511

J. Phycol. 34, 511–514 (1998)

STEROLS OF SOME MARINE PRYMNESIOPHYCEAE1

Parthasarathi Ghosh, Glenn W. Patterson2

Department of Plant Biology, University of Maryland, College Park, Maryland 20742

and

Gary H. WikforsNational Oceanic and Atmospheric Administration (NOAA), National Marine Fisheries Service,

Northeast Fisheries Science Center, Milford, Connecticut 06460

ABSTRACT

Sterols were identified in six marine prymnesiophyte iso-lates, some of which appear to have value as bivalve food.The principal sterol in Pleurochrysis carterae (Milford#961) and an unidentified prymnesiophyte (CCMP1215)was 24-methylcholesta-5,22-dienol, a common sterol inprymnesiophytes. Isolates CCMP594, CCMP609, andCCMP459 contained either 24-ethylcholesta-5,22-dienol or24-ethylcholest-22-enol as the major sterol. In addition,Pavlova pinguis (CCMP609) and Pavlova sp.(CCMP459) contained the unusual dihydroxysterols 24-methylpavlovol and 24-ethylpavlovol, which have beenfound only in members of the Pavlovales. Prymnesiumparvum contained cholesterol without traces of other ste-rols. Compared to the other isolates, the quantity of sterolswas extremely low in P. parvum.

Key index words: 24-ethylcholest-22-enol; 24-ethylcholes-ta-5,22-dienol; 24-methylcholesta-5,22-dienol; cholesterol;dihydroxysterols; Ochrosphaera; Pavlova; pavlovols;Pleurochrysis; Prymnesiophyceae; Prymnesium; sterol

Several members of the algal class Prymnesiophy-ceae have been valuable as a food source in aqua-culture, especially for bivalves, and this class is ex-tensively represented in the marine environment.Pavlova lutheri and P. gyrans (Pavlovales) and severalstrains of Isochrysis (Isochrysidales) have been shownto promote excellent growth of juvenile oysters inthe laboratory. Studies that include a wider range ofphytoplankton have suggested that bivalve growthrates are related to the kind and amount of sterolspresent in the phytoplankton in the diet (Wikfors etal. 1991). Although the taxonomy of the Prymnesio-phyceae is still in a state of flux, using the treatmentof Chretiennot-Dinet et al. (1993) with the ordersPrymnesiales, Pavlovales, Isochrysidales, and Coccol-ithophorales, the type of sterol found in these or-ders appears to be distinctly correlated to taxonomicplacement. For example, the Prymnesiales containcholesterol as a principal sterol (Marlowe et al.1984), and the Pavlovaceae contain primarily D5,C29-sterols as principal sterols (usually 24-ethylcho-lesta-5,22-dienol) and also contain very unusual ste-

1 Received 9 September 1997. Accepted 20 December 1997.2 Author for reprint requests; e-mail GlennWP@AOL.com.

rols called pavlovols, which contain two hydroxylgroups (Gladu et al. 1991, Patterson et al. 1993,Volkman et al. 1997). These sterols have only beenfound in the Pavlovaceae. The most frequently ex-amined order within the Prymnesiophyceae is Iso-chrysidales (Patterson 1992), which appears to con-tain 24-methylcholesta-5,22-dienol as the principalsterol in all species except for those in the familyOchrosphaeraceae, where cholesterol is the princi-pal sterol in the two species examined (Marlowe etal. 1984). Coccolithus pelagicus, which contains 24-methylcholesta-5,22-dienol as its principal sterol, isthe only species examined for sterols in the Coccol-ithophorales. This report describes the amount andidentity of sterols present in six marine isolates fromthe Prymnesiophyceae. None of these isolates havebeen previously examined for sterol composition, al-though the sterol composition has been studied inother isolates of Pavlova pingus, Ochrosphaera verru-cosa, and Pleurochrysis carterae.

MATERIALS AND METHODS

Algal culture and harvest. Algal strains were obtained from theProvasoli-Guillard Center for the Culture of Marine Phytoplank-ton (CCMP), West Boothbay Harbor, Maine, except the strains‘‘Prym’’ and Milford #961, which were in the Milford MicroalgalCulture Collection (Northeast Fisheries Science Center, Milford,Connecticut). Strain designations and taxonomic affinities of theprymnesiophyte strains studied are shown in Table 1.

All algal strains were cultured in Milford seawater enriched with‘‘E medium’’ nutrients (Ukeles 1973). Strains ‘‘Prym’’ and Mil-ford #961 were grown in seawater diluted with distilled water to15-ppt salinity; all others were grown in 30-ppt salinity enrichedseawater. Algal biomass of all strains (except CCMP1215) forchemical analyses was produced in 20-L glass carboys that weremaintained with sterile technique, so that bacteria-free culturesremained so (CCMP459 and CCMP609 contained bacterial con-taminants on receipt, and no effort was made to remove the con-taminants). Carboy cultures were kept in a temperature-con-trolled room at 208 C with cool-white fluorescent lights that pro-vided 300 mmol photons·m22·s21 at the culture surface. Sterilizedair enriched with 3% carbon dioxide was provided to the cultures.Carboy cultures were operated semicontinuously by harvestingand replenishing one-half the total volume of 16 L every 7 days.Algal biomass for sterol analyses was harvested 5 days after themost recent harvest and dilution; we have found cultures har-vested on this schedule to be in an N-limited stationary phase. Anewly-isolated colonial prymnesiophyte from Antarctic waters,CCMP1215, was batch-cultured in Fernbach flasks within a 58 Clighted incubator; cultures for chemical analyses were harvestedafter about 5 weeks of growth.

Algal cells harvested for chemical analysis were concentrated bycold centrifugation (1000 3 g for 10 min); the pellet was resus-

512 PARTHASARATHI GHOSH ET AL.

TABLE 1. Strains of phytoplankton examined in this study.

Name Strain Order

Prymnesium parvum CarterPleurochrysis carterae (Braarud et Fagerlund) Christensena

Pleurochrysis carterae (Braarud et Fagerlund) ChristensenUnidentified colonialPavlova pinguis GreenPavlova sp. Butcher

PrymMilford #961CCMP594CCMP1215CCMP609CCMP459

PrymnesialesCoccolithophoralesCoccolithophoralesUnknownPavlovalesPavlovales

a Listed as Ochrosphaera verrucosa in the CCMP collection.

TABLE 2. Sterols of some prymnesiophytes (as percentage of total sterol). CCMP594 5 Pleurochrysis carterae, CCMP1215 5 unidentified prymesioph-yte, CCMP609 5 Pavlova pinguis, CCMP459 5 Pavlova sp., Milford #961 5 Pleurochrysis carterae, Prym 5 Prymnesium parvum, 11 5 notreported, NA 5 data not available (strain is colonial)

Sterol

Species

Prym Milford #961 CCMP594 CCMP1215 CCMP609 CCMP459 P. gyransa P. lutheria

Cholesterol 100 2 4 4 8 724-Methylcholesta-5,22-dienol 45 16 75Ergosterol 1624-Methylcholesterol 1 1 823,24-Dimethyl-cholesta-5,22-dienol 10 824-Ethylcholesta-5,22-dienol 35 51 1 27 56 29 304-Methylergostanol 524-Ethyl-cholest-22-enol 3 33 14 724-Ethylcholesterol 2 1 3 2 6 3 164-Methyl-24ethyl-cholest-22-enol 9 17 164-Methyl-24-ethyl-cholestanol 16Methylpavlovol 15 1 5 22Ethylpavlovol 13 4 19 4Total sterol (fg/cell) 1 104 99 NA 181 107 11 11

a Gladu et al. (1991).

pended in a minimal volume of isotonic NaCl, and the resus-pended sample was transferred into a lyophilizer vial. A subsam-ple of the material transferred into the lyophilizer vial was re-tained for microscope cell counts using a hemocytometer. Theconcentrated algal sample in the lyophilizer vial was frozen in abath of 2258 C methanol and lyophilized overnight. Lyophilizervials were melt-sealed while still under vacuum and stored in afreezer until shipment to the University of Maryland for sterolanalysis.

Sterol extraction and analysis. A known quantity of 7-stigmastenolacetate (24-ethylcholest-7-enyl acetate) was added to each lyoph-ilized algal sample as a quantitative internal standard before ex-traction with chloroform-methanol (2:1, v/v) in a Soxhlet appa-ratus overnight. Sterols were isolated and analyzed quantitativelyby capillary gas chromatography using the method of Ghosh etal. (1997) on a Varian 3500 gas chromatograph equipped with anSPI injector and a Varian 4400 integrator. The injector receivedthe sample at 958 C and was programmed to increase in temper-ature to 3008 C at 1808 C/min. The column was a 0.25-mm 3 30-m fused silica column with a 0.25-mm film of SPB-1 (Supelco,Bellefonte, Pennsylvania). During chromatography, the columnwas programmed from 1508–2608 C at 108 C/min and held for 15min. Retention times of steryl acetates relative to cholesteryl ac-etate were obtained on the same column that was run isother-mally at 2408 C. These data were used in conjunction with gaschromatography–mass spectroscopy for sterol identification. Massspectra were obtained with a Finnigan-MAT Model 4512 gas chro-matograph–mass spectrometer equipped with a 30-m 3 0.32-mmi.d. fused silica capillary column coated with a 0.25-mm film ofDB-1 (Supelco). Spectra were collected at 70 eV and a sourcetemperature of 1508 C. Data were recorded and analyzed with anINCOS Data System.

RESULTS AND DISCUSSION

The six marine prymnesiophyte isolates examinedcontained numerous sterols frequently encountered

in algae and marine invertebrates. The identity andrelative abundance of sterols from these marineprymnesiophytes are shown in Table 2. Cholesterol,24-methylcholesterol, ergosterol, 24-methylcholesta-5,22-dienol, 24-ethylcholesta-5,22-dienol, and 24-ethylcholesterol were identified on the basis of theirgas chromatographic retention times and mass spec-tra, which were essentially identical to those report-ed in the literature (Knights 1967, Patterson 1971).The principal mass spectral fragmentations and gaschromatographic relative retention times of the oth-er sterols identified are shown in Table 3. The spec-tra of 4-methylergostanol, 4-methyl-24-ethylcholest-22-enol, 4-methyl-24-ethylcholestanol, and 24-ethyl-cholest-22-enol, methylpavlovol, and ethylpavlovolwere essentially identical to those obtained from thesterols isolated from Pavlova gyrans (Gladu et al.1991, Patterson et al. 1993). 4-Methylsterols such asthese are common in the dinoflagellates (Piretti etal. 1997), but the dihydroxysterols of Pavlova haveyet to be identified in dinoflagellates (Patterson1992).

Prymnesium parvum was unusual in that it con-tained very low levels of sterol (1 fg·cell21), and onlycholesterol was detected (Table 2). The sterols ofonly two species have been examined in the Prym-nesiaceae; in both Chrysochromulina polypeptis andPrymnesium patellifera, cholesterol is also the princi-pal sterol (Marlowe et al. 1984). The only otherprymnesiophyte family reported to contain choles-

513PRYMNESIOPHYCEAN STEROLS

TABLE 3. Gas chromatographica and mass spectralb data for some prymnesiophyte sterols. 1 5 24-ethylcholest-22-enol, 2 5 4-methyl-24-ethylcholest-22-enol, 3 5 4-methyl-24-ethylcholestanol, 4 5 methylpavlovol, 5 5 ethylpavlovol

Fragmentation

Sterol

1 2 3 4 5

[M]1

[M2CH3]1

[M2H2O]1

[M233]1

[M243]1

[M259]1

[M243218]1

[M275]1

[M2(C222C29)]1

[M2SC(12H)]1

[M2SC118(12H)]1

[M2SC1D ring]1

[M2SC160]1

Base peakRRT

414 (20)

353 (14)

353 (16)

302 (27)273 (29)257 (25)233 (7)215 (13)

551.46

428 (22)

316 (28)287 (39)271 (27)245 (15)229 (11)

551.69

430 (55)415 (11)

397 (14)

290 (28)271 (5)247 (79)229 (95)

951.98

432 (15)417 (51)414 (8)399 (12)

373 (36)

357 (48)

952.04

446 (12)431 (57)428 (7)413 (12)

387 (38)

371 (44)

952.50

a Reported as the retention time of the steryl acetate relative to cholesteryl acetate at 1.00.b Reported as m/z (relative intensity).

terol as a principal sterol is Ochrosphaeraceae inthe Isochrysidales (two isolates; Marlowe et al.1984). Cholesterol is the principal sterol in wild oys-ters (Berenberg and Patterson 1981), but nearly 40other sterols are found in the oyster (Teshima andPatterson 1980). The oyster must get its sterols fromits diet since it is unable to synthesize sterols (Hold-en and Patterson 1991). Growth rates of culturedoysters showed the greatest positive response withincreases in dietary unsaturated fatty acids and ste-rols (Wikfors et al. 1991). Some high-cholesterolphytoplankton give the most rapid oyster growthrates reported, making cholesterol an attractivecomponent in phytoplankton being considered forfeeding oysters (Wikfors et al. 1996). However, inthe case of P. parvum, toxicity to finfish and shellfishprecludes its use as an aquaculture feed.

Isolates Milford #961 (Pleurochrysis carterae) andCCMP594 (P. carterae, but listed at CCMP as Ochros-phaera verrucosa) had a similar sterol composition(with the exception of the presence of ergosterol inCCMP594) in that both had 24-methylcholesta-5,22-dienol and 24-ethylcholesta-5,22-dienol as principalsterols (both common in prymnesiophytes; Patter-son 1992). In addition, both strains contained therather rare sterol 23,24-dimethylcholesta-5,22-dien-ol, found previously in algae only in the prymne-siophytes Hymenomonas carterae (P. carterae), (Volk-man et al. 1981, Marlowe et al. 1984), Ochrosphaeraneopolitana, and O. verrucosa (Marlowe et al. 1984);in the dinoflagellate Prorocentrum cordatum (Nicholset al. 1984, Robinson et al. 1984); and in the un-identified alga FCRG51 (Nichols et al. 1983). In aprevious report, cholesterol was reported to be theprincipal sterol in O. neopolitana and O. verrucosa(Marlowe et al. 1984).

Isolate CCMP1215 is a colonial prymnesiophyteisolated from Antarctica. It had 24-methylcholesta-5,22-dienol as its major sterol, whereas the rare

methyl sterol, 4-methyl-24-ethylcholestanol, com-prised 16% of the sterol fraction. However, this algadid not contain any of the dihydroxysterols that ac-company the 4-methyl sterols in Pavlova.

The sterols of Pavlova pinguis (CCMP609) andPavlova sp. (#459) are compared in Table 2 to thesterols of Pavlova gyrans and P. lutheri (Gladu et al.1991). All four isolates contained large amounts of24-ethylcholesta-5,22-dienol and both methylpavlo-vol and ethylpavlovol, which are dihydroxy sterolsthat have been found only in several species of Pav-lova (Gladu et al. 1991, Veron et al. 1996, Volkmanet al. 1997) and in Diacronema vlkianum, all from theorder Pavlovales (Volkman et al. 1997). IsolateCCMP459 has been considered to be a Dicrateria,Imantonia, or Pavlova (Internet website: http://ccmp.bigelow.org). Dicrateria has a simple sterolcomposition, with nearly all its sterol in the form of24-methyl-22-dehydrocholesterol (Gladu et al.1990). All Pavlova strains examined thus far containa complex mixture of C28, C29, and C30 sterols, in-cluding the unusual dihydroxysterols, called pavlo-vols. There have been no reports on the sterols ofImantonia. An examination of the sterols of Pavlovain Table 2 shows that although each contained pav-lovols, there were substantial differences betweenspecies and/or strains. For example, CCMP609 con-tained 33% (of total sterol) 24-ethylcholest-22-enol,which was not detected in P. lutheri. On the otherhand, P. lutheri contained 22% methylpavlovol,whereas CCMP 459 contained 1%. Using the inter-nal standard method of sterol analysis (Ghosh et al.1997), the standard error in these studies was lessthan 5%. From the above facts, sterol compositionis consistent with the current CCMP identificationof isolate CCMP459 as Pavlova.

More so than with most algal classes, members ofthe Prymnesiophyceae appear to show a strong cor-relation between sterol composition and taxonomic

514 PARTHASARATHI GHOSH ET AL.

position within the class. Prymnesium contains cho-lesterol, Pavlova contains dihydroxysterols, Isochrysiscontains 24-methylcholesta-5,22-dienol, and Coccoli-thus contains a mixture of 24-methylcholesta-5,22-dienol and 24-ethylcholesta-5,22-dienol (Patterson1992). As a consequence, sterol profiles may help tosolve some of the difficult taxonomic problems inthis class.

We thank Dawn Harrison for the mass spectra. This work wassupported by the Maryland Agricultural Experiment Station.

Berenberg, C. & Patterson, G. W. 1981. The relationship betweendietary phytosterols and the sterols of wild and cultivated oys-ters. Lipids 16:276–8.

Chretiennot-Dinet, M-J., Sournia, A., Ricard, M. & Billard, C.1993. A classification of the marine phytoplankton of theworld from class to genus. Phycologia 32:159–79.

Ghosh, P., Patterson, G. W. & Wikfors, G. H. 1997. Use of animproved internal standard method in the quantitative sterolanalyses of phytoplankton and oysters. Lipids 32:1011–4.

Gladu, P. K., Patterson, G. W., Wikfors, G. H., Chitwood, D. J. &Lusby, W. R. 1990. The occurrence of brassicasterol and epi-brassicasterol in the Chromophycota. Comp. Biochem. Physiol.97B:491–4.

Gladu, P. K., Patterson, G. W., Wikfors, G. H. & Lusby, W. R. 1991.Free and combined sterols of Pavlova gyrans. Lipids 26:656–9.

Holden, M. J. & Patterson, G. W. 1991. Absence of sterol biosyn-thesis in oyster tissue culture. Lipids 26:81–2.

Knights, B. A. 1967. Identification of plant sterols using combinedGLC/mass spectrometry. J. Gas Chromatogr. 5:273–82.

Marlowe, I. T., Green, J. C., Neal, A. C., Brassel, S. C., Eglinton,G. & Course, P. A. 1984. Long chain (n-C37-C39) alkenonesin the Prymnesiophyceae, distribution of alkenones and oth-er lipids and their taxonomic significance. Br. Phycol. J. 19:203–16.

Nichols, P. D., Jones, G. J., deLeeuw, J. W. & Johns, R. B. 1984.The fatty acid and sterol composition of two marine dino-flagellates. Phytochemistry 23:1043–7.

Nichols, P. D., Volkman, J. K. & Johns, R. B. 1983. Sterols andfatty acids in the marine unicellular alga, FCRG 51. Phyto-chemistry 22:1447–52.

Patterson, G. W. 1971. Relation between structure and retentiontime of sterols in gas chromatography. Anal. Chem. 43:1165–70.

1992. Sterols of Algae. In Patterson, G. W. & Nes, W. D.[Eds.] Physiology and Biochemistry of Sterols. American OilChemists Society, Champaign, Illinois, pp. 118–57.

Patterson, G. W., Gladu, P. K., Wikfors, G. H., Parish, E. J., Livant,P. D. & Lusby, W. R. 1993. Identification of two novel dihy-droxysterols from Pavlova. Lipids 28:771–3.

Piretti, V. M., Pagliuca, G., Boni, L., Pistocchi, R., Diamante, M.& Gazzotti, T. 1997. Investigation of 4-methyl sterols fromcultured dinoflagellate algal strains. J. Phycol. 33:61–7.

Robinson, N., Eglinton, G., Brassel, S. C. & Cranwell, P. A. 1984.Dinoflagellate origin for sedimentary 4a- methyl steroids and5a(H)-stanols. Nature 308:439.

Teshima, S. & Patterson, G. W. 1980. Sterols of the oyster Cras-sostrea virginica. Lipids 15:1004–11.

Ukeles, R. 1973. Continuous culture—a method for the produc-tion of unicellular algal food. In Stein, J. [Ed.] Handbook ofPhycological Methods: Culture Methods and Growth Measurements.Cambridge University Press, Cambridge, pp. 233–54.

Veron, B., Dauguet, J. C. & Billard, C. 1996. Sterolic biomarkersin marine phytoplankton. I. Free and conjugated sterols ofPavlova lutheri (Haptophyta). Eur. J. Phycol. 31:211–5.

Volkman, J. K., Farmer C., Barrett S. M. & Sikes E. L. 1997. Un-usual dihydroxysterols as chemotaxonomic markers for mi-croalgae from the order Pavlovales (Haptophyta). J. Phycol.33:1016–23.

Volkman, J. K., Smith, D. J., Eglinton, G., Forsberg, T. E. V. &Corner, E. D. S. 1981. Sterol and fatty acid composition offour marine haptophycean algae. J. Mar. Biol. Assoc. UK 61:509–27.

Wikfors, G. H., Gladu, P. K. & Patterson, G. W. 1991. In searchof the ideal diet for oysters: Recent progress with emphasison sterols. J. Shellfish Res. 10:292 (Abstract).

Wikfors, G. H., Patterson, G. W., Ghosh, P., Lewin, R. A., Smith,B. C. & Alix, J. H. 1996. Growth of post-set oysters, Crassostreavirginica, on high-lipid strains of algal flagellates Tetraselmisspp. Aquaculture 143:411–9.