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Bacteria associated with the sponge Spongia officinalis as indicators of contamination
Ecological Indicators 2 (2003) 339–343
Bacteria associated with the sponge Spongia officinalis as indicators of contamination Efthimios Kefalas a , Joanna Castritsi-Catharios a,∗ , Helen Miliou b a
Department of Biology, Sector of Zoology and Marine Biology, National and Kapodistrian University of Athens, University Campus, Athens 157 84, Greece b Laboratory of Applied Hydrobiology, Faculty of Animal Production, Agricultural University of Athens, Lera Odos 75, Athens 11855, Greece To the honour of Asst. Prof. S. Marakis
Abstract A lot of micro-organisms can been found in the sponge body. In the present study, bacteria and fungi were isolated from the massive commercial sponge Spongia officinalis Linnaeus, 1759 (Porifera, Demospongiae). Samples (sponge extract and sea water) were collected from several areas of the Aegean Sea (ports, beaches, eutrophic gulf and open sea) with different levels and types of contamination. Bacteria identified in sponge extract belong to the genera Escherichia, Morganella, Proteus, Pasteurella, Aeromonas, Pseudomonas and Acinetobacter. Two fungal genera, Trichosporum and Fisarium, were identified in sponge extract. Bacterial load was greater in sponge extract than in proximal sea water, reflecting the sponge ability to concentrate bacteria in its body. The assessment of bacteria associated with the sponge S. officinalis as indicators of contamination is proposed. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Bacteria; Fungi; Sponges; Contamination; Indicator; Aegean Sea
1. Introduction Bacteria and fungi can be used as biological indicators of broad ecological significance (Azam et al., 1983). They are usually present in waters in direct proportion to the amount of organic matter available and are numerous in harbours, near the coasts and in areas of high productivity (Hawker and Linton, 1979). Bacteria in sea water have been used as biological indicators of contamination (Papapetropoulou and Rodopoulou, 1994; Baudisova et al., 1997; Nocciolini et al., 2000; Skanavis and Yanko, 2001).
∗ Corresponding author. Tel.: +30-210-727-4622; fax: +30-210-727-4604. E-mail address: [email protected] (J. Castritsi-Catharios).
The survival of bacteria in a coastal marine environment, and consequently the validity of using them as bio-indicators of contamination, depends on solar radiation exposure and other oceanographic factors (Jeffrey et al., 1996; Reed, 1997). So, if we could choose populations of micro-organisms that are not affected by those factors, the results should be more reliable. These populations could be found in well-protected areas, such those in the body cavities of immobile, filter-feeding benthic organisms. The assessment of shellfish-associated bacteria has been suggested for the evaluation of short temporal and spatial variations of the bacteriological quality of coastal water (Grouhel et al., 1995). Dale and Beyeler (2001) claimed that there is a challenge to derive a manageable set of indicators for considering the full complexity of the ecological system.
1470-160X/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1470-160X(03)00002-5
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E. Kefalas et al. / Ecological Indicators 2 (2003) 339–343
Sponges are dominant filter-feeders in marine ecosystems, obtaining their nutrients from particles, which are carried as drift from sea currents or as suspended components. Bacteria alone can constitute a major food source for sponges and could potentially fulfil their total dietary requirements, especially in coastal waters with high organic content (Reiswig, 1971; Wilkinson, 1978a,b; Hummel et al., 1988; Becerro et al., 1994). Sponge life involves two categories of microorganisms. The first is represented by those microorganisms that come directly from the ambient sea water, remaining in the sponge body structures for a short time after their capture by specific tissues (pinacoderm, choanocyte chambers) (Reiswig, 1971, 1974; Simpson, 1984; Wilkinson et al., 1984; Koukouras et al., 1996). The second is represented by symbiotic micro-organisms found in the tissues of sponges (the mesohyl, where it represents up to 40% of living tissue), between the cells (extracellular) and in the cells, frequently in vacuoles (intracellular) (Vacelet and Donadey, 1977; Southward, 1987; Gaino and Pronzato, 1989; Colwell, 1990; Vacelet et al., 1994). In sponge extract, a clear distinction between these two categories of micro-organisms is not practicable. However, the isolates obtained could be considered, for the major part of them, as micro-organisms with provenience from the aquiferous system of sponges (Müller et al., 1981; Southward, 1987). The objective of the present study was the identification and quantitative determination of microorganisms (bacteria and fungi) isolated from the body of the commercially important sponge Spongia officinalis Linnaeus, 1759 (Porifera, Demospongiae) sampled from areas of Aegean Sea with different degree and type of contamination. The assessment of these micro-organisms as indicators of contamination was evaluated. The selection of S. officinalis was based on the presence of this sponge in almost all coastal ecosystems of Aegean Sea (Castritsi-Catharios, 1998).
2. Materials and methods During summer, S. officinalis samples were obtained from six stations in Aegean Sea (A.S.), by diving to depths of 2–30 m. The sampling stations were: (a) Agathonisi: coast of the small island Agathonisi
in eastern A.S., which can be considered as a control site (no sewage discharges, no tourism); (b) Sitia 1: beach of the small city Sitia in Crete, an island in southern A.S.; (c) Sitia 2: port (pier) of the adjacent city; (d) Kalloni: gulf, eutrophic and closed, of the island Lesvos in north-eastern A.S.; (e) Kalafatis: beach of Myconos, a tourist island in central A.S.; and (f) Kimi: port of the big city Kimi in Evia, an island in north-western A.S. At every station, six samples were taken from various sponges, each approximately 30 cm3 in volume. The pieces of sponges were immediately stored aseptically in sterilised vials. In laboratory, the sponge extract was obtained by squeezing gently with a glass stick the sponge piece. A portion (1 ml) of each sponge extract was subjected to a series of dilutions (10−1 , 10−2 , 10−3 ). Five replicates were taken for each sponge extract. A quantity (1 ml) of the dilutions was mixed with 15 ml of culture medium into petri dishes, which were incubated at 30 ◦ C. Three replicates were prepared for each dilution. Seawater samples were collected simultaneously to the sponge pieces (at a distance of up to 10–20 cm) and stored in 500 ml sterilised vials. Four culture media, Nutrient agar, MacConkey agar (Oxoid), Malt Extract agar and Czapek-Dox agar, were used for bacteria and fungi isolation from sponge extract and sea water according to standard microbiological plating techniques (Norris and Ribbons, 1970; Robina, 1972; Jones, 1974; APHA, 1989; Collins et al., 1989). Classification tables were used for the identification of bacteria (Buchanan and Gibons, 1974; APHA, 1989; Collins et al., 1989) and fungi (Ainsworh et al., 1973; Jones, 1974). For the complete identification of the isolated enterobacteria the API 20E was used.
3. Results Tables 1 and 2 present the mean values and standard deviation (S.D.) of Colony Forming Unit (CFU)/ml, counted on different culture media for bacteria and fungi isolated from sponge extract and sea water collected at six stations. Bacteriological media (Table 1), Nutrient agar and MacConkey agar, showed a high microbial load, especially the former. Moreover, the number of bacterial colonies
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Table 1 Mean values (S.D.) of CFU/ml enumerated on two culture media for the bacteria isolated from sponge extract and sea water Sampling station
Nutrient agar
Agathonisi Sitia 1 Sitia 2 Kalloni Kalafatis Kimi
MacConkey agar
Sponge extract
Sea water
Sponge extract
Sea water
3 2100 2400 66000 130000 400000
1 176 16 75 720 2200
3 860 25 72 7400 9500
0 80 10 12 560 1800
(3.0) a (953.9) b (400.0) b (12165.5) b (21633.3) b (91651.5) b
(0.5) a (43.1) a (11.3) a (9.9) a (268.7) a (282.8) a
(2.0) a (255.3) b (7.5) b (11.1) b (2029.7) b (793.7) b
a (22.6) a (4.2) a (1.4) a (42.4) a (339.4) a
Mean values for the same station and agar having different letters are significantly different (P < 0.05); N = 6. Table 2 Mean values (S.D.) of CFU/ml enumerated on two culture media for the fungi isolated from sponge extract and sea water Sampling station
Malt Extract agar
Agathonisi Sitia 1 Sitia 2 Kalloni Kalafatis Kimi
Czapek-Dox agar
Sponge extract
Sea water
Sponge extract
Sea water
0 1 5 0 1700 0
0 1 10 15 12 78
17 1 10 0 60 0
0 2 43 57 14 96
(1.7) a (2.0) a a (269.1) b a
(1.4) a (0.9) b (5.7) b (5.7) a (11.3) b
(4.3) b (1.0) a (10.0) a a (17.7) b a
a (2.8) a (4.2) b (7.1) b (2.8) a (11.3) b
Mean values for the same station and agar having different letters are significantly different (P < 0.05); N = 6.
Table 3 Bacterial and fungal species isolated from the sponge S. officinalis Sampling station Bacteria Sitia 1
E. coli
Sitia 2
E. coli Aeromonas salmonicida
Kalloni
Pseudomonas aeruginosa E. coli Proteus vulgaris Morganella morganii
Kalafatis
E. coli M. morganii P. vulgaris Proteus mirabilis Pasteurella spp.
Kimi
E. coli A. salmonicida Acinetobacter spp. Pseudomonas spp. Pseudomonas pancimobilis
Fungi
Fisarium spp.
isolated from sponge extract was significantly higher than that from the corresponding proximal sea water, enumerated on the same agar. Fungal media (Table 2), Malt Extract agar and Czapek-Dox agar, presented a relatively low microbial load. In addition, in three stations the number of fungal colonies isolated from sea water was significantly higher than that from sponge extract, counted on both fungal media. Table 3 lists the species of bacteria and fungi, which were identified in sponge extract from five stations.
4. Discussion Trichosporum spp.
Present results indicated that the bacterial load of sponge extract was greater than that of the proximal sea water. A possible explanation could be given is that sponge has the ability to concentrate bacteria in its body. On the contrary, the fungal burden counted in the present study was negligible in most of the samples, especially in sponge extract. It seems that fungi occur in sponges at very low frequency, failing to indicate the contamination degree in different areas.
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This is probably due to an antifungal activity of sponges (Amade et al., 1987; Pettit, 1999), or/and of sponge-associated bacteria (Ivanova et al., 2000). Possible adverse physicochemical conditions in the environment of sponge structures may also influence the fungal populations. Many micro-organisms that are typically found in the intestinal canal of human or terrestrial animals survive for considerable periods of time in sea water (Hawker and Linton, 1979). Sponge-associated bacterial flora from Kalafatis station belong to Enterobacteriaceae, with the exception of Pasteurella. Enterobacteriaceae are small gram-negative rods, among which some are pathogenic, such as Proteus. The aerobic or facultative anaerobic thermotolerant bacterium Escherichia coli, typical representative of the coliform group, appeared in all samplings. Sponge-associated bacterial species isolated from Kimi station (except E. coli) do not belong to Enterobacteriaceae and constitute aerobic gram-negative rods. The motile Aeromonas and Pseudomonas are typically waterborne bacterial genera and include opportunistic pathogenic species to crustaceans, fish and other aquatic organisms, as well as to humans (Aguado and Bashirullah, 1996; Bahlaoui et al., 1997; Pettibone, 1998; Kong et al., 1999; Starliper and Morrison, 2000). Thus, the sponge body represent an area of emerging heterotrophic gram-negative bacteria, enteric and non-enteric. Enterobacteriaceae, and especially E. coli, are considered as indicators of faecal contamination (Baudisova et al., 1997). The abundance of Aeromonas, Pseudomonas and Acinetobacter may be indicative of organic contamination and eutrophication, as a result of an anthropogenic impact on aquatic ecosystems (Rippey and Cabelli, 1989; Cenci et al., 1998; Lemke and Leff, 1999). Thingstad et al. (1999) observed in experimental mixed marine microbial communities that phosphate enrichment had a stimulatory effect on bacterial production, resulting from transfer of added phosphorus into ciliate biomass, while in turn bacterial production depended upon the fulfilment of carbon demand. In the present study, the differentiation among stations in the presence of the bacterial groups identified, may be explained by the kind of contamination that the sampling areas were subjected; e.g. Enterobacteriaceae/Pasteurella in the tourist site of Kalafatis (mainly faecal contamination)
and E. coli/Aeromonas, Pseudomonas, Acinetobacter in the port of Kimi with increased sewage discharges (faecal and organic contamination). The seawater bacterial load enumerated on Nutrient agar was 6 times greater than that on MacConkey agar for the Kalloni station and about 1.5 times for the other stations. Concerning sponge extract, the difference in bacterial colonies between the two culture media became greater, particularly for the samples taken from the eutrophicated gulf of Kalloni and the two ports, Sitia and Kimi. These stations were characterised by the presence of Aeromonas or/and Pseudomonas. It seems that Nutrient agar is more sensitive than MacConkey in isolating these bacterial genera. In addition, proliferation of the non-enteric bacteria in sponge structures is greater than that of the enteric ones. This permits a better discrimination of sites, reflecting the impact of both anthropogenic disturbances, faecal and organic. It is concluded that bacteria, but not fungi, benefited the environment of the body of S. officinalis. It is proposed that sponge-associated bacteria can be used as indicators of contamination in marine ecosystems. Acknowledgements This study was a part of the research program “Sponge Species in the Mediterranean: Contribution to the Sponge Fisheries”, which was financially supported by EEC D.G.-XIV-Fisheries (CL/MED/92/024). References Aguado, N., Bashirullah, A.K.M., 1996. Shell diseases in wild penaeid shrimps in eastern region of Venezuella. J. Aquaricult. Aquat. Sci. 8, 1–6. Ainsworh, G.C., Sparrow, F.K., Susman, A.S., 1973. The Fungi, vol. IVA. Academic Press, New York. Amade, P., Charroin, C., Baby, C., Vacelet, J., 1987. Antimicrobial activities of marine sponges from the Mediterranean Sea. Mar. Biol. 94, 271–275. APHA, 1989. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Azam, F., Fenchel, T., Field, J.C., Grey, J.S., Meyer-Reil, L.A., Thingstad, F., 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257–263. Bahlaoui, M.A., Baleux, B., Troussellier, M., 1997. Dynamics of pollution-indicator and pathogenic bacteria in high-rate oxidation wastewater treatment ponds. Water Res. 31, 630–638.
E. Kefalas et al. / Ecological Indicators 2 (2003) 339–343 Baudisova, D., Morris, R., Grabow, W.O.K., Jofre, J., 1997. Evaluation of Escherichia coli as the main indicator of faecal pollution. Water Sci. Technol. 35, 333–336. Becerro, M.A., Lopez, N.I., Turon, X., Uriz, M.J., 1994. Antimicrobial activity and surface bacterial film in marine sponges. J. Exp. Mar. Biol. Ecol. 179, 195–205. Buchanan, R.E., Gibons, N.E., 1974. Bergey’s Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore. Castritsi-Catharios, J., 1998. Kalymnos and the Secrets of the Sea: Contribution to the Sponge Fisheries, Athens. Cenci, P., Vitaioli, M., Prati, L., 1998. Association between Aeromonas and Pseudomonas genera contaminating surface waters evaluated by means of computing and simulating algorithms. Ig. Mod. 109, 13–27. Collins, C.H., Lyne, P.M., Grange, J.M., 1989. Microbiological Methods. Butterworths, London. Colwell, R.R., 1990. Comparison of bacterial communities associated with the Caribbean sclerosponge Ceratoporella nicholsoni and ambient sea water. Mar. Ecol. Prog. Ser. 67, 73–82. Dale, V.H., Beyeler, S.C., 2001. Challenges in the development and use of ecological indicators. Ecol. Indicators 1, 3–10. Gaino, E., Pronzato, R., 1989. Ultrastractural evidence of bacterial damage to Spongia officinalis fibers (Porifera, Demospongiae). Dis. Aquat. Org. 6, 67–74. Grouhel, A., Treguier, C., Marco, F., 1995. Coastal bacteriological quality assessment by rapid faecal coliforms count in shellfish— use of conductimetric method. Tech. Sci. Methodes Genie Urbain Genie Rural 6, 471–476. Hawker, L.E., Linton, A.H., 1979. Micro-Organisms Function, Form and Environment. Edward Arnold Ltd. Hummel, H., Sepers, A.B.J., de Wolf, L., Melissen, F.W., 1988. Bacterial growth on the marine sponge Halichondria panicea induced by reduced waterflow rate. Mar. Ecol. Prog. Ser. 42, 195–198. Ivanova, E.P., Kuznetsova, T.A., Mikhailov, V.V., 2000. Antifungal compounds produced by bacterial populations of the sea sponge Verongia sp. Russ. J. Mar. Biol. 26, 118–119. Jeffrey, W.H., Pledger, R.J., Aas, P., Hager, S., Coffin, R.B., Von-Haven, R., Mitchell, D.L., 1996. Diel and depth profiles of DNA photodamage in bacterioplankton exposed to ambient solar ultraviolet radiation. Mar. Ecol. Prog. Ser. 137, 283–291. Jones, G.E.B., 1974. Methods in Microbiology. Academic Press, London. Kong, R.Y.C., Pelling, A., So, C.L., Wu, R.S.S., 1999. Identification of oligonucleotide primers targeted at the 16s–23s rDNA intergenic spacers for genus- and species-specific detection of Aeromonads. Mar. Pollut. Bull. 38, 802–808. Koukouras, A., Russo, A., Voultsiadou-Koukoura, E., Arvanitidis, C., Stefanidou, D., 1996. Macrofauna associated with sponge species of different morphology. Mar. Ecol. 17, 569–582. Lemke, M.J., Leff, L.G., 1999. Bacterial populations in an anthropogenically disturbed stream: comparison of different seasons. Microb. Ecol. 38, 234–243. Müller, W.E., Zahn, R.K., Kurelec, B., Lucu, C., Müller, I., Unlenbruck, G., 1981. Lectin, a possible basis for symbiosis between bacteria and sponges. J. Bacteriol. 145, 548–557.
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Nocciolini, S., Spadafina, L., Varci, M.R., Bacci, E., 2000. A simple bacterial index for relative water quality: preliminary application in the Orbetello lagoon (Tuscany, Italy). Chemosphere 41, 1065–1069. Norris, J.R., Ribbons, D.W., 1970. Methods in Microbiology. Academic Press, London. Papapetropoulou, M., Rodopoulou, G., 1994. Occurrence of enteric and non-enteric indicators in coastal waters in southern Greece. Bull. Mar. Sci. 54, 63–70. Pettibone, G.W., 1998. Population dynamics of Aeromonas spp. in an urban river watershed. J. Appl. Microbiol. 85, 723–730. Pettit, G.R., 1999. Antifungal activity of the spongistatins. US Patent 5,883,120. Reed, R.H., 1997. Solar inactivation of faecal bacteria in water: the critical role of oxygen. J. Appl. Microbiol. 24, 276–280. Reiswig, H.M., 1971. Particle feeding in natural populations of three marine Demosponges. Biol. Bull. 141, 568–591. Reiswig, H.M., 1974. Bacteria as food for temperate water sponges. Can. J. Zool. 53, 582–589. Rippey, S.R., Cabelli, V.J., 1989. Use of thermotolerant Aeromonas group for the trophic state classification of freshwaters. Water Res. 23, 1107–1114. Robina, A.G., 1972. Methods in Aquatic Microbiology. Butterworths, Baltimore. Simpson, L.T., 1984. The Cell Biology of Sponges. Springer, New York. Skanavis, K., Yanko, W.A., 2001. Clostridium perfigens as a potential indicator for the presence of sewage solids in marine sediments. Mar. Pollut. Bull. 42, 31–35. Southward, E.C., 1987. Contribution of symbiotic chemoautotrophs to the nutrition of benthic invertebrates. In: Sleigh, M.A. (Ed.), Microbes in the Sea. pp. 83–118. Starliper, C.E., Morrison, P., 2000. Bacterial pathogen contagion studies among freshwater bivalves and salmonid fishes. J. Shellfish Res. 19, 251–258. Thingstad, T.F., Perez, M., Pelegri, S., Dolan, J., Rassoulzadegan, F., 1999. Trophic control of bacterial growth in microcosms containing a natural community from northwest Mediterranean surface waters. Aquat. Microb. Ecol. 18, 145–156. Vacelet, J., Donadey, C., 1977. Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Biol. Ecol. 30, 301–314. Vacelet, J., Gaino, E., Gallissian, M.F., Vacelet, E., 1994. Bacterial attack of spongin skeleton during the 1986–1990 Mediterranean sponge disease. In: Van-Soest, R.W.M., Van-Kempen, T.M.G., Braekman, J.C., (Eds.), Sponges in Time and Space: Biology, Chemistry, Paleontology. Balkema, Rotterdam, pp. 355–362. Wilkinson, C.R., 1978a. Microbial associations in sponges. I. Ecology, physiology and microbial populations of coral reef sponge. Mar. Biol. 49, 161–167. Wilkinson, C.R., 1978b. Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations. Mar. Biol. 49, 169–176. Wilkinson, C.R., Garrone, R., Vacelet, J., 1984. Marine sponges discriminate between food bacteria and bacterial symbiots: Electron microscope radioautografy and in situ evidence. Proc. R. Soc. Lond. Ser. B: Biol. Sci. 220, 519–528.
Bacteria associated with the sponge Spongia officinalis as indicators of contamination Efthimios Kefalas a , Joanna Castritsi-Catharios a,∗ , Helen Miliou b a
Department of Biology, Sector of Zoology and Marine Biology, National and Kapodistrian University of Athens, University Campus, Athens 157 84, Greece b Laboratory of Applied Hydrobiology, Faculty of Animal Production, Agricultural University of Athens, Lera Odos 75, Athens 11855, Greece To the honour of Asst. Prof. S. Marakis
Abstract A lot of micro-organisms can been found in the sponge body. In the present study, bacteria and fungi were isolated from the massive commercial sponge Spongia officinalis Linnaeus, 1759 (Porifera, Demospongiae). Samples (sponge extract and sea water) were collected from several areas of the Aegean Sea (ports, beaches, eutrophic gulf and open sea) with different levels and types of contamination. Bacteria identified in sponge extract belong to the genera Escherichia, Morganella, Proteus, Pasteurella, Aeromonas, Pseudomonas and Acinetobacter. Two fungal genera, Trichosporum and Fisarium, were identified in sponge extract. Bacterial load was greater in sponge extract than in proximal sea water, reflecting the sponge ability to concentrate bacteria in its body. The assessment of bacteria associated with the sponge S. officinalis as indicators of contamination is proposed. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Bacteria; Fungi; Sponges; Contamination; Indicator; Aegean Sea
1. Introduction Bacteria and fungi can be used as biological indicators of broad ecological significance (Azam et al., 1983). They are usually present in waters in direct proportion to the amount of organic matter available and are numerous in harbours, near the coasts and in areas of high productivity (Hawker and Linton, 1979). Bacteria in sea water have been used as biological indicators of contamination (Papapetropoulou and Rodopoulou, 1994; Baudisova et al., 1997; Nocciolini et al., 2000; Skanavis and Yanko, 2001).
∗ Corresponding author. Tel.: +30-210-727-4622; fax: +30-210-727-4604. E-mail address: [email protected] (J. Castritsi-Catharios).
The survival of bacteria in a coastal marine environment, and consequently the validity of using them as bio-indicators of contamination, depends on solar radiation exposure and other oceanographic factors (Jeffrey et al., 1996; Reed, 1997). So, if we could choose populations of micro-organisms that are not affected by those factors, the results should be more reliable. These populations could be found in well-protected areas, such those in the body cavities of immobile, filter-feeding benthic organisms. The assessment of shellfish-associated bacteria has been suggested for the evaluation of short temporal and spatial variations of the bacteriological quality of coastal water (Grouhel et al., 1995). Dale and Beyeler (2001) claimed that there is a challenge to derive a manageable set of indicators for considering the full complexity of the ecological system.
1470-160X/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1470-160X(03)00002-5
340
E. Kefalas et al. / Ecological Indicators 2 (2003) 339–343
Sponges are dominant filter-feeders in marine ecosystems, obtaining their nutrients from particles, which are carried as drift from sea currents or as suspended components. Bacteria alone can constitute a major food source for sponges and could potentially fulfil their total dietary requirements, especially in coastal waters with high organic content (Reiswig, 1971; Wilkinson, 1978a,b; Hummel et al., 1988; Becerro et al., 1994). Sponge life involves two categories of microorganisms. The first is represented by those microorganisms that come directly from the ambient sea water, remaining in the sponge body structures for a short time after their capture by specific tissues (pinacoderm, choanocyte chambers) (Reiswig, 1971, 1974; Simpson, 1984; Wilkinson et al., 1984; Koukouras et al., 1996). The second is represented by symbiotic micro-organisms found in the tissues of sponges (the mesohyl, where it represents up to 40% of living tissue), between the cells (extracellular) and in the cells, frequently in vacuoles (intracellular) (Vacelet and Donadey, 1977; Southward, 1987; Gaino and Pronzato, 1989; Colwell, 1990; Vacelet et al., 1994). In sponge extract, a clear distinction between these two categories of micro-organisms is not practicable. However, the isolates obtained could be considered, for the major part of them, as micro-organisms with provenience from the aquiferous system of sponges (Müller et al., 1981; Southward, 1987). The objective of the present study was the identification and quantitative determination of microorganisms (bacteria and fungi) isolated from the body of the commercially important sponge Spongia officinalis Linnaeus, 1759 (Porifera, Demospongiae) sampled from areas of Aegean Sea with different degree and type of contamination. The assessment of these micro-organisms as indicators of contamination was evaluated. The selection of S. officinalis was based on the presence of this sponge in almost all coastal ecosystems of Aegean Sea (Castritsi-Catharios, 1998).
2. Materials and methods During summer, S. officinalis samples were obtained from six stations in Aegean Sea (A.S.), by diving to depths of 2–30 m. The sampling stations were: (a) Agathonisi: coast of the small island Agathonisi
in eastern A.S., which can be considered as a control site (no sewage discharges, no tourism); (b) Sitia 1: beach of the small city Sitia in Crete, an island in southern A.S.; (c) Sitia 2: port (pier) of the adjacent city; (d) Kalloni: gulf, eutrophic and closed, of the island Lesvos in north-eastern A.S.; (e) Kalafatis: beach of Myconos, a tourist island in central A.S.; and (f) Kimi: port of the big city Kimi in Evia, an island in north-western A.S. At every station, six samples were taken from various sponges, each approximately 30 cm3 in volume. The pieces of sponges were immediately stored aseptically in sterilised vials. In laboratory, the sponge extract was obtained by squeezing gently with a glass stick the sponge piece. A portion (1 ml) of each sponge extract was subjected to a series of dilutions (10−1 , 10−2 , 10−3 ). Five replicates were taken for each sponge extract. A quantity (1 ml) of the dilutions was mixed with 15 ml of culture medium into petri dishes, which were incubated at 30 ◦ C. Three replicates were prepared for each dilution. Seawater samples were collected simultaneously to the sponge pieces (at a distance of up to 10–20 cm) and stored in 500 ml sterilised vials. Four culture media, Nutrient agar, MacConkey agar (Oxoid), Malt Extract agar and Czapek-Dox agar, were used for bacteria and fungi isolation from sponge extract and sea water according to standard microbiological plating techniques (Norris and Ribbons, 1970; Robina, 1972; Jones, 1974; APHA, 1989; Collins et al., 1989). Classification tables were used for the identification of bacteria (Buchanan and Gibons, 1974; APHA, 1989; Collins et al., 1989) and fungi (Ainsworh et al., 1973; Jones, 1974). For the complete identification of the isolated enterobacteria the API 20E was used.
3. Results Tables 1 and 2 present the mean values and standard deviation (S.D.) of Colony Forming Unit (CFU)/ml, counted on different culture media for bacteria and fungi isolated from sponge extract and sea water collected at six stations. Bacteriological media (Table 1), Nutrient agar and MacConkey agar, showed a high microbial load, especially the former. Moreover, the number of bacterial colonies
E. Kefalas et al. / Ecological Indicators 2 (2003) 339–343
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Table 1 Mean values (S.D.) of CFU/ml enumerated on two culture media for the bacteria isolated from sponge extract and sea water Sampling station
Nutrient agar
Agathonisi Sitia 1 Sitia 2 Kalloni Kalafatis Kimi
MacConkey agar
Sponge extract
Sea water
Sponge extract
Sea water
3 2100 2400 66000 130000 400000
1 176 16 75 720 2200
3 860 25 72 7400 9500
0 80 10 12 560 1800
(3.0) a (953.9) b (400.0) b (12165.5) b (21633.3) b (91651.5) b
(0.5) a (43.1) a (11.3) a (9.9) a (268.7) a (282.8) a
(2.0) a (255.3) b (7.5) b (11.1) b (2029.7) b (793.7) b
a (22.6) a (4.2) a (1.4) a (42.4) a (339.4) a
Mean values for the same station and agar having different letters are significantly different (P < 0.05); N = 6. Table 2 Mean values (S.D.) of CFU/ml enumerated on two culture media for the fungi isolated from sponge extract and sea water Sampling station
Malt Extract agar
Agathonisi Sitia 1 Sitia 2 Kalloni Kalafatis Kimi
Czapek-Dox agar
Sponge extract
Sea water
Sponge extract
Sea water
0 1 5 0 1700 0
0 1 10 15 12 78
17 1 10 0 60 0
0 2 43 57 14 96
(1.7) a (2.0) a a (269.1) b a
(1.4) a (0.9) b (5.7) b (5.7) a (11.3) b
(4.3) b (1.0) a (10.0) a a (17.7) b a
a (2.8) a (4.2) b (7.1) b (2.8) a (11.3) b
Mean values for the same station and agar having different letters are significantly different (P < 0.05); N = 6.
Table 3 Bacterial and fungal species isolated from the sponge S. officinalis Sampling station Bacteria Sitia 1
E. coli
Sitia 2
E. coli Aeromonas salmonicida
Kalloni
Pseudomonas aeruginosa E. coli Proteus vulgaris Morganella morganii
Kalafatis
E. coli M. morganii P. vulgaris Proteus mirabilis Pasteurella spp.
Kimi
E. coli A. salmonicida Acinetobacter spp. Pseudomonas spp. Pseudomonas pancimobilis
Fungi
Fisarium spp.
isolated from sponge extract was significantly higher than that from the corresponding proximal sea water, enumerated on the same agar. Fungal media (Table 2), Malt Extract agar and Czapek-Dox agar, presented a relatively low microbial load. In addition, in three stations the number of fungal colonies isolated from sea water was significantly higher than that from sponge extract, counted on both fungal media. Table 3 lists the species of bacteria and fungi, which were identified in sponge extract from five stations.
4. Discussion Trichosporum spp.
Present results indicated that the bacterial load of sponge extract was greater than that of the proximal sea water. A possible explanation could be given is that sponge has the ability to concentrate bacteria in its body. On the contrary, the fungal burden counted in the present study was negligible in most of the samples, especially in sponge extract. It seems that fungi occur in sponges at very low frequency, failing to indicate the contamination degree in different areas.
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This is probably due to an antifungal activity of sponges (Amade et al., 1987; Pettit, 1999), or/and of sponge-associated bacteria (Ivanova et al., 2000). Possible adverse physicochemical conditions in the environment of sponge structures may also influence the fungal populations. Many micro-organisms that are typically found in the intestinal canal of human or terrestrial animals survive for considerable periods of time in sea water (Hawker and Linton, 1979). Sponge-associated bacterial flora from Kalafatis station belong to Enterobacteriaceae, with the exception of Pasteurella. Enterobacteriaceae are small gram-negative rods, among which some are pathogenic, such as Proteus. The aerobic or facultative anaerobic thermotolerant bacterium Escherichia coli, typical representative of the coliform group, appeared in all samplings. Sponge-associated bacterial species isolated from Kimi station (except E. coli) do not belong to Enterobacteriaceae and constitute aerobic gram-negative rods. The motile Aeromonas and Pseudomonas are typically waterborne bacterial genera and include opportunistic pathogenic species to crustaceans, fish and other aquatic organisms, as well as to humans (Aguado and Bashirullah, 1996; Bahlaoui et al., 1997; Pettibone, 1998; Kong et al., 1999; Starliper and Morrison, 2000). Thus, the sponge body represent an area of emerging heterotrophic gram-negative bacteria, enteric and non-enteric. Enterobacteriaceae, and especially E. coli, are considered as indicators of faecal contamination (Baudisova et al., 1997). The abundance of Aeromonas, Pseudomonas and Acinetobacter may be indicative of organic contamination and eutrophication, as a result of an anthropogenic impact on aquatic ecosystems (Rippey and Cabelli, 1989; Cenci et al., 1998; Lemke and Leff, 1999). Thingstad et al. (1999) observed in experimental mixed marine microbial communities that phosphate enrichment had a stimulatory effect on bacterial production, resulting from transfer of added phosphorus into ciliate biomass, while in turn bacterial production depended upon the fulfilment of carbon demand. In the present study, the differentiation among stations in the presence of the bacterial groups identified, may be explained by the kind of contamination that the sampling areas were subjected; e.g. Enterobacteriaceae/Pasteurella in the tourist site of Kalafatis (mainly faecal contamination)
and E. coli/Aeromonas, Pseudomonas, Acinetobacter in the port of Kimi with increased sewage discharges (faecal and organic contamination). The seawater bacterial load enumerated on Nutrient agar was 6 times greater than that on MacConkey agar for the Kalloni station and about 1.5 times for the other stations. Concerning sponge extract, the difference in bacterial colonies between the two culture media became greater, particularly for the samples taken from the eutrophicated gulf of Kalloni and the two ports, Sitia and Kimi. These stations were characterised by the presence of Aeromonas or/and Pseudomonas. It seems that Nutrient agar is more sensitive than MacConkey in isolating these bacterial genera. In addition, proliferation of the non-enteric bacteria in sponge structures is greater than that of the enteric ones. This permits a better discrimination of sites, reflecting the impact of both anthropogenic disturbances, faecal and organic. It is concluded that bacteria, but not fungi, benefited the environment of the body of S. officinalis. It is proposed that sponge-associated bacteria can be used as indicators of contamination in marine ecosystems. Acknowledgements This study was a part of the research program “Sponge Species in the Mediterranean: Contribution to the Sponge Fisheries”, which was financially supported by EEC D.G.-XIV-Fisheries (CL/MED/92/024). References Aguado, N., Bashirullah, A.K.M., 1996. Shell diseases in wild penaeid shrimps in eastern region of Venezuella. J. Aquaricult. Aquat. Sci. 8, 1–6. Ainsworh, G.C., Sparrow, F.K., Susman, A.S., 1973. The Fungi, vol. IVA. Academic Press, New York. Amade, P., Charroin, C., Baby, C., Vacelet, J., 1987. Antimicrobial activities of marine sponges from the Mediterranean Sea. Mar. Biol. 94, 271–275. APHA, 1989. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Azam, F., Fenchel, T., Field, J.C., Grey, J.S., Meyer-Reil, L.A., Thingstad, F., 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257–263. Bahlaoui, M.A., Baleux, B., Troussellier, M., 1997. Dynamics of pollution-indicator and pathogenic bacteria in high-rate oxidation wastewater treatment ponds. Water Res. 31, 630–638.
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