Spongia Officinalis Fibres (Porifera, Demospongiae)

DISEASES OF AQUATIC ORGANISMS Vol. 6: 67-74, 1989 Published February 27 Dis. aquat. Org. I

Ultrastructural evidence of bacterial damage to Spongia officinalis fibres (Porifera, Demospongiae)

Elda Gaino, Roberto Pronzato

Istituto di Zoologia, Via Balbi 5, 16126 Genova, Italy

ABSTRACT: Skeletons of healthy and damaged specimens of Spongia officinaliswere examined by scanning and transmission electron microscopy. Comparisons of the fibre net demonstrated that bacterial invasion was responsible for the decay of spongin fibres in affected sponges. Bacteria were located both inside and on the surface of fibres. Irregularly shaped spaces and canals together with many orifices were visible scattered over the fibre surfaces. Tissue reparation consisted of isolation of affected areas and permitted complete recovery and regeneration of many specimens.

INTRODUCTION 1960) as well as isolating damaged portions of the body (Connes 1967) to allow the recovery of many speci- Even though microbial diseases have been reported mens. Nevertheless, widely distributed populations in sponges of commercial interest, the etiology of these have disappeared because of devasting diseases (Storr devastating maladies is poorly understood. Fungi and 1957). bacteria are reported to cause mass ~nortalities that We found diseased Spongia officinalis along the may completely destroy wide populations (see review coast of Portofino, Italy (Eastern Ligurian Sea) and in Lauckner 1980). Studies on bacterial diseases of demonstated by scanning and transmission electron Porifera are particularly difficult because sponges har- microscopy (SEM, TEM) that the spongin fibres were bour numerous micro-organisms within the mesohyl invaded by bacteria. These micro-organisms damage (Sara & Vacelet 1973, Vacelet 1975). Microflora com- the skeleton causing it to become brittle; thereby position varies considerably within sponge species reducing the commercial value of these sponges. (Vacelet & Donadey 197?), and reflects the species specificity of microbial symbionts within the sponge body (Wilkinson 1978, Wilkinson et al. 1981). METHODS In an early paper, a mucoid coat covering the exter- nal surface of dying Hippospongia equina was reported Damaged and dying Spongja officinalis were to be related to bacterial invasion (Allemand-Martin observed from April to December 1987 within popula- 1906). We observed a white coat consisting principally tions in 4 to 18 m depth. Photographic surveys were of bacteria in Chondrilla nucula under experimental performed to monitor these organisms, and specimens conditions (Gaino & Pronzato 1987). Electron micros- collected for microscopic examination. copic examination of these diseased specimens Immediately after collection, fragments for SEM revealed that exogenous micro-organisms were able to were fixed in 2.5 % glutaraldehyde in artificial sea- penetrate into the sponge body and that some gave rise water for 2 h, washed, and dehydrated in a graded to sporogenous forms (Gaino & Pronzato 1987). Fur- ethanol series. Samples were critical-point dried in CO2 thermore, some sporogenous bacteria possessed dam- and then coated with gold-palladium using coating aged cell walls suggesting the presence of an active unit E5000 (Polaron Equipment Ltd, Watford, Herts, internal defence mechanism of the sponge. Sponges England) and observed in an IS1 DS 130 scanning elec- appear capable of inactivating pathogenic micro- tron microscope. For TEM, fragments were fixed in organisms (Nigrelli et al. 1959, Jakowska & Nigrelli 2.5 % glutaraldehyde in artificial seawater for 1 h,

O Inter-Research/Printed in F. R. Germany 68 Dis. aquat. Org. 6 67-74, 1989

rinsed, and post-fixed for l h in 1 % osmium tetroxide openings belong to the aquiferous system and assure in seawater. Fragments were dehydrated in a graded water flow in the recovering sponges. series of ethanol and embedded in an Epon-Araldite mixture. Ultrathin sections were cut on a Reichert ultramicrotome, double-stained with uranyl acetate DISCUSSION and lead citrate, and examined in a Zeiss EM 109 electron microscope. Spongia officinalis, like most species of Porifera, har- bours symbiotic bacteria in the mesohyl. Healthy specimens, however, contain no bacteria within the skeletal RESULTS framework; fibres are smooth and homogeneous. Dead specimens with exposed fibres and damaged ones, in Mortality in Spongia officiaahs populations was evi- which healthy tissue had isolated the affected network dent in specimens in which the fibre skeleton was of fibres, possessed bacteria-f1.1led fibres. This feature exposed to the environment. In some cases the whole has not been reported previously, even in other sponge body was reduced to a mesh of brittle fibres or moribund species of Porifera whose destruction was only a small portion of living tissue was retained considered in relation to pathogenic micro-organisms (Fig. 1). In others, the damage was limited to a few (Dosse 1940). In S. officinalis much of the bacterial areas (Fig. 2). surface is in close contact with the spongin matrix; this Healthy and damaged specimens contained structur- suggests an actlve lnvolvement of these microorgan- ally different fibres. The skeleton of healthy Spongia isms in its dissolution. The bacteria may release en- officinalis seen by SEM showed a framework of pri- zymes able to excavate the spongin meshwork, similar mary fibres, protruding outside to form conuli and to collagen resorption in sponges (Garrone 1975, Wil- linked together by a network of connecting fibres kinson et al. 1979). The porous internal nature of the (Fig. 3). Healthy fibres had a smooth appearance and a fibres and the canaliculi of the outermost coat could homogeneous organization, with a concentric cortex both arise from enzymic digestion by such micro- and medulla consisting of layers (Fig. 4). organisms. As a consequence the skeleton becomes Damaged specimens showed a discontinuous net- brittle and unsuitable for commercial purposes. work of broken fibres (Fig. 5). The outermost surface of Pathogenic fungi have been described as being these fibres shows laminar projections irregularly involved in the mortality of Porifera but in that case the arranged along the major axis. This gives them a damage was related almost entirely to the soft parts of squamous texture (Fig. 6). Broken fibres revealed a the sponge body (Lauckner 1980). loose core (Fig. 6). The cause of mortality in Spongia officinalis cannot At the TEM level, the fibres contained canaliculi be verified, but fibre-invading bacteria appear to be opening to the outside (Fig. 7). The core was very the most probable agents for this event. It is, however, porous wlth bacteria inside irregularly shaped spaces impossible to identify them, on a morphological basis, delimited by thin strands of the fibrillar matrix (Fig. 8). from other symblonts in the mesohyl of the sponge. It is Bacteria averaged from 0.5 to 0.8 ltm diameter, and possible that under certain physiological conditions, sometimes adhered to the fibrillar matrix with appar- sponge tissues may become unable to control prolifera- ently more than 50 % of their surface (Fig. 9). Bacteria tion of symbionts. This hypothesis is consistent with were also visible adhering to the outermost surface of data on the recently investigated mass mortalities of the fibres which showed many orifices and grooves the marine sponge Halichondria panicea, caused by 2 (Fig. 10). common symbiotic bacteria (Hummel et al. 1988). In In spring 1987, at the beginning of our observations, this speci.es bacterial growth increased significantly about 60 % of the specimens livlng in the examined with increasing temperature and decreasing water- area (6000 m') were affected. In the winter of the same flow. In S. officinalis, bacterial invasion extended from year, about 10 % of the specimens died while survivors the living sponge mass into the skeleton. The openings were able to recover by isolating damaged areas from on the fibre surface would facilitate the spreading of the living tissue. This process resulted in the formation bacteria. In contrast, degeneration of sponges of the of pocket-like cavities whose walls were delimited by genus Aplysina occurs when exogenous bacteria epithelia1 cells forming a colourless 'callus'. This was replace the associated population (Vacelet 1979). clearly evident over the pigmented surface of the Likewise, 1.n Chondrilla nucula exogenous bacteria sponge where it coexisted with the diseased areas penetrate into the sponge body and show atypical (Fig. 2). morphology (Gaino & Pronzato 1987). The surface of the cavities possessed openings of Symbiont proliferation in Spongia officinalis may be about 20 um with a central orifice (Fig. 11). These responsible for fibre destruction. It is worth stressing

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Gaino & Pronzato: Bacterial damage to Spongia offic~naljs 7;

Fig. 11. Spongia officinalis. Pocket deriving from reparative processesin a recovered specimen.Aquiferous systemopenings (op) are visible. SEM; bar = 100 Wm. Stars enc~rclethe opening enlargedin the inset (bar = 12 ~cm) that degeneration of collagen in Chondrosia reniformis press). Despite this, they may be considered very sus- is due to the specific bacteria of that sponge (Wilkinson ceptible to environmental conditions and their mortal- et al. 1979). ity may represent a large-scale epizootic event. In fact, Marine sponges are capable of distinguishing bac- dying S. officinalis specimens are not limited to the terial symbionts from other bacteria and appear to be Ligurian Sea since diseased populations have also able to control them (Wilkinson et al. 1984). They also been found along other Mediterranean coasts, such as produce lectins that promote the growth of bacteria in Tunisia, Greece, Lampedusa and Sicily. In Marsala isolated by the sponge (Miiller et al. 1981). High mor- Lagoon (northwest Sicily) a rich population of S. talities in sponges presumably relate to bacterial lnva- officinalis (Corriero 1984) has vanished in the last few sion even though many species of sponges produce years (G. Corriero pers. comm.). antimicrob~allyactive substances (Nigrelli et al. 1959, Spongia officinalis appears unable to synthesize anti- Jakowka & Nigrelli 1960).More recently, products with biotic products against bacterial invasion, but displays antibiotic properties have been localized within a defence mechanism capable of isolating the damaged spherulous cells of Aplysina fistularis (Thompson et al. area from the sponge body. The onset of this reparative 1983). These products appear to play a role in the mechanism is characterized by the formation of colour- protective system to reduce surface fouling. Biologi- less 'calluses' containing the openings of the inhalant cally active metabolites are released by this species canal systems. Tissue reorganization excludes the into seawater and are believed to reduce tissue dam- damaged areas and allows the water currents to flow age resulting from larval settlement or overgrowth by again within the sponge. This agrees well with the other organisms (Thompson 1985, Walker et al. 1985). autodigestion of fibres recently observed in S. Despite the absence of spherulous cells, Spongia officinalis as a part of the skeletal spongin remodelling officinalis displays an efficient self-defence mechanism that plays an important role in modifying the canal against epibionts (M. Pansini pers, comm.). Horny system and in tissue organization (Vacelet et al. 1988). sponges in the Ligurian Sea reach very large dlmen- No bacteria were observed around or within the fibre sions and become very old (Pansini & Pronzato in network of recovered areas, thus indicating a mechan- 74 Dis. aquat. Org. 6: 67-74, 1989

ism for separating damaged tissues before bacterial Uhlenbruck, G (1981) Lectln. a posstble basis for sym- invasion can spread to the whole living mass. This biosis between bacteria and sponges. J. Bacteriol. 145: represents a successful host response adaptative 548-558 N~grelli,R F., Jakowska, S., Calventi, J. (1959). Ectyonin, an strategy. The isolation of portions of affected sponge antlmicrob1.al agent from the sponge M~crocionaprol~fera tissues is also known in Tethya lyncurium(Connes Verr~el.Zoologica N. Y. 44: 273-176 1967) and in horny sponges associated with pathogenic Pansini, M., Pronzato, R. (in press). Observations on the fungi (Smith 1941). This defence mechanism allows the dynamics of a Mediterranean sponge community. In: Riitz- ler, K., Hartman, W. H. (eds ) New perspectives in sponge complete recovery of many affected Spongia officinalis. biology. Smithsonian Inst. Press, Washington D.C. Even though mortalities did occur, many other speci- Sara, M., Vacelet, J. (1973). Ecologie des demosponges. In: mens were able to eliminate diseased areas and sur- Grasse, P. P. (ed.) Traite de Zoologie, Vol. 111. Spongiaires. vive. Mason, Paris, p. 462-576 Smith, F G. W (1941). Sponge disease in British Honduras, and its transmission by water currents. Ecology 22: Acknowledgements. We express our sincere gratitude to Dr C. 415421 Wilkinson of the Australian Institute of Marine Science, Storr. J. F. (1957). The sponge industry of Florida. Educ. Ser. Townsville, for his critical review of the paper and his valuable Fla St. Bd Conserv. 9: 28 suggestions We are also very grateful to Sal.r.a:a:r >!arccddu Thompson, J. E. (1985). Exudation of biologically-active for his kind technical assistance. metabolites in the sponge Aplysina fistularis. I. Biological evidence. Mar. Biol. 88: 23-26 Thompson, J. E., Barrow, K. D., Faulkner, D. J. (1983). Locali- LITERATURE CITED zation of two brominated metabolites, Aerothionin and Homoaerothionin, in spherulous cells of the marine sponge Nlemand-Martin, A. (1906). Etude de physiologie appliquee Aplysina fistulans (= Verongia thlona). Acta Zool. sur la spongiculture sur les cBtes de Tunisie. These, Univ. (Stockh.) 64: 199-210 Lyon Vacelet, J. (1975). Etude en rnicroscopie electronique de I'as- Connes, R. (1967). Reaction de defense de I'eponge Tethya sociation entre bacteries et spongiaires du genre Verongia lyncurium vis-a-vls des micro-organismes et de I'am- (Dyctyoceratida) J. Microscopie Biol. Cell. 23: 271-288 phipode Leucothoe spinicarpa Abildg. Vie Milieu (Ser A) Vacelet, J. (19791. La place de spongiaires dans les ecosys- 18: 281-289 temes trophiques marins. In: Levi, C., Boury-Esnault, N. Corriero, G. (1984). Note sul popolamento di Poriferi del10 (eds.) Biologie des spongiaires, C.N.R.S. Paris, p. 259-270 stagnone di Marsala (Sicha).Nova Thalassia 6: 213-223 Vacelet, J., Donadey, C. (1977). Electron microscope study of Dosse, G. (1940). Bakterien- und Pilzbefunde sowie the association between some sponges and bacteria. J. pathologische und Faulnisvorgange in Meeres- und SuO- exp. mar. Biol. Ecol. 30: 301-314 wasserschwammen. Untersuchungen im Zusammenhang Vacelet, J., Verdenal, B., Perinet, G. (1988). The iron minerali- mit dem gegenwartigen Sterben der Badeschwamme in zation of Spongia officinalis L. (Porifera, Dictyoceratida) Westindien. 2. Parasitkde 11: 331-356 and its relationships with the collagen skeleton. Biol. Cell Gaino, E., Pronzato, R. (1987). Ultrastructural observations of 62: 189-198 the reaction of Chondnlla nucula (Porifera, Demospon- Walker, R. P., Thompson, J. E., Faulkner, D. J. (1985). Exuda- giae) to bacterial invasion during degenerative processes. tion of biologically-active metabolites in the sponge Aply- Cah. Biol. mar 28: 33-46 sina fistulans 11. Chemical evidence. Mar. Biol. 88: 27-32 Garrone, R. (1975). Collagen resorption in sponges: involve- Wilkinson, C. R (1978). Microbial association in sponges. 11. ment of bacteria and macrophages. In: Peeters. H. (ed.) Numerical analys1.s of sponge and water bacterial popula- 22nd Colloquium Protides of the Biological Fluid. Perga- tions. Mar. Biol. 49: 169-136 mon Press, Oxford. p. 59-63 Wilkinson, C. R., Garrone, R., Herbage, D. (1979). Sponge Hummel, H., Sepers. A. B. J., de Wolf, L., Melissen, F. W. collagen degradation in vitro by sponge-specific bacteria. (1988). Bacterial growth on the marine sponge Halichon- In Levi, C., Boury-Esnault, N. (eds.) Blologie des spon- dria panicea induced by reduced waterflow rate. Mar. glalres. C.N.R.S. Paris, p. 361-364 Ecol. Prog. Ser. 42: 195-198 Wilkinson, C. R.. Garrone, R., Vacelet, J. (1984). Marine Jakowska, S., Nigrelli. R. F. (1960). Antimicrobial substances sponges discriminate between food bacteria and bacterial from sponges. Ann. N. Y Acad. Sci. 90: 913-916 symbionts: electron microscope radioautography and in Lauckner, G. (1980). Diseases of Porifera. In IOnne, 0. (ed ) situ evidence Proc. R. Soc (Ser. B) 220. 519-528 Diseases of marine animals, Vol. I, General aspects, Pro- Wilkinson, C. R., Novak, M., Austin, B., Colwell, R. R. (1981). tozoa to Gastropoda. Wiley & Sons. Chichester. p. 139-165 Specificity of bacterial symbionts in Mediterranean and Miiller, W. E. G., Zahn. R. K., Kurelec, B., Lucu, C., Muller. I., Great Barrier Reef sponges. Microb. Ecol. 7: 13-21

Responsible Subject Editor Dr A. K. Sparks; accepted for printing on Novcmber 15, 1988