Detection of distinct endocytotic and phagocytotic activities in epithelial cells (pinacocytes) of freshwater sponges (Porifera, Spongillidae)

 Light and electron microscopical investigations using externally applied fluorescent and gold-labeled markers have revealed the existence of distinct endocytotic and phagocytotic activities in basal epithelial cells (pinacocytes) of the freshwater sponges Spongilla lacustris and Ephydatia es) of the f. The total rate of endocytotic membrane uptake, ascertained by the application of the cationic lipid probe TMA-DPH, was found to be 3.2% of the cell surface area/h. A typical fluid-phase endocytosis, demonstrated by the use of the water-soluble membrane-impermeable tracers BCECF-dextran and LY-CH, participates in endocytotic activity at a rate of 0.7% of the cell surface area/h and results in the formation of endosomes measuring 0.8–1 μm in diameter. Moreover, the application of labeled BSA succeeded in the detection of a receptor-mediated endocytosis amounting to a concentration-dependent uptake of 2.3–2.8% of the cell surface area/h. Coated pits and coated vesicles conveying the adsorbed BSA measure 0.3 μm in diameter and are covered on the cytoplasmic face with a clathrin-like protein (HC, 180 kDa; LC, 30 kDa). To facilitate phagocytotic activities, a series of fluorescent–labeled and chemically treated particles such as bacteria or latex beads have been successfully employed. Accordingly, the measured values of phagocytic membrane uptake between 1 and 8% of the cell surface area/h depend on the variety of size as well as the chemical nature of the different bioparticles and clearly point to phagocytosis as a key mechanism for providing freshwater sponges with nourishment. Accepted: 3 April 1997     

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 filtration apparatus for food collection in freshwater sponges (Porifera,Spongillidae)

Summary The freshwater sponges (Spongillidae) feed by filtering out small particles from the water passing through them by means of strainer devices in the flagellated chambers. These are filamentous, fine-meshed structures at the distal ends of the choanocyte collars formed of a mucous material similar to that in the glycocalyx. Each strainer separates its flagellated chamber into an outer and an inner zone. The strainers are an extremely efficient filtering mechanism.     

Condensation rhythm of fresh-water sponges (Spongillidae, Porifera)

The mesenchyme continuum of spongillids exhibits rhythmic changes that at first glance appear to be contractions. Actually, however, the process is a condensation initiated by the formation of punctate cell contacts and a rapid swelling of all mesenchymal cells. As the cells come into closer contact and the spaces between them are constricted, the volume of the mesenchyme shrinks, giving the impression of a contraction. It seems likely that rhythmic mesenchyme condensation assists the choanocyte chambers in pumping water through the sponge.     

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.     

Abundance and microhabitats of freshwater sponges (Spongillidae) in a Danubean floodplain in Austria

1. This study examined the abundance and distribution of freshwater sponges (Spongillidae) at 32 sites in a floodplain on the Danube within the ‘Donau‐Auen’ National Park east of Vienna, Austria. Ranked from abundant to rare, the species inventory comprised Ephydatia fluviatilis, Spongilla lacustris, Ephydatia mülleri, Eunapius fragilis and Trochospongilla horrida. 2. The presence of hard substratum was essential for the growth of sponges. Timber stands near the water and drifting dead wood increased the abundance of E. fragilis, E. fluviatilis and E. mülleri, whereas stony substrata were important for S. lacustris. A small fraction of E. fluviatilis was collected from macrophytes (Phragmites). 3. Based on the area colonised, the abundance of S. lacustris, E. fragilis and E. fluviatilis was highest (94.2–100% of the total) in floodplain waters where hydrological connectivity with the Danube was low (0–6 days year⁻¹), whereas E. mülleri and T. horrida made up 20.3–35.9% of the total at sites connected for up to 179 days year⁻¹. Moreover, the area colonised by T. horrida at a current velocity >0.20 m s⁻¹ was larger than in the remaining species. Sites with E. mülleri and T. horrida had a higher silicon concentration (0.9 mg L⁻¹) than sites where the remaining three species were collected (0.4–0.6 mg L⁻¹). 4. In most species, the length of macroscleres (the larger spicules) was positively correlated with conductivity and negatively with pH. With respect to aberrant macroscleres, hooks were observed most frequently, whereas the proportion of centrotylotes (ie with the one on more globular swellings along the spicule) was lowest. 5. Freshwater sponges have a great deal of potential as bioindicators and restoration measures that improve floodplain connectivity will favour rare species, such as E. mülleri and T. horrida, while impairing others (e.g. E. fragilis, S. lacustris and E. fluviatilis).     

Lipid compounds of freshwater sponges: family Spongillidae,class Demospongiae

More than 100 novel, unusual and rare fatty acids, lipids and sterols have been isolated from freshwater sponges. The structures, biogenesis, synthesis and bioactivity of some lipid compounds of freshwater sponge species are reviewed.     

Oogenesis and larval development of Ephydatia fluviatilis (Porifera,Spongillidae)

Summary In Ephydatia fluviatilis young oocytes already appear in autumn. They pass the winter in the highly reduced sponge, but vitellogenesis and further development do not take place before following spring. The fact that the young oocytes appear before the normal period of reproduction makes E. fluviatilis different from all other local freshwater sponges, which reduce totally in autumn. E. fluviatilis seems to be a gonochorist. The oocytes originate from archaeocytes and during the first growth phase they reach a diameter of approximately 40 m. In the second growth phase the oocyte is enclosed in a single-layered follicle epithelium and grows to 170–180 m by phagocytosis of trophocytes. The fully developed egg cell finally shows a distinct layering of the incorporated yolk material. Cleavage is totally equal to unequal so that macro- and micromeres appear in some cleavage stages. Cleavage leads to a solid embryo consisting of uniform cells. At this stage of development the first scleroblasts appear. As the cells develop they are surrounded by companion cells, managing the transport of the scleroblasts. The further development to the larva is marked by the appearance of the larval cavity, typical for larvae of Spongillids, which finally occupies about half the volume of the larva at emergence. The periphery of the larva consists of a single-layered ciliated epithelium. After emergence the larva forms flagellated chambers, which are integrated into the primordia of the excurrent canal system. This system connects with the larval cavity and ensures that it becomes part of the excurrent canal system of the young sponge. Particularly in the region of the larval cavity the ciliated epithelium of the free larva is reduced. Here a new larval surface epithelium is formed by pinacocytes.     

Microtubule- and microfilament-based dynamic activities of the endoplasmic reticulum and the cell surface in epithelial cells ofSpongilla lacustris (Porifera,Spongillidae)

Summary Dynamic activities of the endoplasmic reticulum (ER) and of the cell surface were analyzed in living epithelial cells (pinacocytes) ofSpongilla lacustris by contrast-enhanced video microscopy with the AVEC-DIC or the ACE equipment. Long term sequences revealed the ER to be a highly unstable system undergoing permanent alterations of the reticular patterns in that tubules merge and split or polygons open and close again. Treatment with colcemid or colchicine causes distinct changes of the typical motile phenomena, whereas cytochalasin D has no influence. On the other hand, the dynamic behavior of the cell surface is characterized by distinct ruffling activities as well as the formation and retraction of spiky filopodia. In contrast to the described ER dynamics, cell surface phenomena are clearly influenced by cytochalasin D but not by colcemid or colchicine. Altogether, results of the present paper are similar to correspondent observations on mammalian cells and point to microtubules and microfilaments as the cytoskeletal elements being responsible for ER and cell surface dynamics, respectively.     

Molecular systematics of sponges (Porifera)

The first application of molecular systematics to sponges was in the 1980s, using allozyme divergence to dis-criminate between conspecific and congeneric sponge populations. Since this time, a fairly large database has been accumulated and, although the first findings seemed to indicate that sponge species were genetically more divergent than those of other marine invertebrates, a recent review of the available dataset indicates that levels of interspecific gene identities in most sponges fall within the normal range found between species of other invertebrates. Nevertheless, some sponge genera have species that are extremely divergent from each other, suggesting a possible polyphyly of these genera. In the 1990s, molecular studies comparing sequences of ribosomal RNA have been used to reappraise the phylogenetic relationships among sponge genera, families, orders and classes. Both the 18S small subunit and the 28S large subunit rRNA genes have been sequenced (41 complete or partial and 75 partial sequences, respectively). Sequences of 18S rRNA show good support for Porifera being true Metazoa, but they are not informative for resolving relationships among genera, families or orders. 28S rRNA domains D1 and D2 appear to be more informative for the terminal nodes and provide resolution for internal topologies in sufficiently closely related species, but the deep nodes between orders or classes cannot be resolved using this molecule. Recently, a more conserved gene, Hsp70, has been used to try to resolve the relationships in the deep nodes. Metazoan monophyly is very well supported. Nevertheless, the divergence between the three classes of Porifera, as well as the divergence between Porifera, Cnidaria and Ctenophora, is not resolved. Research is in progress using other genes such as those of the homeodomain, the tyrosine kinase domain, and those coding for the aggregation factor. For the moment the dataset for these genes is too restricted to resolve the phylogenetic relationships of these phyla. However, whichever the genes, the phylogenies obtained suggest that Porifera could be paraphyletic and that the phylogenetic relationships of most of the families and orders of the Demospongiae have to be reassessed. The Calcarea and Hexactinellida are still to be studied at the molecular level.     

Detection and cytoplasmic localization of two different microtubule motor proteins in basal epithelial cells of freshwater sponges

Summary Epithelial sponge cells (pinacocytes) contain a set of 50 to 60 microtubules radiating from the nuclear region to the cell periphery. Vacuoles of the endocytic pathway (endosomes, lysosomes) and mitochondria move along single microtubules in both directions; moreover, the ring-like arrangement of the Golgi apparatus around the nucleus and the net-like organization of the endoplasmic reticulum in the cytoplasmic matrix are also maintained, in an energy-dependent manner, by the microtubular system. Significant changes in the velocities of retrograde and anterograde transport as well as distinct differences in the sensitivity of organelle dynamics to ATPase inhibitors and ATP analogues indicate the existence of two microtubule-based motor proteins. Ion exchange chromatography of pinacocyte homogenates resulted in the enrichment of a 97 kDa kinesin-like protein (SKLP) with the ability to cross-react with antibodies against the kinesin heavy chain. Two other polypeptides, with molecular mass of 75 and 400 kDa, apparently belonging to a cytoplasmic dynein-like protein (SDLP) could be recognized in immunoblots with antibodies against the intermediate and heavy chains of cytoplasmic dynein. In addition, three MAP-like polypeptides (SMAPLPs), with molecular mass of 280, 250 and 70 kDa, obviously related to the MAP-2 and tau-family, have been identified. Immunocytochemical studies at the light and electron microscopical level localized SKLP, SDLP, and SMAPLPs at endocytic vacuoles and mitochondria, whereas the endoplasmic reticulum has SKLP and SMAPLPs, but the Golgi apparatus only SDLP.Abbreviations AMP-PNP 5-adenylylimidodiphosphate - ATP adenosinetriphosphate - DiOC₆ (3) 3,3-dihexyloxacarbocyanine iodide - DTT 1,4-dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine - EM electron microscope - ER endoplasmic reticulum - FPLC fast protein liquid chromatography - GA Golgi apparatus - GTP guanosinetriphosphate - HC heavy chain - HSS high-speed supernatant - IC intermediate chain - LC light chain - MAP microtubule-associated protein - MT microtubule - PIPES 1,4-piperazine-N, N-bis-(2-ethanesulfonic) acid - PMSF phenylmethylsulfonyl fluoride - SDLP sponge dynein-like protein - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide gel electrophoresis - SKLP sponge kinesin-like protein - SMAPLPs sponge MAP-like proteins - UTP uridinetriphosphate     

Scanning electron microscopy of taxonomic diagnostic criteria of the freshwater sponge,Heteromeyenia tubisperma (Potts, 1881) (Porifera : Spongillidae)

Taxonomic diagnostic criteria of the spongillid freshwater sponge, Heteromeyenia tubisperma (Potts, 1881) were examined using scanning electron microscopy. The species is characterized by a gemmule which bears an unusually long, prominent porous tube. The application of SEM to the systematic studies of the freshwater sponges provides diagnostic capabilities not available with the light microscope. It is desirable that a key, coupled with a reference atlas of scanning electron micrographs illustrating taxonomic diagnostic criteria of freshwater sponge species, particularly utilizing type specimens, be developed.     

The metamorphosis of the parenchymula-larva of Ephydatia fluviatilis (Porifera,Spongillidae)

Summary The sexual development of Ephydatia fluviatilis involves a ciliated parenchymula-larva. The mature larva leaves the body of the mother sponge through the excurrent canal system and arrives eventually in the outside world by way of the osculum. At this stage the types of cells found in the adult sponge are already present in the larva. The released larva swims around for a while and then, after a period of between 3 and 48 hours, it attaches, usually with the anterior, larval cavity-bearing pole, onto the substratum. While it is attaching and spreading itself out, the larva undergoes a metamorphosis. The most notable stages of this metamorphosis are as follows: (a) disintegration of the ciliated epithelium from the anterior pole of the larva and its substitution by a pinacocyte epithelium, (b) splitting of the larval cavity and (c) integration of the remains into the developing canal system together with the creation and further development of the organic features of a functioning sponge.     

The sterols of calcareous sponges (Calcarea, Porifera)

Sponges are sessile suspension-feeding organisms whose internal phylogenetic relationships are still the subject of intense debate. Sterols may have the potential to be used as independent markers to test phylogenetic hypotheses. Twenty representative specimens of calcareous sponges (class Calcarea, phylum Porifera) with a broad coverage within both subclasses Calcinea and Calcaronea were analysed for their sterol content. Two major pseudohomologous series were found, accompanied by some additional sterols. The first series encompassing conventional C(27) to C(29)Delta(5,7,22) sterols represented the major sterols, with ergosterol (ergosta-5,7,22-trien-3beta-ol, C(28)Delta(5,7,22)) being most prominent in many species. The second series consisted of unusual C(27) to C(29)Delta(5,7,9(11),22) sterols. Cholesterol occurred sporadically, mostly in trace amounts. The sterol patterns did not resolve intraclass phylogenetic relationships, namely the distinction between the subclasses, Calcinea and Calcaronea. This pointed towards major calcarean lipid traits being established prior to the separation of subclasses. Furthermore, calcarean sterol patterns clearly differ from those found in Hexactinellida, whereas partial overlap occurred with some Demospongiae. Hence, sterols only partly reflect the phylogenetic separation of Calcarea from both of the other poriferan classes that was proposed by recent molecular work and fatty acid analyses.     

Space as a limiting resource in freshwater systems: competition between zebra mussels (Dreissena polymorpha) and freshwater sponges (Porifera)

Species interactions between two types of sessile benthic invertebrates, the zebra mussel (Dreissena polymorpha) and freshwater sponges (Porifera), were evaluated in Michigan City IN Harbor in southern Lake Michigan during 1996. The study objective was to define whether competition plays a role in structuring benthic communities using experimental techniques commonly employed in marine systems. Sponges were uninhibited by zebra mussel presence and overgrew zebra mussel shells on hard vertical substrata. In contrast, zebra mussels did not overgrow sponge colonies, but did show an ability to re-capture hard substrata if relinquished by the sponge. The negative affect of sponges on zebra mussels through overgrowth and recruitment suggests interactions that could eventually displace zebra mussels from these benthic communities. However, seasonal reduction of sponge biomass from autumn through winter appears to allow the zebra mussel a periodic respite from overgrowth, preventing exclusion of zebra mussels from the community and allowing these two taxa to co-exist.     

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.     

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