Biochemical Systematics and Eco/ogy, Vol. 21, No. 6/7, pp. 655-660, 1993. 0305-1978/93 $6.00+ 0.00 Printed in Great Britain. © 1993 Pergamon Press Ltd.

The Chemistry of Three Species of Demospongiae Collected from the Lagoon of Venice: a Comparison with some Ecological Implications*

ANNA AIELLO,t ERNESTO FATTORUSSO, t$ MARIALUISA MENNAt and MAURIZIO PANSlNI§

tDipartimento di Chimica delle Sostanze Naturali, Via D. Montesano 49, 1-80131 Napoli, Italy; §lstituto di Zoologia, via Balbi 5, 16126 Genova, Italy

Key Word Index--Tedania anhelans; Halichondria bowerbanki; Hymeniacidon sanguinea; Porifera; sterols; eicosapentaenoic acid.

Al~lzact--The hitherto undescribed chemical composition of specimens of the sponges Tedania anhelans, Hyrneniacidon sanguinea and Halichondria bowerbanki collected in the lagoon of Venice, was investigated. This study revealed some differences in their chemical composition when compared with samples of the same sponges collected in open sea. This points to a planktonic origin of some metabolites of the specimens coming from the lagoon.

Introduction The lagoon of Venice is a large basin of salty and brackish water located in the Adriatic Sea between the mouths of the Brenta and Sile rivers, covering an area of 55 ha and comprising stretches of water, canals and dry land. It is connected with the sea through three large mouths. It represents a unique ecosystem which includes a large number of aquatic plants and animals.

Urban and industrial settlements located in this area have caused environmental damage, the residential and industrial pollutants adversely affecting the flora and fauna of the lagoon. We are now studying the chemical composition of some marine invertebrates present in the lagoon basin and the impact of the peculiar environmental conditions on their metabolism. In this respect the Porifera appear to be quite interesting organisms on account of their high filtering activity. Sponges are typically marine organisms since only three families (out of a total of more than 80) live in fresh water. Brackish habitats such as estuaries, river mouths, coastal lagoons, etc., harbour several species of marine origin, some of which are able to endure very low salinity.

The three species of Porifera, Tedania anhelans, Halichondria bowerbanki and Hymeniacidon sanguinea, on which we present a chemical investigation, are by far the most common sponges in the zone explored, which covers all of the basin except its northern part. In the inner part of the lagoon, these species can withstand salinity values as low as 20%o (Sara, 1960). In addition, H. sanguinea, which tolerates short periods of immersion, may colonize the intertidal zone. Halichondria bowerbanki is reported to withstand salinity better than its close relative and sympatric species (in the northeast Atlantic and Mediterranean Sea) H. panicea, which, in contrast, better endures exposure to air (Vethaak et al., 1982). Tedania anhelans, which is common in the Northern Adriatic Sea on rocky outcrops and soft bottoms, is quite abundant in the lagoon where it often shows its "digitate" form, characterized by high oscular chimneys. These function by preventing the mixing of exhalant and inhalant currents,

*This work was performed within the scientific project "Sistema Lagunare Veneziano", research line n. 3.02. ~Author to whom correspondence should be addressed.

(Received 9 February 1993)

655

656 A. AIELLO ETAL

which may represent a reaction of the sponge to the weak water movement of the lagoon (Corriero, 1989). No sponge species are known from the so called "dead lagoon", where salinity drops to under 20%0. The ability to adapt to the unusual and very severe environmental conditions of the lagoon enables the three species studied and several others to survive in this selective habitat.

Materials and Methods Genera/experimentalprocedures. Heirms were obtained at 70 eV on a Kratos MS 50 mass spectrometer. The 1H- and 13C-NMR spectra were recorded on a Bruker AMX-500 spectrometer, and the solvent was used as an internal standard (CDCI3: 1H 5 7.26, 13C 6 77.0). Medium pressure liquid chromatography (MPLC) was performed on a Buchi apparatus. Preparative chromatography was performed on Si-gel plates, Merck, 2 mm.

Combined GLC-MS analysis was performed on a Hewlett-Packard 5890 gas chromatograph with a mass selective detector MSD HP 5970 MS and a split injector for capillary columns, using a fused-silica column, 25 m×0.2 mm HP 5 (cross-linked 5% PhMe Silicone, 0.33 ~m film thickness).

Collection and extraction o f the sponges. Sponges were collected by scraping the solid surfaces present in the lagoon (particularly the submerged portions of "briccole", the wood markers present along the canals) in the locations indicated on Fig. 1 during two expeditions (March 1992; October 1992). They were all frozen immediately after collection; a voucher specimen of each sample is deposited in the Dipartimento di Chimica Delle Sostanze Naturali, University of Naples.

Samples of T. anhelans, H. bowerbanki and H. sanguinea collected during spring 1992, or those collected during autumn, were thawed, homogenized and extracted at room temperature with MeOH. Solvent was removed under reduced pressure and the residues were extracted with EtOAc and, then, with n-BuOH. The EtOAc extracts were chromatographed by MPLC on Si-gel columns eluting with solvents of increasing polarity (hexane~EtOAc~MeOH). The n-BuOH extracts were fractionated on Sephadex LH 20 columns eluting with CHCI 3 with increasing amounts of MeOH (2-100%). The fractions obtained were analyzed by chromatographic and spectroscopic methods. Fractions obtained from the n-BuOH extract chromatography were purified further by SiO 2 PLC, eluting with n-BuOH:AcOH:H20 60:20:20, and were analyzed by ~H and 13C-NMR spectroscopy, which showed them to contain common purine and pyrimidine bases, their nucleosides and proteic amino acids.

Analysis o f the sterol lipid fractions. Fractions eluted with hexane:EtOAc 7:3 from the Si gel columns of the EtOAc extracts provided the crude sterol mixture ( T. anhelans = 0.9% dry wt.; H. bowerbanki = 0.7% dry wt.; H. sanguinea = 0.3% dry wt.) Sterols were characterized by GLC-MS analysis of their acetate derivatives prepared with acetic anhydride and pyridine (1:1) for 18 h at room temp. The steryl acetates were filtered on Si-gel columns, eluted with hexane containing increasing amounts of Et20. The fractions eluted with Et20:hexane (8:2) were analyzed further by GLC-MS. The identification of the steryl acetates were based on comparison of the GC-MS spectra with those of authentic specimens. The quantification of sterols was performed by a programmable integrator using 5c(-cholestane as an internal standard. Individual sterols and their percentage for each sponge are reported in Table 1.

5Z,8Z,11Z14Z,17Z-Eicosapentaenoic acid (EPA). This compound was the major component of the free fatty acid fraction in all samples of the sponges examined. EPA was obtained pure from the Si-gel chromatography of the EtOAc extracts, eluting with hexane:EtOAc 1:1. It was identified by comparison of its spectroscopic data with that reported in the literature (Ciminiello et al., 1991). Additional amounts were obtained from more lipophilic fraction, eluted with hexane:EtOAc 9:1, as the methyl ester. Percentages of eicosapentaenoic acid in the different specimens of sponges are reported in Table 2.

5Z,8Z,11Z,14Z,17Z-Eicosapentaenoic acid: HEIRMS: m/z 302.2241 calcd. 302.2247 for C20H3002 . 1H-NMR: 2.36 (t, J= 7.5 Hz, H-2); 1.69 (q, J= 7.5 Hz, H-3); 2.06 (m, H-3 and H-19 overlapped); 5.35-5.55 (olefinic protons); 2.75-2.90 (diallylic protons) and 0.94 (t, J= 7.5 Hz, H-20).

13C-NMR: 8 179.0 (C-1); 33.1 (C-2); 24.6 (C-3); 26.4 (C-4); 128.8-128.9 (C-5 and C-6); 25.6 (C-7, C-10, C-13, C-16, C-19); 127.9--128.3 (C-8, C-9, C-11, C-12, C-14, C-15, C-17); 132.2 (C-18); 13.8 (C-20).

Sphingolipids. Fractions eluted with EtOAc from Si-gel columns of the EtOAc extracts of the samples of T. anhelans, after evaporation under reduced pressure, afforded the mixture of sphingolipids l a - l e (0.2% for spring samples; 0.1% for autumn samples).

l a - l e mixture: HEIRMS: m/z 589.5805 calcd. 589.5801 for C39H7sNO2 ([M-H20]~); 603.5962 calcd. 603.5958 for C40H77NO2 ([M-HzO]+); 617.6119 calcd. 617.6115 for C41H79NO2 ([M-H20]+); 631.6277 calcd. 631.6272 for C42H81NO 2 ([M-H20]]+); 645.6433 calcd. 645.6429 for C43H83NO 2 ([M-H20]+). 1H-NMR: ~ 6.28 (d, J= 7.5 Hz); 5.79 (dt, J= 15, 7.5 Hz); 5.51 (dd, J= 15, 7.5 Hz); 4.31 (bs); 3.95 (dd, J= 10, 7.5 Hz); 3.90 (m); 3.70 (bd, J= 10 Hz); 2.75 (bs, OH); 2.50 (m); 2.18 (t, J = 7 Hz); 1.24 (large signal); 0.85 (t, J = 7 Hz).

Acid hydrolysis o f l a - l e . A solution of 10 mg of the l a - l e mixture in MeOH (2 rot) was treated with an excess of 6N HCI and stirred at reflux temperature for about 12 h. After cooling and addition of water, the suspension was extracted with diethyl ether. Excess ethereal CH2N 2 was added to the ethereal solution. After 1 h the solution was evaporated to dryness and the residue, analyzed by GC-MS (temperature program 1 min 100°C; 100-250°C, 3 ° min 1; 250_290oc, 5 o min -~, final time 10 rain; total time 70 min), gave five peaks with retention times and fragmentation patterns identical to those of the methyl esters of heneicosanoic acid

MAP OF THE LAGOON OF VENICE

'.,'-m

insul in- z a ~ c s m ~ a

s h o d s

=~ cantls ( ~ > lm)

FIG. 1. MAP OF THE LAGOON OF VENICE.

1. Borgo S. Giovanni 2. Canale Perognola 3. Fondi dei Sctte Morti 4. Canale Valgrande 5. Bocca di Porto di Malamocco

6. Isola Poveglia 7. Isola Sacca Sessola 8. Venezia 9. Bocca di Porto di Lido 10. Murano

TABLE 1. STEROL COMPOSITION OF THE SPONGES (%)

T. anhelans 77 anhelans H. sanguinea H. sanguinea H. bowerbanki H. bowerbanki (lagoon) (open sea) (lagoon) (open sea) (lagoon) (open sea)

oo

24-norcholesta-5, 22-dien-3~-ol 6.8 7.9 1.0 1.2 1.6 1.4

cholesta-5, 22-dien-3~-ol 2.1 2.4 5.1 5.3 4.4 4.4

cholesterol 35.1 31.8 3.6 3.9 29.9 21.0 24-methylchotesta-5, 22-dien-3~-ol 7.1 6.8 6.0 6.3 3.4 19.5 24-methylcholesta-5, 24(28)-dien-3~]-ol 10.0 11.4 4.5 4,9 7.7 5.0

24- methylcholest-5-en-3(~-ol 3.6 3.8 2.1 2.4 3.3 2,0 24-ethylcholesta-5, 22-dien-31~-ol 2.5 2.2 2.2 2.6 - - - -

24-ethylcholesta-24(28)-dien-3~-ol . . . . 1.6 1.4 24-ethylcholest-5-en-3~-ol 10.2 9.5 3.5 4.1 5.2 1.8 24-norcholest-22-e n-318-ol - - - - 0.7 - - - - - -

cholest-22-en-31~-ol - - - - 2.3 2.6 1.4 2.4 cholestanol 19.4 20.1 60.1 60.6 39.1 38.4

24-methylcholest-22-en-3~-ol - - - - 3.1 3.4 - - 1.0 24-methyl-3~-cholestanol 0.9 1.2 2.1 2.3 1.2 1.5 24-ethyl-31]-cholesta nol 2.3 2.7 3.9 - - 1.3 - -

TABLE 2. COMPARISON OF THE METABOLIC COMPOSITION OF THE SPONGES

Sponges Samples collected in the lagoon (spring 1992) Samples collected in the lagoon (autumn 1992) Open-sea samples

Tedania anhelans (Lieberkt~hn)

Halichondria bowerbanki Burton

Hymeniacidon sanguinea (Grant)

Sterol fraction: 0.90% dry wt.

EPA: 0.40% dry wt. Sphingolipids: 0.20% dry wt.

Sterol fraction: 0.70% dry wt.

EPA: 0.35% dry wt.

Sterol fraction: 0.30% dry wt. EPA: 0.43% dry wt. 1-methyl-6-iminopurine: absent 1,9-dimethyl-6-imino-8-oxopudne: absent

4-hydroxyphenylpyruvic acid oxime: absent

Sterol fraction: 0.90% dry wt. EPA: 0.20% dry wt.

Sphingolipids: 0.09% dry wt.

Sterol fraction: 0.75% dry wt.

EPA: 0.20% dry wt.

Sterol fraction: 0.30% dry wt,

EPA: 0.29% dry wt. 1-methyl-6-iminopurine: absent

1,9-dimethyl-6-imino-8-oxopurine: absent 4-hydroxyphenylpyruvic acid oxime: absent

Sterol fraction: 0.80% dry wt.

EPA: absent Sphingolipids: absent

Steroi fraction: 1.20% dry wt.

EPA: absent

Sterol fraction: 0.60% dry wt. EPA: absent.

1-methyl-6-iminopurine: 0.5% dry wt. 1,9-dimethyl-6-imino-8-oxopurine: 0.5% dry wt. 4-hydroxyphenylpyruvic acid oxime: 1.0% dry wt.

~>

F- O

CHEMISTRY AND ECOLOGY OF DEMOSPONGIAE 659

(retention time: 52.5 min), docosanoic acid (55.3 min), tricosanoic acid (56.5 min), tetracosanoic acid (59.6 min), and pentacosanoic acid (62.0 min).

Results and Discussion We carried out an extensive chemical analysis on the three sponges, T. anhelans, H. sanguinea and H. bowerbankl; which revealed that these organisms did not contain peculiar secondary metabolites, typical of a number of species belonging to the phylum Porifera. Therefore, our investigation was turned to the analysis of the most abundant common lipophylic metabolites which were found to be sterols and fatty acids. Remarkable amounts of sphingolipids were also found in T. anhelans.

To determine the sterol pattern, the ethyl acetate extract of each sponge was chromatographed on a Si-gel column and the crude sterol bands were acetylated and subjected to a further separation by chromatography on Si-gel columns, eluting with hexane containing increasing amounts of diethyl ether. Analysis of each fraction by GC-MS allowed identification of the individual sterol acetates. Quantitative determination was made using integrated areas of peaks in the gas-chromatograms. As reported in Table 1, the results obtained showed that/~s compounds were the most common sterols in the sponge T. anhelans, while cholestanol was found to be the major sterol in the H. sanguinea. Both stanols and/~s sterols co-occur in H. bowerbankL

The lipophylic fraction of all the species examined had an extremely high content of 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid (EPA, see Table 2) which was isolated by SiO 2 chromatography, eluting with hexane:ethyl acetate 1:1. Spectral data are reported in the experimental section.

The ethyl acetate extract of T. anhelans contained a further major metabolite (sphingolipids) which was eiuted from the SiO 2 column with a more polar eluent (ethyl acetate). This fraction exhibited five peaks in the mass spectrum at rn/z 589.5805, 603.5962, 617.6169, 631.6277, 645.6433 which corresponded with the molecular formulae: C39H75NO 2, C40H77NO 2, C41H79NO2, C42Hs1NO 2 and C43H83NO 2, and the loss of H20 from the molecular ions of five homologues ( l a - le ) which were not separated further. The NMR data clearly indicated l a - l e to be normal ceramides differing only in the acyl moieties, which were characterized by acid hydrolysis of the mixture to give an acidic fraction identified as their methyl esters by GC-MS analysis.

The chemical analysis of the hydrophilic fraction of T. anhelans, H. sanguinea, and H. bowerbankJ has also been carried out. It revealed that all the sponge extracts contained only common hydrophilic compounds like protein amino acids and free purine and pyrimidine bases, together with their nucleosides.

In order to examine the influence of the lagoon environment on the metabolism of these organisms, the above results were compared with those reported for the corresponding open-sea species. Previous papers report the sterol composition of H. bowerbankiand H. sanguinea (Sica eta/., 1978) which parallel those determined for the sponges coming from the lagoon (see Table 1). It is also reported that a sample of H. sanguinea collected near Taranto in the Mediterranean Sea contained remarkable quantities of 1-methyl-6-iminopurine, 1,9-dimethyl-6-imino-8-oxopurine (Cimino et al., 1985), and 4-hydroxyphenylpyruvic acid oxime (Cimino et a/., 1975). These compounds were absent in our collection of H. sanguinea and this might be ascribed to the environmentally very different areas where the animals lived.

No chemical data were available on the sponge T. anhelans. Therefore, we examined a specimen collected along the northern coasts of Sicily. Analogous to the lagoon samples, the open-sea specimen did not contain unusual metabolites and showed a similar sterol composition (see Table 1). An interesting finding is that in the open sea species EPA and sphingolipids were absent. This makes it likely that they were produced not by the sponge itself but by the microflora and/or microfauna of the lagoon of Venice. It is to be noted that the eicosapentaenoic acid is a characteristic

660 A. AIELLO ETAL.

m e t a b o l i t e o f d i a t o m s (Vo lkman e t al., 1980; Gi l lan etal . , 1981) w h i c h are ve ry a b u n d a n t du r i ng the sp r ing in the l agoon sea wate rs .

In o rde r to ob ta in s o m e s u p p o r t to th is hypo thes i s w e e x a m i n e d a s a m p l e o f T. anhe lans co l lec ted in a u t u m n , w h e n the p lank ton i c b i o m a s s is genera l l y lower . The s a m e analys is w a s e x t e n d e d to H. sangu inea and H. b o w e r b a n k i w h i c h also con ta i ned s ign i f i can t a m o u n t s o f EPA. The o b s e r v e d dec rease o f EPA in all the s p o n g e s and tha t o f sph ingo l i p i ds in T. anhe lans (see Table 2) appea rs to subs tan t ia te the e x o g e n o u s or ig in o f these m e t a b o l i t e s in the l agoon spec imens .

Acknowledgements--Mass spectra were provided by "Servizio Spettrometria di Massa del CNR e dell'Univer- sit& di Napoli". NMR spectra were performed at "Centro Interdipartimentale di Analisi Strumentale", University of Naples "Federico I1".

References Ciminiello, P., Fattorusso, E., Magno, S., Mangoni, A., lalenti, A. and Di Rosa, M. (1991) Furan fatty acid steryl

esters from the marine sponge Dictyonella incisa which show inflammatory activity. Experientia 47, 739-743. Cimino, G., De Giulio, A., De Rosa, S., De Stefano, S., Puliti, R. Mattia, C. A. and Mazzarella, L. (1985) Isolation

and X-ray crystal structure of a novel 8-oxopurine compound from a marine sponge. J. Nat. Prod. 48, 523- 528.

Cimino, G., De Stefano, S. and Minale, L. (1975) Occurrence of 4-hydroxyphenyl pyruvic acid oxime in the marine sponge Hymeniacidon sanguinea. Experienda 35, 756.

Corriero, G. (1989) The sponge fauna from the Stagnone di Marsala (Sicily): taxonomic and ecological observa- tions. Boll. Mus. Ist. Biol. Univ. Genova 53, 101-113.

Gillan, F. T., McFadden, G. I., Wetherbee, R. and Johns, R. B. (1981) Sterols and fatty acids of an antarctic sea ice diatom, Stauroneis amphioxis. Phytochemistr~, 20, 1935-1937.

Sara, M. (1960) Osservazioni sulla composizione, ecologia e differenziamento della fauna di Poriferi di acqua salmastra. Ann. Ist. Mus. Zool. Univ. Napoli12, 1-9.

Sica, D., De Simone, F., Ramundo, E. and Zollo, F. (1978) Sterols from sponges Biochem. Syst. Ecol. 6, 77-79. Vethaak, A. D., Cronie, R. J. A. and Van Soest, R. W. M. (1982) Ecology and distribution of two sympatric, closely

related sponge species, Halichondria panicea (Pallas, 1766) and H. bowerbanki Burton, 1930, Porifera, Demospongiae with remarks on their speciation. Bijdr. Dierk. 52, 82-102.

Volkman, J. K., Englinton, G. and Corner, E. D. S. (1980) Sterols and fatty acids of the marine diatom Biddulphia sinensis. Phytochemistry 19, 1809-1813.