponges left in still water for over 30 min. Choanocytes, and in particular collar microvilli, varied in size and shape, depending on their location in the choanocyte chamber. Although some of the odd shapes of choanocytes and their collars can be explained by the development of large embryos first beneath and later on top of the choanocytes, the presence of many fused collar microvilli on choanocytes may reflect peculiarities of the hydrodynamics in large syconoid choanocyte chambers. The unusual formation of a hollow blastula larva and its inversion through the choanocyte epithelium are suggestive of epithelial rather than mesenchymal cell movements. These details illustrate that calcareous sponges have characteristics that allow comparison with other metazoans—one of the reasons they have long been the focus of studies of evolution and development.     

Isolation of the choanocyte in the fresh water sponge, Ephydatia fluviatilis and its lineage marker, Ef annexin

In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.     

Bau und Funktion des Süßwasserschwamms Ephydatia fluviatilis L. (Porifera)

Zusammenfassung Die Kragengeißelkammern von Ephydatia fluviatilis entstehen frei im Mesenchym. An den Entstehungsorten trifft man auf Anhäufungen rundlicher Zellen, die allem Anschein nach von Archäocyten stammen, jedoch kleiner sind als diese und einen nukleoluslosen Kern besitzen. Hierbei handelt es sich um Choanoblasten, die zunächst eine Geißel, später den Kragen ausbilden und sich als Choanocyten zu Kragengeißelkammern zusammenfügen.Die im Mesenchym vorläufig fertiggestellten Kragengeißelkammern gelangen an das Endopinacocytenepithel des ausführenden Kanalysystems. Daraufhin bilden sich die tangierten Choanocyten zu Konuszellen um. Das Endopinacocytenepithel antwortet seinerseits mit der Ausbildung einer Poruszelle pro Kragengeißelkammer. Die Porocyten gehen mittels der konfrontierten Konuszellen dauerhafte Verbindungen mit den zugehörigen, nunmehr funktionstüchtigen Kragengeißelkammern ein.

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Structure and function of the fresh-water sponge Ephydatia fluviatilis L. (Porifera)VIII. The origin and development of the flagellated Chambers and their junction with the excurrent canal system

Summary The flagellated chambers of Ephydatia fluviatilis arise at scattered sites within the mesenchyme. Each such site is marked by an accumulation of rounded cells, which appear to be derived from archaeocytes in most respects except that they are smaller than the latter and have no nucleoli in the nucleus. These are choanoblasts, which first develop a flagellum and later a collar; eventually, as choanocytes, they become arranged so as to form a flagellated chamber.Having reached this preliminary stage of completion in the mesenchyme, the flagellated chambers migrate to the endopinacocyte epithelium of the excurrent canal system. Then the choanocytes at the contact point are converted to cone cells. The endopinacocyte epithelium in turn responds by developing one pore cell for each flagellated chamber. The porocytes become permanently joined to the chamber by way of the adjacent cone cells, and from this time on the flagellated chamber is functional.

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Abkürzungen A Archäocyte - aK ausführender Kanal - B Bakterien - Ch Choanocyte - eK einführender Kanal - G Geißel - GK Kragengeißelkammer - GK-A Anlage von Kragengeißelkammern - K Zellkern - Kr Kragen - KZ Konuszelle - M Mesenchym - N SiO₂-Nadel - PC Endopinacocyten - PD Pinacoderm - PV pulsierende Vakuole - PZ Porenzelle - S Gemmulaschale - Sk Skleroblast - Sp Spongin - SR Subdermalraum     

Canal systems and choanocyte chambers in freshwater sponges (Porifera,Spongillidae)

Summary The spongillid species Spongilla lacustris and Ephydatia fluviatilis possess choanocyte chambers of the classical eurypylous type. They are surrounded by the mesenchymal tissue and connected to the incurrent canal system by prosopyles and to the excurrent canal system by wide apopyles. Each apopyle is sealed against spaces between the basal choanocyte collar parts by a ring of uniflagellated cone cells. The functional aspects of the choanocyte chamber and canal structure are discussed.Dedicated to Prof. Dr. K.E. Wohlfarth-Bottermann, Bonn, in honor of his 65th birthday     

The spermatogenesis ofHalichondria panicea (Porifera,Demospongiae)

Summary Spermatogenesis of the marine spongeHalichondria panicea begins with the break up of choanocyte chambers, choanocytes constituting the origin of spermatogonia. The transition from choanocytes to spermatogonia is direct, without cell division. Already the spermatogonia are flagellated. The ensuing large aggregates of spermatogonia are enclosed by spermatocyst-building cells. Further development takes place within the spermatocysts, mostly arranged in fields which, however, lack any developmental gradient. Within a single spermatocyst development is mostly synchronous. Spermatogonia transform into first order spermatocytes directly. The transition from spermatid to spermatozoon is characterized by an unusual prolongation of the chromatin, often resulting in a helical form of the chromosome material and a strong enlargement of the mitochondria which align with the nucleus, leading to an irregular shape of the spermatozoon. Another exceptional feature is the virtual absence of a Golgi apparatus during all stages of spermatogenesis. TheH. panicea investigated here contained only male reproductive elements, thus appear to be gonochorists. Some features of the spermatogenesis ofH. panicea, such as dissolving choanocyte chambers, the enclosure of spermatogonia by spermatocyst-building cells and the formation of a synaptonemal complex in first order spermatocytes occur in other sponge species as well; however, the early presence of flagella in spermatogonia, the absence of the Golgi apparatus and the later irregular development of nuclei, mitochondria and the spermatozoa themselves represent features hitherto not observed in sponges.     

Ultrastructural investigation of spermatogenesis in Spongilla lacustris and Ephydatia fluviatilis (Porifera,Spongillidae)

Summary Spermatogenesis of the spongillids investigated here is similar in Spongilla lacustris and Ephydatia fluviatilis and proceeds, on the whole, as in other Eumetazoa. Sponges however lack true sex organs, the germ cells developing from somatic cells. The male germ cells originate in spongillids from choanocytes and the female ones from archaeocytes. In Spongilla lacustris single choanocytes leave the flagellated chambers and transform into spermatogonia; in Ephydatia fluviatilis they result from differential cell division. The spermatogonia gather in distinct mesenchyme regions and are surrounded by cyst-building cells. Thus spermatocysts are built in which spermatogenesis proceeds. The spermatogonia in the spermatocysts differentiate into flagellated spermatocytes of I. order. In this process, the early appearance of the flagellum and its mode of formation are uncommon. The following meiotic divisions generate spermatocytes of II. order in the first step and spermatids in the second. In both developmental stages the cells remain connected by cytoplasmic bridges. In the subsequent spermiocytogenesis the cytoplasm of the spermatids is reduced. The reduced parts of the cytoplasm appear as cell fragments in the lumen of the spermatocysts and are eventually ingested by the cystwall cells. The mature spermatozoa arrange in the spermatocysts in a characteristic pattern. Later the spermatocysts open into the excurrent canal system and the spermatozoa leave the sponge with the egestive water stream.     

STRUCTURE AND FORMATION OF THE COLLAR IN CHOANOCYTES OF TETILLA SERICA (LEBWOHL), DEMOSPONGE

There are two types of collar in the choanocytes of adult Tetilla serica : one type is a continuous cytoplasmic tube and the other consists of discontinuous microvilli. The former is found in the small flagellated chamber and is considered to belong to a young choanocyte in the process of differertiation. To confirm this idea, very young choanocytes which are about to differentiate the collar were examined during embryogenesis.
The youngest choanocytes are noticed forming aggregations of small cells in 3-day larvae. Around the flagellum in each choanocyte, there is a depression which will become wider. At first, the collar is observed as a ring of cytoplasm; next this extends outward and becomes thinner, and finally it divides into microvilli. The microvillous collar is formed by the opening of vesicles and fusion of their membranes. These vesicles are considered to be derived from the Golgi complex. The process of collar formation through fusion of vesicles is discussed.     

Choanocyte ultrastructure in Halisarca dujardini (Demospongiae,Halisarcida)

Understanding poriferan choanocyte ultrastructure is crucial if we are to unravel the steps of a putative evolutionary transition between choanoflagellate protists and early metazoans. Surprisingly, some aspects of choanocyte cytology still remain little investigated. This study of choanocyte ultrastructure in the halisarcid demosponge Halisarca dujardini revealed a combination of minor and major distinctive traits, some of them unknown in Porifera so far. Most significant features were 1) an asymmetrical periflagellar sleeve, 2) a battery of specialized intercellular junctions at the lateral cell surface complemented with an array of lateral interdigitations between adjacent choanocytes that provides a particular sealing system of the choanoderm, and 3) a unique, unexpectedly complex, basal apparatus. The basal apparatus consists of a basal body provided with a small basal foot and an intricate transverse skeleton of microtubules. An accessory centriole, which is not perpendicular to the basal body, is about 45°. In addition, a system of short striated rootlets (periodicity = 50–60 nm) arises from the proximal edge of the basal body and runs longitudinally to contact the nuclear apex. This is the first flagellar rootlet system ever found in a choanocyte. The accessory centriole, the rootlet system, and the nuclear apex are all encircled by a large Golgi apparatus, adding another distinctive feature to the choanocyte cytology. The set of distinct features discovered in the choanocyte of H. dujardini indicates that the ultrastructure of the poriferan choanocyte may vary substantially between sponge groups. It is necessary to improve understanding of such variation, as the cytological features of choanocytes are often coded as characters both for formulation of hypotheses on the origin of animals and inference of phylogenetic relationships at the base of the metazoan tree. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Hydroxyurea: An Inhibitor of the Differentiation of Choanocytes in Fresh-Water Sponges and a Possible Agent for the Isolation of Embryonic Cells

During the development of a fresh-water sponge from its gemmules, most cell types originate from the undifferentiated archaeocytes through a few divisions, whereas each choanocyte chamber, composed of several tens of choanocytes, arises from a single archaeocyte through repeated mitoses.
This process was studied on gemmules incubated in various concentrations of hydroxyurea.
A concentration of 100 μg/ml postponed the hatching by about two days, and blocked the differentiation of the choanocytes and the morphogenesis of the aquiferous system. The resulting organism was a hollow dome of pinacoderm, stretched on spicules, the bottom of which was strewn with embryonic archaeocytes. After washing and incubation in mineral medium, the sponge differentiated its choanocytes and achieved normal development.
The incorporation of ³H-thymidine into DNA was compared throughout the development of normal and hydroxyurea-treated gemmules. Hydroxyurea delayed the first peaks of incorporation and abolished the large peak that normally occurs around 90 h, just before the formation of choanocyte chambers.
When added after 96 h incubation, hydroxyurea did not affect the differentiation of the choanocytes.
These results suggest that the differentiation of the choanocytes and the further morphogenesis of the aquiferous system depend on the repetitive divisions of the archaeocytes that normally occur around 90 h.
Furthermore, hydroxyurea-blocked sponges provide a suitable source for the isolation of pure populations of embryonic archaeocytes.     

Embryo development of Corticium candelabrum (Demospongiae: Homosclerophorida)

Abstract. Corticium candelabrum is a homosclerophorid sponge widespread along the rocky Mediterranean sublittoral. Scanning and transmission electron microscopy were used to describe the gametes and larval development. The species is hermaphroditic. Oocytes and spermatocytes are clearly differentiated in April. Embryos develop from June to July when the larvae are released spontaneously. Spermatic cysts originate from choanocyte chambers and spermatogonia from choanocytes by choanocyte mitosis. Oocytes have a nucleolate nucleus and a cytoplasm filled with yolk granules and some lipids. Embryos are surrounded by firmly interlaced follicular cells from the parental tissue. A thin collagen layer lies below the follicular cells. The blastocoel is formed by migration of blastomeres to the morula periphery. Collagen is spread through the whole blastocoel in the embryo, but is organized in a dense layer (basal lamina) separating cells from the blastocoel in the larva. The larva is a typical cinctoblastula. The pseudostratified larval epithelium is formed by ciliated cells. The basal zone of the ciliated cells contains lipid inclusions and some yolk granules; the intermediate zone is occupied by the nucleus; and the apical zone contains abundant electron-lucent vesicles and gives rise to cilia with a single cross-striated rootlet. Numerous paracrystalline structures are contained in vacuoles within both apical and basal zones of the ciliated cells. Several slightly differentiated cell types are present in different parts of the larva. Most cells are ciliated, and show ultrastructural particularities depending on their location in the larvae (antero-lateral, intermediate, and posterior regions). A few smaller cells are non-ciliated. Several features of the C. candelabrum larva seem to support the previously proposed paraphyletic position of homoscleromorphs with respect to the other demosponges.     

Ultrastructure of the trophic chamber and nutritive cord of Aspidiotus hederae (Homoptera,coccoidea)

Summary Each ovariole of the coccidian Aspidiotus hederae contains a single oocyte connected by means of a nutritive cord to the trophic chamber. The trophic chamber consists of three nurse cells characterized by an enlarged, ramified nucleus with a prominent nucleolus. The perinuclear cytoplasm contains nuage material, large amounts of free ribosomes, and scattered mitochondria. Occasional cisternae of the rough endoplasmic reticulum and bacteroids are found in trophocyte cytoplasm. The nutritive cord contains many microtubules in parallel array interspersed with numerous free ribosomes and a few mitochondria. The nutritive cord is strengthened by trophocyte projections which surround it. Microtubules in the projections are oriented perpendicular to the long axis of the cord.     

Tissue organization ofFarrea occa (Porifera,Hexactinellida)

Summary The tissue organization ofFarrea occa has been examind by light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). It was found to agree closely with the hexactinellid model established forRhabdocalyptus dawsoni by Mackie and Singla (1983) in consisting of a thin general syncytium incorporating few discrete cellular components, several of which share membrane continuity with the general syncytium by distinctive plug junctions. The general syncytium, supported by a thin collagenous mesolamella, is specialized regionally as dermal membrane, gastral membrane, peripheral trabecular strands, and primary reticulum (R1) of flagellated chambers. Extensions of the syncytium, which lack mesolamella support, form the distinctive secondary reticulum (R2) inside chambers and a newly discovered structure, the inner membrane, which occupies the central region of flagellated chambers. The choanosyncytia are enucleate networks of collar bodies and stolons embedded in R1 and plugged to R1 and choanoblasts. The discrete cell population consists of choanoblasts and archeocytes located in the thin mesohyle space and plugged to syncytial elements, cystencytes and vacuolar cells also located in the mesohyle but lacking plug connections, and granular cells emergent on R1 and apparently not bearing plug connections. The status of scleroblast syncytia has not been resolved. Large populations of rod-shaped bacteria occupy the mesohyle space; intracellular ovoid bodies, possible symbiotic prokaryotes, are common in R1 and R2. The previously unknown inner membrane probably functions to control flagellar activity on a very localized scale and to accumulate and release egesta in packages.Abbreviations ac archeocyte congeries - ap apopyle - ar archeocyte - b bacterium - c collagen fibrils - cb collar body - cbl choanoblast - cbs collar body socket - ch choanosome - cm collar microvilli - co choanocyte collar - cr crystalloid - cs connecting strand - dm dermal membrane - dv debris vacuole - e exhalant opening - ex exhalant space - f flagellum - fn filamentous network - Gb Golgi body - gf glycocalyx filaments - gm gastral membrane - im inner membrane - is inhalant space - ml mesolamella - ms mesohyle space - mt mitochondrion - n1 nucleus of R1 - o ostium - ob ovoid body - os osmiophilic body - pm plasma membrane - pr prosopyle - pt peripheral trabeculae - R1 primary reticulum - R2 secondary reticulum - s spicule space - ser smooth endoplasmic reticulum - ts trabecular strand     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

Ultrastructural study of spermatogenesis in Oscarella lobularis (Porifera,Demospongiae)

Summary

The various phases of spermatogenesis in the demosponge Oscarella lobularis were studied by electron microscopy. Spermatogenesis occurs within spermatic cysts, which are presumed to derive from choanocyte chambers by transformation of choanocytes into spermatogonia. Germ cells develop asynchronously within spermatocysts, and cytoplasmic bridges, indicating incomplete cells division, connect several germ cells. Attached spermatogonia suggest gonial generations. Spermatocytes I typically show the presence of synaptonemal complexes indicating meiotic divisions. Spermatocytes II have a small size probably because of the meiotic divisions of spermatocytes I. Spermatids are characterized by an acrosome, a big mitochondrion and a peripheral sheath of condensed chromatin surrounding a clearer central area in the nucleus. The mature spermatozoon shows a lateral flagellum and a flattened acrosome capping the nucleus. The phylogenetic implications of some features of the spermatozoon are suggested.     

Experimental metamorphosis of Halisarca dujardini larvae (Demospongiae, Halisarcida): evidence of flagellated cell totipotentiality

The potency of flagellated cells of Halisarca dujardini (Halisarcida, Demospongiae) larvae from the White Sea (Arctic) was investigated experimentally during metamorphosis. Two types of experiments were conducted. First, larvae were maintained in Ca2+ free seawater (CFSW) until the internal cells were released outside through the opening of the posterior pole. These larvae that only composed of flagellated cells (epithelial larvae) were then returned to sea water (SW) to observe their metamorphosis. The posterior aperture closed before they settled on a substratum and started a metamorphosis similar to intact larvae. Secondly, epithelial larvae were, first, further treated in CFSW and then mechanically dissociated. Separated cells or groups of cells were returned to SW, where they constituted large friable conglomerates. After 12-17 h in SW, flagellated cells showed the first steps of dedifferentiation, and regional differentiation was noticeable within conglomerates after approximately 24-36 h. External cells differentiated into pinacocytes while internal cells kept their flagella and became united in a layer. Within 48-72 h, internal cells of the conglomerates formed spherical or ovoid clusters with an internal cavity bearing flagella. These clusters further fused together in a rhagon containing one or two large choanocyte chambers. The sequence of cellular processes in epithelial larvae and in flagellated cell conglomerates was similar. Previous observations indicating the totipotentiality of larval flagellated cells during normal metamorphosis of H. dujardini are thus confirmed.