Sterol Composition of the Peridinin-Containing Dinoflagellate Gertia stigmatica, a Member of the Kareniaceae without a Canonical Haptophyte-Derived Plastid.

Gertia stigmatica is a recently described member of the Kareniaceae with a peridinin-containing plastid rather than the aberrant, haptophyte-derived, tertiary plastid found in canonical Kareniaceae genera such as Karenia, Karlodinium, and Takayama. G. stigmatica provides a unique opportunity to compare biochemical traits, such as sterol composition, between these two fundamentally different types of Kareniaceae. To this point, canonical members of the Kareniaceae have been observed to typically produce a set of 4α-methyl-substituted, Δ8(14)-nuclear-unsaturated major sterols, such as (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3ß-ol (gymnodinosterol) and 27-nor-(24R)-4α-methyl-5α-ergosta-8(14),22-dien-3ß-ol (brevesterol), which are very uncommon throughout other members of the class Dinophyceae. Our objective was to compare the sterols of G. stigmatica to canonical Kareniaceae to elucidate whether these same distinctive sterols are found, with our hypothesis being that they would because G. stigmatica is indeed a member of the Kareniaceae. Contrary to our hypothesis, G. stigmatica lacks gymnodinosterol and brevesterol, with its sterols instead dominated by 4-desmethyl sterols, such as cholesterol, 24-methylcholesta-5,22E-dien-3ß-ol, and the unusual tri-unsaturated sterols ergosta-5,8(14),22E-trien-3ß-ol and cholesta-5,8(14),22E-trien-3ß-ol. No sterols were found to possess a 4α-methyl substituent or a single Δ8(14) nuclear unsaturation. Thus, G. stigmatica's sterol composition as a member of the Kareniaceae is atypical.

Sterols of morphologically distinct, okadaic acid-producing Prorocentrum texanum var. texanum and var. cuspidatum isolated from the Gulf of Mexico.

Prorocentrum texanum var. texanum and its morphologically distinct yet genetically identical (as based on the sequences of five genes) variety P. texanum var. cuspidatum represent a species of Prorocentrum recently isolated from the Gulf of Mexico. Together, these two varieties represent a sister species to Prorocentrum micans. P. micans has had its sterols, which are ringed lipids common to eukaryotic cell membranes, shown in some studies to be comprised of cholesterol (cholest-5-en-3ß-ol), 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and 4α,23,24-trimethyl-5α-cholestan-3ß-ol (dinostanol) as major sterols, thus placing it within a previously identified cluster of dinoflagellates characterized by the predominance of cholesterol and dinosterol. In this study we have determined the sterol compositions of these two varieties of P. texanum to be abundant in cholesterol, 23,24-dimethyl-cholesta-5,22-dien-3ß-ol, 23,24-dimethyl-5α-cholest-22E-en-3ß-ol, dinosterol, and dinostanol such that the varieties are virtually indistinguishable from each other, making them both in general agreement with the sterols of P. micans, its closest species relative. This expands our knowledge of the sterols of this environmentally important dinoflagellate genus.

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8 (14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae.

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ8(14) sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3ß-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ8(14) sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3ß-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3ß-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8 (14) sterols previously considered Amphidinium-specific biomarkers.

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ8(14) -nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3ß-ol (C28:1 ) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3ß-ol (amphisterol; C29:2 ) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3ß-ol; C27:1 ), which is found in many other dinoflagellate genera, rather than Δ8(14) sterols. While the Δ8(14) sterols 4α-methyl-5α-cholest-8(14)-en-3ß-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3ß-ol (C30:2 ) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3ß-ol (dinosterol; C30:1 ), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ8(14) sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ8(14) sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.

Sterol Composition of the Peridinioid Dinoflagellate Zooxanthella nutricula, A Symbiont of Polycystine Radiolarians.

Some dinoflagellates, such as Symbiodinium, are able to form symbiotic relationships with larger marine organisms. An important aspect of dinoflagellate symbiosis involves the exchange of lipids, namely sterols, from the symbiont to the host. Much research has explored the lipid biochemistry of the symbiotic relationship between cnidarians and Symbiodinium dinoflagellates. However, no research has addressed the sterol biochemistry of the symbiosis between radiolarians and dinoflagellates such as Zooxanthella nutricula. To this end, we have provided the first sterol characterization of Z. nutricula isolated from a spumellarian polycystine radiolarian. Fifteen sterols and one steroidal ketone were observed where the major sterol identified was C27 22-dehydrocholesterol, which does not tend to be a dominant sterol among dinoflagellates, including closely related peridinioid species in the genus Heterocapsa. However, C30 dinosterol and dinostanol were major sterols in both Z. nutricula and Heterocapsa spp., thus indicating common sterols between closely related taxa. Major sterols of the distantly related genus Symbiodinium, a symbiont of foraminifera and cnidarians, have included C27 cholesterol and C30 gorgosterol, whereas in Z. nutricula these sterols were minor and absent, respectively. Our results indicate potentially different sterol pools available to cnidarian and radiolarian symbiont hosts during their respective relationships with symbiotic dinoflagellates.

First examination of sterols in the marine dinoflagellate genus Vulcanodinium.

Vulcanodinium is an ecologically relevant dinoflagellate genus due to its production of neurotoxins known as pinnatoxins. We present here the first examination of the sterols of a Vulcanodinium rugosum isolate. Sterols are ringed lipids that assist in maintaining rigidity of cellular membranes, and the Dinophyceae are well-studied for their ability to produce a diverse array of sterols, many of which have chemotaxonomic utility. We have determined that V. rugosum produces a set of major sterols, namely cholesterol, dinosterol, 4α,24-dimethyl-5α-cholest-22E-en-3ß-ol, and 4α,24-dimethyl-5α-cholestan-3ß-ol, common to the Dinophyceae. However, this displayed marked differences from those studied members of the genera Scrippsiella and Peridinium, the closest phylogenetic relatives. Included in these differences is production by V. rugosum of a much lower percentage of dinostanol, a saturated form of dinosterol.

Sterols of the Toxic Marine Dinoflagellate, Pyrodinium bahamense.

Pyrodinium bahamense is a dinoflagellate of concern in subtropical and tropical coastal environments. To date, there is only a single published study on its fatty acids, but no published data on its sterol composition. Sterols, which are membrane-reinforcing lipids in eukaryotes, display a great diversity of structures in dinoflagellates, with some serving as chemotaxonomic markers. We have examined the sterol compositions of two isolates of P. bahamense from Indian River Lagoon and Tampa Bay, Florida, and have found both to produce three sterols: cholesterol, dinosterol, and 4α-methylgorgostanol. All three sterols are found in closely related, armored taxa.

Plastid Transcript Editing across Dinoflagellate Lineages Shows Lineage-Specific Application but Conserved Trends.

Dinoflagellates are a group of unicellular protists with immense ecological and evolutionary significance and cell biological diversity. Of the photosynthetic dinoflagellates, the majority possess a plastid containing the pigment peridinin, whereas some lineages have replaced this plastid by serial endosymbiosis with plastids of distinct evolutionary affiliations, including a fucoxanthin pigment-containing plastid of haptophyte origin. Previous studies have described the presence of widespread substitutional RNA editing in peridinin and fucoxanthin plastid genes. Because reports of this process have been limited to manual assessment of individual lineages, global trends concerning this RNA editing and its effect on the biological function of the plastid are largely unknown. Using novel bioinformatic methods, we examine the dynamics and evolution of RNA editing over a large multispecies data set of dinoflagellates, including novel sequence data from the peridinin dinoflagellate Pyrocystis lunula and the fucoxanthin dinoflagellate Karenia mikimotoi. We demonstrate that while most individual RNA editing events in dinoflagellate plastids are restricted to single species, global patterns, and functional consequences of editing are broadly conserved. We find that editing is biased toward specific codon positions and regions of genes, and generally corrects otherwise deleterious changes in the genome prior to translation, though this effect is more prevalent in peridinin than fucoxanthin lineages. Our results support a model for promiscuous editing application subsequently shaped by purifying selection, and suggest the presence of an underlying editing mechanism transferred from the peridinin-containing ancestor into fucoxanthin plastids postendosymbiosis, with remarkably conserved functional consequences in the new lineage.

Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3ß-ol, and a minor unknown sterol which appeared to be a C(28:4) compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted.

Sterols of the green-pigmented, freshwater raphidophyte, Gonyostomum semen, from Scandinavian lakes.

Sterols are a class of membrane-reinforcing, ringed lipids which have a long history of examination in algae as a means of deriving chemotaxonomic relationships and as potential lipidic biomarkers. The Raphidophyceae represent a class of harmful, bloom-forming, marine and freshwater algae. To date, there have been four published examinations of their sterol composition, focusing primarily on brown-pigmented, marine species within the genera, Chattonella, Fibrocapsa, and Heterosigma. Lacking in these examinations has been the species Gonyostomum semen Ehrenb., which is a green-pigmented, freshwater raphidophyte with a worldwide distribution. The goal of this study was to examine the sterol composition of this nuisance alga, determine the potential of using its sterol profile as a biomarker, and finally to determine if there is any intraspecific variability between isolates. We have examined 21 isolates of G. semen from a number of Scandinavian lakes, and all were found to produce two major sterols, 24-ethylcholesta-5,22E-dien-3ß-ol and 24-ethylcholest-5-en-3ß-ol, and 24-methylcholest-5-en-3ß-ol as a minor sterol; the presence of 24-ethylcholesta-5,22E-dien-3ß-ol differentiates G. semen from brown-pigmented, marine raphidophytes which generally lack it. The results of this study indicate that isolates of G. semen from geographically separate lakes across Finland and Scandinavia have the same sterol biosynthetic pathway, and that there is no evolutionary divergence between the isolates with regard to sterol composition. The sterols of G. semen are not considered to be useful biomarkers for this particular organism because they are commonly found in other algae and plants.

Structural analysis and cellular localization of polyunsaturated C27 hydrocarbons in the marine dinoflagellate, Pyrocystis lunula (Dinophyceae).

A neutral lipid fraction obtained from two strains of the permanently sheathed dinoflagellate, Pyrocystis lunula, was found to contain three polyunsaturated C27 hydrocarbons as abundant lipid components. A combination of mass spectrometry techniques was used to identify these compounds as n-heptacosa-3,6,9,12,15,18-hexaene (C27:6), approx. 0.7 ng/sheathed cell), n-heptacosa-3,6,9,12,15,18,21-heptaene (C27:7), approx. 2 ng/sheathed cell), and n-heptacosa-3,6,9,12,15,18,21,24-octaene (C27:8), approx. 2 ng/sheathed cell). Polyunsaturated C21, C23, and C25 hydrocarbons were also found at lesser amounts of approximately 0.2-0.5 ng/sheathed cell. Fluorescent microscopy revealed Nile red staining in both the vegetative cell and structures within the outer sheath surrounding the cell. These hydrocarbons were not present in two other species of Pyrocystis, P. fusiformis and P. noctiluca. Although their function(s) is not known, previous studies have shown and hypothesized that similar hydrocarbons function in carbon storage, buoyancy regulation, or signaling.

Sterol composition and biosynthetic genes of the recently discovered photosynthetic alveolate, Chromera velia (chromerida), a close relative of apicomplexans.

Chromera velia is a recently discovered, photosynthetic, marine alveolate closely related to apicomplexan parasites, and more distantly to perkinsids and dinoflagellates. To date, there are no published studies on the sterols of C. velia. Because apicomplexans and perkinsids are not known to synthesize sterols de novo, but rather obtain them from their host organisms, our objective was to examine the composition of the sterols of C. velia to assess whether or not there is any commonality with dinoflagellates as the closest taxonomic group capable of synthesizing sterols de novo. Furthermore, knowledge of the sterols of C. velia may provide insight into the sterol biosynthetic capabilities of apicomplexans prior to loss of sterol biosynthesis. We have found that C. velia possesses two primary sterols, 24-ethylcholesta-5,22E-dien-3ß-ol, and 24-ethylcholest-5-en-3ß-ol, not common to dinoflagellates, but rather commonly found in other classes of algae and plants. In addition, we have identified computationally three genes, SMT1 (sterol-24C-methyltransferase), FDFT1 (farnesyl diphosphate farnesyl transferase, squalene synthase), and IDI1 (isopentenyl diphosphate Δ-isomerase), predicted to be involved in sterol biosynthesis by their similarity to analogous genes in other sterol-producing eukaryotes, including a number of algae.

Sterols of the green-pigmented, aberrant plastid dinoflagellate, Lepidodinium chlorophorum (Dinophyceae).

Lepidodinium chlorophorum is a green-pigmented dinoflagellate with an aberrant, tertiary plastid of chlorophyte ancestry rather than the typical red algal, secondary endosymbiont found in the vast majority of photosynthetic dinoflagellates. To date, only one published study exists on the galactolipids of L. chlorophorum, with nothing known about other lipid classes, including sterols. Our objectives were to examine the sterol composition of L. chlorophorum to determine if it produces any unique sterols with the potential to serve as biomarkers, and to compare it to members of the Chlorophyceae to determine if it has inherited any signature green algal sterols from its chlorophyte-derived endosymbiont. We have found that L. chlorophorum produces 6 sterols, all with a 4α-methyl substituent and none of which are known to occur in the Chlorophyceae. Rather, the sterols produced by L. chlorophorum place it within a group of dinoflagellates that have the common dinoflagellate sterols, dinosterol and dinostanol, as part of their sterol composition.

Free sterol composition of species in the dinoflagellate genus Pyrocystis: a spectrum of sterol diversity.

The dinoflagellate genus Pyrocystis includes a small number of marine species, which spend the majority of their life cycles as nonmotile cells within a carbohydrate sheath, and which are found ubiquitously throughout the world's oceans. The biochemistry of this model dinoflagellate genus has been widely studied due to its ability to bioluminesce. However, Pyrocystis has been comparatively understudied with respect to its lipid biochemistry, in particular that of sterols. To date, examination of the sterols of Pyrocystis has focused primarily upon Pyrocystis lunula, which produces cholesterol and 4,24-dimethyl-5α-cholestan-3ß-ol as its predominant sterols, while it lacks the common dinoflagellate sterol, dinosterol. We have examined the sterol composition of the two other commercially available species of Pyrocystis, Pyrocystis fusiformis and Pyrocystis noctiluca. Pyrocystis noctiluca possesses dinosterol as its most abundant sterol, while P. fusiformis possesses dinosterol and 4,24-dimethyl-5α-cholestan-3ß-ol as the predominant sterols, placing it at an intermediate position between P. lunula and P. noctiluca, as based on sterol composition. The potential limitations of the dinoflagellate sterol biomarker dinosterol are also explored in this study due to its notable absence in P. lunula.

MONO- AND DIGALACTOSYLDIACYLGLYCEROL COMPOSITION OF RAPHIDOPHYTES (RAPHIDOPHYCEAE): A MODERN INTERPRETATION USING POSITIVE-ION ELECTROSPRAY/MASS SPECTROMETRY/MASS SPECTROMETRY(1).

Raphidophyte algae (Raphidophyceae) can be divided according to pigment composition and plastid ancestry into two categories, brown- and green-pigmented taxa. We sought to examine if there are any biochemical differences in plastid lipid composition between the two groups. To this end, the composition and positional distribution of fatty acids of the chloroplast lipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), were examined using positive-ion electrospray/mass spectrometry (ESI/MS) and electrospray/mass spectrometry/mass spectrometry (ESI/MS/MS). Brown-pigmented strains from the genera Chattonella, Fibrocapsa, and Heterosigma primarily consisted of 20:5/18:4 (sn-1/sn-2) MGDG and 20:5/18:4 DGDG, while isolates of the green-pigmented raphidophyte Gonyostomum semen (Ehrenb.) Diesing contained these as well as 18:3/18:4 MGDG and DGDG, thus underscoring its green algal plastid lineage. Although previously unseen without the regiochemical information provided by ESI/MS/MS, Chattonella subsalsa Biecheler possessed 20:5/18:3 DGDG as a major form, a potential biosynthetic intermediate in the production of 20:5/18:4 DGDG. These results provide a modern interpretation of the fatty acid regiochemistry of MGDG and DGDG.

A data mining approach to dinoflagellate clustering according to sterol composition: correlations with evolutionary history.

This study examined the sterol compositions of 102 dinoflagellates using clustering and cluster validation techniques, as a means of determining the relatedness of the organisms. In addition, dinoflagellate sterol-based relationships were compared statistically to 18S rDNA-based phylogenetic relationships using the Mantel test. Our results indicated that the examined dinoflagellates formed six clusters based on sterol composition and that several, but not all, dinoflagellate genera, which formed discrete clusters in the 18S rDNA-based phylogeny, shared similar sterol compositions. This and other correspondences suggest that the sterol compositions of dinoflagellates are explained, to a certain extent, by the evolutionary history of this lineage.

Sterols of the syndinian dinoflagellate Amoebophrya sp., a parasite of the dinoflagellate Alexandrium tamarense (Dinophyceae).

Several harmful photosynthetic dinoflagellates have been examined over past decades for unique chemical biomarker sterols. Little emphasis has been placed on important heterotrophic genera, such as Amoebophrya, an obligate, intracellular parasite of other, often harmful, dinoflagellates with the ability to control host populations naturally. Therefore, the sterol composition of Amoebophrya was examined throughout the course of an infective cycle within its host dinoflagellate, Alexandrium tamarense, with the primary intent of identifying potential sterol biomarkers. Amoebophrya possessed two primary C(27) sterols, cholesterol and cholesta-5,22Z-dien-3beta-ol (cis-22-dehydrocholesterol), which are not unique to this genus, but were found in high relative percentages that are uncommon to other genera of dinoflagellates. Because the host also possesses cholesterol as one of its major sterols, carbon-stable isotope ratio characterization of cholesterol was performed in order to determine whether it was produced by Amoebophrya or derived intact from the host. Results indicated that cholesterol was not derived intact from the host. A comparison of the sterol profile of Amoebophrya to published sterol profiles of phylogenetic relatives revealed that its sterol profile most closely resembles that of the (proto)dinoflagellate Oxyrrhis marina rather than other extant genera.