Jeffrey D. Leblond

A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS, KARENIA MIKIMOTOI, AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. I. Peridinin-containing taxa

Two glycolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are components of chloroplast membranes. This study addresses the lack of information regarding the major intact forms of these glycolipids in photosynthetic dinoflagellates through the use of a full-scan positive-ion electrospray ionization/mass spectrometry (ESI/MS) survey of MGDG and DGDG in 35 peridinin-containing taxa from the class Dinophyceae. Further analysis using positive-ion electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS) was performed to determine the positional distribution of fatty acids associated with MGDG and DGDG. The dinoflagellates examined were divided into two clusters based on the forms of MGDG and DGDG present. The first possessed 18:5/18:4 MGDG (sn-1/sn-2), 18:5/18:5 MGDG, 18:4/18:4 DGDG, and 18:5/18:4 DGDG as major forms, and the second possessed 20:5/18:4 MGDG, 20:5/18:5 MGDG, 20:5/18:4 DGDG, and 20:5/18:5 DGDG as major forms. The differentiation between C18/C18 and C20/C18 glycolipids indicates differing biosynthetic pathways, as well as a possible evolutionary divergence between the two dinoflagellate clusters.

Betaine lipids in chlorarachniophytes

Evolving from the endosymbiosis of a green algal cell by a filose amoeba or amoeboflagellate, the chimearic chlorarachniophytes combine unique features retained from both of their ancestral units. They have preserved from the endosymbiont only the nucleomorph and chloroplast. Four strains from three genera of this algal class were studied to identify a set of non‐phosphorous‐containing polar lipids and their associated fatty acids using the techniques of positive‐ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Fourteen non‐phosphorous‐containing polar lipids, classified as betaine lipids were primarily identified as forms of diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) and its structural isomer diaclyglycerylhydroxymethyl‐N,N,N‐trimethyl‐β‐alanine (DGTA). Though the number of forms of DGTA and DGTA were roughly equal, DGTS composed more of the polar lipid portion present in three of the strains tested, while the fourth, Lotharella globosa, was dominated by forms of DGTA. In addition, a lipid tentatively identified as diacylglycerylcarboxyhydroxymethylcholine (DGCC) was observed twice in minor amounts. The polar lipid‐associated fatty acids of the aforementioned algal strains generally included dodecanoic acid (12:0), tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), and eicosapentaenoic acid [20:5(n‐3)]. The differences in betaine lipid content among the species studied may allow for further conclusions to be drawn regarding the taxonomy of chlorarachniophytes.

The long and short of it: temperature-dependent modifications of fatty acid chain length and unsaturation in the galactolipid profiles of the diatoms Haslea ostrearia and Phaeodactylum tricornutum

The purpose of this study was to examine the effect of different growth temperatures on the fatty acid compositions of the photosynthetically important galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), of the “blue” pennate diatom, Haslea ostrearia, and the model pennate diatom, Phaeodactylum tricornutum, with the hypothesis that their sn-2 fatty acids would be modulated in the same manner as for dinoflagellates. Positive-ion electrospray ionization/mass spectrometry/mass spectrometry was used to characterize the galactolipids of each diatom. At 20°C, H. ostrearia and P. tricornutum were rich in eicosapentaenoic acid (EPA; C20:5) at the sn-1 position and in C16 fatty acids at the sn-2 position of MGDG and DGDG. At 30°C, however, H. ostrearia and P. tricornutum contained no EPA or other C20 fatty acids, but rather contained higher percentages of C18 fatty acids at sn-1. At 30°C, no galactolipid in either diatom contained more than three unsaturations on any of its fatty acids. While these two species differ in galactolipid composition, they both possess a similar method of acclimating their galactolipids to a higher growth temperature: reducing the numbers of the longest and shortest fatty acid chains, as well as decreasing the total number of unsaturations.

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. IV. Temperature-induced modulation of fatty acid regiochemistry as observed by electrospray ionization/mass spectrometry

Members of the dinoflagellate genus Pyrocystis possess forms of mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) that have the C20 fatty acid eicosapentaenoic acid [20:5(n–3)] at the sn-1 position and either octadecapentaenoic acid [18:5(n–3)] or octadecatetraenoic acid [18:4(n–3)] at the sn-2 position. We have examined the effect of growth at 15°C, 20°C, and 25°C on modulation of the fatty acids associated with MGDG and DGDG in two strains of each of three species of Pyrocystis, P. fusiformis, P. lunula, and P. noctiluca and have observed using positive-ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS) that modulation of the sn-2 fatty acid of DGDG was the only consistent, statistically significant temperature response across all three species. Only one strain of P. lunula displayed a statistically significant temperature-induced modulation of MGDG. In addition, the effect of growth temperature on two forms of the recently identified lipid, trigalactosyldiacylglycerol (TGDG), was examined; TGDG displayed a statistically significant temperature response in only one strain of P. noctiluca. The results presented herein demonstrate how DGDG is modified preferentially over MGDG and TGDG in response to growth temperature by modulating the sn-2 fatty acid of DGDG between the 18:5(n–3) and 18:4(n–3) forms, while the sn-1 fatty acid, 20:5(n–3), remained constant.

Fatty acid and sterol composition of two evolutionarily closely related dinoflagellate morphospecies from cold Scandinavian brackish and freshwaters

Little is known about the lipid composition of cold-adapted dinoflagellates. Here, we investigate Peridinium aciculiferum and Scrippsiella hangoei, two cold-adapted dinoflagellate morphospecies that present significant differences in general morphology, physiology and habitat, but which share identical ribosomal DNA (rDNA) sequences, indicating a recent evolutionary divergence between them. Despite their present taxonomic names, it is now known that these two genetically isolated morphospecies are evolutionarily related to the so-called Pfiesteria-like species. We have found that chloroplast-associated glycolipids from P. aciculiferum and S. hangoei contained a higher relative percentage of two polyunsaturated C18 fatty acids (octadecapentaenoic [18:5(n-3)] and octadecatetraenoic [18:4(n-3)] acids), and a lower relative percentage of hexadecanoic acid (16:0) than typically observed in previously examined warm-adapted dinoflagellate species. This suggests that these two fatty acids provided the requisite membrane fluidity for chloroplasts under a cold growth condition in these two organisms. A similar increase in 18:5(n-3) and 18:4(n-3) over warm-adapted species was also found in the phospholipids and triglycerides. P. aciculiferum and S. hangoei were also examined for the production of unique sterols that may serve as chemical biomarkers. Both were found to have a very similar sterol composition, consisting of two common dinoflagellate sterols, cholestanol and dinostanol, as dominant sterols, along with a number of other common dinoflagellate sterols as minor sterols. This demonstrated the absence of a specific steroidal biomarker but, more importantly, showed that these two morphospecies still produce very similar sterols despite having diverged in several other traits.

Mono- and digalactosyldiacylglycerol composition of glaucocystophytes (Glaucophyta): A modern interpretation using positive-ion electrospray ionization/mass spectrometry/mass spectrometry

Glaucocystophytes are freshwater algae that possess an almost‐intact cyanobacterium, referred to as a cyanelle, as their photosynthetic organelle. Because the cyanelle represents an intermediate state in plastid evolution, glaucocystophytes have been the subject of several studies to characterize the genetics and biochemistry of their cyanelles. However, only a small handful of older studies exist on the composition of their lipids, particularly two major plastid lipids, mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively), found in all photosynthetic life. Our study has used a modern mass spectrometry approach, namely positive‐ion electrospray ionization/mass spectrometry/mass spectrometry, to provide a fresh interpretation of the MGDG and DGDG composition of the species, Cyanophora paradoxa Korshikov and Glaucocystis nostochinearum Itzigsohn, representing two glaucocystophyte genera. We have found that the major forms of MGDG and DGDG (with sn‐1/sn‐2 regiochemistry) are 20:5/16:0 MGDG, 20:5/20:5 MGDG, 20:5/16:0 DGDG, and 20:5/20:5 DGDG. A comparison of these four forms, along with other more minor forms of MGDG and DGDG, to two examples of cyanobacteria has revealed that glaucocystophytes do not share intact forms of MGDG and DGDG with extant cyanobacteria, but may have maintained certain C16 and C18 cyanobacterial fatty acids.

LIPID COMPOSITION OF CHLORARACHNIOPHYTES (CHLORARACHNIOPHYCEAE) FROM THE GENERA BIGELOWIELLA, GYMNOCHLORA, AND LOTHARELLA1

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast‐associated glycolipids, storage triglycerides, and cytoplasmic membrane‐associated polar lipids were characterized. Glycolipid‐associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0). Triglyceride‐associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n‐3)] dominant along with a number of minor C18 and C20 fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans Moestrup. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences were transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.

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

Abstract 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 octadecapentaenoic acid [18:5(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, (24S)-4α-methyl-5α-ergosta-8(14),22-dien-3β-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 examinations 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 mini review on the biochemistry of dinoflagellate lipids, as studied in both the laboratory and the environment.

Sterols of the heterotrophic dinoflagellate, pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.