LIPID, FATTY ACID, AND STEROL COMPOSITION OF EIGHT SPECIES OFKARENIACEAE (DINOPHYTA): CHEMOTAXONOMY AND PUTATIVE LIPID

PHYCOTOXINS1

Ben D. Mooney2

School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia

Peter D. Nichols

CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, Tasmania 7001, Australia

Miguel F. de Salas and Gustaaf M. Hallegraeff

School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia

The lipid class, fatty acid, and sterol compositionof eight species of ichthyotoxic marine gym-nodinioid dinoflagellate (Karenia, Karlodinium,and Takayama) species was examined. The majorlipid class in all species was phospholipid(78%–95%), with low levels of triacylglycerol(TAG; 0%–16%) and free fatty acid (FFA; 1%–11%). The common dinoflagellate polyunsaturatedfatty acids (PUFA), octadecapentaenoic acid (OPA18:5x3), and docosahexaenoic acid (DHA 22:6x3),were present in all species in varying amounts(14%–35% and 8%–23%, respectively). The very-long-chain PUFA (VLC-PUFA) 28:7x6 and 28:8x3were present at low levels (<1%), and the ratio ofthese fatty acids may be a useful chemotaxonomicmarker at the species level. The typical dinoflagel-late sterol dinosterol was absent from all speciestested. A predominance of the 4-methyl and 4-des-methyl D8(14) sterols in all dinoflagellate species in-cluded 23-methyl-27-norergosta-8(14),22-dien-3b-ol(Karenia papilionacea A. J. Haywood et Steid, 59%–66%); 27-nor-(24R)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol, brevesterol, (Takayama tasmanica deSalas, Bolch et Hallegraeff 84%, Takayama helix deSalas, Bolch, Botes et Hallegraeff 71%, Kareniabrevis (C. C. Davis) G. Hansen et Moestrup 45%,Karlodinium KDSB01 40%, Karenia mikimotoi(Miyake et Kominami ex Oda) G. Hansen etMoestrup 38%); and (24R)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol, gymnodinosterol, (K. mikimotoi48%, Karenia umbella de Salas, Bolch et Hallegraeff59%, Karlodinium veneficum (D. L. Ballant.) J. Lar-sen 71%–83%). In Takayama species, five steroidketones were identified, including for the first timethe 3-keto form of brevesterol and gymnodinosterol.These results indicate a biochemical link betweensterol and steroid ketone biosynthesis, suggestingthat selected dinoflagellates can make a significant

contribution to ketones in marine sediments. Thepresence of steroid ketones, specific sterols, andfatty acids, and the ratio of VLC-PUFA may prove tobe a useful chemotaxonomic tool for distinguishingbetween morphologically similar species. The rela-tive levels of the PUFA, OPA, and DHA, coupledwith the potential inhibitory action of D8(14) sterols,may provide an insight into the ichthyotoxicity ofthese bloom-forming dinoflagellates.

Key index words: chemotaxonomy; dinoflagellate;fatty acid; ichthyotoxicity; lipid; sterol

Abbreviations: DHA, docosahexaenoic acid; FFA,free fatty acid; OPA, octadecapentaenoic acid;ROS, reactive oxygen species; TAG, triacylglycerol;VLC-PUFA, very-long-chain polyunsaturated fattyacids

Gymnodinioid dinoflagellates belonging to what isnow known as the family Kareniaceae form a well-defined grouping characterized by the possession of fu-coxanthin and derivative chloroplast pigments and theircommon involvement in wild or aquaculture fish kills.Initially, these species were accommodated within Gym-nodinium or Gyrodinium, but Daugbjerg et al. (2000),based on a combination of molecular sequencing andultrastructural studies, redefined the genus Karenia forspecies with straight apical grooves, and Karlodinium forspecies with a short straight groove and small ventralpore. De Salas et al. (2003) later added the genusTakayama for species with a sigmoid apical groove.

The first taxon to be recognized as having fucoxan-thin instead of peridinin pigments was what is nowknown as Karlodinium veneficum (also referred toas Karlodinium micrum, Gyrodinium galatheanum, orGyrodinium sp. A; Bj�rnland and Tangen 1979). Thisfeature was subsequently also confirmed for Kareniabrevis (also referred to as Gymnodinium breve; Bj�rnlandand Tangen 1979) and Takayama species (de Salas et al.2003). Such chloroplast pigments have derived from a

1Received 16 May 2006. Accepted 27 September 2006.2Author for correspondence: e-mail ben.mooney@utas.edu.au.

101

J. Phycol. 43, 101–111 (2007)r 2007 by the Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2006.00312.x

long-established symbiotic association with a hap-tophyte endosymbiont (Yoon et al. 2005).

The main interest in species of Kareniaceae relatesto their potential ichthyotoxicity. Fish kills caused byK. brevis in Florida have been documented since the1700s (Steidinger et al. 1998) and are widely attributedto the production of the neurotoxin brevetoxin, whichis the cause of neurotoxic shellfish poisoning (NSP) inhuman shellfish consumers. Karenia mikimotoi (alsoknown as Gyrodinium aureolum) has been widely associ-ated with salmonid aquaculture fish kills in Europe(Tangen 1978). More recently, K. papilionaceain New Zealand (Haywood et al. 2004) and K. umbellain Tasmania, Australia, have also been associated with asalmonid aquaculture mortality (de Salas et al. 2004b).Karlodinium veneficum (as K. micrum) is increasingly be-ing associated with wild fish kills in the Swan River,Western Australia, and North American estuaries(Deeds et al. 2002). Finally, Takayama species (previ-ously referred to as Gymnodinium pulchellum orGymnodinium type 84K) have caused fish kills inFlorida, Japan, and Australia.

Analysis of lipid composition of dinoflagellates canprovide a valuable clue to chemotaxonomy and iden-tify biomarkers for use in field monitoring, but it hasalso revealed candidate lipid phycotoxins responsiblefor fish kills. Previous lipid studies on Kareniaceaehave been limited to K. brevis, K. mikimotoi, andK. veneficum. In the present survey, we reexaminedfurther strains of these three taxa as well as newlyanalyzed K. papilionacea, K. umbella, Takayama tasmanica,Takayama helix, and an as yet undescribed Karlodiniumspecies (referred to as KDSB01 and to be described infull elsewhere).

MATERIALS AND METHODS

Algal strains and growth conditions. Details of algal culturesare summarized in Table 1. Cultures were grown in duplicateor triplicate in 250 mL polycarbonate screw-top containers,inoculated with 50 mL stock culture and made up to 200 mLwith GSe medium (Blackburn et al. 2001). The cultures were

grown at either 171C or 201C (Table 1), consistent with thetemperature at which the stock culture was maintained. A12:12 light:dark (L:D) cycle of 80 mmol photons �m�2 � s�1 ofcool-white fluorescent light was constant throughout the ex-periment. Cultures were grown to the late-exponentialgrowth phase, between 2 and 4 weeks depending on the spe-cies, subsampled for cell counts, and harvested in their en-tirety onto precombusted glass-fiber filters (Whatman GF/F47 mm diameter, Whatman GFC, Maidstone, UK). Sampleswere stored at � 201C in the dark until analysis.

Lipid extraction and analysis. Filters were extracted using amodified Bligh and Dyer (1959) method with an initialchloroform/methanol/water (1:2:0.8, v/v/v) single-phasesolution. Phase separation was performed by addition ofchloroform and purified water (Milli-Qs system, Millipore,Sydney, NSW, Australia) to yield a final chloroform/metha-nol/water of 1:1:0.9 (v/v/v). The lipid-containing lowerchloroform layer was concentrated in vacuo by rotary evap-orator and transferred to tared vials. Lipid samples werestored at � 201C in the dark until analysis.

Lipid class composition was determined by an IatroscanMK V thin-layer chromatography-flame ionization detector(TLC-FID) analyzer (Iatron Laboratories, Tokyo, Japan).Samples were spotted onto silica gel SIII Chromarods (5mmparticle size) and developed in a glass tank lined with pre-extracted filter paper. The solvent system used for the lipidseparation was hexane/diethyl ether/acetic acid (60:17:0.1, v/v/v; Volkman and Nichols 1991). After development for 25 min,the chromarods were oven dried and analyzed immediately tominimize adsorption of atmospheric contaminants. Lipidclasses were quantified by DAPA software (Kalamunda, WA,Australia). The FID was calibrated for each compound class:phosphatidylcholine, cholesterol, cholesteryl ester, oleic acid,hydrocarbon (squalene), wax ester (derived from fish oil), tri-acylglycerol (TAG; derived from fish oil), and diacylglycerylether (DAGE) purified from shark liver oil.

An aliquot of the total lipid was saponified using 5% KOH inaqueous MeOH and heated for 3 h at 801C. Free sterols andsteroids were extracted three times with hexane/chloroform(4:1, v/v) and converted to their trimethylsilyl ethers(TMS) using N,O-bis(trimethylsilyl)trifluoroacetamide (BST-FA; 501C, overnight) before gas chromatography-flame ioniza-tion detection (GC-FID) and gas chromatography-massspectrometry (GC-MS) analysis. Another aliquot was trans-methylated using MeOH/HCl/CHCl3 (10:1:1, v/v/v; 1001C,60 min), and after addition of water, the fatty acid methyl es-ters (FAME) were extracted three times with hexane/chloro-

TABLE 1. Algal species screened in the present study.

Species Culture codeTemperature

(1C) Synonyms Locality Isolator and date

Karenia brevis KBFL718 20 Gymnodinium breve Florida Wilson, 1958Karenia mikimotoi KMNZ63 17 Gymnodinium mikimotoi;

cf. Gyrodiniumaureolum (Europe)

New Zealand Haywood, 1994

Karenia papilionacea PL01 20 Port Lincoln (PL), SouthAustralia

de Salas, May 15, 2003

AB01 20 Ansons Bay (AB), Tasmania de Salas, May 25, 2003Karenia umbella LV01 20 Port Lincoln de Salas, March 15, 2002Karlodinium veneficum SR01 20 Karlodinium micrum; cf. Swan River, Western Australia de Salas, March 9, 2001

DE01 20 Gyrodiniumgalatheanum

Derwent Estuary, Tasmania de Salas, February 21, 2003

HU01 20 Huon River, Tasmania de Salas, January 10, 2005Karlodinium sp. SB01 17 de Salas, May 18, 2005Takayama helix NWB01 17 North West Bay, Tasmania de Salas, May 14, 2001Takayama tasmanica DE01 17 Gymnodinium

pulchellumDerwent Estuary, Tasmania de Salas, February 7, 2001

B. D. MOONEY ET AL.102

form (4:1, v/v). All samples were made up to a known volumewith an internal injection standard (23:0 or 19:0 FAME) andanalyzed by GC using an Agilent Technologies 6890N GC (PaloAlto, CA, USA) equipped with an HP-5 cross-linked methylsilicone fused silica capillary column (50 m � 0.32 mm i.d.),and an FID. Helium was used as the carrier gas. Samples wereinjected, by a split/splitless injector and an Agilent Technolo-gies 7683 Series autosampler in splitless mode, at an oventemperature of 501C. After 1 min, the oven temperature wasincreased to 1501C at 301C �min� 1, then to 2501C at21C �min�1, and finally to 3001C at 51C �min�1. Peaks werequantified by Agilent Technologies GC ChemStation software.The GC-MS analyses were performed on all samples using aFinnigan Thermoquest GCQ GC-MS fitted with an on-columninjector and using Thermoquest Xcalibur software (Austin,TX, USA). The GC was fitted with a capillary column similar tothat described above.

The FAME, derivatized sterols, and steroidal ketones wereidentified by comparison with retention time of laboratorystandards, mass spectral data, and relative retention times pub-lished in the literature (Harvey et al. 1988, Jones et al. 1994,Mansour et al. 1999b). Peak areas were used to attribute rela-tive proportions of fatty acids, sterols, and steroidal ketones.Gas chromatography results are typically subject to an error of�5% of individual component area.

Statistics. The mean values are reported as plus or minusthe SE of the mean. Bray-Curtis cluster analysis and principalcomponents analysis (PCA) were performed using Primer 5,Version 5.2.9. (PRIMER-E, Plymouth, UK).

RESULTS

Lipid class composition. The cell counts and lipidclass composition of eight species of marine dinoflag-ellates are presented in Table 2. The major lipid classin all species was polar lipid (84%–95%), with theexception of Takayama species containing lowerlevels (78%). Free fatty acid (FFA) was the highest inK. umbella (11%), T. helix (10%), and the Swan Riverisolate of K. veneficum (9%). The relative level of TAGwas the highest in Takayama species (T. tasmanica 16%,T. helix 6%), while all other species had <5% TAG.Sterols and steryl esters were present at low levels(<5% per lipid class).

Fatty acid composition. Karenia: The major satur-ated fatty acids (SFA) in the five Karenia species were16:0 (21%–34%), 14:0 (4%–21%), and 18:0 (2%–4%;Table 3). Monounsaturated fatty acids (MUFA) werefound in lower levels ranging from 13% to 22% of thetotal fatty acids and were dominated by 18:1o9,18:1o7, and 16:1o9. The main polyunsaturated fat-ty acids (PUFA) were 18:5o3 and 22:6o3, whichranged from 14% to 23% and from 8% to 15%, re-spectively. Karenia umbella had the highest levels of16:0 (32%) and 22:6o3 (15%) among the Karenia spe-cies studied. Moderate levels of 16:4o3 (6%) and18:2o6 (7%) were present in K. mikimotoi and K. um-bella, respectively. The very-long-chain PUFA (VLC-PUFA) 28:7o6 and 28:8o3 were present at low levels(� 1%) in all species. The ratio of 28:7o6 to 28:8o3 inKarenia ranged from 0.2 to 1, except for K. mikimotoi,in which 28:7o6 was not detected.

Karlodinium: The three isolates of K. veneficumand an undescribed Karlodinium, KDSB01, contained T

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B. D. MOONEY ET AL.104

as the major SFA present 16:0 (16%–33%), 14:0(4%–16%), and 18:0 (2%–4%; Table 3). In all Karl-odinium species, MUFA were dominated by 18:1o9(3%–8%) and 16:1o7 (3%–4%). The major PUFA was18:5o3, present at 19%–35%, followed by 22:6o3(10%–18%). The VLC-PUFA 28:7o6 and 28:8o3were present at low levels with a ratio of 1–2.5.

Takayama: The two species of Takayama containedthe main SFA as 16:0 (23%–24%), 14:0 (5%–8%), and18:0 (2%–3%). The MUFA 18:1o9 and 16:1o7 werepresent at low levels (<5%). The PUFA 22:6o3 wasthe dominant component (23%), followed by 18:5o3(15%–22%; Fig. 1). Low relative levels of 28:7o6 and28:8o3 were present in T. helix (ratio of 0.8), with28:7o6 absent in T. tasmanica.

Sterols and steroid ketone composition. Karenia: Thesterol profiles of K. mikimotoi and K. brevis were bro-adly similar, with the major sterols (24R)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol (gymnodinosterol; 33%–48%), 27-nor-(24R)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol (brevesterol; 38%–43%), and cholesterol (7%–14%;Table 4). Lower levels (�4%) of minor unidentifiedC28 and C29 unsaturated sterols were present. The twostrains of K. papilionacea had very similar profiles, butdiffered markedly from the other Karenia species.High levels of 23-methyl-27-norergosta-8(14), 22-dien-3b-ol (59%–66%), and an unknown C28 sterolwith three double bonds (TMS ether Mþ 468, 16%–17%) were present in K. papilionacea, with lower levelsof a suite of unknown C28 unsaturated sterols present.The sterol profile of K. umbella comprised gymnodi-nosterol (59%) with lower levels of unidentified C28,C29, and C30 sterols. The stanol of gymnodinosterol,(24R)-4a-methyl-5a-ergosta-22-en-3b-ol (Fig. 2), is re-ported for the first time, present at low levels (1%) inK. umbella.

Karlodinium: The sterol profiles of the three iso-lates of K. veneficum were similar and dominated bygymnodinosterol (71%–83%), an unknown C29 sterolwith three double bonds (8%–13%), cholesterol (7%–

9%), and brevesterol (4%–6%, not present in theSwan River strain). The sterol profile of KDSB01contained brevesterol (40%) and gymnodinosterol(35%), with lower levels of a C29 sterol with threedouble bonds (9%) and cholesterol (4%) present.

Takayama: Both Takayama species had similarsterol profiles, with a predominance of brevesterol(62%–70%), gymnodinosterol (9%–13%), an un-known C30 sterol with two double bonds (4%–11%),and cholesterol (2%–9%). These species also con-tained the steroid ketone of brevesterol (49%–89%of total steroids) and gymnodinosterol (9%–16%) andother unidentified C30, C31, and C32 3-keto steroids.The distribution of steroidal ketones in Takayama andmass spectral data are shown in Table 5.

DISCUSSION

Lipid class. The main lipid class in all dinoflagellatespecies studied was polar lipid (78%–95%), with lowlevels of FFA and TAG. The polar lipid fraction indinoflagellates is known to contain similar propor-tions of glycolipid and phospholipid (Parrish et al.1993, Bodennec et al. 2000, Leblond and Chapman2000, Leblond et al. 2003). In photosynthetic algae,such as dinoflagellates, glycolipids are found in thechloroplast, whereas phospholipids have a role instructure and fluidity in the cell membrane. We in-vestigated the levels of FFA due to suspected inter-action with reactive oxygen species (ROS) as theproposed mechanism of fish kills (Marshall et al.2003). The latter authors demonstrated with the rap-hidophyte Chattonella marina that low concentrationsof the PUFA eicosapentaenoic acid (EPA) in combi-nation with ROS are more toxic to damselfish thanwhen tested individually or compared with whole al-gal cells. Juttner (2001) showed that low levels of FFAwere released by diatom biofilms, which providedprotection against grazing by fairy shrimp. Our re-sults indicate variable levels of FFA in the speciesstudied (1%–11%) and suggest that algal growthconditions may also influence production of FFA.Further experiments examining more precisely theproduction of FFA under nutrient deficient andother conditions, as well as ROS production, areplanned.

Fatty acid. The fatty acid profiles of the gym-nodinioid species studied exhibited similarities tothose observed in other investigations into lipids ofmarine dinoflagellates. The prominent PUFA in allspecies were 18:5o3 and 22:6o3, ranging from 14%to 35% and from 8% to 23%, respectively. The highlevels of these two PUFA, and the relatively low levelsof other PUFA, such as octadecatetraenoic acid (OTA)and EPA, suggest that these two fatty acids may beuseful biomarkers for these dinoflagellates. To sep-arate dinoflagellate inputs from other algal groups,the ratio of the two PUFA can be used, as most otheralgal groups do not contain 18:5o3, thereby distin-guishing dinoflagellates from other genera.

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FIG. 1. Representative gas chromatogram of fatty acid methylesters from Takayama helix.

LIPID COMPOSITION OF KARENIACEAE 105

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B. D. MOONEY ET AL.106

Isolates of K. veneficum had similar levels of all fattyacids compared with previous reports (Deeds et al.2000). Studies of K. brevis and K. mikimotoi reportedlevels of octadecapentaenoic acid (OPA) and DHA inNorth American isolates of this species (Parrish et al.1993, Bodennec et al. 2000, Leblond and Chapman2000, Leblond et al. 2003) comparable with thosefound in the present study. Karenia mikimotoi was theonly species to contain appreciable levels of 16:4o3(6.3%), which is in agreement with the levels reportedby Parrish et al. (1993) and Bodennec et al. (1995).This fatty acid is likely to reside in the glycolipid frac-tion (Bodennec et al. 2000).

The VLC-PUFA 28:7o6 and 28:8o3 were observedin all strains apart from K. mikimotoi and T. tasmanica(28:7o6 absent; Table 3), consistent with previousreports for marine dinoflagellates (Mansour et al.1999a, b, Leblond and Chapman 2000, Leblondet al. 2003). The ratio of 28:7o6 and 28:8o3 wasbetween 0.2 and 1.0 in Karenia species but higher inKarlodinium, ranging from 1.0 to 2.5, and in T. helix(0.8). The VLC-PUFA have been reported in the phos-pholipid fraction of K. brevis, but their cellular functionremains unclear (Leblond et al. 2003). The ratio of

28:7o6 and 28:8o3 for K. brevis agrees with thatreported by Leblond et al. (2003) and suggests thatthese fatty acids are useful lipid biomarkers.

Sterol and steroid ketones. The predominance of thesterols brevesterol and gymnodinosterol in all speciesof Karenia, Karlodinium, and Takayama (with theexception of K. papilionacea) confirms previously re-ported levels of these sterols (Leblond and Chapman2002a, b, Giner et al. 2003, Leblond et al. 2003).While brevesterol and gymnodinosterol have beenobserved as minor components in other dinoflagel-lates (Leblond and Chapman 2002b), the combinedcomposition of these components in the total sterolsin Karenia, Karlodinium, and Takayama species is>60%. The notable absence of dinosterol in all spe-cies of the family Kareniaceae is atypical for dino-flagellates.

The sterol profiles for K. papilionacea strains weredominated by 23-methyl-27-norergosta-8(14), 22-dien-3b-ol. This C28 4-desmethyl sterol with D8(14) unsatu-ration was previously identified as a minor componentin K. brevis (Giner et al. 2003) but was not detected inthe current study of this taxon, nor confirmed by Le-blond and Chapman (2002a, b) for K. brevis. Theabsence of brevesterol and gymnodinosterol inK. papilionacea is inconsistent with the phylogenetic re-lationship of this species with other Karenia and Karl-odinium species. However, de Salas et al. (2004a) placeK. papilionacea as a taxonomically distinct group fromother Karenia species. Karenia and Karlodinium areclosely related with similar chemotaxonomic proper-ties, but K. papilionacea is an exception.

The five steroid ketones present in Takayama are ofchemotaxonomic significance as these compounds arerarely found in other algae. This is, to our knowledge,the first report of the 3-keto steroids of brevesterol andgymnodinosterol, with combined percentages of 61%and 62% of the total ketones in T. tasmanica and T. helix,respectively. The presence of these compounds inTakayama supports the claim that algae contribute ke-tones directly into marine sediments (Jones et al. 1983,Harvey et al. 1988, Volkman et al. 1998). Steroid ke-tones have previously been reported in Scrippsiella

FIG. 2. Representative mass spectrum of (24R)-4a-methyl-5a-ergosta-22-en-3b-ol (gymnodinostanol) from Karenia umbella.

TABLE 5. Relative retention time and mass spectral data for 3-keto steroids in the Takayama species.

Steroid RRT

Abundance

Mass spectral dataMþ Base peak

27-nor-(24R)-4a-methyl-5a-ergosta-8(14),22-dien-3-one

0.95 396 (46) 283 381 (55), 367 (10), 339 (18), 311 (28), 285 (84),271 (39), 258 (48), 241 (37), 91 (60)

C291 1.07 408 (36) 295 393 (28), 379 (7), 351 (18), 339 (22), 323 (15),310 (12), 297 (71), 283 (34), 270 (39), 253 (30),91 (63)

(24R)-4a-methyl-5a-ergosta-8(14),22-dien-3-one

1.27 410 (46) 283 395 (45), 381 (8), 367 (14), 339 (17), 311 (32),285 (86), 267 (44), 258 (47), 91 (61)

C311 1.57 440 (49) 327 425 (30), 411 (7), 393 (44), 355 (17), 329 (84),315 (55), 91 (60)

C321 1.86 454 (43) 327 439 (25), 355 (22), 329 (92), 315 (62), 91 (62)

RRT, relative retention time, with cholesterol at 1.00 and 24-ethylcholesterol at 1.63 (Jones et al. 1994), CXY, where CX is thenumber of carbon atoms, and Y is the number of double bonds.

LIPID COMPOSITION OF KARENIACEAE 107

(Harvey et al. 1988, Volkman et al. 1998, Mansouret al. 1999b), Crypthecodinium and Peridinium (Withers1987), and the calcified dinoflagellate Thoracosphaeraheimii (Lohman) Kampter (Jones et al. 1983). The ster-ol composition is usually mirrored by that of the ster-oid ketones (Harvey et al. 1988, Mansour et al. 1999b)as was observed in Takayama species.

Chemotaxonomy. A PCA of the most abundant fattyacids (17) revealed a close association between strainsof K. veneficum, from the Huon and Derwent estuar-ies in Tasmania and the Swan River in Western Aus-tralia, and those of K. papilionacea from Port Lincoln,South Australia, and Ansons Bay, Tasmania, as well asa grouping of T. tasmanica with KDSB01 (Fig. 3). Thisis most likely due to the levels of 18:5o3 and 22:6o3.Little association was observed with the remainingKarenia and Takayama species.

Sterol composition expressed as a dendrogramusing Bray-Curtis cluster analysis showed a similargrouping of dinoflagellate species and strains (Fig. 4).Karenia papilionacea clearly separated from other spe-cies studied here due to the high levels of 23-methyl-27-norergosta-8(14), 22-dien-3b-ol, and the absence ofbrevesterol and gymnodinosterol. This is in agreementwith the results documented by de Salas et al. (2004a),which clearly separate K. papilionacea from other flat-tened Karenia species, as it has a high level of ribosomalgene sequence divergence. This species is likely to beevolving at a faster rate than the rest of the genusKarenia and could be in the process of forming adivergent lineage. Strains of K. veneficum, like thoseof K. papilionacea, formed clearly distinct groups, con-firming that sterol composition is well preserved inculture. Both the Takayama species grouped togetherand were present in the same section of the dendro-gram as K. mikimotoi, K. brevis, and KDSB01. The Baye-sian phylogenetic tree of the species in the familyKareniaceae that are examined in this study (Fig. 5)groups Takayama and Karlodinium species together, in agroup distinctly separated from all Karenia species.Other studies are in agreement (de Salas et al. 2003,de Salas et al. 2004b, 2005). In general terms, the stat-istical analysis of fatty acid and sterol profiles using

Bray-Curtis cluster dendrograms and PCA indicatesthe separation of some morphologically similar dino-flagellate species and close grouping of isolates fromdifferent sources. While not in perfect agreement withthe Bayesian phylogeny presented in Fig. 5, chemo-taxonomic (Figs. 3 and 4) distribution of the familyKareniacea has some potential as a complementarytool for species identification.

The distinctive lipid profiles of the genera Karenia,Karlodinium, and Takayama exhibit a number of within-species chemotaxonomic classifiers. Of particular noteare the D8(14) sterols brevesterol, gymnodinosterol, and23-methyl-27-norergosta-8(14), 22-dien-3b-ol; the fat-ty acids OPA, DHA, and VLC-PUFA; and the steroidketones in Takayama species. Future studies on thesespecies aim to investigate the effect of culture condi-tions on specific lipids, including FFA, with a view tounderstanding and identifying possible triggers fortheir production and further examining potentialichthyotoxicity.

Takayama helix (NWB)

Karenia brevis (FL)

Karenia papilionacea (AB)

Karenia mikimotoi (NZ)

Karenia umbella

Karenia papilionacea (PL)

K. veneficum (DE)

K. veneficum (HU)

K. veneficum (SR) Takayama tasmanica (DE)

KDSB01

PC1

PC2

4

3

2

1

0

0 1 2 3 4

–1

–2

–3–4 –3 –2 –1

FIG. 3. Principal component analysis of fatty acids fromKarenia, Karlodinium (K.), and Takayama species. PC1 and PC2refer to principal components 1 and 2.

FIG. 4. Dendrogram (Bray-Curtis cluster analysis—single linkage) of sterols from Karenia, Karlodinium (K.), and Takayama species.

B. D. MOONEY ET AL.108

Ichthyotoxicity. The importance of understandingthe chemical ecology of marine dinoflagellates is in-creasing due to research on HABs affecting aquacul-ture. The ichthyotoxicity of K. brevis (brevetoxin) iswell established; however, the mechanism forichthyotoxicity for the remaining species of theKareniaceae family is yet to be conclusively deter-mined. Except for K. brevis, brevetoxin analysis of allspecies in this study was below detection level (PbTx-2 <0.006 pg � cell� 1) (McNabb et al. 2006), suggest-ing another means of ichthyotoxicity. Hemolytic gly-colipids and toxic PUFA have previously beenidentified in K. mikimotoi (Yasumoto et al. 1990, Par-rish et al. 1998, Fossat et al. 1999, Sola et al. 1999,Arzul et al. 2000). Similarly, hemolytic glycolipidsand toxic PUFA were detected in the haptophyteChrysochromulina polylepis Manton et Parke (Yasumotoet al. 1990); however, analysis of toxic and nontoxicstrains of this species suggests that lipids do not playan exclusive role in toxicity (John et al. 2002).Ichthyoxicity is also not confined to fucoxanthin-containing Kareniaeceae, as peridinin-containing di-noflagellates include the marine species Cochlodiniumpolykrikoides Margalef (Hallegraeff 2002) and thefreshwater species Peridiniopsis polonicum (Wo"osz.)Bourr. (Roset et al. 2002), which are known to betoxic to fish. The toxicity of K. veneficum (as K. micrum)is currently not attributed to OPA or glycolipids, butto a newly defined karlotoxin that is yet to be fullychemically characterized (Deeds et al. 2002). Whilethere exists a large body of work confirming the toxicpotential of PUFA from microalgae (Kozakai et al.

1982, Yasumoto et al. 1987, Smolowitz and Shumway1997, Arzul et al. 1998, Sola et al. 1999, Sellem et al.2000, Juttner 2001, Hiraga et al. 2002, Leblond andChapman 2002a, Marshall et al. 2002, 2003, Cem-bella 2003, Ikawa 2004), the exact nature of PUFAtoxicity remains to be fully explained because thesecompounds are equally present in nontoxic dinoflag-ellates and other microalgae (Volkman et al. 1998,Mansour et al. 1999b).

The toxicity of some algae may be linked to lipidperoxidation of PUFA by ROS. Hemolytic activity ofOPA and DHA from K. mikimotoi decreased under lowoxygen conditions (He or N2 bubbling; Arzul et al.1998), and in the raphidophyte Chattonella marina(Subrahm.) Y. Hara et Chihara, PUFA combined withelevated levels of ROS enhanced the toxicity of thisspecies to damselfish (Marshall et al. 2003). In theseexperiments, PUFA and ROS alone exhibited lowerlevels of toxicity, and a synergistic mechanism of thesechemicals was suggested. The level of superoxide pro-duced by Chattonella, Gymnodinium, Takayama, and Ka-renia species is an order of magnitude greater than thatof nontoxic algae, with K. veneficum also producing lowlevels (Marshall et al. 2005a, b). The relationship be-tween PUFA, ROS, and toxicity remains unclear; how-ever, the lipid results for the species studied herereflect the possibility that fatty acids and ROS mayplay a role in the observed ichthyotoxicity.

The observed ichthyotoxity of unarmoured dino-flagellates may also be associated with the abundantD8(14) unsaturated sterols present in the lipid. The roleof these unsaturated sterols is unclear; however, they

FIG. 5. Bayesian phylogeny GTRþ IþG model, based on LSU rDNA of the species of Kareniaceae studied.

LIPID COMPOSITION OF KARENIACEAE 109

could be a significant contributor to reduced predationby invertebrates and promote bloom formation, as theunusual structural side-chain modifications may ren-der these sterols nonnutritious for planktivorous in-vertebrates (Giner et al. 2003). In K. brevis andK. veneficum, the role of D8(14) unsaturated sterols inprotecting the alga from their own toxin has also beenproposed (Place and Deeds 2003), with weak bindingof karlotoxin to gymnodinosterol compared with thatof cholesterol or ergosterol (Deeds and Place 2006).The relationship between lipids and ROS as the mech-anism for ichthyotoxicity will continue to be the subjectof further investigation.

This work was supported by Australian Research Council Dis-covery Project DP0557820. We thank Helen Bond at theSchool of Plant Science, University of Tasmania, for culturingassistance and advice on algal growth. From CSIRO Marineand Atmospheric Research, Danny Holdsworth managed theGC-MS facility, and John Volkman and Peter Mansour pro-vided invaluable advice on algal fatty acid identification andsterol structural elucidation.

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