Pergamon

PII: S0306-1978(96)00068-0

Biochemical Systematlc$ and Ecology, VoI. 24, N(~ 6, pp. 521-529,1996 Copyright © 1996 Published by Bsevier Science Ltd

Printed in Great Britain. AJI rights reserved 0305-1978/96 $15,00+0.00

Low Molecular Weight Metabolites of Three Species of Ascidians Collected in the Lagoon of Venice*

ANNA AIELLO, ERNESTO FATTORUSSOt and MARIALUISA MENNA Dipartimento di Chimica delle Sostanze Naturali, Via D. Montesano 49, 1-80131, Napoli, Italy

Key Word Index--Aplidium nordmani;, Styela partita; Botrillus leachii; Tunicata; carotenoids; sterols; eicosapentaenoic acid; glycosphingolipid; homarine.

Abst ract - -The chemistry of the three species of ascidians (Aplidium nordmani, SWela partita, Botrillus leachii) collected from the lagoon of Venice has been investigated. This led to the isolation of a phyto- sphingosine type cerebroside from B. leachii, which represents the first example of a glycosphingolipid from a tunicate, and of two new carotenoid pigments from A. nordmani. Also reported is an analysis of the most abundant low molecular weight metabolites of these organisms. Copyright © 1996 Published by Elsevier Science Ltd

Introduction Because of the historic and environmental interest in the lagoon of Venice and considering the damage induced by urban and industrial pollutants to its eco- system, we have recently undertaken a systematic survey of the biochemistry of the invertebrates present in this area in order to gain information of the influence that its peculiar habitat exerts on the lagoon's living creatures. Our attention has been turned to the organisms with high filter activity, like sponges and ascidians, whose metabolism is presumably affected to a large extent by the environmental condi- tions.

The results obtained from the chemical analysis of the most common sponges in the lagoon pointed to an exogenous origin some of their important metabolites (Aiello et al., 1 993, 1995, 1 996).

We report here the results obtained from an investigation of the chemistry of three ascidians, Aplidium nordmani (Milne Eduards), Styela partita ( Stimpson ) and Botrillus leachi (Savigny), benthic invertebrates belonging to the Phylum Chordata, Subphylum Urochordata. The three species investigated appear to be well adapted to survive in the selective habitat of the lagoon of Venice, being the most common tunicates present in the area explored, which covers almost all the lagoon basin. They are commonly found attached to submerged solid surfaces such as wharf pilings, bottoms of boats and shells of mussels and colonize mostly the zone adjacent to the open sea. Ascidians, in fact, cannot tolerate long periods of immersion in fresh water or places where salinity is too low, so they colonize only salty and brackish waters. No ascidians were found in the so-called "dead lagoon" characterized by a low salinity and along the coasts in areas where the most important industrial settlements are established.

This study revealed the absence in the three ascidians of unusual secondary metabolites, which, generally, play a critical role in the behavioural and ecological

*This work was performed within the scientific project "Sistema Lagunare Veneziano", research line n. 3.02.

~Author to whom correspondence should be addressed.

(Received 23 March 1996; accepted 31 May 1 996)

521

522 A. AIELLO ETAL.

interactions of predators and their prey in marine communities. This lack could be ascribed to the low predation existing in the lagoon habitat.

Experimental General experimental procedures HEIRMS (70 eV) and FAB MS were obtained on a Fisons VG Prospec 1000 mass spectrometer. The 1 H- and 13C_NM R spectra were recorded on a Bruker AMX-500

2

OH

,.,~ ,,~O H

H O,..."-.v-"~ :3 _ ~ . , , , , , O H . . , . x ,', .~.

~,- HO CH 3

4

H o ~ O H

CH3 5

~ C O 0 " I CH 6

3

CIR R1 R 2

• 2"

3 I . v_ ] ; " A 3 ~ (,%1 H H OR ~ (15%)

(16%) ~ (25%)

7 R = H ~ (30%)

8 R = Ac ~ (13%)

FIG. 1. METABOLITES ISOLATED FROM THE LAGOONAL COLLECTIONS OF THE TUNICATES A, NORDMANI, B. LEACHII AND S. PARTITA.

METABOLITES OF ASCIDIANS SPECIES 523

spectrometer and the solvent was used as an internal standard (CDCI3: 1H 6 7.26, 13C 6 77.0; CD3OD: 1H ~ 3.34, 13C 6 49.0). 1H connectivities were determined by COSY experiments. Optical rotations were measured on a Perkin-Elmer 192 polarimeter equipped with a sodium lamp (589nm) and a 10 cm microcell. Medium pressure liquid chromatography (MPLC) was performed on a Buchi apparatus. Droplet counter current chromatography (DCCC) was performed on a DCC B-670 Buchi chromatograph equipped with 294 tubes. High pressure liquid chromatography was performed on a Varian H PLC Model 5000 with Merck Si60 and RP-18 LiChrospher columns (5 l~m) using a dual cell refractometer detector. 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, 25m x 0.2mm HP 5 (crosslinked 5% Ph Me silicone, 0.33 I~m film thickness).

Collection and extraction of the biological material Ascidians were collected by scraping the submerged solid surfaces and the

mussels in several locations of the lagoon (Borgo San Giovanni, San Pietro in Volta, Isola Poveglia, Isola S. Giacomo in Palude, Canale di Mazzorbo, Burano) during two expeditions (March 1992, October 1993). They were all frozen imme- diately after collection; a voucher specimen of each sample is deposited in the Dipartimento di Chimica delle Sostanze Naturali, University of Naples.

Thawed organisms were homogenized and extracted at room temperature with MeOH. The solvent was removed under reduced pressure and the residues were partitioned between EtOAc and H20 and, subsequently, between n-BuOH and H20.

The EtOAc extracts were chromatographed by MPLC on SiO2 columns eluting with solvents of increasing polarity (n-hexane~EtOAc~MeOH). The n-BuOH extracts were fractionated by DCCC using n-BuOHlacetonelH20 (45:15:75) in the ascending mode (the lower phase was the stationary phase). Fractions (I 0 ml) were monitored by TLC on SiO2 with n-BuOHIAcOHIH20 (60:20:20). On the basis of a preliminary chromatographic (TLC) and spectroscopic ( IH-NMR) ana- lysis, the fractions obtained from MPLC and DCC chromatographies were fairly combined.

Isolation of the terpenehydroquinones I and 2 SiO2 chromathography of the EtOAc extracts of A. nordmani and S. partita,

eluting with hexane/EtOAc (7:3), yielded yellow oils. They were further chroma- tographed by HPLC on a RP-18 column eluting with MeOH to give two com- pounds, the geranylhydroquinone I (A. nordmani: 0.14%, S. partita: 0.08% dry wt) and 2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-ol (2, A. nordmani: 0.07% dry wt). These compounds were identified by comparison of their spectral data with those reported in literature (Guella et aL, 1987; Manners and Jurd, 1 977).

Isolation of carotenoids 3-5 Carotenoid fractions were obtained by SiO2 flash chromatography of the EtOAc

extracts of A. nordmani and S. partita, eluting with hexane/AcOEt, 1:1. They were concentrated (A. nordmani: 0.04%, S. partita: 0.01% dry wt) and separated by HPLC on a RP-18 column (MeOH/H20, 95:5) thus affording compounds 3-5 in a pure form.

(3S, 3"S)-7,8,7',8'-Tetradehydro-/],j~-carotene-3,3'-diol (3) (A. nordmani: 33% of total carotenoids). UV Zmax (Et20) 426, 452, 480 rim; IR Vmax (KBr) 3300, 2160 and 960 cm-1; HREIMS: observed m/z 564.3949, C4oH5202 requires 564.3954; CD (Et20/isopentane/EtOH, 5:5:2) nm (As) 231 (+0.5), 253 (+0.70), 280

524 A. AIELLO ETAL.

(+ 1.0); 1H- and 1 3 C _ N MR data were identical with those reported for alloxanthin (Moss, 1976; Davies et al., 1984).

(3R, 5S, 6R, 3"S)-5,6-Epoxy-3,3"-dihydroxy-7",8"-didehydro-5,6,7,8-tetra- hydro-/3,j~-caroten-8-one (4) (A. nordmani: 36% of total carotenoids). UV Zmax (Et20) 405, 425, 452 nm; IR Vmax (KBr) 3300, 1645, 2160; HREIMS: observed m~ z 598.4001, C4oHs404 requires 598.4008; CD (Et20/isopentane/EtOH, 5:5:2) nm (As) 232 (+0.8), 260 (+3.0), 282 (+2.8); 1H-NMR data were identical with those reported for halocynthiaxanthin (Matsuno and Ookubo, 1 981 ).

5,6-Epoxy-3,3",5"-trihydroxy-6",7"-didehydro-5,6,7,8,5",6"-hexahydro-f3,fJ-car- oten-8-one (5) (A.nordmanL" 6.7%, S, partita: 53% of total carotenoids). UV Z_m~lx (Et20) 410 (sh), 445, 476 nm; IR Vmax (KBr) 3350, 1930, 1660 and 960 cm , HREIMS: observed m/z 616.4113, C4oHseO5 requires 616.4108; 1H-NMR data were identical with those reported for fucoxanthinol (Galasko et al., 1969).

Analysis of the sterol lipid fractions Fractions eluted with n-hexane/EtOAc, 7:3 from SJO 2 columns of the EtOAc

extracts provided the crude sterol mixtures (A. nordmani:1.0%, S. partita: 0.6%, B. leachii: 0.54% dry wt). Sterols were characterized by GLC-MS analysis of their acetate derivatives prepared by treating with acetic anhydride/pyridine (1:1 ) for 18 h at room temperature. The steryl acetates were filtered on SiO2 columns, eluting with hexane containing increasing amounts of Et20. The fractions eluted with Et20/ hexane (8:2) were further analyzed by GLC-MS. The identification of the steryl acetates was based on comparison of the GC-MS spectra with those of authentic specimens. The quantitation of sterols was performed by a program- mable integrator using 5¢-cholestane as an internal standard. Individual sterols and their percentage for each ascidian are reported in Table 1.

(5Z, 8Z, 11Z, 14Z, 17Z)-Eicosa-5,8,11,14,17-pentaenoic acid (EPA) EPA was obtained pure from SiO2 chromatography of the EtOAc extracts,

eluting with hexane/EtOAc, 1:1. It was identified by comparison of its spectro- scopic data (Mass, NMR) with those reported in the literature (Ciminiello et al., 1991; Aiello et al., 1993). Additional amounts were obtained from more lipophilic fractions eluted with hexane/EtOAc, 9:1 as methyl ester; they, probably, derived from the extraction with methanol. Percentages of EPA in the three ascidians are reported in Table 2.

TABLE 1. STEROL COMPOSITION OF ASCIDIANS (%)

A. nordmani B. leachii S. partita

24- Norcholesta-5, 22-dien-3~-ol 6.0 4.8 0.7

Cholesta-5, 22-dien-3~-ol 14.1 6.7 17.7 Cholesterol 61,7 33.0 57.6 24- Methylcholesta-5, 22-dien-3~-ol - - 4.9 4.6

24- Methylcholesta-5, 24(28)-dien-3~-ol - - 15.2 1.2 24- Methylcholest -5-en-3~-ol - - 3.9 0.4 24- Ethylcholesta-5. 22-dien-313-ol 2.9 - - 24- Ethylcholest-5-en-3~-ol 3.7 3.3 - - Cholestanol 11.3 23.3 17.3 24-Methylcholest-22-en-3~-ol - - - - 0.3 24- Met hyl-3~ -cholestanol 1.5 0.9 0.2

24- Ethyl- 3[~-cholestanol 1.7 1.1 - -

METABOLITES OF ASCIDIANS SPECIES

TABLE 2. COMPARISON OF THE METABOLIC COMPOSITION OF ASCIDIANS =

525

Aplidium nordmani (Milne Eduards)

Sterol fraction: 1% EPA: 0.21%; EPA methyl ester: 0.37%. Carotenoids: 0.04% Terpene hydroquinones: 0.21% Homarine: 0.35%

BolTillu$ leachii Sterol fraction: 0.54% (Savigny) EPA: 0.22%; EPA methyl ester: 0.11%

G licosphigolipids 0.15% Homarine: 0.4%

Styela partita Sterol fraction: 0.6%. (Stirnpeon) EPA: 0.28%; EPA methyl ester: 0.22%

Carotenoids 0.01% Terpene hydroquinones 0.08%

=Percentages are expressed with respect to the dry weights of the organisms.

Isolation of homarine (6) Fractions containing pure homarine were obtained from DCC chromatography

of n-BuOH extracts of A. nordmani and B. leachii. This compound (A. nordmani: 0.35%, B. leachii: 0.4% dry wt) was identified by comparison of its spectroscopic features (Mass, NMR) with those reported in the literature (Quast and Schmitt, 1970; Sciuto et al., 1988).

Isolation of cerebroside 7 as its peracetate (8) DCC chromatography of the n-BuOH extract of B. leachii gave a glyco-

sphingolipids fraction. It was evaporated to give a residue (0.15% dry wt) which was dissolved in pyridine (500/11) and allowed to react with Ac20 (200/d) at room temperature (12 h). The reaction mixture was then concentrated and filtered on a SiO2 column eluting with a linear gradient of CH3OH (2-50%) in CHCl 3. Fractions eluted with CHCI3/CH3OH, 95:5 were further purified to a main com- pound (8, 2.7 mg) by HPLC using a LiChrospher Si-60 column 5 I~m (4 x 250 mm) with the mobile phase hexane/AcOEt, 55:45.

(2S, 3S, 4R)-O-(~-D-Glucopyranosyl)-(1-~ 1)-2-[(R)- 1-hydroxyalk- ylcarbonylamino]- 1,3,4-alkane-triol peracetate (8)

[~]25D= +12 (C=0.002 in CHCI3) Negative FAB MS, m/z 1152, 1138, 1124, 1110, 1096, 1082, 1068 and 1054 [ M - H I series. 1H-NMR (CDCI3): ~ 6.68 (1 H, d , J = 9 Hz, NH), 5.18 (1H, t, J=9.3 Hz, 3'-H), 5.12 (1H, overlapped, 3-H), 5.11 (1 H, overlapped, 2"-H), 5.06 (1 H, t, J=9.3 Hz, 4'-H), 4.88 (1 H, dd, J = 9.2, 8.8 Hz, 2'- H), 4.84 (1 H, m, 4-H), 4.48 (1 H, d, J = 8.8 Hz, I ' -H), 4.23 (1 H, overlapped, 6'-Ha), 4.23 (1H, overlapped, 2-H), 4.12 (1H, dd, J=3.5, 2.5 Hz, 6'-Hb), 3.82 (1 H, dd, J = 10.2, 3.0 Hz, 1 -Ha), 3.67 (1 H, overlapped, 1 -Hb), 3.67 (1 H, over- lapped, 5'-H), 1.99-2.24 (7 singlets, Ac's), 1.80 (2H, m, 3"-H2) and 1.59 (2H, overlapped, 5-H2).

Methanolysis of 8 A solution of the glycosphingolipid peracetate 8 (2 mg) in 1N HCI-MeOH (1

ml) was kept for about 12 h at 80 ° in a sealed tube. The reaction mixture was dried under nitrogen, dissolved in CHCI3 and passed through a SiO2 column. Elution with 1 5 ml of 0.1% o pyridine in CHCI3 gave a mixture of =-hydroxy acid methyl esters [fraction A: [a ]2So=-2 (c=0.001 in CHCI3); 1H-NMR (CDCI3) $ 4.19

526 A. AIELLO ETAL,

(1H, t, J=6.5Hz, 2-H), 3.78 (3H, s, OMe), 1.77 (2H, m, 3-H2), 1.25 (br, alkyl- chain protons), 0.86 (3H, t, J=7Hz, CH2-CH3) ]. Subsequent elution with 0.1% o pyridine in MeOH afforded a mixture of sphinganines and methyl glycosides. The mixture was partitioned between CHCI3 and H20/MeOH (8:2), and the organic and aqueous layers were separated and concentrated to give sphinganines (fraction B) and the methyl glycosides (fraction C), respectively. Fraction A was analyzed by GC-MS and the components were identified by comparison of their retention times and mass spectra with those of authentic samples. An aliquot of fraction B was acetylated as reported above to give sphinganine tetraacetates [1H- N MR (CDCI3): 5 5.92 (1 H, d, J= 8 Hz, 2-NH), 5.10 (1 H, dd, J = 8.5 and 3 Hz, 3- H), 4.93 (1H, ddd, J = 9 , 3, and 3 Hz, 4-H), 4.47 (1H, m, 2-H), 4.29 (1H, dd, J=11.5 and 5 Hz, 1b-H), 4.00 (1H, dd, J = 1 1 . 5 and 5 Hz, 1a-H), 2.08 (3H, s, Ac), 2.06 (6H, s, Ac), 2.03 (3H, s, Ac), 1.69 (2H, m, 5-H2), 1.24 (br, alkyl-chain protons), 0.86 (t, J = 7 Hz, CH2-CH3), 0.84 (d, J=6.5 Hz, CH2(CH3)2]. The alkyl chains of the phytosphingosines were characterized by oxidative cleavage with KMnO4/NalO4 and GC-MS according to the described procedure (Costantino et al., 1994).

Results and Discussion No chemical data were available in the literature for the three species of ascidians, A. nordmani, B leachii and S. partita. A preliminary analysis of the metabolic content, carried out on several specimens of the three ascidians collected from different lagoonal zones, showed no significant variations in their chemistry. This indicated that their metabolism is not affected by different microhabitats which undoubtedly exist within the lagoonal area. We performed an analytical investi- gation of the hydrophilic and lipophilic low molecular weight metabolites of these organisms, which were isolated by a combination of several chromatographic procedures. The fractions obtained from lipophilic (EtOAc) and hydrophilic (BuOH) extracts, which showed to contain similar metabolites on the basis of their chromatographic and 1 H- N M R properties, were appropriately combined.

The composition of the sterol fractions of the ascidians has been determined. Crude sterol mixtures were obtained by fractionation of the ethyl acetate extracts on Si-gel columns and were acetylated. After successive chromatography on Si gel columns, each fraction was analysed by GC-MS to identify the individual sterol acetates. Quantitative determination was made using integrated areas of peaks in the gas-chromatograms. Table 1 reports the sterol profile of each ascidian and the relative percentages.

Analogously to what has been observed from analysis of the sponges collected in the lagoon of Venice (Aiello et aL, 1993, 1995, 1996), the major component of the fatty acids fractions of the ascidians was found to be 5Z, 8Z,11Z,14Z,17Z- eicosapentaenoic acid (EPA). The planktonic origin of EPA in sponges was established by observing that it is the major metabolite of lagoonal planktonic samples (Aiello et al., 1996); in addition, the seasonal variation of the EPA content in specimens of lagoonal sponges was shown to parallel the variation of microflora and/or microfauna in this area (Aiello et al., 1993, 1996). An analogous dietary origin for EPA most likely occurs in the ascidians.

The ethyl acetate extracts of A. nordmani and S. partita showed antibacterial activity against Gram + bacteria; the active principles were isolated in a pure form by separation on Si gel columns followed by reversed phase HP/C. They were identified as the geranyl-hydroquinone I (A. nordrnani: 0.14%, S. partita: 0.08% dry wt) and its cyclized derivative, 2-methyl-2-(4-methylpent-3-enyl)-2H-chromen- 6-ol (cordiachromen A, 2, A. nordmani: 0.07% dry wt) by comparison of their spectroscopic features with those reported in the literature. The geranyl-hydro-

METABOLITES OF ASCIDIANS SPECIES 527

quinone 1 was first isolated from a non-identified Mexican Aplidium (Fenical, 1976); it was then isolated from several plants belonging to the families Borragi- naceae and Hydrophylaceae (Manners and Jurd, 1977a; Manners, 1983; Rey- naulds and Rodriguez, 1979). Cordiachromen A (2) was previously found Cordia alliodora (fam. Borraginacea) (Manners and Jurd, 1977b) as well as in the ascidian Aplidium constellatum (Targett and Keeran, 1984).

An investigation of the carotenoids present in the three species of Tunicata has been carried out. B. leachii did not contain detectable amounts of these pig- ments, while the principal carotenoid isolated from S. partita was fucoxanthinol (5). The separation of the total carotenoid fraction of A. nordmani led to the iso- lation, in addition to fucoxanthinol, of two carotenoids (3 and 4) whose UV, MS and NMR data were identical with those reported for alloxanthin and halo- cynthiaxanthin, respectively. These last two compounds are commonly found in ascidians (Matsuno et al., 1984; Matsuno and Ookubo, 1 981, 1984), but surpris- ingly, CD spectra of the metabolites 3 and 4 showed opposite features to those reported for alloxanthin and halocynthiaxanthin, respectively (Matsuno et al., 1 985, 1 984). Thus, the stereostructures of the two new carotenoids isolated from A. nordmani have been assigned as (3S, 3'S)-7,8,7',8"-tetradehydro-~,jS-carotene- 3,3'-diol (3) and (3R, 5S, 6R, 3'S)-5,6-epoxy-3,3"-dihydroxy-7',8'-didehydro- 5,6,7,8-tetrahydro-jS, jS-caroten-8-one (4). It is worthy of note that we recently reported the isolation from the sponge Suberites massa, collected in the lagoon of Venice, of a new acetylenic carotenoid also possessing opposite chirality to that of structurally-related compounds isolated from non-lagoon organisms (Aiello et al., 1 995).

A chemical analysis of the hydrophilic fractions obtained from DCC chromato- graphy of the n-BuOH extracts of A. nordmani, S. partita and B. leachii has also been carried out. All three ascidians were shown to contain the common DNA- encoded amino acids and free purine and pyrimidine bases together with their nucleosides, n-BuOH extracts of B. leachii and A. nordmani contained large amounts of homarine (6), a quaternary ammonium metabolite of marine algae (Sciuto et al., 1988, 1989), which has also been detected in other marine organisms and whose antifouling activity against the benthic marine diatom Navicula salinicola has been reported (Targett et al., 1983).

A cerebroside mixture was obtained from the water-insoluble lipid fraction of the n-BuOH extract of B. leachii; the glycolipid nature of the isolated material was deduced from the features of its 1H-NMR spectrum which showed a large signal at 6 1.25 and a series of overlapping signals at ~ 3.5-5.5. This mixture was fractio- nated, after acetylation with acetic anhydride/pyridine, on a Si gel column, eluting with a linear gradient of methanol in chloroform. It was finally purified by HPLC thus affording the peracetate (8) of the original cerebroside 7. Its structure was identified as follows. The FAB mass spectrum (negative-ion mode) of 8 suggested that it was a mixture of homologues, showing eight ion peaks at rn/z 1152, 1138, 1124, 1110, 1096, 1082, 1068 and 1054 ( [M-H]-) corresponding to C56H96NO17+n CH2, n= 0-7.1H-NMR signals of 8 (see experimental) relative to the sugar moiety were identical to those of an authentic sample of (2S, 3S,4R)-O- (p- D- glucopyranosyl) - (1 -~1 ) -2- [ (R) - I - hydroxyalkylcarbonylamino] -1,3,4- alkane-triol tetraacetate obtained by treatment with acetic anhydride/pyridine of the natural glycosphingolipid isolated from the Caribbean sponge Agelas cla- throdes (Costantino et al., 1995). This established the presence of a ~-D-gluco- pyranosyl unit in compound 8. The composition of fatty acids and sphinganines of 8 was determined by degradative analysis. Compound 8 was subjected to acidic methanolysis, which, after Si gel chromatography, followed by solvent partition, afforded three fractions containing fatty acid methyl esters (fraction A), long-chain

528 A. AIELLO ETAL.

bases (fraction B), and methyl glycosides (fraction C), respectively. The 1H-NMR and optical rotation of fraction A showed that it was a mixture of (2R)-2-hydroxy acid methyl esters (Higuchi et aL, 1990b), whose composit ion was determined by GC-MS analysis. An al iquot of fraction B was acetylated wi th acetic anhydr ide/ pyridine. Comparison of 1H-NMR spectrum of the acetylated material wi th l i t- erature data (Natori et al., 1993) showed that it was a mixture of (2S, 3S, 4R)- phytosphingosine tetraacetates, differing from each other in their terminal alkyl chain. Their structures were ascertained by permanganate/periodate oxidative cleavage of the C3/C4 bond and methylation of the resulting carboxylic acids, fol- lowed by G C- MS analysis.

The above evidence al lowed the structure 7 to be assigned. To the best of our knowledge, compound 7 is the first glycosphingol ipid isolated from a tunicate. Its structure resembles that of glycosphingol ipids recently isolated from marine invertebrates (Higuchi et al., 1990a,b; Kawano et aL, 1988; Kochetkov and Smirnova, 1986; Schmitz and McDonald, 1 974).

The analysis of the chemistry, particularly the high content of EPA of the three lagoon ascidians A. nordrnani , S. part i ta and B. leachii, suggested that the impact of tha lagoon enviroment on the metabolism of sponges (Aiello et aL, 1993, 1995) can extend even to the metabolism of ascidians and, consistently, to the fi lter feeding organisms. In order to confirm this hypothesis we aim to extend the ana- lysis to other lagoon tunicates and to perform a chemical comparative study of the corresponding open-sea ascidian species.

Acknowledgements--We wish to thank Professor Angelo Tursi (Istituto di Zoologia e Anatomia Com- parata, University of Bari, Italy) for identifying the ascidians. Mass and NMR spectra were performed at "Centro Interdipartimentale di Analisi Strumentale", University of Naples, "Federico I1".

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M ETABOLITES OF ASCIDIANS SPECIES 529

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