Marine Biology (2005) 146: 869–875 DOI 10.1007/s00227-004-1489-1 R ES E AR C H A RT I C L E Alexander V. Ereskovsky Æ Elizaveta Gonobobleva Andrey Vishnyakov Morphological evidence for vertical transmission of symbiotic bacteria in the viviparous sponge Halisarca dujardini Johnston (Porifera, Demospongiae, Halisarcida) Received: 12 March 2004 / Accepted: 8 October 2004 / Published online: 26 November 2004
Springer-Verlag 2004 Abstract All stages of vertical transmission of symbiotic bacteria, from the penetration into oocytes to the formation of rhagon, were investigated in the White Sea (Arctic) representatives of Halisarca dujardini Johnston (Demospongiae). Small populations of free-living specific symbiotic bacteria inhabit the mesohyl of H. dujardini. They are represented by a single morphotype of small spiral gram-positive bacteria. Vertical transmission of symbiotic bacteria between generations in sponges may occur in different ways. In the case of H. dujardini the bacteria penetrate into growing oocytes by endocytosis. A part of the bacteria plays a trophic role for oocytes and the other part remains undigested in membrane-bound vacuoles within the cytoplasm. In cleaving embryos bacteria are situated between the blastomeres or in the vacuoles. In the blastula all bacteria are disposed in the blastocoel. The symbionts are situated in intercellular spaces in free-swimming larvae and during metamorphosis. Symbiotic bacteria do not play any trophic role in the period of embryonic and postembryonic development of H. dujardini. No signs of destruction and digestion of bacteria were revealed at any stage of development. Introduction One of the specific features of the sponges (Porifera) is the presence of obligate species-specific endosymbiotic photosynthesizing or nonautotrophic bacteria in their body (Sarà and Vacelet 1973; Simpson 1984; Althoff Communicated by O. Kinne, Oldendorf/Luhe A. V. Ereskovsky (&) Æ E. Gonobobleva Æ A. Vishnyakov Department of Embryology, Biological Faculty, St. Petersburg State University, Universitetskaja nab. 7/9, 199034 St. Petersburg, Russia E-mail: aereskovsky@pisem.net et al. 1998; Sarà et al. 1998; Lopes et al. 1999). Researchers enumerating and characterizing sponge– microbial interactions have shown that bacterial biomass can reach over 50% in some marine sponges, with corresponding phenomenally huge diversity of bacteria (Wilkinson 1987; Fuerst et al. 1999). The number of symbiont morphotypes in a sponge varies from one to eight (Fuerst et al. 1999; Muricy et al. 1999). An important function of symbiotic bacteria is considered to be their involvement in the physiology of sponges by recycling insoluble proteins and their participation in structural rearrangement of the sponge mesohyl (Wilkinson et al. 1979). Antimicrobial compounds have been isolated from bacteria collected from sponges, which could indicate that these bacteria may also play a role in the defense mechanism of these invertebrates (Stierle and Stierle 1992; Shigemori et al. 1992; Jayatilake et al. 1996). Symbiotic bacteria can serve as a supplementary nutritive source, the sponges either utilizing them by direct phagocytosis or using the products of their metabolism (Vacelet 1975, 1979; Vacelet et al. 1995). Marine sponges are of profound interest to chemists and biologists alike in view of their pharmaceutical potential (Garson 1994; Schmitz 1994). The bioactive chemicals may be sponge derived (Garson et al. 1998; Uriz et al. 1996), of symbiotic origin (Unson et al. 1994; Bewley et al. 1996), or isolated from culture of associated microorganisms (Shigemori et al. 1992; Kobayashi et al. 1993). How do bacteria penetrate into sponges? Two hypotheses have been proposed: (1) the capture of bacteria by the adult sponge from water by filtrating activity (Reiswig 1971; Pile et al. 1996), and (2) the transfer of symbiotic bacteria from maternal sponge to the next generation (vertical transmission) through eggs (in oviparous sponges) or through larvae (in viviparous sponges). The transfer of symbionts represents an important step of sponge biology. The possibility of vertical transmission of symbiotic bacteria in the sponges was first proposed by Lévi and 870 Porte (1962). They demonstrated the presence of bacteria inside the larvae of Oscarella lobularis (Homoscleromorpha). Lévi and Lévi (1976) first showed the vertical transmission for oviparous sponge Chondrosia reniformis. There are only two studies of viviparous sponges with evidence of vertical transmission (Kaye and Reiswig 1991; Ereskovsky and Boury-Esnault 2002). The cleavage, embryonic development, and ultrastructure of the larva of Halisarca dujardini (Demospongiae, Halisarcida) were described at the electron microscopic level (Sizova and Ereskovsky 1997; Ereskovsky and Gonobobleva 2000; Ereskovsky 2002; Gonobobleva and Ereskovsky 2004). It was shown that symbiotic bacteria were present inside the embryos and larvae. The aim of the present research was to investigate the full cycle of vertical transmission of symbiotic bacteria from the oocyte to the rhagon in viviparous species H. dujardini from the White Sea on the basis of scanning (SEM) and transmission (TEM) electron microscopy. Materials and methods Reproducing specimens of Halisarca dujardini Johnston, 1842 were collected in the Chupa inlet, 3305¢E, 6615¢N (the Kandalaksha Bay, the White Sea, the Arctic) from a depth of 1.5–5 m in June–July 1999–2003. Embryonic development of H. dujardini is synchronous both in single sponges and in local populations (Ereskovsky 2000). For electron microscopic investigations the sponges were cut into cubes of about 1 mm. The samples were prefixed in 1% OsO4 for 10 min and fixed in 2.5% glutaraldehyde in phosphate buffer at room temperature for 1 h. After fixation, sponge samples were washed in phosphate buffer and postfixed in 1% OsO4 in phosFig. 1 Symbiotic bacteria of Halisarca dujardini: a TEM micrograph of the symbiotic bacteria (B) in the mesohyl and inside a mesohylar nucleolated (N) amoebocyte. Scale bar 2 lm. b TEM micrograph of the symbiotic bacteria (B) in the mesohyl of the sponge. Scale bar 15 nm. c SEM micrograph of symbiotic bacteria (B) inside the larva. Scale bar 1 lm phate buffer for 1 h. Samples were dehydrated through a graded ethanol series and embedded in Epon-Araldite. Semi-thin sections were stained with toluidine blue. Ultrathin sections were stained with uranyl-acetate and lead citrate and observed under a JEM 100 CX TEM. For SEM the specimens were fractured in liquid nitrogen, critical-point dried, sputter coated with gold palladium, and observed under a Hitachi S 570 SEM. Results Halisarca dujardini is a widespread Atlantic boreal species, which inhabits very diverse shallow-water environments with fluctuating conditions (Ereskovsky 1993, 1994). In the White Sea H. dujardini dwells at depths of 1.5–8 m. Its size varies from 5 to 40 mm in width and from 2 to 6 mm in height. The body surface is smooth and slimy. Oscules are small, one or several per individual (Ereskovsky 1993). H. dujardini is a dioecious, viviparous sponge. Maturation and cleavage in sponges from the White Sea normally take place in late June to early July (Ereskovsky 2000). Symbiotic bacteria of H. dujardini are represented by a single morphotype both in the mesohyl of adult sponges and in all stages of embryonic development. Bacteria are almost evenly distributed in the mesohyl and do not form accumulations. Sometimes bacteria are situated in vacuoles of archaeocytes. They have a curved spiral form, characteristic of the group of spirills (Fig. 1). Their length is about 0.45 lm, their thickness 0.18 lm, and the cell wall 0.05 lm thick. The features of its composition allow one to consider them to be grampositive. No flagella or piles at the surface of the bacterial wall were revealed. The cytoplasm is heterogeneous. Its peripheral part has medium electronic density, then comes a small electron-transparent part. The cen- 871 tral part of the cell is electron dense, possibly corresponding to the nucleoid. The oocytes are scattered in the mesohyl of the choanosomal region (Fig. 2a). They are 80–90 lm in diameter. The surface of oocytes is irregular, with numerous pseudopodia. Distal parts of late oocytes contained the phagosomes and yolk granules at different stages of maturation. Before the onset of the maturation division the oocyte is oval, about 129·105 lm, without any pseudopodia. The nucleus is round, 29 lm in diameter, and located in the middle of the egg. There is a prominent nucleolus (8–12 lm in diameter). The yolk granules are distributed evenly through the bulk of the egg (Fig. 2b). During vitellogenesis of H. dujardini, symbiotic bacteria, characteristic of the mesohyl of adult sponges, are incorporated in the oocyte. The bacteria were often contained in vacuoles, their entry into the oocyte occurring by endocytosis, which takes place on the whole oocyte surface (Fig. 2c). Some phagosomes of the oocyte contained bacteria at various stages of digestion, strongly electron-opaque areas with fibrous structure suggesting residual bacterial membranes (Fig. 2d). The cleavage of H. dujardini is equal, asynchronous, and has a polyaxial pattern (Ereskovsky 2002). Cleavage results in formation of a coeloblastula (Fig. 3a, c). The few internal cells of the prelarva are derived from multipolar migration of external cells. During early embryogenesis symbiotic bacteria are present in the space between the cells of the follicular envelope and the blastomeres, between the blastomeres, and inside the blastomeres. Inside the blastomeres the bacteria are always surrounded by a vacuole (Fig. 3b). At the eight-cell stage the bacteria start to concentrate in the forming blastocoel (Fig. 3b, d). Larvae are ovoid, 125–130 lm in diameter, and completely ciliated, although the cilia are sparse on the Fig. 2 a Semi-thin micrograph of oocyte during the stage of vitellogenesis of H. dujardini. N Nucleus. Scale bar 30 lm. b Semi-thin micrograph of mature egg. N Nucleus. Scale bar 30 lm. c TEM micrograph of symbiotic bacteria (B) inside the vacuole of oocyte. Y yolk granule. Scale bar 0.5 lm. d TEM micrograph of symbiotic bacteria (B) inside the vacuole and digested bacteria (Db) inside the phagosome (Ph) of oocyte. Y Yolk granule. Scale bar 0.5 lm posterior pole (Fig. 4a). Within the population of H. dujardini under study variability of larval morphotypes was observed. Besides disphaerula, coeloblastula-like and parenchymella-like types of larvae also occur (Gonobobleva and Ereskovsky 2004). After termination of an embryogenesis the bacteria are retained in the central part of the larva and in the intercellular space of basal parts of ciliated cells until metamorphosis (Fig. 4b). Quantity of bacteria increases during development. The symbionts inside blastomeres, embryos, and larvae of H. dujardini show no morphological signs of digestion, many of them undergoing division. After attachment to the substrate, the anterior hemisphere of a larva is spread along the substratum and the posterior one keeps its shape (Fig. 4c). During metamorphosis, the regularity of larval epithelium is disturbed. Most ciliated cells sink into the postlarva formed; the rest stay on the surface, differentiating into exopinacocytes. In 3–6 h after settlement and attachment, a larva is finally transformed into a cell aggregate with a length of 230 lm. Then the cell differentiation and the formation of the aquiferous system of a rhagon occur. At the final stage of metamorphosis, an oscular tube is formed and the aquiferous system begins to function. During the metamorphosis bacteria are present in extracellular spaces in different parts of the pupa and the rhagon (Fig. 4d). The symbionts inside the rhagon of H. dujardini show no morphological signs of digestion, and many of them undergo division. Discussion Endosymbiotic associations are well documented in all adult sponges. These relationships may involve pro- 872 Fig. 3 a SEM micrograph of cleaving embryos of H. dujardini. Bl Blastomeres, F follicle. Scale bar 25 lm. b TEM micrograph of symbiotic bacteria (B) inside and between blastomeres of cleaving embryos. Bl Blastomeres, Y yolk granules. Scale bar 1 lm. c SEM micrograph of a coeloblastula of H. dujardini. Bl Blastomeres, F follicle. Scale bar 25 lm. d TEM micrograph of symbiotic bacteria (B) in the coeloblastula cavity. Bc Basal parts of blastular cells, Y yolk granules. Scale bar 5 lm Fig. 4 The larva and the metamorphic stages of H. dujardini and symbiotic bacteria inside these stages. a Semi-thin micrograph of disphaerula larva of H. dujardini. Ic Internal cavity, Pp posterior pole. Scale bar 50 lm. b TEM of symbiotic bacteria (B) inside a larva. Lc larval cells. Scale bar 2 lm. c SEM micrograph of a metamorphic larva of H. dujardini. Ex Exopinacocytes. Scale bar 50 lm. d TEM photo of symbiotic bacteria (B) inside a metamorphic larva. Ex Exopinacocytes, C cells of metamorphic larva, N nucleus. Scale bar 2 lm karyotic or eukaryotic organisms as well as photosynthetic and nonautotrophic symbionts that may be intercellular or extracellular or both (Simpson 1984; Rützler 1990). There are three broad categories of bacterial symbiosis in sponge bodies: (1) small populations of the same nonspecific bacteria as in the surrounding seawater; (2) large mesohyl populations of symbiotic bacteria, which appear to constitute a specific flora; (3) small populations of specific symbiotic intracellular bacteria (Vacelet 1975). For Halisarca dujardini a fourth broad 873 category of bacterial symbiosis can be added: small populations of specific symbiotic extracellular bacteria. Symbiotic bacteria can be associated with marine sponges in three ways: most are free living in the mesohyl; large aggregates occur in bacteriocytes; and a few are present in digestive vacuoles (Wilkinson 1978a, 1978b). In H. dujardini symbiotic bacteria are free living in the mesohyl. Vertical transmission of symbiotic bacteria between generations is an important feature of sponge biology and reproduction. This transmission can occur in different ways. The time and method of penetration also varies in bacteria from different sponge species. At present four modes of incorporation of the symbiotic bacteria from maternal mesohyl into the egg or the embryos are known (Fig. 5): 1. Oocytes gain bacteria directly from the adult mesohyl by phagocytosis (Fig. 5a). This has been shown for oviparous sponges: Verongia cavernicola (Gallissian and Vacelet 1976), Chondrilla nucula (Gaino 1980), Erylus discophorus (Sciscioli et al. 1989), Stelletta grubii (Sciscioli et al. 1991), Geodia cydonium (Sciscioli et al. 1994), Tethya tenuisclera and T. seychellensis (Gaino and Sarà 1994), Chondrilla australiensis (Usher et al. 2001). Our study of H. dujardini has demonstrated this pathway of incorporation of the symbiotic bacteria for the viviparous Demospongiae for the first time. Fig. 5 Diagram of different incorporation modes of the symbiotic bacteria from maternal mesohyl into the egg or embryos of sponges. a Phagocytosis of bacteria (B) by oocyte directly from the adult mesohyl. b Incorporation of bacteria from maternal follicle bacteriocytes (Bc) in the embryo of Chondrosia reniformis. c Transfer of bacteria (B) from parent to embryo along a mucous umbilicus (U) in some Dictyoceratida. F Follicle. d Penetration of symbiotic bacteria (B) in the space between follicle (F) and egg before closing of the follicle in Homoscleromorpha 2. The embryo of Chondrosia reniformis incorporates bacteria from maternal follicle cells (Fig. 5b; Lévi and Lévi 1976). 3. In four species of Dictyoceratida, Spongia barbara, S. cheisis, S. graminea, and Hippospongia lachne, bacteria are apparently transferred from parent to embryo along a mucous umbilicus during development (Fig. 5c; Kaye 1991; Kaye and Reiswig 1991). 4. In Homoscleromorpha penetration of symbiotic bacteria through the follicle occurs during late oogenesis in the space between follicle and egg before closing of the follicle (Fig. 5d; Ereskovsky and Boury-Esnault 2002). In an early TEM investigation of Halisarca dujardini oogenesis bacteria were not shown inside the egg (Aisenstadt and Korotkova 1976). The authors postulated that the yolk originates mainly from phagocytosis of mesohylar cells, and partly from authosynthetical processes. Nevertheless we have shown the bacteria in the eggs of these species, both undigested inside the vacuoles and digested inside the yolk granules, to be formed. It is evident that the symbiotic bacteria participate in the formation of reserve material for embryonic development in the eggs of H. dujardini. In H. dujardini the transposition of bacteria between blastomeres up to the blastocoel is possible. Transport of the vacuoles with bacteria to the blastocoel starts in the period of polarization of blastomeres and compaction of the embryo. This process may be controlled by intercellular transport. Presently, symbiotic bacteria have been found in embryos or larvae of all classes of Porifera: Demospongiae—Alectona millary, A. wallichii, and A. mesatlantica (Garrone 1974; Vacelet 1999), Hamigera hamigera (Boury-Esnault 1976), Aplysina cavernicola (Gallissian and Vacelet 1976), Hemectyon ferox (Reiswig 1976), Chondrosia reniformis (Lévi and Lévi 1976), Vaceletia crypta (Vacelet 1979), Spongia barbara, S. graminea, Hippospongia lachne (Kaye 1991; Kaye and Reiswig 1991), Haliclona tubifera (Woollacott 1993), Cladorhiza sp. (Vacelet et al. 1995, 1996), Halisarca dujardini (Sizova and Ereskovsky 1997; Ereskovsky and Gonobobleva 2000), Chondrilla australiensis (Usher et al. 2001), Swenzea zeai (Rützler et al. 2003), Ircinia oros (Ereskovsky and Tokina 2004), different species of Homoscleromorpha (Ereskovsky and Boury-Esnault 2002; Boury-Esnault et al. 2003), Calcarea—Grantia compressa (Lufty 1957; Gallissian 1983), Sycon ciliatum (Franzen 1988), Hexactinellida—Oopsacas minuta (Boury-Esnault et al. 1999). Gallissian and Vacelet (1976) excluded a trophic function of symbiotic bacteria in the egg of Verongia. Sciscioli et al. (1994) accepted that the participation of the bacteria in the formation of reserve material is minimal in the oocyte of Geodia cydonium. According to our results the symbiotic bacteria in Halisarca dujardini can participate in the formation of reserve material only during oogenesis. The function of symbiotic bacteria in 874 embryos, larvae, rhagons, and adult sponges of H. dujardini is so far unknown. Acknowledgements The authors thank Dr. Jean Vacelet and Nicole Boury-Esnault for helpful discussions, and Chantal Bézac for help during the SEM preparation. This work was funded by grant INTAS-YSC 02-4441, by the program Universities of Russia no. 07.01.017, and by grant RFBR no. 03-04-49773; A. Ereskovsky received a special grant from the Federal Scientific Policy of Belgium, intended to promote collaboration S&T with oriental and central Europe. References Aisenstadt TB, Korotkova GP (1976) A study of oogenesis in the marine sponge Halisarca dujardini II. Phagocytic activity of the oocytes and vitellogenesis. Tsitologiia 18:818–823 Althoff K, Schott C, Steffen R, Batel R (1998) Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea: harbour also for putitatively toxic bacteria? Mar Biol 130:529–536 Bewley CA, Holland ND, Faulkner DJ (1996) Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experimentia 52:716–722 Boury-Esnault N (1976) Ultrastructure de la larve parenchymella d’Hamigera hamigera (Shmidt) (Démosponge, Poecilosclerida). Origine des céllules grises. Cah Biol Mar 17:9–20 Boury-Esnault N, Efremova SM, Bezak C, Vacelet J (1999) Reproduction of a hexactinellid sponge: first description of gastrulation by cellular delamination in the Porifera. Invert Reprod Dev 35:187–201 Boury-Esnault N, Ereskovsky AV, Bézac C, Tokina DB (2003) Larval development in Homoscleromorpha (Porifera, Demospongiae): first evidence of a basement membrane in sponge larvae. Invert Biol 122:187–202 Ereskovsky AV (1993) Addition to the fauna of sponges (Porifera) of the White Sea. Vestnik St. Petersburg Univ Ser 3 2(10):3–12 Ereskovsky AV (1994) Materials to the faunistic study of the White Sea and Barents Sea sponges 2. Biogeographical and comparative faunistic analysis. Vestnik St. Petersburg Univ Ser 3 1(3):13–26 Ereskovsky AV (2000) Reproduction cycles and strategies of coldwater sponges Halisarca dujardini (Demospongiae, Dendroceratida), Myxilla incrustans and Iophon piceus (Demospongiae, Poecilosclerida) from the White Sea. Biol Bull (Woods Hole) 198:77–87 Ereskovsky AV (2002) Polyaxial cleavage in sponges (Porifera): a new pattern of metazoan cleavage. Doklady Biol Sci 386:472–474 Ereskovsky AV, Boury-Esnault N (2002) Cleavage pattern in Oscarella species (Porifera, Demospongiae, Homoscleromorpha), transmission of maternal cells and symbiotic bacteria. J Nat Hist 36:1761–1775 Ereskovsky AV, Gonobobleva EL (2000) New data on embryonic development of Halisarca dujardini Johnston, 1842 (Demospongiae: Halisarcida). Zoosystema 22:355–368 Ereskovsky AV, Tokina DB (2004) Morphology and fine structure of the swimming larvae of Ircinia oros (Porifera, Demospongiae, Dictyoceratida). Invert Reprod Dev 45:137–150 Franzen W (1988) Oogenesis and larval development of Scypha ciliata (Porifera, Calcarea). Zoomorphology 107:349–357 Fuerst JA, Webb RI, Garson MJ, Hardy L, Reiswig HM (1999) Membrane-bounded nuclear bodies in a diverse range of microbial symbionts of Great Barrier Reef sponges. Mem Queensl Mus 44:193–203 Gaino E (1980) Indagine ultrastrutturale sugli ovociti maturi di Chondrilla nucula Schmidt (Porifera, Demospongiae). Cah Biol Mar 21:11–22 Gaino E, Sarà M (1994) An ultrastructural comparative study of the eggs of two species of Tethya (Porifera, Demospongiae). Invert Reprod Dev 26:99–106 Gallissian MF (1983) Ètude ultrastructurale du developpement embryonaire chez Grantia compressa F. (Porifera, Calcarea). Arch Anat Microsc Morphol Exp 1:59–75 Gallissian MF, Vacelet J (1976) Ultrastructure de quelques stades de l’ovogénèse des spongiaires du genre Verongia (Dictyoceratida). Ann Sci Nat Zool Biol Anim 18:381–404 Garrone R (1974) Ultrastructure d’une ‘‘gemmule armée’’ planctonique d’Èponge clionidae. Arch Anat Microsc Morphol Exp 63:163–182 Garson MJ (1994) The biosynthesis of sponge secondary metabolites: why it is important. In: Soest RWM van, Kempen TMG van, Braekman JC (eds) Sponges in time and space. Balkema Press, Rotterdam, 427–440 Garson MJ, Flowers AE, Webb RI, Charan RD, McCafferty EJ (1998) A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation. Cell Tissue 293:365–373 Gonobobleva EL, Ereskovsky AV (2004) Polymorphism in freeswimming larvae of Halisarca dujardini (Demospongiae, Halisarcida). In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds) Sponge sciences in the new millennium. Boll Mus Ist Biol Univ Genova 68:349–356 Jayatilake GS, Thornton MP, Leonard AC, Grimwade JE, Baker GJ (1996) Metabolites from an Antarctic sponge associated bacterium Pseudomonas aerugenosa. J Nat Prod 59:293–296 Kaye HR (1991) Sexual reproduction in four Caribbean commercial sponges II. Oogenesis and transfer of bacterial symbionts. Invert Reprod Dev 19:13–24 Kaye HR, Reiswig HM (1991) Sexual reproduction in four Caribbean commercial sponges III. Larval behaviour, settlement and metamorphosis. Invert Reprod Dev 19:25–35 Kobayashi M, Aoki S, Kawazoe K, Kihara N, Sasaki T, Kitagawa I (1993) Altohyrtin A: a potent anti-tumor macrolide from the Okinawan marine sponge Hyrtios altum. Tetrahedron Lett 34:2795–2798 Lévi C, Lévi P (1976) Embryogénèse de Chondrosia reniformis (Nardo), demosponge ovipare, et transmission des bactéries symbiotiques. Ann Sci Nat Zool Biol Anim 18:367–380 Lévi C, Porte A (1962) Ètude au microscope électronique de l’éponge Oscarella lobularis Schmidt et de sa larve amphiblastula. Cah Biol Mar 3:307–315 Lopes JV, McCarthy PJ, Janda KE, Willoughby R, Pomponi S (1999) Molecular techniques reveal wide phyletic diversity of heterotrophic microbes associated with Discodermia spp. (Porifera, Demospongiae). Mem Queensl Mus 44:329–341 Lufty RG (1957) On the origin of the so-called mesoblast cells in the amphiblastula larvae of calcareous sponges. Cellule (Belgique) 58:231–236 Muricy G, Bezac C, Gallissian MF, Boury-Esnault N (1999) Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida). J Nat Hist 33:159–176 Pile AJ, Patterson MR, Witman JD (1996) In situ grazing on plankton <10 lm by the boreal sponge Mycale lingua. Mar Ecol Prog Ser 141:95–102 Reiswig HM (1971) Particle feeding in natural populations of three marine demosponges. Biol Bull (Woods Hole) 141:568–591 Reiswig HM (1976) Natural gamete release and oviparity in Caribbean Demospongiae. In: Harrison FM, Cowden RR (ed) Aspects of sponge biology. Academic Press, New York, pp 99– 112 Rützler K (1990) Associations between Caribbean sponges and photosynthetic organisms. In: Ruetzler K (ed) New perspectives in sponge biology. Smithsonian Institution Press, Washington, D.C., pp 455–466 Rützler K, Soest RWM van, Alvarez B (2003) Swenzea zeai, a Caribbean reef sponge with a giant larva, and Scopalina ruetzleri: a comparative fine-structural approach to classification (Demospongiae, Halichondrida, Dictyonellidae). Invert Biol 122:203–222 Sarà M, Vacelet J (1973) Écologie des Demosponges. In: Grassé PP (ed) Traité de Zoologie, vol 1. Masson, Paris, pp 462–576 875 Sarà M, Bavestrello G, Cattaneo-Vietti R, Cerrano C (1998) Endosymbiosis in sponges—relevance for epigenesis and evolution. Symbiosis 25:57–70 Schmitz FJ (1994) Cytotoxic compounds from sponges and associated microfauna. In: Soest RWM van, Kempen TMG van, Braekman JC (eds) Sponges in time and space. Balkema Press, Rotterdam, pp 485–496 Sciscioli M, Lepore E, Scalera-Liaci L, Gherardi M (1989) Indagine ultrastrutturale sugli ovociti di Erylus discophorus (Schmidt) (Porifera, Tetractinellida). Oebalia 15:939–941 Sciscioli M, Scalera-Liaci L, Lepore E, Gherardi M, Simpson TL (1991) Ultrastructural study of the mature egg of the marine sponge Stelletta grubii (Porifera Demospongiae). Mol Reprod Dev 28: 346–350 Sciscioli M, Lepore E, Gherardi M, Scalera-Liaci L (1994) Transfer of symbiotic bacteria in the mature oocyte of Geodia cydonium (Porifera, Demospongiae): an ultrastructural study. Cah Biol Mar 35:471–478 Shigemori H, Bae MA, Yazava K, Sasaki T, Kobayashi J (1992) Alteramide A, a new tetracyclic alkaloid from a bacterium Alteromonas sp. associated with the marine sponge Halichondria okadai. J Org Chem 57:4317–4320 Simpson TL (1984) The cell biology of sponges. Springer, Berlin Heidelberg New York Sizova NA, Ereskovsky AV (1997) Ultrastructural peculiarities of the early embryogenesis in a White Sea sponge Halisarca dujardini (Demospongiae, Dendroceratida). In: Ereskovsky A, Keupp H, Kohring R (eds) Modern problems of Poriferan biology. (Sonderheft der Berliner Geowissenschaftlichen Abhandlungen E 20) Freie Universitat, Berlin, pp 103– 113 Stierle DB, Stierle AA (1992) Pseudomonic acid derivatives from a marine bacterium. Experimentia 48:1165–1169 Unson MD, Holland ND, Faulkner DJ (1994) A brominated secondary metabolite synthesized by the cyanobacterial symbiont of marine sponge and accumulation of the crystalline metabolite in the sponge tissue. Mar Biol 119:1–11 Uriz MJ, Becerro MA, Tur JM, Turton X (1996) Location of toxicity within the Mediterranean sponge Crambe crambe (Demospongiae, Poecilosclerida). Mar Biol 124:583–590 Usher KM, Kuo J, Fromont J, Sutton DC (2001) Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae). Hydrobiologia 461:15–23 Vacelet J (1975) Étude en microscopie électronique de l’association entre bactéries et spongiaires du genre Verongia (Dictyoceratida). J Microsc Biol Cel 23:271–288 Vacelet J (1979) Quelques stades de la reproduction sexuée d’une éponge sphinctozoaire actuelle. In: Lévi C, Boury-Esnault N (eds) Biologie des Spongiaires. Coll Int CNRS Paris 291:95–111 Vacelet J (1999) Planktonic armoured propagules of the excavating sponge Alectona (Porifera: Demospongiae) are larvae: evidence from Alectona wallichii and A. mesatlantica sp. nov. Mem Queensl Mus 44:627–642 Vacelet J, Boury-Esnault N, Fiala-Médioni A, Fischer CR (1995) A methanotrophic carnivorous sponge. Nature 377:296 Vacelet J, Fiala-Médioni A, Fisher CR, Boury-Esnault N (1996) Symbiosis between methane-oxidising bacteria and a deep-sea carnivorous cladorhizid sponge. Mar Ecol Prog Ser 145:77–85 Wilkinson CR (1978a) Microbial association in sponges I. Ecology, physiology and microbial populations of coral reef sponges. Mar Biol 49:161–167 Wilkinson CR (1978b) Microbial association in sponges II. Numerical analysis of sponge and water bacterial populations. Mar Biol 49:169–176 Wilkinson CR (1987) Interocean differences in size and nutrition of coral reef sponge population. Science 236:1654–1657 Wilkinson C, Garrone R, Herbage D (1979) Sponge collagen degradation in vitro by sponge-specific bacteria. In: Lévi C, Boury-Esnault N (eds) Biologie des Spongiaires. Coll Int CNRS Paris 291:361–364 Woollacott RM (1993) Structure and swimming behavior of the larva of Haliclona tubifera (Porifera: Demospongiae). J Morphol 218:301–321