J Appl Phycol (2009) 21:295306 DOI 10.

1007/s10811-008-9367-8

Metabolite profiling of the benthic diatom Cocconeis scutellum by GC-MS

Michela Nappo & Strahil Berkov & Carles Codina & Conxita Avila & Patrizia Messina & Valerio Zupo & Jaume Bastida

Received: 12 March 2008 / Revised and accepted: 25 July 2008 / Published online: 11 September 2008 # Springer Science + Business Media B.V. 2008

Abstract Cocconeis scutellum is a benthic diatom producing one or more compounds responsible for the early programmed cell death (apoptosis) of the male gonad and the androgenic gland of the protandric shrimp Hippolyte inermis Leach. The metabolite composition of both the ether and butanol extracts was studied by gas chromatography-mass spectrometry (GC-MS) in both electron impact (EI) and chemical ionization (CI) mode. The compounds were identified as trimethylsilyl (TMSi) derivatives. The structure of the fatty acids (FA) was confirmed after conversion to their methyl esters. The ether fraction of C. scutellum contained FA (76%), of which 30% were saturated (SFA), 24% monounsaturated (MUFA) and 22% polyunsaturated (PUFA). Mono- (8%) and diglycerides (3%) as well as sterols (5%) and isoprenoid compounds (4%) were also found in this fraction. The butanol extract consisted of FA (45%), carbohydrates (25%), amino acids and N-containing metabolites (10%), fatty alcohols
M. Nappo : S. Berkov : C. Codina : J. Bastida (*) Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Catalunya, Espanya e-mail: jaumebastida@ub.edu C. Avila Departament de Biologia Animal (Invertebrats), Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Catalunya, Espanya P. Messina : V. Zupo Stazione Zoologica A. Dohrn, Laboratorio di Ecologia del Benthos, Punta S. Pietro, 80077 Ischia, Napoli, Italia

(9%), glycerides (4%) and organic acids (3%). In the literature, many reports deal with the chemistry and the chemical ecology of planktonic diatoms; contrastingly, benthic species are still less studied, especially with respect to their chemical composition, due to their difficult cultivation. Hence, our investigation represents a preliminary approach to clarify on chemical bases the ecological role of Cocconeis on decapods in the marine benthos, as well as a description of the metabolic pattern of this benthic diatom. Keywords Cocconeis scutellum . Hippolyte inermis . Apoptosis . Metabolites . GC-EI/MS . GC-CI/MS

Introduction Diatoms are eukaryotic unicellular algae characterized by a siliceous cell wall (Lebeau and Robert 2003) and belonging to the class Bacillariophyceae. They are widely distributed in most aquatic habitats, and they are responsible for more than 50% of marine primary production (Nelson et al. 1995). Furthermore, they play a key role in the global carbon cycle (Pohnert 2005), constituting a rich diet for many herbivores (Newell and Newell 1963; Raymont 1983) both in the marine benthos and in the water column, due to their high concentration of lipids (Sicko-Goad and Andresen 1991). This study is based on previous investigations dealing with the influence of benthic diatoms (i.e., living close to the sea bottom) of the genus Cocconeis on the sex reversal of the shrimp Hippolyte inermis Leach 1915 (Zupo 1994, 2000, 2001). This small decapod lives in shallow waters of the Mediterranean Sea and along the Atlantic coast of Spain (Zariquey 1968), where it forms stable populations in seagrass meadows (Gambi et al. 1992). H. inermis is a protandric crustacean (i.e., individuals undergo

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a male stage before switching to female about a year after hatching), with a life cycle characterized by 2-yearly periods of recruitment: autumn and spring. Autumn-born decapods experience a male phase before reverting to females ( females, according to Zupo 1994) in the next spring. The sex change naturally occurs in H. inermis about 712 months after birth, while the female gonad derives from undifferentiated tissues only after the whole disruption of the testes (Reverberi 1950). Spring recruits, instead, show a different sexual maturation: they immediately produce in the field both male and female individuals. The seasonal difference has been explained by the influence of various species of benthic diatoms upon which the crustaceans feed. These diatoms are epiphytes of Posidonia oceanica leaves and bloom mainly in spring, while they are scarcely represented in the periphyton community in autumn (Buia et al. 2000; Zupo 2001). It was hypothesized that dietary diatoms contain one or more compounds able to destroy selectively the male gonad of H. inermis during its development, thus producing primary () females (Zupo 1994, 2000; Zupo et al. 2007). The effect of diatoms would be triggering the cell death in postlarvas male gonad, a process that in absence of the microalgae would happen about 1 year after birth in mature male individuals (Zupo 2000). The action of the diatom apoptotic compound(s) is species-specific and extremely selective for the male gonad and the androgenic gland of H. inermis (Zupo and Messina 2007). This shrimp is largely adapted to P. oceanicas life cycle (dUdekem dAcoz 1996), and the effect of the dietary microalgae could be a spring signal for the development of females, whose presence is crucial for keeping a constant sex ratio (Zupo 1994; Buia et al. 2000). Our recent studies indicated that C. scutellum is the species with the major effect in the sex reversal of H. inermis (authors, unpublished data). This diatom plays a key function in the ecology of the crustacean, resulting in a large number of females ( and ) for the next autumn mating period. Such a mechanism of regulation is unknown for planktonic diatoms (i.e., free-floating organisms) which, contrastingly, exercise deleterious effects towards their crustacean predators (Miralto et al. 1995, 1999; Ianora et al. 2004). In fact, diatoms like Skeletonema costatum and Thalassiosira rotula have abortive and teratogenic effects on copepods by production of oxylipins, mainly aldehydes, derived from the oxidation of polyunsaturated fatty acids (PUFA) (Fontana et al. 2007). The chemical ecology of planktonic diatoms is well documented (Pohnert and Boland 2002; Pohnert 2005; Fontana et al. 2007); in contrast, few studies are reported about benthic diatoms and even fewer about their metabolite composition (Simental et al. 2001) and effects on other organisms (Taylor et al. 2007). One of the reasons for the limited information in this field is that benthic diatoms are much more difficult to sample, quantify and cultivate than

planktonic ones. In particular, Cocconeis is characterized by low growth rates and strong adhesion to the substratum, which makes the isolation of the diatoms and the manipulation of their cultures difficult (Raniello et al. 2007). In the related literature on diatom chemistry, great attention is paid to the lipid fractions due to their nutritional value in the marine food web (Newell and Newell 1963; Raymont 1983; Gordon et al. 2006). Sterol fractions are mainly analyzed as TMSi derivatives or without derivatization (Gladu et al. 1991; Souchet and Laplante 2007) while fatty acids (FA) are usually identified by GC-MS as their methyl esters (FAME) (Mjos and Pettersen 2003; Rousch et al. 2003; Dayhuff and Wells 2005; Gordon et al. 2006). Various analytical methods, including HPLC-MS and GC-MS, have been reported for analysis of free amino acids and carbohydrates in algae (Hokputsa et al. 2003; Gordon et al. 2006). Several papers on metabolite profiling have proved that GC-MS is a reliable, reproducible, rapid, selective and sensitive method requiring a small biomass for simultaneous identification of plant metabolites from various classes, such as amino acids, phenolic compounds, saccharides, organic acids, FA, sterols, glycerides, etc. after derivatization (Roessner et al. 2000; Benkeblia et al. 2007; Schliemann et al. 2008). To our knowledge, no detailed reports on the metabolite profiling of benthic diatoms have been published. Hence the objective of this study was to characterize the metabolic pattern of these diatoms as a basis to clarify their ecological role on selected decapods in the marine benthos.

Materials and methods Cocconeis scutellum diatoms were isolated and cultivated at the Stazione Zoologica A. Dohrn of Ischia, Naples, Italy. Axenic cultures were cultivated in sterilized Petri dishes (14 cm diameter) containing 100 mL of F2 medium (Sigma Aldrich) and incubated in a thermostatic chamber (18C, 12/12 h photoperiod). After 16 days, the Petri dishes were opened, the culture medium was drained and the dishes were washed twice with distilled water, then freeze-dried and scraped with a metal blade, to collect the produced biomass (Zupo et al. 2007; Raniello et al. 2007). Extraction The extraction of freeze-dried diatoms of C. scutellum (1.5 g dry wt) was carried out in agreement with the protocol described by Gavagnin et al. (2005). The material was previously reconstituted in distilled water (50 mL) and then treated with acetone (3100 mL) at room temp. in an ultrasound bath for 23 min, in order to increase the membrane breakdown and the release of compounds from the cells. The

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suspension was filtered and the filtrate was concentrated by rotary evaporator. The residual water was extracted with diethyl ether (3150 mL) by means of a separatory funnel. After separation, the ether phase was treated with anhydrous Na2SO4 and evaporated to dryness under reduced pressure, obtaining 182 mg of dry extract. The aqueous residue was further extracted with n-butanol (100 mL). The n-butanol soluble part was evaporated until dryness and yielded 74 mg. The extraction with diethyl ether and n-butanol allows for a repartition of the acetone-extracted compounds in apolar and polar fractions. The remaining water phase was lyophilized and then weighted to obtain 8 mg of residue. In order to search for aldehydes, another diatom sample (1.0 g dry wt) was sonicated in distilled water for 1 min and left on the bench for 30 min. Acetone was added and the resulting suspension was centrifuged three times at 4,000g for 10 min. The supernatant was exhaustively extracted with CHCl3. The organic layers were combined, dried over anhydrous Na2SO4 and then evaporated under reduced pressure, affording 91 mg of dry extract. Derivatization Ether fraction (ca. 23 mg) was silylated with 50 L of N, O-bis-(trimethylsilyl)trifluoro-acetamide (BSTFA) in 50 L of pyridine for 2 h at 50C. Butanol fraction (ca. 23 mg) was first methoxymated with 100 L of methoxyamine hydrochloride (MOA, 20 mg mL1 in pyridine) for 90 min at 50C to stabilize the carbonyl moiety and prevent ring formation in sugars, then silylated by addition of 100 L of BSTFA and heating at 50C for 1 h. The mixtures were evaporated under a stream of N2 and redissolved in 100 L of chloroform for further GC-MS analysis. In parallel, FAME were obtained from ca. 5 mg of dry ether fraction dissolved in 2 mL of 1.5% solution of HCl in absolute methanol and heated overnight (12 h) at 60C. After cooling, 2 mL of water containing 5% of NaCl were added to the reaction mixture and the acid solution was extracted with n-hexane (23 mL). The organic layer was separated and washed with 3 mL of water containing 5% of Na2CO3 and dried over anhydrous Na2SO4. After evaporation of the n-hexane, the residue was dissolved in 100 L of chloroform for further GC-MS analysis. The chloroform extract (ca. 3 mg) was treated with (carbetoxyethylidene)-triphenylphosphorane (CETtriphenylphosphorane) in order to converse eventual unstable aldehydes into ethyl esters by Witting reaction (dIppolito et al. 2002). GC-MS analysis The data were recorded on a Hewlett Packard 6890+MSD 5975 (Hewlett Packard, Palo Alto, CA, USA) operating in

EI and CI mode with a HP-5 MS capillary column (30 m 0.25 mm0.25 m). The temperature program was: 100 180C at 15C min1, 1 min at 180C, 180300C at 5C min1, and 1 min held at 300C. Injector temperature was 280C. The flow rate of carrier gas (helium in EI mode and methane in CI mode) was 0.8 mL min1 and the electron potential was 70 eV. In order to detect aldehydes, the GCMS conditions were the same as those reported in the literature (dIppolito et al. 2002). Identification of metabolites The metabolites of the ether extract were identified as TMSi derivatives comparing their mass spectra and Kovats Indexes (RI) with those of an on-line available plantspecific database (The Golm Metabolome Database; http:// csbdb.mpimp-golm.mpg.de/csbdb/gmd/home/gmd_sm. html) and the NIST 05 database. Additionally, the structure of FA was confirmed after their conversion to FAME considering the characteristic mass spectral fragmentation of each subgroup, molecular ion, retention time and mass spectra available in the on-line lipid library (http://www. lipidlibrary.co.uk/ms/ms01/index.htm), NIST 05 database and literature data as indicated in Table 1. Peaks in the GC-MS of the butanol extract were identified as TMSi and/ or MOA derivatives with the help of the NIST 05 database and on-line available plant-specific database (The Golm Metabolome Database) as well as with literature data on the basis of the match of mass spectra and RI as indicated in Table 1. The measured mass spectra were deconvoluted by the Automated Mass Spectral Deconvolution and Identification System (AMDIS), before comparison with the databases. Then, the spectra of individual components were transferred to the NIST Mass Spectral Search Program MS Search 2.0 where they were matched against reference compounds of the NIST Mass Spectral Library 2005 and the Golm Metabolome Database. RI of the compounds were recorded with standard n-hydrocarbon calibration mixture (C9-C36) (Restek, Cat no. 31614, supplied by Teknokroma, Spain) using AMDIS 3.6 software. The search for aldehydes was performed after derivatization of the chloroform extract with CET-phosphorane. The MS data were deconvoluted by AMDIS as above, transferred to the NIST program and compared with the library of derivatized standards which we previously built.

Results

The GC-MS analysis of both the ether and butanol fractions resulted in the detection of more than 150 metabolites, about 100 of which were identified. The metabolite pattern of C. scutellum is presented in Tables 1 and 2. The extraction

298 Table 1 Metabolites of C. scutellum presented as % of TIC Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 L-Alanine a, b L-Valine a, b Urea a, b UC L-Leucine a, b Glycerol a, b Phosphoric acid a, b L-Isoleucine a, b Succinic acid a, b UC UC L-Serine a, b L-Threonine a, b Glutaric acid a, b Decanoic acid (10:0) a, b, c Aspartic acid a, b Dodecanol a, b, c Hexadecane a, b, c Tridecanoic acid ME (13:0) h Ornithine a, b 1-Tetradecanol a, b, c L-Phenylalanine a, b Pentonic acid-1,4-lactone b Dodecanoic acid (12:0) a, b, c Arabinose 1 a, b, d Arabinose 2 a, b, d UM Heptadecane a, b, c 1-Pentadecanol a, b, c UM Glycerol-2-phosphate a, b Hydroxycinnamic acid a, b Glycerol-3-phosphate a, b UC Octadecane a, b, c UC 2-Ketogluconic acid a, b Hypoxanthine a, b 9-Tetradecenoic acid (14:19) a, b, c UC Tetradecanoic acid (14:0) a, b, c Galactofuranose a, b, d Adenine a, b UM 9,12-Hexadecadienoic acid ME (16:2 9,12) h UM UC Talose a, b, d Pentadecanoic acid (15:0) a, b, c L-Tyrosine a, b 1-Hexadecanol a, b, c UC 6,9,12,15-Hexadecatetraenoic acid (16:46,9,12,15) 6,9,12-Hexadecatrienoic acid (16:36,9,12) b, c Glucose a, b, d 9-Hexadecenoic acid (16:19) a, b, c Rt 4.00 5.16 5.33 5.71 5.72 5.75 5.79 5.95 6.09 6.14 6.38 6.59 6.86 6.94 7.40 7.65 8.49 8.82 9.10 9.11 9.20 9.30 9.43 9.44 9.50 9.68 9.88 10.01 10.34 10.65 10.67 10.94 11.17 11.23 11.33 11.44 11.50 11.63 11.78 11.93 12.13 12.42 12.54 12.67 12.70 13.00 13.01 13.19 13.55 13.68 13.75 14.22 14.53 14.60 14.61 14.96 Key ions (m/z)* 116, 147, 180 144, 218, 246 147, 189

J Appl Phycol (2009) 21:295306

Ether extract

Butanol extract 0.14 0.23 2.34

0.12 158, 205, 299, 158, 147, 232, 260 147, 218 314 218 247 0.07 0.01 0.33 8.62 0.26 0.20 0.96 0.40 0.07 0.03 0.07 0.04 0.01

204, 218, 278 218, 219, 291 147, 216, 158 117, 229, 244** 232, 334 243***, 75, 103 57, 71, 226** 74, 87, 228** 142, 204, 348** 271***, 75, 103 218, 192, 294*** 217, 147, 231 117, 257***, 272** 217, 230, 191 217, 230, 191 57, 71, 240** 285***, 103, 75 243, 299, 445*** 179, 192, 310** 357, 299, 445*** 57, 71, 254** 292, 217, 305 265***, 280** 117, 298***, 283** 117, 285***, 300** 217, 191, 204 264***, 279** 81, 95, 266**

0.02 0.02 0.01 tr

0.08 0.01 0.27 0.28 0.04 0.15 0.53 0.41 0.01 tr 3.56 0.24 0.02 0.01 0.39 1.78 0.28 6.94 6.94 0.34 0.46 0.06 0.09 0.07

0.02 0.40 7.18

0.12 tr 0.59 0.45

204, 191, 217 117, 299***, 314** 218, 280, 382*** 75, 103, 299*** 79, 305***, 320** 79, 307***, 322** 204, 191, 217 117, 311***, 326**

6.89 0.10

3.47 0.44 0.32 0.10

4.16 0.92 21.74 3.99 5.64

J Appl Phycol (2009) 21:295306 Table 1 (continued) Compounds 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 11-Hexadecenoic acid (16:111) a, c Hexadecanoic acid (16:0) a, b, c UC Heneicosane a, b, c Inositol a, b, d Heptadecanoic acid (17:0) a, b, c Octadecanol a, b, c Phytol a, b Octadecanoic acid polyene (18:?5) 6,9,12-Octadecatrienoic acid (18:36,9,12) a, b, c 6,9,12,15-Octadecatetraenoic acid (18:46,9,12,15) c 9,12-Octadecadienoic acid (18:29,12) a, b, c cis-11-Octadecenoic acid (18:111) a, b, c trans-11-Octadecenoic acid (18:111) a, b, c Octadecanoic acid (18:0) a, b, c UC UM Tricosane a, b, c 4,8,12,16-Tetramethylheptadecane-4-olide a, b 2-O-Glycerol-D-galactopyranoside (floridoside) a, b, e Arachidonic acid (20:4 5,8,11,14) a, c 5,8,11,14,17-Eicosapentaenoic acid (20:55,8,11,14,17) c Eicosatrienoic acid (20:3) c Tetracosane a, b, c 1-Monotetradecanoylglycerol (14:0) a, c 11-Eicosenoic acid (20:111) a, b, c Eicosanoic acid (20:0) a, b, c Uridine a, b Pentacosane a, b, c 2-Monohexadecenoylglycerol (16:1) c UC 2-Monohexadecanoylglycerol (16:0) a, c 1-Monohexadecenoylglycerol (16:1) c Hexacosane a, b, c 1-Monohexadecanoylglycerol (16:0) a, b, c Docosanoic acid (22:0) a, b, c Adenosine a, b Sucrose a, b, d 2-Monooctadecenoylglycerol (18:1) c 1-Monooctadecenoylglycerol (18:1) a, c 1-Monooctadecanoylglycerol (18:0) a, b, c Tetracosenoic acid (24:1) c Squalene a, b Tetracosanoic acid (24:0) a, b, c UC UC Sterol A a, c 1-Monoeicosanoylglycerol (20:0) a, c Triacontane a, b, c Hexacosanoic acid (26:0) a, b, c -Tocopherol a -Tocopherol a, b Cholesterol a, b, c Brassicasterol a, c UC 24-Methylenecholesterol f Rt 15.03 15.23 15.34 15.92 16.34 16.67 16.87 17.23 17.31 17.47 17.59 17.76 17.86 17.97 18.26 18.36 18.69 19.08 19.99 20.16 20.28 20.51 20.61 20.66 20.86 21.07 21.36 21.78 22.17 23.03 23.29 23.30 23.54 23.67 23.80 24.30 24.70 25.26 25.81 26.28 26.51 26.76 26.95 27.08 27.58 28.19 28.50 29.12 29.18 29.71 29.94 31.22 31.29 31.86 31.96 32.57 Key ions (m/z)* 117, 311***, 326** 117, 313***, 328** 79, 91, 108 57, 71, 296** 305, 217, 191 117, 327***, 342** 327***, 75, 103 143, 123, 353*** 79, 91, 117 79, 335***, 350** 79, 333***, 348** 79, 337***, 352** 117, 339***, 354** 117, 339***, 354** 117, 341***, 356** Ether extract 0.37 12.29 0.28 0.02 0.01 0.08 0.74 0.06 0.34 0.78 0.31 0.69 1.39 0.70 0.15 0.13 0.02 2.68 12.44 0.05 0.10 0.36 0.04 1.51 0.11 1.05 1.30 2.05 0.05 2.94 0.11 11.22 0.20 1.33

299

Butanol extract

16.59

0.12 0.19 0.34

0.11 1.06 0.76 0.25

57, 71, 324** 99, 114, 324** 204, 337, 671*** 117, 361***, 376** 117, 359***, 374** 129, 363***, 378** 57, 71, 338** 343, 147, 431** 117, 367***, 382** 117, 369***, 384** 217, 259, 445*** 57, 71, 352** 129, 457***, 472** 129, 218, 459*** 369, 457***, 472** 57, 71, 366** 371, 459***, 474** 117, 397***, 412** 230, 445***, 460** 361, 217, 437 103, 339, 485*** 397, 485***, 500** 399, 487*** 117, 423***, 438** 69, 81, 410** 117, 425***, 440**

0.22 2.88 0.44 0.06 0.57 0.12 0.40 1.12 0.25 4.27 0.87

0.10 0.08 0.03 0.07 0.15 0.70 0.33 0.27 0.03 0.02 0.01 tr 2.63 0.07 0.10 3.33

1.33 0.61 0.56

380**, 255 427, 515*** 57, 71, 422** 117, 453***, 468** 151, 416** 502**, 237 329, 368, 458** 255, 380, 470** 314, 271, 398**

0.06 0.82 0.08 0.49 0.77 1.26

300 Table 1 (continued) Compounds 113 114 115 116 117 118 119 120 121 122 123 124 Campesterol a, b, c UC 1-Tetradecanoyl-2-hexadecenoylglycerol (14:0; 16:1) c 1-Tetradecanoyl-2-hexadecanoylglycerol (14:0; 16:0) c 1-Tetradecanoyl-3-hexadecenoylglycerol (14:0; 16:1) c 1-Tetradecanoyl-3-hexadecanoylglycerol (14:0; 16:0) c 1,2-Dihexadecenoylglycerol (16:1; 16:1) c 1-Hexadecanoyl-2-hexadecenoylglycerol (16:0; 16:1) c 1,2-Dihexadecanoylglycerol (16:0; 16:0) a, b 1,3-Dihexadecenoylglycerol (16:1; 16:1) c 1-Hexadecanoyl-3-hexadecenoylglycerol (16:0; 16:1) c 1,3-Dihexadecanoylglycerol (16:0; 16:0) a, b, c Rt 32.63 33.93 39.91 40.16 40.80 41.07 45.21 45.62 45.96 46.55 46.94 47.27 Key ions (m/z)* 342, 382, 472** 129, 145, 343, 343, 129, 129, 129, 369, 371, 371, 357, 129, 369, 371, 383, 385, 385, 385, 369, 385, 595*** 597*** 595*** 597*** 621*** 623*** 625*** 621*** 623*** 625***

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Ether extract 0.07 0.08 0.23 0.28 0.09 0.09 0.22 1.00 0.43 0.07 0.39 0.06

Butanol extract 0.04

* First key ion (m/z) is the base peak ** Molecular ion [M] + *** [M-15]+ ion UC Unidentified compound, UM unidentified monosaccharide Identification: a NIST Mass spectral library 2005 b The Golm Metabolome Database c Recursive procedure for metabolite assignment d Medeiros and Simoneit 2007 e Nagashima and Fukuda 1981 f Souchet and Laplante 2007 (as TMSi-derivative) and Gladu et al. 1991 (as non-derivatized compound) h The Lipid Library

procedure allowed for partitioning the diatom compounds into an apolar ether fraction (12% of the total diatom biomass) and a polar butanol fraction (5% of the original sample). Representative chromatograms of both the ether and butanol fractions are shown in Fig. 1. All the identified compounds and those still to be characterised (but comprising more than 0.2% of the total ion current, TIC) are included in the tables. The chemical pattern of the ether fraction consisted of FA (75.8%), glycerides (10.8%), sterols (4.7%), isoprenoid compounds (3.6%) as well as alkanes, fatty alcohols and phosphates (Table 2). It was characterized by FA from C10 to C26, dominated (expressed as percent of total FA) by C16 and C20: 16:0 (16.2%), 16:19 (29.2%), 16:46,9,12,15 (5.5%) and 20:55,8,11,14,17 (16.4%). Relatively high levels of 14:0 (9.5%) and 15:0 (9.1%) FA were found as well. FA pattern was dominated by saturated FA (SFA) (38.9%), followed by monounsaturated FA (MUFA) (32.1%) and PUFA (28.6%). The glyceride pattern was dominated by monoglycerides with a ratio mono-diglycerides of 2.8:1. The glycerol-esterified FA were 14:0, 16:0, 16:1, 18:0 and 18:1. The main esterified FA (presented as % of total glycerides) in monoglycerides are 16:0 (53.3%) and 16:1 (39.0%). The diglycerides were esterified mainly with 14:0 and 16:1 or 16:0 (24.3%) and 16:1 and/or 16:0 (75.7%) FA. Five sterols were detected in C. scutellum. 24-methylenecholesterol (51.9% of total sterols) was the main compound followed by sterol A (23.0%), brassicasterol (20.0%), cholesterol (3.4%) and campesterol (1.6%).

The profile of the butanol fraction is dominated by FA (44.6%) followed by low molecular carbohydrates (24.5%), amino acids and other N-containing compounds (9.5%), alcohols (8.8%), glycerides (4.4%), organic acids (2.5%),

Table 2 Metabolite groups of C. scutellum presented as % of TIC Compounds Amino acids and N-containing metabolites Organic acids Phosphates Fatty acids Saturated Monoenes Dienes Polyenes Alcohols Alkanes Carbohydrates Monosaccharides Disaccharides Glycerides Monoglycerides Diglycerides Sterols Isoprenoids Total identified compounds (% of TIC) Ether extract Butanol extract 9.51 2.51 0.10 44.59 29.30 13.76 1.53 8.81 24.54 23.67 0.87 4.35 1.93 2.42 1.17 95.58

0.27 75.82 29.47 24.32 0.31 21.72 0.28 0.47

10.8 7.95 2.85 4.74 3.55 95.93

J Appl Phycol (2009) 21:295306 Fig. 1 GC-MS chromatograms of the ether (A) and butanol fractions (B) of C. scutellum. The numbers are in accordance with Table 1. a diethyl ether stabilizers

301

56 58 41 78 a 91 108 112

123

B
6 a 41 58 76

93

isoprenoid compounds (1.2%), and phosphates (0.1%). GCMS analysis resulted in the identification of several monosaccharides (96.5% of total carbohydrates) and one disaccharide (sucrose, 3.5% of total carbohydrates). The most abundant low molecular carbohydrate was floridoside [2-O-glycerol-D-galactopyranoside (No. 76 in Table 1), 45.7% of all carbohydrates]. Amino acids (10 identified compounds) comprised 18.1% of all detected N-containing compounds, while the nucleosides adenosine and uridine together with urea were 44.9%, 4.7%, and 24.6%, respectively. Alcohols were represented mainly by glycerol (8.6% of the polar fraction). Several organic acids, including the phenolic hydroxycinnamic acid, monoglycerides, two isoprene compounds (tocopherol and phytol), and glycerol-3-phosphate, were also detected in trace amounts in the fraction. Aldehydes like 2,4-heptadienal, 2,4-octadienal and 2,4decadienal were not detected in the studied diatoms.

Discussion Preliminary studies indicated that the diethyl ether fraction of Cocconeis scutellum was the more active in provoking the sex reversal of H. inermis and apoptosis in human cancer cell lines (authors, unpublished data). The lipid chemistry of algae, and of diatoms in particular, relies mainly on analysis of the FA as FAME by GC-EI/MS (Mansour et al. 2005). In our work, the FA of both the ether and the butanol fractions were analyzed as FAME in parallel to their TMSi derivatives in order to obtain additional information using their specific MS fragmenta-

tion ( and ions) to determine the position of double bonds in PUFA (Christie 2003; Mjos and Pettersen 2003). In the EI/MS of FA, [M]+ and [M-15]+ for the TMSi derivatives and [M]+ and [M-31]+ for the FAME were easily distinguishable, and this permitted, together with the other characteristic ions, their unambiguous identification. In other cases, such as long-chain PUFA, these ions were hardly detectable and GC-MS analysis in CI mode was used to display their molecular weight (MW). The [M+1]+ of FA in CI/MS spectra was the most abundant ion (base peak), while in EI/MS spectra the [M]+ was decreasing with the increase of the number of carbons and double bonds. An example is given in Fig. 2 in which 16:46,9,12,15 and 16:29,12 co-eluted, and their separation, MS deconvolution, and identification in EI/MS mode was problematic due to the similarity of their fragmentation pattern, low amount of 16:29,12 and very low abundance of their [M]+ and [M15]+ ions. However, GC-CI/MS analysis provided unambiguous identification and separation by their base peak ([M+1]+ at m/z 321 for 16:46,9,12,15 and at m/z 325 for 16:29,12). Recently, such an approach was used for the analysis of FA in samples of fish origin (Dayhuff and Wells 2005). The FA composition of C. scutellum was similar to that published for other diatoms (Rousch et al. 2003; Pistocchi et al. 2005). The presence of the odd-chain 15:0 FA should be noted, which has been rarely reported for diatoms (Parrish et al. 1991; Gordon et al. 2006). The identification of mono- and diglycerides was based on comparison of their fragmentation pattern with reference compounds and defining their [M]+ (when detectable) and [M-15]+ ion fragments in EI/MS mode. The 1-

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m/z 321 m/z 325

m/z 323

[M+1]+

[M+29]+ [M+41]+

100

73

D
91 105 117 129 147 161 80 100 120 140 160 189 201 213 225 239 252 180 200 220 240 260 280 305 320 300 320

50 41 0 40 55

67

[M-15]+

[M]+

60

Fig. 2 GC-CI-MS separation of 16:46,9,12,15 ([M+1]+ at m/z 321), 16:29,12 ([M+1]+ at m/z 325) and 16:36,9,12 ([M+1]+ at m/z 323). TIC chromatogram (A), selected ion chromatogram (B), GC-CI-MS of 16:46,9,12,15 (C) and GC-EI-MS of 16:46,9,12,15 (D)

monosubstituted glycerides showed an intensive [M-103]+ (often as base ion) and detectable [M-15]+ and [M]+. The 2monosubstituted glycerides showed a base peak ion at m/z 103 or 129 and characteristic losses [M-15]+, [M-90]+, [M161]+ and [M-acyl]+. In contrast to EI/MS, the CI/MS spectra of 1- or 2-monosubstituted glycerides displayed similar fragmentation pattern with relatively intensive [M+ 1]+ ion, confirming their MW (Fig. 3). Diglycerides possessed relatively intensive [M-15]+ ion and characteristic fragmentation depending on the position of substitution as indicated in Fig. 4. The presence of triglycerides was not

confirmed by GC-MS as their boiling point would be above the highest temperature used in the elution program. The structures of sterols were confirmed by comparing both EI/MS of TMSi derivatives (together with RI) and non-derivatized compounds with those of reference compounds or literature data. Sterols determine the diatom value as food, which can affect predators growth, as reported in oysters (Patterson et al. 1993). Unlike higher plants, algae and microalgae contain a large diversity of sterols (Boutry et al. 1979; Veron et al. 1998) whose profiles can be sometimes characteristic of a particular

J Appl Phycol (2009) 21:295306

303

class, family, genus or even species, and thus used for chemotaxonomic and phylogenetic purposes (Volkman 1986; Patterson 1992). 24-methylenecholesterol (No. 112 in Table 1) was the principal sterol like in other diatoms (Gladu et al. 1991; Pistocchi et al. 2005). It showed a [M]+ at m/z 398, a base peak at m/z 314 (characteristic of the 24-methylene group), and an intensive ion at m/z 271 (loss of side chain) was identified in accordance with literature data (Gladu et al. 1991; Souchet and Laplante 2007). This compound was isolated from plant pollens, like that from Pyrus malus (Hgel 1962), and from the soft coral Sinularia sp. (Ahmed et al. 2006), and is a common constituent not only of algae, but also of clams, silkworms

and oysters. Sterol A (No. 103 in Table 1) showed equal MS spectra in both FAME fraction and TMSi-derivatized sample indicating absence of a hydroxyl group, a molecular ion at m/z 380 (confirmed by CI) and an intensive ion at m/ z 255 which is characteristic for 5,22 sterols (Souchet and Laplante 2007). In addition, [M-18]+, characteristic for compounds with hydroxyl group (at C-3), was not detected. Its MS was similar to that of an ergost-3,5,22trien structure. Several isoprenoid compounds were detected in the ether fraction, including a -lactone-4,8,12,16-tetramethylheptadecane-4-olide (No. 75 in Table 1), previously found in marine sediments (Rontani and Volkman 2005), - and -

A-1
[M-Acyl]
+

[M-161]+

[M-90]

[M-15]

[M-103]

B-1
[M-15]
+

A-2
[M+1]
+

B-2
[M+1]
+

Fig. 3 GC-EI-MS (A) and GC-CI-MS (B) of 1-monohexadecenoylglycerol (1) and 2-monohexadecenoylglycerol (2)

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A
[M-15]
+

B
[M-15]
+

385

A
OC14H27 O

211 OC14H27

211

343

OC16H31

TMSi

357

239

371

TMSi

OC16H31 239

Fig. 4 MS of 1-O-tetradecanoyl-2-O-hexadecanoylglycerol (A) and 1-O-tetradecanoyl-3-O-hexadecanoylglycerol (B)

tocopherols, found in many microalgae (Brown et al. 1999), phytol, and squalene (precursor of sterols). Relatively low amounts (<0.5% of total compounds) of phosphates (phosphoric acid, glycerol-2-phosphate, and glycerol-3phosphate), fatty alcohols (from C12 to C18) as well as nalkanes (from C16 to C30) were detected in the studied ether fraction. Despite the characteristic fragmentation and RI, the n-alkanes showed detectable [M]+ in EI mode. The metabolites in the butanol fraction (amino acids, Ncontaining compounds, organic acids, and saccharides) were identified by matching their mass spectra and RI with the databases and the literature (Medeiros and Simoneit 2007; Nagashima and Fukuda 1981) as indicated in Table 1. The FA composition is dominated by SFA and MUFA, while PUFA were considerably less abundant compared to

the apolar fraction (Table 2). Floridoside (No. 76 in Table 1), found to also be the dominant monosaccharide in other unicellular algae, showed under GC conditions distinguishable [M-15]+ at m/z 671, mass fragmentation pattern and RI identical to those reported in the literature (Nagashima and Fukuda 1981) and databases. Galactose, arabinose, glucose, talose and two unidentified carbohydrates were also found in the butanol fraction, as in other algae (Abdel-Fattah and Edrees 1977; Nagashima and Fukuda 1981). The high relative amount of N-containing compounds in the polar fraction (9.5%) was noteworthy. Aldehydes, responsible for teratogenic and abortive effects in planktonic copepods (dIppolito et al. 2002), were not detected in the studied diatoms. Therefore, this class of compounds is not involved in the sex reversal of H.

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305 Sinularia gibberosa. J Nat Prod 69:12751279 doi:10.1021/ np0601509 Benkeblia N, Shinano T, Osaki M (2007) Metabolite profiling and assessment of metabolome compartmentation of soybean leaves using non-aqueous fractionation and GC-MS analysis. Metabolomics 3:297305 doi:10.1007/s11306-007-0078-y Boutry JL, Saliot A, Barbier M (1979) The diversity of marine sterols and the role of algal biomasses; from facts to hypothesis. Experientia 35:15411543 doi:10.1007/BF01953179 Brown MR, Mular M, Miller I, Farmer C, Trenerry C (1999) The vitamin content of microalgae used in aquaculture. J Appl Phycol 11:247255 doi:10.1023/A:1008075903578 Buia MC, Gambi MC, Zupo V (2000) Structure and functioning of Mediterranean Seagrass ecosystem: an overview. Biol Mar Mediterr 7:167190 Christie WW (2003) Lipid analysis. The Oily Press, Bridgewater, England. http://www.lipidlibrary.co.uk/ms/ms01/index.htm Dayhuff LE, Wells M (2005) Identification of fatty acids in fishes collected from the Ohio River using gas chromatography-mass spectrometry in chemical ionization and electron impact modes. J Chromatogr A 1098:144149 doi:10.1016/j.chroma.2005.08.049 dIppolito G, Iadicicco O, Romano G, Fontana A (2002) Detection of short-chain aldehydes in marine organisms: the diatom Thalassiosira rotula. Tet Lett 43:61376140 dUdekem dAcoz C (1996) The genus Hippolyte Leach, 1814 (Crustacea: Decapoda: Caridea: Hippolytidae) in the East Atlantic Ocean and the Mediterranean Sea, with a checklist of all species in the genus. Zool Verh 303:1133 Fontana A, dIppolito G, Cutignano A, Miralto A, Ianora A, Romano G, Cimino G (2007) Chemistry of oxylipin pathway in marine diatoms. Pure Appl Chem 79:481490 Gambi MC, Lorenti M, Russo GF, Scipione MB, Zupo V (1992) Depth and seasonal distribution of some groups of vagile fauna of the Posidonia oceanica leaf stratum: structural and trophic analyses. PSZNI Mar Ecol 13:1739 Gavagnin M, Carbone M, Nappo M, Mollo E, Roussis V, Cimino G (2005) First chemical study of anaspidean Syphonota geographica: structure of degraded sterols aplykurodinone-1 and 2. Tetrahedron 61:617621 Gladu PK, Patterson GW, Wikfors GH, Chitwood DJ, Lusby WR (1991) Sterols of some diatoms. Phytochemistry 30:23012303 Gordon N, Neori A, Shpigel M, Lee J, Harpaz S (2006) Effect of diatom diets on growth and survival of the abalone Haliotis discus hannai postlarvae. Aquaculture 252:225233 Hokputsa S, Hu C, Paulsen BS, Harding SE (2003) A physicochemical comparative study on extracellular carbohydrate polymers from five desert algae. Carbohydr Polym 54:2732 Hgel MF (1962) tude de quelque constituants du pollen. Ann Abeille 5:97133 Ianora A, Miralto A, Poulet SA, Carotenuto Y, Buttino I, Romano G, Casotti R, Pohnert G, Wichard T, Colucci-DAmato L, Terrazzano G, Smetacek V (2004) Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom. Nature 429:403407 Lebeau T, Robert JM (2003) Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales. Appl Microbiol Biotechnol 60:612623 Mansour MP, Frampton DMF, Nichols PD, Volkmann JK, Blackburn SI (2005) Lipid and fatty acid yield of nine stationary-phase microalgae: Applications and unusual C24-C28 polyunsaturated fatty acids. J Appl Phycol 17:287300 Medeiros PM, Simoneit BRT (2007) Analysis of sugars in environmental samples by gas chromatography-mass spectrometry. J Chromatogr A 1141:271278 Miralto A, Ianora A, Poulet SA (1995) Food type induces different reproductive responses in the copepod Centropages typicus. J Plankton Res 17:15211534

inermis, according to in vivo experiments carried out by Zupo et al. (2007). However, the participation of other kinds of oxylipins cannot yet be excluded and further analyses of this topic should be performed. Bio-guided assays on H. inermis postlarvae with the active ether fraction and its subfractions are currently in progress in order to identify the compound(s) that are responsible, alone or synergistically, for the sex change in the model shrimp Hippolyte inermis. Parallel tests with diatom extracts on human cancer cell lines will also allow the establishing of the presence of a possible apoptotic activity on mammalian cells, in the perspective of eventual pharmacogical applications. In vitro and in vivo studies with C. scutellum extracts have also been carried out on commercially important crustaceans in order to evaluate their possible use in aquaculture.

Conclusions This paper represents not only one of the few reports about natural products from benthic diatoms, but the first chemical report about Cocconeis scutellum. It also adds information about apoptotic compounds deriving from benthic microalgae (Zupo et al. 2007). Moreover, our study demonstrates the advantage of GC-MS analysis for rapid and simultaneous identification of metabolites from various groups. As the method is proven to be highly reproducible, it could be used for studies on metabolite dynamics and distribution or influence of different abiotic factors on their synthesis. Further work on the chemistry of the benthic diatom Cocconeis scutellum may reveal a chemoecological functionality and reveal potential apoptotic compound(s) for application in different fields.
Acknowledgements This study was carried out within the PHARMAPOX project, supported by the European Community (FP62003NEST-A/STREP 4800). We are grateful to all the participants of the PHARMAPOX research team, Dr. A. Sagi (Ben Gurion University, Beer Sheva, Israel), Dr. S. Zupo (National Institute of Cancer, Genoa, Italy) and their groups, as well as Mr. S. Taboada (CEAB-CSIC, Blanes, Spain) The authors thank Generalitat de Catalunya (2005SGR-00020) for financial support and Dr. A. Marn of the Serveis Cientificotcnics, University of Barcelona (Faculty of Pharmacy) for performing the GC-MS analyses.

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