Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.     

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.     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.     

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.     

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.     

Ultrastructural evidence of extruding exocytosis of residual bodies in the freshwater sponge Ephydatia fluviatilis

Exocytosis of residual bodies by choanocytes, archeocytes and endopinacocytes lining the aquiferous system of Ephydatia fluviatilis has been demonstrated using calibrated latex beads and Escherichia coli as tracers. In passing into the mesohyl or the lumen of the exhalant aquiferous canals, beads, and altered bacteria were enveloped by the plasma membrane of the cell containing them. The membrane constricted at a neck region to form extruding vacuoles. This process appeared first in choanocytes and later in other cell types. The occurrence of these buds increased with the length of incubation time, as did the number of particles they contained. Acid phosphatase activity was frequently associated with the particles budding from the cell membrane, confirming that this process followed digestive activity. Membranous vacuoles were recovered from the external medium and observed by TEM and those adhering to the substratum were seen by SEM. These observations proved that vacuoles were released from the sponges. This membrane-consuming mechanism of exoctyosis implies intense membrane replacement in the digestive cells of the sponge.     

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.     

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.     

Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti

The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1). To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed. Results show that choanocytes and archaeocytes are the major sponge cell types and that H. vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria. Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes. This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1. The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes. The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production. Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge. The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge.     

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.     

First observations on egg release in the oviparous sponge Chondrilla nucula (Demospongiae, Chondrosida, Chondrillidae) in the Mediterranean Sea

Abstract. The reproduction of the demosponge Chondrilla nucula in Portofino (Ligurian Sea, Italy) was studied during August 2001. Eighteen individuals were sampled and examined with light microscopy for the presence of gametes, and 5 individuals carrying oocytes were found. In addition to microscopic observations, reproductive individuals could be easily identified as female even at the macroscopic level because of the presence of a grayish layer in the mesohyl where oocytes were concentrated. Oogenesis resulted in modifications of the external sponge morphology and of the aquiferous system. Approximately one‐third of the sponge body was filled with oocytes with the consequent disappearance of choanocyte chambers in the reproductive portion of the sponge. Under laboratory conditions, we obtained fertilized eggs from females and observed the first stages of embryonic development. Our observations suggest that fertilization in specimens of Chondrilla nucula occurs internally and not in the water. During the 2 years following these observations, no reproductive specimens were found among the same population during the reported reproductive period.     

Distinct histomorphology for growth arrest and digitate outgrowth in cultivated Haliclona sp. (Porifera: Demospongiae)

The use of sponges in biotechnological processes is limited by the supply problem, and sponge biomass production is becoming a current topic of research. The distinction between characteristics for growth and growth arrest is also important for environmental monitoring. In this study, we analyze the morphology of the digitate outgrowths from the sponge Haliclona sp . The sponge Haliclona sp . was successfully cultivated for 14 months in a closed system. The morphological characterization of growth arrest was performed after submitting explants to starvation‐stress for approximately 2 weeks, to correlate morphology with growth and growth arrest. The digitate outgrowth showed three distinct regions: mature (MR), transition (TR) and immature (IR). Our data suggest a growth developmental program, with collagen fascicles guiding axial growth in IR, followed by progressive development of choanocyte chambers and large aquiferous systems at the more mature proximal region (choanosome). The intercalation of choanocyte chambers and small aquiferous systems inside collagen fascicles previously originated at the IR region can be responsible for thickening expansion and conversion of the collagen fascicles into columnar choanosome in MR. The growth arrest after starvation‐stress assay showed morphological changes in the IR corroborating collagen in the extreme tip of the digitate outgrowth as an important role in guiding of axial growth of Haliclona sp . The identification of distinct morphologies for growth and growth arrest suggest a growth developmental program, and these data could be useful for further investigations addressing sponge biomass gain and environmental monitoring.     

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.     

The aquiferous systems of three marine demospongiae

The aquiferous systems of three common, coastal, marine Demospongiae, Halichondria panicea (Pallas), Haliclona permollis (Bowerbank) and Microciona Prolifera (Ellis and Solander), are analyzed by measurements of cross-sectional areas of conducting elements. The patterns in demosponges of extremely different organizational morphologies are found to be quantitatively similar. The porocyte nature of the ostia is established for all three species. Choanocyte chamber densities range from 1 to 1.8 × 10⁷ chambers ml⁻¹ with 57 to 95 choanocytes per chamber (means). Cross-sectional area of the intervillar space of the choanocyte collars is calculated to be 12 to 56 times the lateral surface area of the specimen. Velocities of water movement through specific elements of the aquiferous system are calculated from cross-sectional area data and measured oscular flow of Haliclona permollis. The calculated Reynolds numbers lie below the critical value and fluid flow is thus considered laminar throughout the aquiferous systems of these sponges.     

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.     

The influence of an eucaryotic intranuclear cell parasite on the production of gemmules inEphydatia fluviatilis (Porifera,Spongillidae)

Summary A prerequisite for the production of gemmules is the presence of intact archaeocytes and trophocytes, which give rise to the thesocytes with which the gemmule is eventually filled. The coat enclosing the gemmule requires spongioblasts for its formation and incorporates amphidisk spicules, which develop in amphidiskoblasts. The cell parasite, the development of which is described here, infects mainly archaeocytes but also spongioblasts and amphidiskoblasts. Even a moderate infection results in significant malformation of the gemmule covering. In the thesocyte nucleus, the parasite can survive the resting phase of the gemmule. After the gemmule has hatched, the parasite, again in the virulent form, is present in the young, developing sponge. The parasite may be a microsporidian of the primitive type, close to the genusMetchnikovella, which typically occurs in gregarines.Abbreviations for the microscopic procedures PhM phase-contrast microscopy - TEM transmission electron microscopy - SEM scanning electron microscopy     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

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.     

Cytological and cytochemical investigations of development from dormant gemmules of the marine sponge,Haliclona loosanoffi

Vernalized gemmules of the marine sponge Haliclona loosanoffi were cultured at 20°C, fixed at 24-hour intervals (0–11 days), and processed for light microscopy by using a variety of absorption and fluorescent staining methods. The cytochemistry and morphology of development were compared to the well-studied developmental patterns of freshwater sponges and to the patterns described in the marine sponge Suberites domuncula. The precocious development of H. loosanoffi gemmules involves early morphogenesis occurring within the unhatched gemmule, as opposed to the patterns in freshwater sponges, where most development occurs after the gemmule hatches. Definitive sponge tissue surrounding a single osculum is present 9 days after release from dormancy.