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.     

Feeding in a calcareous sponge: particle uptake by pseudopodia

Sponges are considered to be filter feeders like their nearest protistan relatives, the choanoflagellates. Specialized "sieve" cells (choanocytes) have an apical collar of tightly spaced, rodlike microvilli that surround a long flagellum. The beat of the flagellum is believed to draw water through this collar, but how particles caught on the collar are brought to the cell surface is unknown. We have studied the interactions that occur between choanocytes and introduced particles in the large feeding chambers of a syconoid calcareous sponge. Of all particles, only 0.1-microm latex microspheres adhered to the collar microvilli in large numbers, but these were even more numerous on the choanocyte surface. Few large particles (0.5- and 1.0-microm beads and bacteria) contacted the collar microvilli; most were phagocytosed by lamellipodia at the lateral or apical cell surface, and clumps of particles were engulfed by pseudopodial extensions several micrometers from the cell surface. Although extensions of the choanocyte apical surface up to 16 microm long were found, most were 4 microm long, twice the height of the collar microvilli. These observations offer a different view of particle uptake in sponges, and suggest that, at least in syconoid sponges, uptake of particles is less dependent on the strictly sieving function of the collar microvilli.     

Choanoflagellates, choanocytes, and animal multicellularity

Abstract. It is widely accepted that multicellular animals (metazoans) constitute a monophyletic unit, deriving from ancestral choanoflagellate‐like protists that gave rise to simple choanocyte‐bearing metazoans. However, a re‐assessment of molecular and histological evidence on choanoflagellates, sponge choanocytes, and other metazoan cells reveals that the status of choanocytes as a fundamental cell type in metazoan evolution is unrealistic. Rather, choanocytes are specialized cells that develop from non‐collared ciliated cells during sponge embryogenesis. Although choanocytes of adult sponges have no obvious homologue among metazoans, larval cells transdifferentiating into choanocytes at metamorphosis do have such homologues. The evidence reviewed here also indicates that sponge larvae are architecturally closer than adult sponges to the remaining metazoans. This may mean that the basic multicellular organismal architecture from which diploblasts evolved, that is, the putative planktonic archimetazoan, was more similar to a modern poriferan larva lacking choanocytes than to an adult sponge. Alternatively, it may mean that other metazoans evolved from a neotenous larva of ancient sponges. Indeed, the Porifera possess some features of intriguing evolutionary significance: (1) widespread occurrence of internal fertilization and a notable diversity of gastrulation modes, (2) dispersal through architecturally complex lecithotrophic larvae, in which an ephemeral archenteron (in dispherula larvae) and multiciliated and syncytial cells (in trichimella larvae) occur, (3) acquisition of direct development by some groups, and (4) replacement of choanocyte‐based filter‐feeding by carnivory in some sponges. Together, these features strongly suggest that the Porifera may have a longer and more complicated evolutionary history than traditionally assumed, and also that the simple anatomy of modern adult sponges may have resulted from a secondary simplification. This makes the idea of a neotenous evolution less likely than that of a larva‐like choanocyte‐lacking archimetazoan. From this perspective, the view that choanoflagellates may be simplified sponge‐derived metazoans, rather than protists, emerges as a viable alternative hypothesis. This idea neither conflicts with the available evidence nor can be disproved by it, and must be specifically re‐examined by further approaches combining morphological and molecular information. Interestingly, several microbial lin°Cages lacking choanocyte‐like morphology, such as Corallochytrea, Cristidiscoidea, Ministeriida, and Mesomycetozoea, have recently been placed at the boundary between fungi and animals, becoming a promising source of information in addition to the choanoflagellates in the search for the unicellular origin of animal multicellularity.     

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.     

Hydrodynamics in early animal evolution

Choanoflagellates and sponges feed by filtering microscopic particles from water currents created by the flagella of microvillar collar complexes situated on the cell bodies of the solitary or colonial choanoflagellates and on the choanocytes in sponges. The filtering mechanism has been known for more than a century, but only recently has the filtering process been studied in detail and also modelled, so that a detailed picture of the water currents has been obtained. In the solitary and most of the colonial choanoflagellates, the water flows freely around the cells, but in some forms, the cells are arranged in an open meshwork through which the water can be pumped. In the sponges, the choanocytes are located in choanocyte chambers (or choanocyte areas) with separate incurrent and excurrent canals/pores located in a larger body, which enables a fixed pattern of water currents through the collar complexes. Previous theories for the origin of sponges show evolutionary stages with choanocyte chambers without any opening or with only one opening, which makes separation of incurrent and excurrent impossible, and such stages must have been unable to feed. Therefore a new theory is proposed, which shows a continuous evolutionary lineage in which all stages are able to feed by means of the collar complexes.     

Microscopical aspects on symbiosis of Spongilla lacustris (Porifera,Spongillidae) and green algae

Summary When growing in the sunlight, some specimens of Spongilla lacustris are coloured green due to the presence of symbiotic unicellular chlorellae. The algae live inside most sponge cells. The chlorellae were extracted from green sponges, cultivated, added to algae-free sponges and fixed after different incubation times. In this way the uptake of the algae, their distribution and their final whereabouts in the mesenchymatic cells could be followed by in vivo microscopy, phase-contrast microscopy and electron microscopy. A few minutes after addition, the chlorellae can be found inside the choanocyte chambers. Here they are taken up by the cell bodies and collars of the choanocytes. Pinacocytes are also involved in the uptake. The distribution of algae results from a specific transmission from the donor cell to the receiver cell. The chlorellae are not released from their host vacuoles until they are extensively enclosed by the cell taking them up. Six hours after addition, all sponge cells contain algae except granulocytes, microscleroblasts, the pinacocytes of the peripheral rim region and those of the pinacoderm. The chlorellae are able to divide inside the sponge cells.Abbreviations StM Stereo-microscopical photograph - PhC Phase-contrast microscopical photograph - EM Electron microscopical photograph     

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.     

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     

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.     

Metamorphosis of coeloblastula performed by multipotential larval flagellated cells in the calcareous sponge Leucosolenia laxa

The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.     

Comparative study of the choanosome of porifera: II. The keratose sponges

The aquiferous system of representatives of the orders Dictyocer-atida, Dendroceratida, and Verongida has been studied to note its relevance to the systematics of the groups. The volume of the choanocyte chamber, the size and shape of the choanocytes, the number of choanocytes per chamber, the relative development of the mesohyl, and the features of endopinacocytes are estimated from scanning and transmission electron microscopic observations of representatives of most families of the three orders. Although the Dysideidae have a reticulate skeleton and were classified in the order Dictyoceratida, they are actually closer to the Aplysillidae (Dendroceratida) than to dictyoceratids. The anatomy and cytology of the Halisarcidae differ profoundly from those of these three orders and are clearly more closely related to nonkeratose sponges. Some changes in classification lead to a pattern with highly homogeneous orders that clearly differ in their anatomic and cytologic features, which does not support the hypothesis of a common origin of the “keratose” sponges.     

Solenoid: a new aquiferous system to Porifera

The aquiferous system is an essential character of poriferans and supports their monophyly. Within the Calcarea, this system displays its greatest variety and traditionally is classified as: asconoid, syconoid, sylleibid, and leuconoid. Species of Leucascus, however, present a different type of aquiferous system composed of anastomosed (interconnected) choanocyte tubes and have an atrium lacking choanoderm. There is such confusion about the classification of the aquiferous system of Leucascus that, depending on the author, it has been classified as asconoid, syconoid, or leuconoid. Therefore, in the present work, we describe a new type of aquiferous system for Leucascus: the solenoid aquiferous system. This new aquiferous system is defined by the presence of anastomosed tubes internally lined by choanocytes and atrium without choanoderm. Although no deep phylogenetic significance has been attributed to the aquiferous system, the solenoid system raises important evolutionary questions about the variety of systems found among the poriferans.     

A New Flow-Regulating Cell Type in the Demosponge Tethya wilhelma – Functional Cellular Anatomy of a Leuconoid Canal System

Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system, flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma, which we studied using scanning electron microscopy. We found a hitherto undescribed cell type, the reticuloapopylocyte, which is involved in flow regulation in the choanocyte chambers. It has a highly fenestrated, grid-like morphology and covers the apopylar opening. The minute opening of the reticuloapopylocyte occurs in an opened, intermediate and closed state. These states permit a gradual regulation of the total apopylar opening area. In this paper the three states are included in a theoretical study into flow conditions which aims to draw a link between functional cellular anatomy, the hydrodynamic situation and the regular body contractions seen in T. wilhelma. This provides a basis for new hypotheses regarding the function of bypass elements and the role of hydrostatic pressure in body contractions. Our study provides insights into the local and global flow conditions in the sponge canal system and thus enhances current understanding of related physiological processes.     

Continuous cell movements rearrange anatomical structures in intact sponges.

Time-lapse cinemicrography was used to record the active movements of cells in living intact sponges. Each of the three main cell types (pinacocytes, mesohyl cells, and choanocytes) continuously moved and rearranged themselves so that the internal anatomy of the sponge was continuously remodeled. The shape and appearance of the sponges anatomical structures often changed substantially within a few hours. The most motile were the mesohyl cells, with many moving as fast as one cell-length per minute (15 microns/min). Mesohyl cell locomotion was often accompanied by displacements of spicules, canals, and choanocyte chambers; the patterns of these displacements suggested that the mesohyl cells were providing the motive forces for these rearrangements. The locomotion of the pinacocytes varied according to position: those along the outer sponge margins were most active, whereas those in other parts of the surface moved relatively little. Choanocytes were never observed to undergo independent locomotion but were always found grouped together in choanocyte chambers. These choanocyte chambers interacted with pinacocytes and mesohyl cells to form excurrent canals, which continuously moved, fused with, and branched from one another. These observations suggest that the experimental phenomenon of sponge cell-reaggregation and reconstitution, discovered by H. V. Wilson, represents an extreme version of morphogenetic processes that normally go on continuously within intact sponges. The results from the present study also suggest that these cellular rearrangements are controlled by active cell movements and behavioral responses that include but are not limited to selective cell adhesion.     

High resolution spatial mapping of brominated pyrrole-2-aminoimidazole alkaloids distributions in the marine sponge Stylissa flabellata via MALDI-mass spectrometry imaging

A number of pharmacologically active brominated pyrrole-2-aminoimidazole (B-P-2-AI) alkaloids have been isolated from several families of marine sponges, including those belonging to the genus Stylissa. In the present study, MALDI mass spectrometry imaging (MALDI-imaging) was applied to determine the spatial distribution of B-P-2-AIs within 20 μm cross sections of S. flabellata. A number of previously characterised B-P-2-AIs were readily identified by MALDI-imaging and confirmed by MS-MS and NMR profiling. Unknown B-P-2-AIs were also observed. Discrete microchemical environments were revealed for several B-P-2-AIs including dibromophakellin which was localised within the external pinacoderm and internal network of choanoderm chambers. Additionally, dibromopalau'amine and konbu'acidin B were also found to be confined to the choanoderm, while sceptrin was found to be highly abundant within the mesohyl. Further brominated compounds of unknown structure were also observed to have distinct localisation in both choanoderm chambers and the pinacoderm. These findings provide insights into the chemical ecology of S. flabellata, as most B-P-2-AIs were found on highly exposed surfaces, where they may act to prevent pathogens, predation and/or biofouling. Moreover this study demonstrates the power of MALDI-imaging to visualise the location of a range of metabolites in situ and to characterise compounds by MS-MS directly from intact specimens without the need for extraction. These methodologies facilitate selective targeting of micro-regions of sponge to screen for symbiotic microbial candidates or genes that may be involved in the production of the correlated compounds, and may represent a change in paradigm for natural product drug 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.     

Formation and construction of asexual buds of the freshwater spongeRadiospongilla cerebellata (Porifera,Spongillidae)

Summary The buds ofRadiospongilla cerebellata are formed asexually. Budding can be induced experimentally by injuring the sponge. The first sign of budding is a slight elevation of some surface areas, which proceed to rise rapidly so that they soon protrude conspicuously from the surface of the sponge. As a bud develops, the broad base joining it to the mother sponge narrows to a stalk, which finally breaks. The free buds drift in the water for 15–20 min and then settle, forming new sessile sponges. The buds, 1.5–2.5 mm in diameter, have an internal organization identical with that of the mother sponge. They are enclosed in a layer of pinacoderm perforated by dermal pores. Under the pinacorderm there is a shallow subdermal space, which is in communication with the incurrent canals leading to the choanocyte chambers. The water sucked into these chambers proceeds into the excurrent canal system and emerges from the sponge through the oscular tube. Spicules projecting radially from the bud bear apical tufts of microscleres. The skeletal spicules of the buds, like their choanocyte chambers, are smaller than those in the mother sponge. The chambers expand to their mature size by choanocyte mitosis. Buds and sponges are colored green by intracellular symbiotic algae of the genusChlorella.     

The choanoderm of Sycettusa hastifera (Calcarea,Porifera) is able to generate new individuals

One of the main characteristics of sponges is their capacity for cell dedifferentiation. This capability can allow an impressive amount of asexual reproduction in these animals, because they are able to develop new individuals from just a few somatic cells. Studies of dedifferentiation, however, have focused mainly on sponges of the class Demospongiae. Therefore, we investigated here whether individuals of three different species of Calcarea are able to reconstitute new individuals following artificial fragmentation. We observed that fragmentation releases clumps of choanoderm able to initiate somatic embryogenesis. In Borojevia brasiliensis (asconoid aquiferous system, subclass Calcinea) and Paraleucilla magna (leuconoid aquiferous system, subclass Calcaronea), these clumps started to develop, but they did not pass through the first developmental phases. In Sycettusa hastifera (syconoid aquiferous system, subclass Calcaronea), the choanoderm was reorganized into primmorphs that fused to each other and formed an exopinacoderm. The first primmorphs’ spicules were triactines. Despite a large mortality rate, the primmorphs developed into olynthus stages. The somatic embryogenesis and the metamorphosis of the olynthus were similar to those observed during the sexual development of this and other calcareous sponge species. Our results show that in S. hastifera, and perhaps in other syconoid calcareous sponges, somatic embryogenesis occurs mainly from choanocytes, at least in vitro. However, primmorph development does not follow the same pattern observed in post‐metamorphic sexual development, as in that case diactines are always the first spicules to be synthesized in calcaronean species.     

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.     

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.