Phycologia (2003) Volume 42 (4), 324-331 Published 4 September 2003

The biochemistry of dinoflagellate lipids, with particular reference to the fatty acid and sterol composition of a Karenia brevis bloom

JEFFREY D. LEBLOND1*, TERENCE J. EVANS2 AND PETER J. CHAPMAN3 'Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA "Southern Regional Research Center, Agricultural Research Service, United States Department ofAgriculture, New Orleans, LA 70124, USA "Ncuional Health Effects and Environment Research Laboratory, Gulf Ecology Division, United States Environmental Protection Agency, Gulf Breeze, FL 32561, USA

J.D. LEBLOND, T.J. EVENS AND PJ. CHAPMAN. 2003. The biochemistry of dinoflagellate lipids, with particular reference to the fatty acid and sterol composition of a Karenia brevis bloom. Phycologia 42: 324-331.

The harmful marine dinoflagellate, Karenia brevis (Dinophyceae), frequently forms large toxic blooms in the waters off the west coast of Florida (USA) and is responsible for massive fish kills and public health concerns. Despite decades of field studies on this organism, no investigation has yet characterized the lipid composition of a K. brevis bloom. To address this lack of information, samples from a 1999 K. brevis bloom from the north-west Florida coast were analysed for their fatty acid and sterol composition. Fatty acids found in different lipid fractions containing membrane phospholipids, chloroplast­ associated glycolipids or storage triglycerides differed significantly. The glycolipid fraction was found to contain octade­ capentaenoic acid [l8:5(n-3)], a fatty acid commonly associated with dinoflagellates. The phospholipid fraction was found to contain small amounts of two recently described, highly unsaturated fatty acids, octacosaoctaenoic acid [28:8(n-3)] and octacosaheptaenoic acid [28:7(n-6)]. Fatty acids from the triglyceride fraction were more abundant than those associated with glycolipids or phospholipids. Sterols were found mainly as free sterols and were dominated by two compounds, (245)­ 4a-methyl-5a-ergosta-8(l4),22-dien-313-ol and its 27-nor derivative. The lipid composition of these samples very closely resembles laboratory-grown cultures .of K. brevis and serves to provide an in situ field validation of past laboratory exam­ inations of this organism. The implications of our data are discussed in the context of the physiological autecology of K. brevis, in the form of a minireview on the biochemistry of dinoflagellate lipids, as studied in both the laboratory and the environment.

INTRODUCTION effects of environmental pressures, such as temperature, irra­ diance, and nutrient status, upon lipid and fatty acid content, Of the more than 30 toxic microalgal species found in the and how this relates to a host of cellular processes, such as Gulf of Mexico, only one frequently produces massive fish photosynthesis, reproduction, and nutrition. Although several kills on both temporal and spatial scales (Halligan 1985; Var­ studies, particularly in the field of aquaculture, have examined go et al. 1987; Steidinger et al. 1998). The causative microa1­ the effects of changes in environmental conditions upon the ga is the Florida red-tide toxic dinoflagellate, Karenia brevis lipid composition of economically important microalgae (Pohl (Davis) Gert Hansen & Moestrup [a new combination for & Zurheide 1979; Parrish & Wangersky 1987; Siron et al. Gymnodinium breve Davis (Daugbjerg et al. 2000)]. The suite 1989; Hodgson et al. 1991; Ahlgren et al. 1992; Thompson of toxins - 'brevetoxins' - produced by K. brevis is respon­ et al. 1992; Dunstan et al. 1993; Reitan et al. 1994; Lombardi sible for cases of neurotoxic shellfish poisoning (NSP), human & Wangersky 1995; Bell & Pond 1996; Fidalgo et al. 1998; respiratory problems, fish kills, and marine mammal mortali­ Lopez Alonso et al. 1998, 2000), very few of these investi­ ties (Viviani 1992; Bossart et al. 1998). The economic and gations have been concerned with toxic dinoflagellates such public health impacts produced by the release of brevetoxins as K. brevis (cf. Parrish et al. 1993, 1994). This is in stark during blooms that may persist for weeks to months can be contrast to the wealth of information available on how terres­ quite significant (Riley et al. 1989; Shumway et al. 1990; trial plant and cyanobacterial fatty acid-containing lipids alter Tester & Fowler 1990; Hallegraeff 1993; Anderson et al. in response to changing environmental conditions (e.g. Gom­ 2000). Consequently, the biogeography of K. brevis has been bos et al. 1992, 1994; Kanervo et al. 1997; Wada & Murata the focus of numerous studies (Marshall 1982; Churchill & Cornillon 1991; Tester & Steidinger 1997). However, it is only 1998; Routabou1 et al. 2000). recently that the physiological autecology of this dinoflagel­ Microalgae of the class Dinophyceae have been document­ late species has been examined in any detail (cf. Paerl 1988; ed extensively as producers of an impressive array of lipo­ Shanley & Vargo 1993; Smayda 1997; Evens et al. 2001); philic chemicals not commonly found in other classes of eu­ thus, essential gaps exist in our understanding of the funda­ karyotic microalgae. These include a substantial number of mental biochemistry and physiology of K. brevis. toxins (Shimizu 1993; Plumley 1997; Rein & Borrone 1999), One of the largest of these gaps is in our knowledge of the pigments (Johansen et al. 1974; Jeffrey et al. 1975; Jeffrey & Vesk 1997), sterols and steroidal compounds (Withers 1983, Corresponding author (jleblondts'mtsu.edu). 1987; Kerr & Baker 1991; Volkman et al. 1998), and fatty Leblond et al.: Karenia brevis bloom lipids 325

acids found as components of acylglycerolipids (see below). eluding many unarmoured species closely related to K. brevis Of these classes of compounds, toxins, particularly brevetox­ [on the basis of morphological and 18S ribosomal DNA se­ ins, are perhaps the most studied, because of their often lethal quence comparisons (Haywood et al. 1996; Daugbjerg et al. effects on aquatic animals and the hazards to human health 2000; Tengs et al. 2001; J. Leblond, unpublished observa­ associated with consumption of toxin-contaminated shellfish. tions)], has shown that ED and NED are of a very limited However, analysis of brevetoxins in water or shellfish samples distribution in this algal class (Leblond & Chapman 2002). is an intricate and often difficult process, which requires high­ They are produced as primary sterols in the morphologically pressure liquid chromatography-electrospray ionization mass similar dinoflagellates, Karenia mikimotoi and Karlodinium spectrometry (Hua et al. 1995, 1996; Pierce & Kirkpatrick micrum [formerly known as Gymnodinium mikimotoi and 2001) because of the high molecular weights and low con­ Gymnodinium galatheanum, respectively: Daugbjerg et al. centrations of these compounds. The K. brevis 'signature' pig­ (2000)], and are found at very low levels as part of complex ment, gyroxanthin-diester, which is found in K. brevis and a sterol profiles in a small number of other unarmoured dino­ few closely related unarmoured dinoflagellates (Johansen et flagellates. They were not found in any of the armoured di­ al. 1974; Jeffrey et al. 1975; Bjornland & Tangen 1979; noflagellates examined. To date, ED and NED have not been Bjornland 1990; Johnsen & Sakshaug 1993; Millie et al. 1995, observed in situ as part of a K. brevis bloom. 1997), has been used in the tracking of this organism in the The intention of this report is to present an initial charac­ environment, and thus may be useful as an indicator of a high terization of the fatty acids and sterols associated with a brevetoxin contamination risk. bloom of K. brevis that occurred off the north-west Florida When one considers the environmental importance of K. coast in the autumn of 1999. The results presented herein will brevis and the number of field studies conducted during the be related to information obtained from previous studies of past several decades (cf. Steidinger et al. 1998), it is some­ dinoflagellate blooms and from laboratory studies with K. what surprising that in situ lipid composition (i.e. fatty acids brevis and other dinoflagellates. and sterols) of K. brevis has not been employed as an addi­ tional means of better understanding its physiological and chemical ecology. This is in contrast to a number of studies MATERIAL AND METHODS that have characterized the fatty acid or sterol composition of water bodies in order to show seasonal heterogeneity of algal Collection of samples from K. brevis bloom populations and physiological changes in the water column On 29 September 1999, a short, opportunistic sampling ex­ organisms during periods of low production or bloom decay cursion was made into a large bloom of K. brevis off the (Mayzaud et al. 1989; Fahl & Kattner 1993; Pond et al. 1998; north-west coast of Florida. Limited access to ship time al­ Najdek et al. 2002). Such information can be obtained from lowed only two sites to be sampled on this date; therefore, the size-fractionated samples as a means of elucidating trophic information presented in this report is expected to represent relationships (Harvey & Johnston 1995; Phleger et al. 1998; the minimum chemical variability of a K. brevis bloom. Site Cotonnec et al. 2001). 1 was located immediately offshore from Navarre Beach The paucity of knowledge concerning the in situ lipid (30 022'40.3"N, 86°51 '21.5"W), and Site 2 was located imme­ chemistry of K. brevis is also remarkable because there have diately offshore from Pensacola Beach (30022'38.5"N, recently been studies of several other dinoflagellates, which 86°51'18.1"W). Surface samples were collected in 4-litre bot­ have broadened the information already accumulated from de­ tles and returned to the laboratory for processing (see below). cades of study on the fatty acid and sterol biochemistry of Examination of the samples using microscopy and flow cy­ laboratory-cultured species. For example, Coolia monotis has tometry revealed the concentrations of K. brevis to be c. 2 X been reported to possess a novel ceramide containing a CIS 107 cells 1-1 at both sites, with an extremely low incidence of fatty acid with a branch point at the C 15 position (Tanaka et other co-occurring phytoplankton or zooplankton species (F. al. 1998; Akasaka et al. 2000). In addition, Mansour et al. Genthner, United States Environmental Protection Agency, (1999a, b) have recently discovered two novel fatty acids in personal communication). Water temperature and salinity at a number of laboratory-cultured dinoflagellates. These com­ both sites were 28.3°C and 34.5%0, respectively. pounds, octacosaoctaenoic acid [28:8(n-3)] and octacosahep­ taenoic acid [28:7(n-6)], have not been reported in any other Lipid extraction, fractionation, derivatization, and gas class of eukaryotic microalgae. Since their discovery, they chromatography-mass spectrometry (GC-MS) analysis have been observed in the nonphotosynthetic, heterotrophic dinoflagellate, Crypthecodinium cohnii (Van Pelt et al. 1999) Cells were harvested using filtration through precombusted and have been shown to be associated with cellular membrane Whatman 934-AH glass fibre filters. Such filters, with a nom­ phospholipids, but not chloroplast-derived glycolipids (Leb­ inal pore size of 1.5 p.m, are unlikely to harvest any free­ lond & Chapman 2000). Field observations of 28:8(n-3) and floating prokaryotes associated with the K. brevis bloom. The 28:7(n-6) have not been reported to date. retentate was freeze-dried and kept frozen at -80°C before The field of dinoflagellate sterol biochemistry has also wit­ lipid extraction. Extraction of lipids from glass fibre filters was nessed exciting advances in recent years. Two novel sterols, performed according to a modified Bligh and Dyer extraction (24S)-4a-methyl-5a-ergosta-8(14),22-dien-3~-ol(ED) and its (Guckert et al. 1985). The total lipid extracts were separated 27-nor derivative (NED), not previously described in the di­ into five component lipid fractions on columns of activated verse array of sterols already known to be produced by di­ Unisil silica (1.0 g, 100-200 mesh, activated at 120°C; Clark­ noflagellates, were found in K. brevis by Faraldos & Giner son Chromatography, South Williamsport, PA, USA) accord­ (1998). A subsequent survey of over 40 dinoflagellates, in- ing to the solvent regime of Leblond & Chapman (2000), viz. 326 Phycologia, Vol. 42 (4), 2003

16:0 Table 1. Relative abundance (in % of total peak area) of selected fatty acid methyl esters (FAMEs) derived from glycolipids (Glyc), phos­ j A. pholipids (Phos), and triglycerides (Tri).' 3000000J 1 14:0 Site I Site 2 1 I 2000000j FAME RTI Glyc. Phos. Tri. Glyc. Phos. Tri. 18 series 14:0 17.0 13.1 2.6 16.1 6.4 1.2 15.5 J 16: 1 (n-7) 21.2 2.0 0.6 6.0 1.8 tr 5.0 1000000j 16:0 21.9 41.4 34.4 39.8 61.1 39.1 39.2 18:5 (n-3) 26.7 16.1 2.0 3.4 3.0 tr 5.8 I 18:4 (n-3) tr 2.3 1 1 26.8 2.9 0.7 3.0 tr 1 11. 18:2 (n-6) 2.2 105j-1.o:r:0"r-r-.,,-~c:ry~~~~~r-rl-c-f~~,.,-~~~~Idl 27.1 2.9 0.8 3.1 tr 25'.00 45'.00 55'00 18: 1 (n-9) 27.5 12.0 4.0 22.6 16.5 6.3 19.4 18:1 (n-7) 27.6 0.5 tr tr 16:0 2.5 18:0 28.4 1.3 12.5 2.5 tr 16.8 2.8 3000000 20:5 (n-3) 33.1 tr 0.5 0.8 tr 0.7 B. 20:0 35.7 0.6 15.3 0.3 20.6 0.9 Q) 22:6 (n-3) 40.3 6.5 24.4 3.8 5.6 16.2 5.9 til l: 24:0 48.9 0.3 tr tr tr o 2000000 28:7 (n-6) 22:6 54.2 0.5 tr Co 28:8 (n-3) 54.4 0.5 tr tr til Q) 18 series 20:0 28:0 55.7 0.3 0::: Others 0.7 0.6 1.7 0 0 0.3 1000000 I RT, retention time in minutes; tr, less than 0.2%; blank spaces, not detected. 28 series 15.00 25.00 35.00 45.00 55.00 Leblond & Chapman (2000). Derivatization and GC-MS anal­ 16:0 yses of sterols (as their trimethylsilyl ethers) found in the ste­ 9000000 rol ester and free sterol fractions, respectively, were performed 14:0 c. according to the procedures described in Leblond & Chapman (2002).

6000000 18 series RESULTS

3000000 Fatty acids associated with glycolipids, phospholipids, 22:6 and triglycerides

I j To assess the relative distribution (according to lipid class) of 25.00 35.00 45.00 55.00 fatty acids found as components of acylglycerolipids, an in­ terfraction comparison was made of the total FAME peak ar­ Retention time (min) eas for fractions 2, 4, and 5, which contained triglycerides, Fig. 1. Fatty acid methyl ester (FAME) profiles from Site 1. Panel A glycolipids, and phospholipids, respectively. At both sampling ~ FAMEs derived from the glycolipid fraction. Panel B- FAMEs sites it was observed that the majority of the fatty acids were derived from the phospholipid fraction. Panel C- FAMEs derived derived from the fraction containing triglycerides. In the Site from the triglyceride fraction. I sample, 72.6% of the fatty acids were found in this fraction, whereas 13.5% and 13.9% of the fatty acids were derived (1) 12 ml methylene chloride (for sterol esters); (2) 15 mlof from the glycolipid and phospholipid fractions, respectively. 5% acetone in methylene chloride with 0.05% acetic acid (for The Site 2 sample displayed a similar distribution, with 86.2% free sterols, tri- and diacylglycerols, and free fatty acids); (3) of the fatty acids found in the triglyceride fraction and 7.6% 10 ml of 20% acetone in methylene chloride (for monoacyl­ and 5.8% of the fatty acids derived from the glycolipid and glycerols); (4) 45 rn1 acetone [for monogalactosyldiacylgly­ phospholipid fractions, respectively. This predominance of tri­ cerol (MGDG), digalactosyldiacylglyccrol (DGDG), and sul­ glyceride-associated fatty acids in both samples is presented foquinovosyldiacylglycerol (SQDG)]; and (5) 15 ml methanol as uncorrected, direct observations from the GC-MS chro­ with 0.1 % acetic acid (for phospholipids). Dinoflagellates of­ matograms; however, it is still evident after correction for the ten have abundant amounts of triacylglycerols (referred to as higher molar ratio of fatty acids associated with triglycerides triglycerides) (Parrish et al. 1993, 1994; Myher et al. 1996), (3 mol-i) than glycolipids or phospholipids (2 mol-i). Con­ but diacylglycerols (diglycerides) are either absent or are pre­ version of the triglyceride-associated fatty acid peak areas to sent in amounts below detection limits. Therefore, fraction 2 2 fatty acids mol" yields 18.4% and 18.0% for glycolipid­ is referred to in the text as the triglyceride fraction. and phospholipid-derived fatty acids, respectively, in the Site

$:: Derivatization and GC-MS analyses of fatty acids [as their I sample, and 10.7% and 8.2% in the Site 2 sample. No ap­ fatty acid methyl esters (FAMEs)] associated with triglycer­ preciable amount of FAMEs was found in fractions I or 3. ides, glycolipids (MGDG, DGDG, and SQDG), and phospho­ An examination of the fatty acid composition of the tri­ lipids were performed according to the methodology used by glyceride fraction (Fig. I, Table I) revealed that the most I~ I ~._...._------Leblond et al.: Karenia brevis bloom lipids 327

Table 2. Relative percentages of sterols found as free sterols and sterol esters.'

Molecular Suggested structure Weight'RT Site 1 Site 2 cholest-fi-cn-Sji-ol (cholesterol) 458 tr 27-nor-(245)-4a-methyl-So-ergosta-8( 14),22-dien-313-01 (NED) 470 17.4 (tr) 10.8 (18.3) cholesta-7,22E-dien-313-01 (stellasterol) 470 2.2 (tr) tr unidentified C28 sterol with two double bonds 470 2.1 (tr) (245)-40'-methy1-5a-ergosta-8(14),22-dien-313-01 (ED) 484 76.2 (100) 89.2 (81.8) unidentified C29 sterol with three double bonds 482 2.0 tr

1 Sterol ester relative percentages are shown in parentheses. RT, retention time in minutes; tr, less than 0.5%; blank spaces, not detected. 2 Molecular weight of sterols as their trimethylsilyl ether derivatives.

abundant compound was hexadecanoic acid (16:0); it consti­ amounts of 0.5% each in the Site 1 sample, and as trace tuted 39.8% and 39.2% of the triglyceride-derived fatty acids amounts in the Site 2 sample. Also present in the phospholipid at Sites 1 and 2, respectively. Other dominant fatty acids were fraction of the Site 1 sample was octacosanoic acid (28:0). octadecenoic acid [18:1(n-9)], which constituted 22.6% and This compound, found as a low proportion (0.3%) of the total 19.4% in Sites 1 and 2, respectively, and tetradecanoic acid FAMEs, was not observed in a previous survey of dinofla­ (14:0), which constituted 16.1 % and 15.5% in Sites 1 and 2, gellate phospholipid fatty acids (Leblond & Chapman 2000). respectively. Both sites were characterized by a small amount of octadecapentaenoic acid [18:5(n-3)]. A trace amount of oc­ Sterols found as free sterols and sterol esters tacosaoctaenoic acid [28:8(n-3)] was also observed in this fraction from the sample from Site 1. Fatty acids of bacterial A comparison of the distribution of sterols present as free sterols vs those present as sterol esters revealed that free ste­ origin, such as CIS or C l7 compounds, were either not detected or found at trace levels in this and other fractions. rols were the predominant form. In the Site 1 sample, 98.2% The fatty acid composition of the glycolipid fraction (Fig. of the sterols were found as free sterols (fraction 2), and the 1, Table 1), which is indicative of the fatty acid composition remaining 1.8% were found as sterol esters (fraction 1). Site of the chloroplast (Harwood 1998), was dominated by 16:0. 2 showed a similar higher abundance of sterols as free sterols It accounted for 41.4% and 61.1 % of the fatty acids in this (92.9%) than of those found as sterol esters (7.1%). fraction from Sites 1 and 2, respectively. In the sample from The free sterol composition of samples from both sites (Ta­ Site 1, the next most abundant fatty acid was 18:5(n-3), which ble 2) was dominated by two compounds, ED and NED. In accounted for 16.1 %. This is in contrast to the sample from the Site 1 sample, ED and NED represented 76.2% and Site 2, where 18:5(n-3) accounted for only 3.0%. Octadeca­ 17.4%, respectively, and in the Site 2 sample, 89.2% and tetraenoic acid [18:4(n-3)], which is often found along with 10.8%. Both sites were also characterized by a small number 18:5(n-3) in the glycolipids of dinoflagellates (Parrish et al. of minor sterols (approximately 2% or less) in the free sterol 1998; Leblond & Chapman 2000), was present at only 2.9% fraction. The unidentified C 28 and C 29 sterols do not appear to in the Site 1 sample and. as a trace amount in the Site 2 sam­ correspond to unidentified sterols previously observed in cul­ ple. In the Site 2 sample, 18:1(n-9) accounted for 16.5%; this tured K. brevis (Leblond & Chapman 2002). particular fatty acid was also a prevalent component of the Site 1 sample (12.0% of the fatty acids). The Site 1 sample also had a higher relative amount of 14:0 than Site 2 (13.1 % DISCUSSION vs 6.4%). The phospholipid fraction of both samples (Fig. 1, Table 1) Harmful algal blooms (HABs) formed by toxic dinoflagel­ was found to be dominated by 16:0; this particular fatty acid lates, particularly K. brevis, have received scant attention with was the major component of all three fractions examined. In respect to their lipid composition. This is in stark contrast to the Site 1 sample, it accounted for 34.4% of the fatty acids, the amount of information available concerning the toxins and and in the Site 2 sample it accounted for 39.1 %. This fraction pigments in cultured and natural populations of K. brevis (cf. also possessed a higher relative amount of docosahexaenoic Millie et al. 1997; Steidinger et al. 1998; Daranas et al. 2001). acid [22:6(n-3)] (24.4% and 16.2% in Sites 1 and 2, respec­ This lack of detailed lipid data is surprising for two reasons. tively) than either the triglyceride or glycolipid fractions. The Firstly, K. brevis is arguably one of the most environmentally phospholipid fraction also had much higher relative amounts and economically important dinoflagellates. In order to predict of eicosanoic acid (20:0) (15.3% and 20.6% in Sites 1 and 2, the dynamics of bloom initiation, maintenance, and decline, it respectively) and octadecanoic acid (18:0) (12.5% and 16.8% is necessary to understand the physiological autecology of this in Sites 1 and 2, respectively) than either of the other two organism. Assessment of the fatty acid and sterol composition fractions. Conversely, the phospholipid fraction had a much of intracellular lipid classes within a bloom is technically rel­ lower relative amount of 18:1(n-9) (4.0% and 6.3% in Sites atively easy and complements more standard oceanographic 1 and 2, respectively) than the other fractions examined. measurements, such as nutrient concentration and pigment The phospholipid fractions from both sites were distin­ composition; together, such data give a more holistic descrip­ guished by the presence of two highly unsaturated C 28 fatty tion of the phytoplankton community (the Introduction cites acids, octacosaoctaenoic acid [28:8(n-3)] and octacosaheptae­ examples). Secondly, study of laboratory cultures of dinofla­ noic acid [28:7(n-6)]. These compounds were found in relative gellates has revealed numerous examples of novel lipid com- 328 Phycologia, Vol. 42 (4), 2003 ponents for which there have been few or no field observa­ age) would have been overlooked. Hence, an important insight tions. By the same token, much is still to be learned about into the physiological status of the cells within a bloom might fatty acid biosynthesis using clonal cultures. For example, the also have gone unrecognized. This would be true for other structure of 18:5(n-3) was confirmed by Joseph (1975) for the studies, such as that of Napolitano et al. (1995), if triglycer­ dinoflagellate Prorocentrum minimum. Since that time, 18: ides were found to be a major lipid class. The abundance of 5(n-3) has been observed as a major glycolipid-associated fat­ triglyceride-derived fatty acids in the K. brevis bloom samples ty acid in many cultured Dinophyceae (Parrish et al. 1998; described here contrasts with that reported in an earlier com­ Mansour et al. 1999b; Leblond & Chapman 2000), Hapto­ munication (Leblond & Chapman 2000) for a cultured K. phyceae (Napolitano et al. 1988; Renaud et al. 1991; Oku­ brevis strain. An interfraction comparison has shown that this yama et al. 1992; Bell & Pond 1996), and Raphidophyceae laboratory-cultured strain yielded approximately 12% of the (Nichols et al. 1987; Bell et at. 1997; Mostaert et al. 1998). total fatty acids as products of triglycerides (J. Leblond, un­ It has been observed in a bloom dominated by species of the published data). The glycolipid and phospholipid fraction dinoflagellate genus Ceratium (Mayzaud et at. 1976); how­ yields were 34% and 54% of the total fatty acids, respectively. ever, 18:5(n-3) is seldom found as a major fatty acid in other Parrish et at. (1994) have noted the presence of triglycerides reported dinoflagellate blooms. Napolitano et al. (1995), in an as a significant lipid class in cultured Gymnodinium cf. na­ examination of a freshwater bloom of Peridiniopsis penardii, gasakiense. reported very low levels (less than 3% of total fatty acids) of The major components of the phospholipid fraction from both 18:5(n-3) and 18:4(n-3). The fatty acid composition of the bloom samples described here were 16:0 and 22:6(n-3), this bloom was dominated by the common dinoflagellate fatty indicating that these two fatty acids constitute much of the acids, hexadecanoic acid (16:0), eicosapentaenoic acid [20: phospholipid bilayer in the cellular membrane of K. brevis. 5(n-3)], and docosahexaenoic acid [22:6(n-3)]. Shamsudin The relative amount of 22:6(n-3) was much higher in this (1996) did not find 18:5(n-3) in a bloom dominated by the fraction than in either the glycolipid or the triglyceride frac­ dinoflagellate Peridinium quinquecorne. The lack of fraction­ tions. In addition, two fatty acids, 18:0 and 20:0, were found ation of component lipid classes in these studies may partially at much higher levels in the phospholipid fraction than in the explain the relatively low abundance of this fatty acid within other two fractions. These results roughly correspond to the their samples (see discussion below). However, some photo­ phospholipid-derived fatty acid composition of cultured K. trophic dinoflagellates appear to produce little or no 18:5(n­ brevis described in Leblond & Chapman (2000). 3), even in culture (Viso & Marty 1993; Carballeira et al. The few previously-mentioned studies of the lipid compo­ 1998; Leblond & Chapman 2000). This may reflect an ab­ sition of dinoflagellate blooms predate the identification of 28: sence of some key enzyme in the 18:5(n-3) biosynthetic path­ 8(n-3) and 28:7(n-6) by Mansour et al. (1999a, b). In the way, or a set of environmental conditions where synthesis is samples from the K. brevis bloom, these two fatty acids were repressed. Presently, it is unclear as to why some photosyn­ found at low relative amounts in the phospholipid fraction. thetic dinoflagellates do not produce this fatty acid. This association with phospholipids corresponds to previous Karenia brevis in culture has been observed to produce 18: observations by Leblond & Chapman (2000). However, the 5(n-3) as a constituent of its glycolipids (Leblond & Chapman laboratory culture of K. brevis examined by Leblond & Chap­ 2000). Within the K. brevis bloom assayed in this work, 18: man did not appear to produce these two compounds, which 5(n-3) constituted approximately 16% of the fatty acids of the could be a result of culturing conditions. The physiological glycolipid fraction in the Site 1 sample (Table 1) and was the significance of these minor compounds is still unclear. The second most abundant fatty acid in this fraction. However, in presence of 28:0 in the Site 1 sample could indicate a pre­ the Site 2 sample, 18:5(n-3) constituted only 3% of the gly­ cursor of these highly unsaturated C 28 fatty acids; however, it colipid fatty acids. Thus, it is unclear how environmental con­ has yet to be reported in laboratory cultures of dinoflagellates. ditions in different bloom patches may affect production of Fractionation of sterols into free sterols and sterol esters this fatty acid. When the fatty acids of the glycolipid, phos­ has shown that the majority of the sterols (>90%) were pre­ pholipid, and triglyceride fractions (total acylglycerolipids) sent as free sterols in samples from both sites. This corre­ are considered together (as these fatty acids would be in stud­ sponds to recent observations on laboratory cultures of K. ies where only a total, unfractionated lipid extract is exam­ brevis (Leblond & Chapman 2002). The free sterols from both ined), 18:5(n-3) represents only 2.2% of all fatty acids present sites contained two principal sterols, ED and NED, which cor­ in the Site I sample; this is largely because of the high abun­ responds closely with results obtained from laboratory cul­ dance of fatty acids derived from the triglyceride fraction tures of K. brevis (Faraldos & Giner 1998; Leblond & Chap­ (72.6% of total fatty acids, discussed below). This low per­ man 2002). The production of only two primary sterols in centage of 18:5(n-3) is similar tothat observed in the Peri­ both laboratory cultures and during a natural bloom is unusual diniopsis penardii bloom by Napolitano et al. (1995). Thus, for a member of the Dinophyceae. A review of the literature the role of 18:5(n-3), which has been observed as an important shows that many dinoflagellates generally produce a more component of the lipids of the chloroplast membranes in many complex profile of sterols, such as that observed in a bloom cultured dinoflagellates, may have gone unrecognized in past of Gymnodinium catenatum by Nichols et al. (1996), in which field studies. the common dinoflagellate sterols cholest-c-en-Sfs-ol (choles­ In both our bloom samples, most fatty acids were found to terol), 4a,23,24-trimethyl-5a-cholest-22E-en-313-01 (dinoster­ be associated with the triglyceride fraction. Without fraction­ 01), and 4a,23,24-trimethyl-5a-cholestan-313-01 (dinostanol) ation into component lipid classes before derivatization and were observed. GC-MS analysis, the high abundance of triglyceride-derived Although the fatty acid and sterol compositions of our nat­ fatty acids (which are often used as a vehicle for carbon stor- ural K. brevis bloom samples closely resemble those observed Leblond et al.: Karenia brevis bloom lipids 329

in laboratory cultures of this organism, the information ob­ in the United States. Woods Hole Oceanographic Institution Tech­ tained from this bloom raises a number of salient questions nical Report, WHOI-2000-11. Woods Hole, Massachusetts. BELL M.V. & POND D. 1996. Lipid composition during growth of about the roles of lipids in the physiology of K. brevis. For motile and coccolith forms of Emiliania huxleyi. Phytochemistry 41: example, how might the glycolipid-associated fatty acids, 465-471. which form the chloroplast membranes, interact with the pig­ BELL M.V., DICK J.R. & POND D.W. 1997. Octadecapentaenoic acid ments (e.g. xanthophyll-cycling) and proteins of the photo­ in a raphidophyte alga, Heterosigma akashiwo. Phytochemistry 45: synthetic apparatus to protect against damage caused by high 303-306. irradiance or ultraviolet radiation (cf. Evens et al. 2001)7 In BJPRNLAND T. 1990. Carotenoid structures and lower plant phylogeny. addition, little is known about how the composition of gly­ In: Carotenoids: chemistry and biology (Ed. by N.l. Krinsky, M.M. MathewsRoss & R.F. Taylor), pp. 21-36. Plenum Press, New York. colipid- and phospholipid-associated fatty acids alters during BJPRNLAND T. & TANGEN K. 1979. Pigmentation and morphology of periods of phosphorus or nitrogen limitation, or during chang­ a marine Gvrodinium (Dinophyceae) with a major carotenoid dif­ es in temperature (Vargo & Shanley 1985; Tester et al. 1993). ferent from peridinin and fucoxanthin. Journal of Phycology 15: It is also not certain whether the amount of triglyceride-as­ 457-463. sociated fatty acids changes during the course of a bloom as BOSSART G.D., BADEN D.G., EWING R.Y., ROBERTS B. & WRIGHT S.D. the nutritional status of K. brevis changes. More specifically, 1998. Brevetoxicosis in manatees from the 1996 epizootic: gross, will this organism accumulate triglycerides as the primary histologic, and immunohistochemical features. Toxicologic Pathol­ ogy 26: 276-282. storage product while it is photosynthetically active in the CARBALLEIRA N.M., EMILIANO A., SOSTRE A., RESTITUYO J.A., GON­ near-surface waters, and then use this energy reserve to fuel ZALEZ l.M., COLON G.M., TOSTESON c.o. & TOSTESON T.R. 1998. metabolism after migration to deeper, nutrient-laden waters Fatty acid composition of bacteria associated with the toxic dino­ (MacIntyre et al. 1997; Kamykowski et al. 1998, 1i;)99)7 flagellate Ostreopsis lenticularis and with caribbean Palythoa spe­ Because HABs occur with a highly unpredictable frequen­ cies. Lipids 33: 627-632. cy, the first step toward answering some of the above ques­ CHURCHILL J.H. & CORNILLON P.e. 1991. Water discharged from the tions is to conduct experiments where individual variables, Gulf Stream north of Cape Hatteras. Journal of Geophysical Re­ search 96: 22227-22243. such as fluctuations in temperature or nutrient concentration COTONNEC G., BRUNET C., SAUTOUR B. & THOUMELIN G. 2001. Nutri­ can be examined under carefully controlled conditions. The tive value and selection of food particles by copepods during a importance of understanding the physiological and genetic ba­ spring bloom of Phaeocystis sp. in the English Channel, as deter­ sis for HABs cannot be underestimated (cf. Millie et al. 1999). mined by pigment and fatty acid analysis. Journal ofPlankton Re­ Information obtained from future laboratory and field exper­ search 23: 693-703. iments will enable a deeper understanding of the physiological DARANAS A, NORTE M. & FERNANDEZ J. 2001. Toxic marine microal­ gae. Toxicon 39: 1101-1132. autecology of K. brevis, which will facilitate prediction and DAUGBJERG N., HANSEN G., LARSEN J. & MOESTRUP 0. 2000. Phylog­ mitigation of this toxic microalga. eny of some of the major genera of dinoflagellates based on ultra­ structure and partial LSU rDNA sequence data, including the erec­ tion of three new genera of unarmoured dinoflagellates. Phycologia ACKNOWLEDGEMENTS 39: 302-317. DUNSTAN G.A., VOLKMAN J.K., BARRETT S.M. & GARLAND C.D. 1993. Changes in the lipid composition and maximization of the poly­ Funding for portions of this work, provided by the National unsaturated fatty acid content of three microalgae grown in mass Research Council while. J.L. was a postdoctoral research as­ culture. Journal ofApplied Phycology 5: 71-83. sociate, is gratefully acknowledged. We are also grateful to EVENS T.J., KIRKPATRICK G.J., MILLIE D.F., CHAPMAN DJ. & SCHOFIELD Rick Greene, Roman Stanley, Lisa Smith, and Michael Mur­ O.M.E. 2001. Photophysiologica1 responses of the toxic red-tide rell for information and assistance in site characterization; Jim dinoflagellate Gymnodinium breve (Dinophyceae) under natural Patrick for transport to the bloom sites; and Gary Kirkpatrick sunlight. Journal of Plankton Research 23: 1177-1193. and two anonymous reviewers for comments on the manu­ FAHL K. & KATTNER G. 1993. Lipid content and fatty acid composi­ tion of algal communities in sea-ice and water from the Weddell script. The views expressed herein are those of the authors, Sea (Antarctica). Polar Biology 13: 405-409. and do not necessarily reflect the views of the supporting gov­ FARALDOS J. & GINER J.L. 1998. Isolation and synthesis of unique ernmental agencies. Mention of commercial products does not sterols from the toxic dinoflagellate Gymnodinium brevis. 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