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Ability of mobile cells of the freshwater sponge Ephydatia fluviatilis (Porifera, Demospongiae) to digest diatoms a

Elda Gaino & Manuela Rebora

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Dipartimento di Biologia Animate ed Ecologia, Via Elce di Sotto, Perugia, I‐06123, Italy E-mail: Published online: 28 Jan 2009.

To cite this article: Elda Gaino & Manuela Rebora (2003) Ability of mobile cells of the freshwater sponge Ephydatia fluviatilis (Porifera, Demospongiae) to digest diatoms, Italian Journal of Zoology, 70:1, 17-22, DOI: 10.1080/11250000309356491 To link to this article: http://dx.doi.org/10.1080/11250000309356491

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Ital. J. Zool., 7O. 17-22 (2003)

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Ability of mobile cells of the freshwater sponge Ephydatia fluviatilis (Porifera, Demospongiae) to digest diatoms

INTRODUCTION

Sponges are typically filter-feeding organisms, the only exception being carnivorous representatives belonging to Asbestopluma (Poecilosclerida) (Vacelet & Boury-Esnault, 1995, 1996). Water flows into and out of the sponge body, passing through the aquiferous system, a ELDA GAINO complex network of canals and choanocyte chambers. MANUELA REBORA Of the physiological functions that the aquiferous sysDipartimento di Biologia Animate ed Ecologia, tem performs, the most relevant is food procurement. Via Elce di Sotto, I-06123 Perugia (Italy) Even though the ensuing pathways of food processing E-mail: [email protected] vary considerably, depending on both ingested particle size and structural organisation of the sponge (see review in Simpson, 1984), food digestion takes place within vacuoles in the cytoplasm. Direct particle entrapment by cells alien to the aquiferous system, but lining the outermost sponge surface (exopinacocytes), has been documented in Ephydatia fluviatilis by using suspensions of calibrated latex beads (Willenz & Van de Vyver, 1982). This work substantiated the possible involvement of this sponge covering in the uptake of particles, a feature also documented during skeletal formation in a keratose sponge (Terragawa, 1986a, b) and in the two Antarctic sponges Phorbas glaberrima and Tedania charcoti. In these species, the exopinacoderm directly takes up benthic diatoms that settle on the sponge surface. The diatoms become arranged beneath the surface layer of cells, thereby constituting a permanent association (Gaino et al, 1994). The nature of associations between sponges and diABSTRACT atoms is still obscure (Sara, 1966; Cox & Larkum, 1983). A recent scanning electron microscopical investigation In Lake Piediluco (Central Italy) the reed Phragmites australis is carried out on an Antarctic hexactinellid sponge sugcolonized by the freshwater sponge Ephydatia fluviatilis, which gested a negative impact of diatoms on sponge tissues, assumes a different shape according to the season: highly expanded in June and July; gradually reducing in August; fairly leading to degenerative processes of the sponge body round and minute (from less than 1 to 10 mm) from September to (Cerrano et al., 2000). However, in Antarctic habitats, March, following the die-back of the reeds. Ultrastructural investicharacterised by quantitatively fluctuating food supplies gations carried out on the minute sponges gave evidence that during the year, stored diatoms may constitute an addithey were functional and contained diatoms both free in the tional food source (Cattaneo-Vietti et al., 2000). Surprissponge mesohyl and enclosed in cytoplasmic vacuoles of the wandering cells. Vacuoles contained a single diatom of different ingly, tests on the concentration of sponge carbohysize (about 9 × 5 urn the largest; about 5.7 × 3.8 μm the smallest) drates and chlorophyll- a carried out on two Antarctic and in various phases of degradation. Although sponges are species provided evidence that endobiotic, autotrophic known to host auto- and heterotrophic symbionts, this is the first diatoms can shift from mutualism to parasitism because report of their progressive digestion of endocellular diatoms up to the final fragmentation of their frustule. the total sugar content of the tissues decreased as the chlorophyll-a increased (Bavestrello et al, 2000). KEY WORDS: Uptake - Ultrastructure - Phagocytosis - Piediluco Heterotrophic bacteria, autotrophic cyanobacteria, Lake. zoochlorellae, and zooxanthellae are commonly found in Porifera, and in many cases make up a majority of the sponge tissue volume. These organisms can be obACKOWLEDGMENTS served both inside cell vacuoles and in the mesohyl maThis work was supported in part by a grant from "Cofinanziatrix (Sara, 1971; Sara & Vacelet, 1973; Wilkinson, 1978; mento Nazionale", and in part by the financial support of the Rosell & Uriz, 1992; Bigliardi et al., 1993; Frost et al., "Progetto d'Ateneo" of the University of Perugia. We would like to 1997; Friedrich et al, 1999; Scalera Liaci et al., 1999). express our gratitude to Dr. Tisza Lancioni, Dr. Barbara Todini, and Mr. Umberto Chiappafreddo for their help in sponge samEven though the bulk of data corroborate the sponges pling. We also thank Dr. Renata Manconi for confirming the speability to capture diatoms (Reiswig, 1971), diatoms assocific attribution of the specimens. ciated with the sponge body have been observed only in the mesohyl. A digestion by sponge cells was suggested by Frost (1981), who examined the condition of (Received 30 May 2002 - Accepted 25 September 2002)

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the cytoplasm of these algae within a sponge suspension. Likewise, the large amounts of diatom fragments in pseudopellets of Dysidea avara were advocated to support sponge digestion (Ribes et al., 1999)- Therefore, knowledge of a direct phagocytosis and of the importance as a food source of these algae is essentially based on experimental tests. This paper represents the first evidence of a progressive digestion of diatoms in sponges.

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MATERIALS AND METHODS The minute forms (from less than 1 to 10 mm) (Fig. IB, C) of the freshwater sponge Ephydatia fluviattiis (L., 1758), the subject of the present investigation, were collected from September 2000 to March 2001 on the reed Phragmites australis (Cav.) Trin. ex Steud from Lake Piediluco, an artificially regulated lake about 20 km SE of Terni, Umbria, Central Italy. A branch of the Nera river flows into the lake as a tributary and effluent water gives rise to the Velino River. Nearly 75% of the riparian vegetation consists of monospecific stands of the reed Phragmites australis. For transmission electron microscopy (TEM), the minute sponges were fixed in the field in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2 for 4 h., rinsed overnight in the same buffer and post-fixed for 1 h. in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.2. The material was then dehydrated in a graded series of ethanols and embedded in an Epon-Araldite mixture. Semithin sections were stained with 1% methylene blue in 1% borax. Silver to pale gold ultrathin sections were cut on a Reichert ultramicrotome, mounted on formvar-coated copper grids, and examined in a Philips EM 208 transmission electron microscope, after staining in uranyl acetate and lead citrate. In addition, in order to ascertain the organisation of the sponges during their life cycle, some plugs were cut off from several specimens collected in Lake Piediluco all year round (June 2000-May 2001) and processed using the routine technique for histological observations.

RESULTS The reed Ph. australis constitutes a favourable substratum for attachment and growth of E. fluviatilis. In fact, a monitoring of the sponge population gave evidence that sponges grow preferentially on the reed belt, where these animals display a typical sleeve-shaped morphology. However, the shape of the sponge varied markedly according to both season and position on the reed. In June and July, sponge bodies were highly expanded, with irregular extensions (Fig. 1A); in August, progressive reduction in body size led to the formation of laminar envelopes; from September to March, following the die-back of reeds, E. fluviatilis was represented mainly by minute, fairly round individuals (Fig. IB). These ranged in size from less than 1 to 10 mm, and typically adhered to small rootlets (Fig. IB), which emerged from the basal region and immersed nodal parts of the reed. As they grew, sponges progressively enveloped adjacent rootlets (Fig. 1C) from which they could expand and colonise new reeds. The colour of these minute sponges varied from slightly brownish to pale green to white.

E. GAINO, M. REBORA

Histology and ultrastructure carried out on these minute sponges gave evidence of their being active. Overall, these sponges had a loose texture, the most obvious feature being the aquiferous system composed of large canals and closely associated choanocyte chambers (Fig. ID). In the mesohyl of these minute sponges, sponge cells — whose general morphology is consistent with their belonging to the category of wandering elements - containing diatoms within cytoplasmic vacuoles (Fig. IE) were observed: 1) just below both exopinacoderm and basopinacoderm; 2) close to the canals of the aquiferous system; 3) scattered in the mesohyl matrix, between the choanocyte chambers. Each vacuole of the wandering elements included a single diatom. A single cell can include multiple vacuoles with diatoms of different size. In fact, the largest diatom, shown in Figure IE, measures about 9 x 5 pm, and the smallest one about 5.7 X 3.8 um. The different shape and size of the diatoms is consistent with their attribution to different species. Long (ca. 15 pm) and narrow (ca. 3 um) diatoms were observed externally to the sponge body, between the basopinacoderm and the rootlet surface (Fig. IF). Diatoms other than those enclosed in vacuoles were scattered within the sponge mesohyl (Fig. 2A). Among these free diatoms, two kinds were recognised on the basis of their ultrastructural morphology shown in cross section: small diatoms (5 x 3.5 pm) with a flat ventral side and a convex dorsal side; and larger diatoms (6 x 5.6 pm) with a fairly round shape, and slightly pierced walls. Both kinds of diatoms, shown in Figure 2A, contained plastids with evident thylakoids. Engulfed diatoms cause a marked deformation of the sponge cell shape, particularly when more than one diatom is ingested. The cytoplasm of the hosting cell greatly extends in such a way that only a narrow strand separates the cell vacuoles from the external milieu. A case in point is represented by cells containing up to three diatoms (Fig. 2B), each differing in shape and size. The example given in Figure 2B shows (a) a fairly round diatom (4 x 3.5 pm); (b) a diatom with a flat base and convex opposite side (5 x 2.5 pm); and (c) a fairly rectangular diatom (7 x 3-7 pm). Whereas the plastid persisted in some endocellular diatoms (Fig. 2C), in others only remnants of the plastid were found within the frustule (Fig. 2D). Frustules of diatoms located freely in the mesohyl or enclosed in sponge cell vacuoles ranged in thickness from 0.6 pm in the former to 0.3 pm or less in the latter. In addition, the walls of engulfed diatoms tended to lose their integrity and break into small segments (Fig. 2E). Changes to diatom cytoplasmic inclusions could be the first step in diatom degradation. Occasionally, large vacuoles including the remains of a diatom wall in the form of electron-dense dots along with the plastid showing thylakoids were also observed (Fig. 2C). Furthermore, some sponge cells exhibited large vacuoles at

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DIATOM DIGESTION BY A FRESHWATER SPONGE

Fig. 1 - Different shapes of the freshwater sponge Ephydatia fluviatilis during growth on the reed Phragmites australis, and diatoms. A, field image of the sponge showing its expanded configuration (scale bar, 2 cm). B, field image of minute sponges (arrows) developing on the small rootlets of the reed (sr) (scale bar, 0.5 cm). C, stereomicroscopic view of minute sponges (s) progressively growing around the small rootlets (sr) (scale bar, 1.5 mm). D, semithin section of a minute sponge developing around a small rootlet (sr); note the loose texture of the sponge with its aquiferous system showing large canals (c), and choanocyte chambers (cc) (scale bar, 125 um). E, transmission electron microscopy view of a minute sponge showing a wandering cell containing diatoms (d) within its cytoplasmic vacuoles; cc, choanocyte chamber (scale bar, 3.6 um). F, transmission electron microscopy view of a minute sponge showing the basopinacoderm (b) separating the sponge body (sb) from the rootlet (r) surface where diatoms (d) can accumulate (scale bar, 2.7 um).

E. GAINO, M. REBORA

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Fig. 2 - Transmission electron microscopy of minute sponges enclosing diatoms. A, free diatoms scattered in the sponge mesohyl. Note the plastid thylakoids (t) (scale bar, 1.3 um). B, a wandering cell containing diatoms of various sizes (a, b, c) in cytoplasmic vacuoles (scale bar, 1.7 um). C, a wandering cell engulfing a well-preserved diatom (a) and another one (b) whose wall displays electron-dense dots (arrows); t, thylakoid (scale bar, 1.4 um). D, a wandering cell containing a diatom with remnants of its plastid (arrow) (scale bar, 2.3 um). E, a wandering cell showing the frustule of the enclosed diatom (arrows) (scale bar, 2 um). F, large vacuole of a wandering cell containing electron-dense debris (arrow and arrowhead) (scale bar, 1.6 um); in the inset the detail pointed to by the arrowhead (scale bar, 0.5 um). G, sponge sclerocyte producing a siliceous sponge spicule (ss) (scale bar, 2.7 um).

DIATOM DIGESTION BY A FRESHWATER SPONGE

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the cell periphery. They were almost completely empty except for some debris (Fig. 2F) whose electron-density and periodicity (inset of Fig. 2F) was comparable to that of the diatom shell. In these minute sponges, spiculogenesis was very intense and sclerocytes characterised by their endocellular siliceous spicules (Fig. 2G) were commonly observed. The histological monitoring of the sponge tissue from the individuals collected throughout the year (June 2000-May 2001) revealed that endocellular diatoms are exclusive to the minute sponges collected from September to March. DISCUSSION One of the characteristic attributes of sponges is their plastic organisation, which allows these animals to assume a vast array of growth forms. Sponges undergo continuous remodelling due to the capacity of some types of adult cells to act in an embryonic-like fashion (Gaino & Burlando, 1990; Gaino et aL., 1995). This feature was particularly evident in specimens of E. fluviatilis developing on the reed Ph. australis in Lake Piediluco. Indeed, the sponge adapts its morphology to the substratuM., in such a way that its maximum growth takes place concomitantly with that of the reeds. Since the first report of E. fluviatilis in Lake Piediluco (Moretti et aL., 1981), this sponge has never before been observed in the form of minute individuals. At present, their origin is unknown. They could derive from newly settled larvae or from the fragmentation of larger sponges, thereby producing new individuals. Fragmentation is common in sponges and, along with sexual reproduction, maximises the dispersion of these animals (Maldonado & Uriz, 1999). The life cycle of E. fluviatilis has been studied intensively (Rader & Winger, 1985; Pronzato et aL., 1993; Corriera et aL., 1994) as well as its ability to react to stimuli (Masuda et aL., 1998). In particular, this freshwater sponge has been also utilised as an experimental model for investigating food incorporation, such as particle uptake rates by using two sizes both of latex beads and bacterial cells in culture (Francis & Poirrier, 1986), while experiments carried by Kilian (1952) showed that particles larger than 50 pm in diameter were taken up directly by the external coat, thereby bypassing the aquiferous system passageway. The size of the diatoms entrapped by the wandering cells of E. fluviatilis, as seen in ultrastructural sections, is of a dimensional range compatible with their entering through the aquiferous system. Their entrance could also be facilitated by the loose texture exhibited by these minute sponges. We can rule out an active participation of the small choanocytes in this process because they are too small to entrap and transfer diatoms to wandering cells. The pinacoderm could have some function in promoting diatom uptake, and in some Antarctic

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sponges, large settled diatoms become enveloped progressively by the pinacoderm and form a coat strengthening the sponge surface (Gaino et aL., 1994). Different reactivity patterns of the pinacoderm to crystalline and amorphous silica were demonstrated experimentally in the marine sponge Chondrosia reniformis in which amorphous opaline spicules elicit a motile response of the exopinacocytes that coalesce to form a cellular coat on a spicule (Bavestrello et aL., 1998). The morphological and structural changes shown by the diatoms inside sponge cell vacuoles constitute the first evidence of a progressive intracellular digestion of these algae. This implies that the vacuoles are equipped with digestive enzymes able to attack the siliceous shell. Consequently, the natural strength of the shell is reduced and its fragmentation enables nutrients to pass from diatoms to the sponge cell. This introduces the possibility that sponges might utilise silica from other living organisms to build up their siliceous skeletons. In conclusion, the occurrence of diatoms inside the wandering cells seems to be exclusive to the minute sponges of E. fluviatilis. In fact, histological sections of the plugs from sponges collected in late spring and summer did not reveal the presence of endocellular diatoms. It can thus be hypothesized that diatom seasonality affects the sponge's trophic strategy. REFERENCES Bavestrello G., Arillo A., Calcinai B., Cattaneo-Vietti R., Cerrano C., Gaino E., Penna A., Sarà M., 2000 - Parasitic diatoms inside Antarctic sponges. Biol. Bull., 198: 29-33. Bavestrello G., Arillo A., Calcinai B., Cerrano C., Lanza S., Sarà M., Cattaneo-Vietti R., Gaino E., 1998 - Siliceous particles incorporation in Chondrosia reniformis (Porifera, Demospongiae). Ital. J. Zool., 65: 343-348. Bigliardi E., Sciscioli M., Lepore E., 1993 - Interactions between prokaryotic and eukaryotic cells in a sponge endocytobiosis. Endocyt. Cell Res., 9: 215-221. Cattaneo-Vietti R., Bavestrello G., Cerrano C., Gaino E., Mazzella L., Pansini M., Sarà M., 2000 - The role of sponges in the Terra Nova Bay ecosystem. In. F. M. Faranda, L. Guglielmo & A. lanosa (eds), Ross Sea ecology. Springer, Heidelberg, pp. 539549. Cerrano C., Arillo A., Bavestrello G., Calcinai B., Cattaneo-Vietti R., Penna A., Sarà M., Totti C., 2000 - Diatom invasion in the Antarctic hexactinellid sponge Scolymastra joubini. Polar Biol., 23: 441-444. Cordero G., Vaccaro P. M. R., Manconi R., Pronzato R., 1994 - Life strategies of Ephydatia fluviatilis (L. 1758) in two different environments. In: R. W. M. Van Soest, T. M. G. Van Kempen & J. Braekman (eds), Sponges in time and space. A. A. Balkema, RotterdaM., pp. 321-326. Cox G., Larkum A. W. D., 1983 - A diatom apparently living in symbiosis with a sponge. Bull. Mar. Sci., 33: 943-945. Francis J. C., Poirrier M. A., 1986 - Particle uptake in two freshwater sponge species, Ephydatia fluviatilis and Spongilla alba (Porifera: Spongillidae). Trans. Am. Microsc. SoC., 105: 11-20. Friedrich A. B., Merkert H., Fendert T., Hacker J., Proksch P., Hentschel U., 1999 - Microbial diversity in the marine sponge Aplysina cavernícola (formely Verongia cavernícola ) analyzed by fluorescence in situ hybridization (FISH). Mar. Biol., 134: 461-470. Frost T. M., 1981 - Analysis of ingested particles within a freshwater sponge. Trans. Am. Microsc. SoC., 100: 271-277.

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