Diacylglycerylcarboxyhydroxymethylcholine Research Articles

Membrane lipid remodeling and autophagy to cope with phosphorus deficiency in the dinoflagellate Prorocentrum shikokuense

Dinoflagellates, which are responsible for more than 80% of harmful algal blooms in coastal waters, are competitive in low-phosphate environments. However, the specific acclimated phosphorus strategies to adapt to phosphorus deficiency in dinoflagellates, particularly through intracellular phosphorus metabolism, remain largely unknown. Comprehensive physiological, biochemical, and transcriptomic analyses were conducted to investigate intracellular phosphorus modulation in a model dinoflagellate, Prorocentrum shikokuense, with a specific focus on membrane lipid remodeling and autophagy in response to phosphorus deficiency. Under phosphorus deficiency, P. shikokuense exhibited a preference to spare phospholipids with nonphospholipids. The major phospholipid classes of phosphatidylcholine and phosphatidylethanolamine decreased in content, whereas the betaine lipid class of diacylglyceryl carboxyhydroxymethylcholine increased in content. Furthermore, under phosphorus deficiency, P. shikokuense induced autophagy as a mechanism to conserve and recycle cellular phosphorus resources. The present study highlights the effective modulation of intracellular phosphorus in P. shikokuense through membrane phospholipid remodeling and autophagy and contributes to a comprehensive understanding of the acclimation strategies to low-phosphorus conditions in dinoflagellates.

Composition of galactolipids, betaine lipids and triglyceride‐associated fatty acids of the symbiotic dinoflagellate Zooxanthella (Brandtodinium) nutricula: A glimpse into polyunsaturated fatty acids available to its polycystine radiolarian host

SUMMARYZooxanthella nutricula is a photosynthetic dinoflagellate symbiont of polycystine radiolarians. As such, it is hypothesized to provide fixed organic carbon, including in the form of acylglycerolipids and sterols, to its non‐photosynthetic host. We have previously characterized the sterols of Z. nutricula that may be transferred to its host and, in the present study, have turned our attention to three classes of fatty acid‐containing lipids, chloroplast‐associated galactolipids, betaine lipids, which are non‐phosphorylated phospholipid analogs present in many eukaryotes, and triglycerides. Zooxanthella nutricula was observed using positive‐ion electrospray/mass spectrometry (ESI/MS) and ESI/MS/MS to produce the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched in octadecapentaenoic (18:5(n‐3)) and octadecatetraenoic (18:4(n‐3)) acid to place it within a group of peridinin‐containing dinoflagellates in a C18/C18 (sn‐1/sn‐2 fatty acid regiochemistry) cluster, as opposed to another cluster with C20/C18 MGDG and DGDG, where the C20 fatty acid is eicosapentaenoic acid (20:5(n‐3)) and the C18 fatty acid is either 18:5(n‐3) or 18:4(n‐3). Zooxanthella nutricula was also observed to produce 38:10 (total number of fatty acid carbons:total number of double bonds), 38:6, and 44:7 diacylglycerylcarboxyhydroxymethylcholine (DGCC) as the sole type of betaine lipid. Although it is more difficult to determine which fatty acids are present in the sn‐1 and sn‐2 positions on the glycerol backbone of DGCC using ESI/MS/MS, gas chromatography/mass spectrometry (GC/MS)‐based examination indicated the putatively DGCC‐associated polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:6(n‐3)). Coupled with the C18 PUFAs of MGDG and DGDG, and fatty acids associated with triglycerides (also examined via GC/MS), Z. nutricula could serve as a rich source of PUFAs for its radiolarian host. These data demonstrate that Z. nutricula produces a similar set of PUFA‐containing lipids as Symbiodinium microadriaticum, a photosynthetic dinoflagellate symbiont of cnidarians, indicating a metabolic commonality in these phylogenetically discrete dinoflagellate symbionts with unrelated host organisms.

Revealing the polar lipidome, pigment profiles, and antioxidant activity of the giant unicellular green alga, Acetabularia acetabulum.

Marine algae are one of the most important sources of high-value compounds such as polar lipids, omega-3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single-celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS), tandem mass spectrometry (MS/MS), and high-performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl-carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis-neoxanthin, violaxanthin, lutein and β,β-carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC50 ) with concentrations of 57.91 ± 1.20 μg · mL-1 (438.18 ± 8.95 μmol Trolox · g-1 lipid) in DPPH and 20.55 ± 0.60 μg · mL-1 in ABTS assays (918.56 ± 27.55 μmol Trolox · g-1 lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds.

Smart utilization of betaine lipids in the giant clam Tridacna crocea

The giant clam Tridacna crocea thrives in poorly nourished coral reef water by forming a holobiont with zooxanthellae and utilizing photosynthetic products of the symbiont. However, detailed metabolic crosstalk between clams and symbionts is elusive. Here, we discovered that the nonphosphorous microalgal betaine lipid DGCC (diacylglycerylcarboxy-hydroxymethylcholine) and its deacylated derivative GCC are present in all tissues and organs, including algae-free sperm and eggs, and are metabolized. Colocalization of DGCC and PC (phosphatidylcholine) evidenced by MS imaging suggested that DGCC functions as a PC substitute. The high content of GCC in digestive diverticula (DD) suggests that the algal DGCC was digested in DD for further utilization. Lipidomics analysis showing the organ-specific distribution pattern of DGCC species suggests active utilization of DGCC as membrane lipids in the clam. Thus, the utilization of zooxanthellal DGCC in animal cells is a unique evolutionary outcome in phosphorous-deficient coral reef waters.

Metabolomics Characterization of Scleractinia Corals with Different Life-History Strategies: A Case Study about Pocillopora meandrina and Seriatopora hystrix in the South China Sea.

Life-history strategies play a critical role in susceptibility to environmental stresses for Scleractinia coral. Metabolomics, which is capable of determining the metabolic responses of biological systems to genetic and environmental changes, is competent for the characterization of species' biological traits. In this study, two coral species (Pocillopora meandrina and Seriatopora hystrix in the South China Sea) with different life-history strategies ("competitive" and "weedy") were targeted, and untargeted mass spectrometry metabolomics combined with molecular networking was applied to characterize their differential metabolic pathways. The results show that lyso-platelet activating factors (lyso-PAFs), diacylglyceryl carboxyhydroxymethylcholine (DGCC), aromatic amino acids, and sulfhydryl compounds were more enriched in P. meandrina, whereas new phospholipids, dehydrated phosphoglycerol dihydroceramide (de-PG DHC), monoacylglycerol (MAG), fatty acids (FA) (C < 18), short peptides, and guanidine compounds were more enriched in S. hystrix. The metabolic pathways involved immune response, energy metabolism, cellular membrane structure regulation, oxidative stress system, secondary metabolite synthesis, etc. While the immune system (lysoPAF) and secondary metabolite synthesis (aromatic amino acids and sulfhydryl compounds) facilitates fast growth and resistance to environmental stressors of P. meandrina, the cell membrane structure (structural lipids), energy storage (storage lipids), oxidative stress system (short peptides), and secondary metabolite synthesis (guanidine compounds) are beneficial to the survival of S. hystrix in harsh conditions. This study contributes to the understanding of the potential molecular traits underlying life-history strategies of different coral species.

Chrysotila pseudoroscoffensis as a source of high-value polar lipids with antioxidant activity: A lipidomic approach

Microalgae are emerging as sustainable sources of a wide range of high-value compounds. However, the knowledge about microalgae polar lipids is still limited, despite their interest due to their chemical diversity and bioactivity. This study shows, for the first time, the polar lipidome of the haptophyte microalga Chrysotila pseudoroscoffensis unveiled by using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS and MS/MS) and gas chromatography–mass spectrometry (GC–MS). Freeze-dried C. pseudoroscoffensis biomass has a lipid content of 6.4 %, containing higher amounts of ash (45.5 %), proteins (11.6 %), and sugars (11.0 %). Uronic acids (53.8 mol%) are the sugars present in higher content, followed by glucose (13.7 mol%) and galactose (12.7 mol%). The polar lipid species identified by HILIC-MS/MS included betaine lipids, glycolipids, and phospholipids, some of them with recognised bioactive properties. Among the lipid classes found from C. pseudoroscoffensis, some are less reported in algae: betaine lipids diacylglycerylcarboxyhydroxymethylcholine (DGCC) and monoacylglycerylcarboxyhydroxymethylcholine (MGCC) (characteristic of haptophyte microalgae); acid glycolipid class glucuronosyldiacylglycerol (GlcADG) (mainly reported in plants with protective effects in phosphate-deprivation conditions); and phospholipid classes monomethylphosphatidylethanolamine (MMPE) and lysomonomethylphosphatidylethanolamine (MMLPE). As estimated by colorimetric assays, glycolipids and phospholipids accounted for 64 and 3 % of the total lipid extracts, respectively. Fatty acid profiling by GC–MS showed that total esterified fatty acids accounted for about 32 % of the total lipid extracts, of which 23 % were omega-3 polyunsaturated fatty acids (PUFA). Four lipid extract concentrations (12.5, 62.5, 125 and 250 μg mL−1 in ethanol) were tested and displayed antioxidant capacity toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. A dose-dependent behaviour was observed with IC50 of 111.9 μg mL−1 for ABTS and IC35 of 234.8 μg mL−1 for DPPH assay. In conclusion, the lipid extracts of C. pseudoroscoffensis may be a source of high-value lipids for the development of novel microalgae-based products, namely nutraceuticals and cosmeceuticals.

The Polar Lipidome of Cultured Emiliania huxleyi: A Source of Bioactive Lipids with Relevance for Biotechnological Applications.

Polar lipids from microalgae have aroused greater interest as a natural source of omega-3 (n-3) polyunsaturated fatty acids (PUFA), an alternative to fish, but also as bioactive compounds with multiple applications. The present study aims to characterize the polar lipid profile of cultured microalga Emiliania huxleyi using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC–MS) and fatty acids (FA) analysis by gas chromatography (GC–MS). The lipidome of E. huxleyi revealed the presence of distinct n-3 PUFA (40% of total FA), namely docosahexaenoic acid (22:6n-3) and stearidonic acid (18:4n-3), which give this microalga an increased commercial value as a source of n-3 PUFA present in the form of polar lipids. A total of 134 species of polar lipids were identified and some of these species, particularly glycolipids, have already been reported for their bioactive properties. Among betaine lipids, the diacylglyceryl carboxyhydroxymethylcholine (DGCC) class is the least reported in microalgae. For the first time, monomethylphosphatidylethanolamine (MMPE) has been found in the lipidome of E. huxleyi. Overall, this study highlights the potential of E. huxleyi as a sustainable source of high-value polar lipids that can be exploited for different applications, namely human and animal nutrition, cosmetics, and pharmaceuticals.

Mass Spectrometry Data Repository Enhances Novel Metabolite Discoveries with Advances in Computational Metabolomics.

Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.

Metabolomic and transcriptomic analyses reveal the effects of ultraviolet radiation deprivation on Isochrysis galbana at high temperature

The microalga Isochrysis galbana exhibits a seasonal fluctuation of biomass referred to as ‘decline’ during summer, leading to a great economic loss in the mariculture industry. In the present study, we aimed to explore the role of ultraviolet radiation (UVR) in I. galbana decline at high temperature by integrated analysis of metabolomic and transcriptomic data. The positive effects of UVR deprivation on the growth and photosynthetic activity of I. galbana were preliminarily evidenced by the increase in biomass concentration, the effective photochemical efficiency of photosystem II (PSII) (Fv′/Fm′), and oxygen evolution rate. Furthermore, we compared the contents of eight organic acids, total lipids and gene expression of I. galbana grown under natural sunlight versus UVR-deprived conditions, as well as. The contents of some tricarboxylic acid (TCA) cycle-related organic acids were higher in the UVR-deprived group. Lipidomic analysis indicated that UVR deprivation resulted in the accumulation of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC), diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), and lyso-lipids, while triacylglycerol (TAG) was decreased. Consistent with these results, the transcriptomic data showed that the expressions of some key genes involved in the TCA cycle and the photosynthesis pathways were clearly induced by UVR deprivation. Besides, the down-regulation of diacylglycerol O-acyltransferases (DGAT) and the up-regulation of TAG lipase collectively resulted in the lower accumulation of TAG, which was consistent with the metabolomic data. Taken together, the decline of I. galbana in summer could be explained by the disorder of a series of biological processes caused by UVR, especially the damage to photosynthesis, the slowdown of TCA cycle, and the disturbance of lipid metabolism. This study allowed us to better understand the responses of I. galbana to UVR stress and provided useful information for the cultivation of I. galbana in summer, greatly expanding our current knowledge on the genetic information of I. galbana.

Low-molecular-mass organic acid and lipid responses of Isochrysis galbana Parke to high temperature stress during the entire growth stage

Isochrysis galbana is an important food source in aquaculture especially in the rearing of bivalve mollusks and usually cultured in summer, when water temperatures in breeding pools may reach 35°C. However, the impact of high temperature stress on the growth and metabolism of I. galbana is poorly understood. In this study, the changes of total superoxide dismutase (T-SOD) activity, organic acid, and lipid levels were analyzed at different growth stages when I. galbana Parke was cultured under normal temperature (20°C) and extreme high temperature (35°C), respectively. The result showed that T-SOD activity reached the highest level at exponential phase at both temperatures but exhibited lower level during growth at 35°C. The lower content of citric acid and α-ketoglutaric acid during late stationary phase indicated that tricarboxylic acid (TCA) cycle, amino acid metabolism, and the synthesis of fatty acids may severely be damaged under high temperature stress. The upregulation of monogalactosyldiacylglycerol (MGDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC), diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), triacylglycerol (TAG), and lyso-lipids was observed from adaptation phase to early stationary phase. But downregulation of glyceroglycolipids (MGDG, DGDG, and SQDG), DGCC, and DGTS were found between early stationary phase and late stationary phase at high temperature. Besides, the level of DHA decreased under high temperature stress, revealing that the nutritional value of I. galbana Parke had been reduced.This is the first report addressing metabolic responses of I. galbana Parke to high temperature stress combining T-SOD activity, organic acid, and lipid analysis, and the results demonstrated that the extreme high temperature (35°C) will not only suppress the activity of T-SOD and the formation of organic acids in the TCA cycle and photorespiration, but also influence the lipid metabolism in I. galbana Parke. This study provided a useful reference value for the cultivation of I. galbana under high temperature condition.

Effect of high temperature on the lipid composition of Isochrysis galbana Parke in logarithmic phase

Isochrysis galbana is a species of marine microalgae that is principally used to feed the larvae and early juveniles of bivalve mollusks (e.g., ark shells, clams, mussels, oysters and scallops). The artificial hatchery stages of most of these larvae are primarily occur during high-temperature seasons, when the water temperature in the breeding pool frequently exceeds 30 °C and even reaches 35 °C, which may change the lipid composition of I. galbana, thus affecting its nutritional value. In this experiment, we investigated changes in the lipid composition of I. galbana Parke at high temperature using ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. The results showed that the sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC) and digalactosyldiacylglycerol (DGDG) contents increased during growth at 20 and 35 °C. Conversely, the lyso-SQDG, lyso-MGDG, lyso-DGDG, lyso-DGCC and triacylglycerol (TAG) contents clearly decreased. Moreover, higher SQDG, MGDG, DGDG and DGCC contents and lower lyso-lipid and TAG contents were observed in I. galbana Parke grown at 35 versus 20 °C. In addition, the content of docosahexaenoic acid (DHA) improved at 20 and 35 °C during growth, and DHA accumulated to greater levels at 35 °C than at 20 °C at the same time points. These results revealed an increase in DHA content as the temperature rose, which indicated that the nutritional value of I. galbana Parke during logarithmic phase improved in the same cultured time with an increasing temperature.

Exploring occurrence and molecular diversity of betaine lipids across taxonomy of marine microalgae

Betaine lipids (BL) from ten microalgae species of the kingdoms Plantae and Chromista were identified and quantified by HPLC/ESI-TOF-MS. Diacylgyceryl-N-trimethylhomoserine (DGTS) was detected in Trebouxiophyceae and Eustigmatophyceae species, whereas Tetraselmis suecica was described as the first green algae containing diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA). DGTA molecular species where also characterized in Cryptophyceae species as well as in the Bacillariophyceae diatom Phaeodactylum tricornutum. The Mediophyceae diatom Chaetoceros gracilis had no DGTA, but contained diacylglyceryl-carboxyhydroxymethylcholine (DGCC). A principal coordinate (PCO) analysis of microalgae species revealed the existence of three main clusters around each BL type. The first PCO axis (43.9% of total variation) grouped Chlorophyceae, Trebouxiophyceae and Eustigmatophyceae species and positively correlated with DGTS. The second PCO axis (27.8% of total variation) segregated DGTA from DGCC containing species. Cryptophyceae, Bacillariophyceae and Chlorodendrophyceae were the more closely associated species to DGTA. Mediophyceae and Dinophyceae species contained DGCC as the only BL. Molecular diversity varied from the simplest DGCC composition in Gyrodinium dorsum to the highest spectrum of ten different molecular species detected for DGTA (Rhodomonas baltica) and DGCC (C. gracilis). The fatty acid profile of DGTS was very dissimilar to that of the whole lipid cell content. DGTS from Nannochloropsis gaditana was highly unsaturated respecting to total lipids, whereas in Picochlorum atomus DGTS unsaturation was nearly one half to that of total lipids. Dissimilarity between DGTA and total lipid fatty acid profile was minimum among all BL and DGTA fatty acid unsaturation was the maximum observed in the study. New DGCC molecular species enriched in 20:5 were described in Mediophyceae diatoms. Multivariate microalgae ordination using BL as descriptors revealed a higher chemotaxonomic potential than that based on their respective BL fatty acid profile. Nevertheless, taxonomic resolution was improved when fatty acids from the whole cell lipid pool were used.

Squishy lipids: Temperature effects on the betaine and galactolipid profiles of a C18/C18 peridinin‐containing dinoflagellate, Symbiodinium microadriaticum (Dinophyceae), isolated from the mangrove jellyfish, Cassiopea xamachana

SummaryPrevious work from our laboratory has shown dinoflagellates, which possess the carotenoid peridinin, have been divided into two clusters based on plastid galactolipid fatty acid composition. In one cluster major forms of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), lipids that comprise the majority of photosynthetic membranes, were C18/C18 (sn‐1/sn‐2), with octadecapentaenoic [18:5(n‐3)] and octadecatetraenoic [18:4(n‐3)] acid as principal fatty acids. The other cluster contained C20/C18 major forms, with eicosapentaenoic acid [20:5(n‐3)] being the predominant sn‐1 fatty acid. In this study, we have found that Symbiodinium microadriaticum isolated from the jellyfish, Cassiopea xamachana, when grown at 30°C, produced MGDG and DGDG with a more saturated fatty acid, 18:4(n‐3), at the sn‐2 carbon than when grown at 20°C where 18:5(n‐3) predominates. This modulation of the sn‐2 fatty acid's level of saturation is mechanistically similar to what has been observed in Pyrocystis, a C20/C18 dinoflagellate. We have also examined the effect of growth temperature on the betaine lipid, diacylglycerylcarboxyhydroxymethylcholine (DGCC), which has been observed by others to be the predominant non plastidial polar lipid in dinoflagellates. Temperature effects on it were minimal, with very few modulations in fatty acid unsaturation as observed in MGDG and DGDG. Rather, the primary difference seen at the two growth temperatures was the alteration of the amount of minor forms of DGCC, as well as a second betaine lipid, diacylglyceryl‐N,N,N‐trimethylhomoserine.

Differences in betaine lipids and fatty acids between Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP isolates (Haptophyta)

Two Haptophytes were isolated from extensive aquaculture ponds at Veta La Palma state (Guadalquivir estuary, SW Spain). They were identified as Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP based on their SSU rDNA homology to other Haptophytes and positioned in the Isochrysidaceae and Pavlovaceae families, respectively. Both Haptophytes had phosphatidilglycerol (PG) as the only phospholipid (PL), representing a low proportion of the total lipid content (0.8% in P. paradoxa VLP and 3.3% in D. vlkianum VLP). Instead, they were found to have different types of betaine lipids (BL) that were identified and characterized by HPLC/ESI-TOF-MS operating in multiple reacting monitoring (MRM) modes. P. paradoxa VLP had 2.2% of total lipids as diacylgyceryl-N-trimethylhomoserine (DGTS): it is the first Haptophyte reported to have this BL. Its total lipid fraction also contained 12.0% of diacylglyceryl-carboxyhydroxymethylcholine (DGCC) as the main BL and no diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) was detected. DGTA was only present (4.6% of total lipids) in D. vlkianum VLP: this was the main difference in BL content relative to P. paradoxa. D. vlkianum VLP also had DGTS (4.1%) and DGCC (7.6%): it is the first microalgae in which the simultaneous presence of these three BL has been demonstrated. The fatty acid profiles of P. paradoxa VLP and D. vlkianum VLP were close to those described for the major part of known members of the Isochrisidaceae and Pavlovaceae families, respectively, with the main differences due to the higher percentages of 18:1n9 (18.5%), 18:4n3 (12.6%) and 22:6n3 (9.3%) in the former. The corresponding fatty acid percentages for D. vlkianum VLP were 3.9%, 3.5% and 3.9%, respectively. D. vlkianum VLP showed higher 16:1n7 (16.1%) and 20:5n3 (9.4%) contents, whereas P. paradoxa VLP had significantly lower percentages of 16:1n7 (1.7%) and 20:5n3 (0.6%). Fatty acids of BL differed between both haptophytes. In DGTS from P. paradoxa VLP, 90.9% of total molecular species consisted of the 14:0-18:1 fatty acid combination, whereas DGTS from D. vlkianum showed a more diverse range of fatty acids. The unsaturation index (UI) of DGTS was lower (55.8) than that of total lipid UI (178.3) in P. paradoxa VLP. In D. vlkianum VLP the UI of DGTS was higher (146.9) and similar to that for total cell lipids (145.9). DGTA from D. vlkianum VLP had the highest UI (321.8) of all BL studied and it contained maximum levels (27.7%) of 22:6n3, representing 7.1 times the proportion of this fatty acid in the whole lipid extract. DGCC was enriched in 20:5n3 by a factor of around four in both microalgae. Due to different levels of this fatty acid in the two microalgae their respective 20:5n3 content in DGCC varied from 2.2% (P. paradoxa VLP) to 41.0% (D. vlkianum VLP) and these concentrations were also associated with UI values of 92.2 and 271.0, respectively. The specific differences in BL and fatty acids described in the present work for two phylogenetic distant Hatophytes is a contribution to a better understanding on the complex relationship between lipid composition and taxonomy of this important Division of microalgae. Present results can also be useful for a more accurate identification of primary producers in food web studies using fatty acids and intact polar lipids as trophic markers.

Molecular Ion‐Independent Quantification of Polar Glycerolipid Classes in Marine Plankton Using Triple Quadrupole MS

Polar glycerolipids are a diverse family of lipid molecules that form the bulk of bacterial and eukaryotic microbial membranes. The earth and ocean sciences has a long history of using fatty acids as biomarkers for microbes, but have only recently begun to examine the intact polar lipids from which they are derived. Current analytical approaches rely on laboriously quantifying the molecular ions of each of these species independently. Thus, we saw a need for a method for quantifying polar glycerolipid classes that was: (i) selective for individual classes, (ii) inclusive of all species within a class, (iii) independent of foreknowledge of the molecular ions of the polar glycerolipid, and (iv) amenable to automated, high-throughput data analysis methods. Our new HPLC-electrospray-ionization triple-quadrupole MS (HPLC-ESI-TQMS) method can be applied to quantify the nine major classes of polar glycerolipid in planktonic communities: the phospholipids phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the glycolipids monoglycosyldiacylglycerol, diglycosyldiacylglycerol and sulfoquinovosyldiacylglycerol; and the betaine lipids diacylglyceryl trimethyl homoserine, diacylglyceryl hydroxymethyl trimethyl-β-alanine, and diacylglyceryl carboxyhydroxymethylcholine. The analyses rely on neutral loss and parent ion scan events that yield one chromatogram for each class of polar glycerolipid, simplifying downstream analysis and increasing sample throughput. The efficacy of the method was demonstrated by analyzing plankton community samples from a variety of marine environments.

Spatial distribution of intact polar lipids in North Sea surface waters: Relationship with environmental conditions and microbial community composition

We characterized and quantified the intact polar lipid (IPL) composition of the surface waters of the North Sea and investigated its relationships with environmental conditions, microbial abundances, and community composition. The total IPL pool comprised at least 600 different IPL species in seven main classes: the glycerophospholipids phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE); the sulfur‐bearing glycerolipid sulfoquinovosyldiacylglycerol (SQDG); and the nitrogen‐bearing betaine lipids diacylglyceryl‐trimethylhomoserine (DGTS), diacylglyceryl‐hydroxymethyltrimethylalanine (DGTA), and diacylglyceryl‐carboxyhydroxymethylcholine (DGCC). Although no significant relationships were found between the IPL composition and environmental parameters, such as nutrient concentrations, distance‐based ordination yielded distinct clusters of IPL species, which could in turn be tentatively correlated with the predominant microbial groups. SQDGs and PGs, as well as PC species containing saturated fatty acid moieties, were related to picoeukaryote abundances and PC species with polyunsaturated fatty acid (PUFA) moieties to nanoeukaryote abundances. The PEs were likely of mixed cyanobacterial–bacterial origin, whereas DGTA and DGCC species were mainly associated with cyanobacteria. DGTSs were likely derived from either pico‐ or nanoeukaryotes, although the DGTS species with PUFAs also showed some relationship with cyanobacterial abundances. Concentrations of the algal‐derived IPLs showed strong positive correlations with chlorophyll a concentrations, indicating they may be used as biomarkers for living photosynthetic microbes. However, direct relationships between the IPLs and microbial groups were relatively weak, implying that the predominant IPLs in marine surface waters are not derived from single microbial groups and that direct inferences of microbial community compositions from IPL compositions should be considered with care.

Betaine lipids in chlorarachniophytes

SUMMARY 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.

Bacterial and eukaryotic intact polar lipids in the eastern subtropical South Pacific: Water-column distribution, planktonic sources, and fatty acid composition

Fatty acids are generally the most abundant lipid molecules in plankton, and thus play a central role in the cycling of organic matter in the upper ocean. These fatty acids are primarily derived from intact polar diacylglycerolipids (IP-DAGs), which compose cell membranes in plankton. The molecular diversity of IP-DAGs in the upper ocean remains to be fully characterized, and the advent of high performance liquid chromatography/electrospray ionization–mass spectrometry (HPLC/ESI-MS) approaches have now provided the opportunity to readily analyze IP-DAGs from marine planktonic communities. We used HPLC/ESI-MS to determine the concentrations of three classes of phospholipids (phosphatidlyglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC)), three classes of betaine lipids (diacylglyceryl trimethylhomoserine (DGTS), diacylglyceryl hydroxymethyl-trimethyl-β-alanine (DGTA), and diacylglyceryl carboxyhydroxymethylcholine (DGCC)), and three classes of glycolipids (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG)) in plankton filtered (>0.2 μm) from seawater collected within the euphotic zone of the eastern South Pacific. The distributions of these IP-DAGs along the cruise transect provided important new insights on their tentative planktonic sources. Complementary data from our cruise, a principle components analysis of our IP-DAG concentrations, observed fatty acid compositions of IP-DAG classes and published IP-DAG distributions in pure cultures of plankton suggest that heterotrophic bacteria were the dominant sources of PG and PE, while MGDG and SQDG originated primarily from Prochlorophytes. The origins of the other classes of IP-DAGs were less clear, although it is likely that PC, DGTS, DGTA, and DGCC were derived primarily from eukaryotic phytoplankton. The molecular distributions of fatty acids attached to the different classes of IP-DAGs were generally distinct from one another, and suggest that reported distributions of total fatty acids (as analyzed by gas chromatography) in the literature should be regarded as homogenized mixtures of distinct molecular pools of fatty acids.

Lipids of Pavlova lutheri: Cellular site and metabolic role of DGCC

Pavlova lutheri contains triacylglycerols (TAG), monogalactosyldiacylglycerols (MGDG), galactosyldiacylglycerols (DGDG), sulfoquivonosyldiacylglycerols (SQDG), diacylglyceryl hydroxymethyl- N,N,N-trimethyl-β-alanine (DGTA), diacylglyceryl carboxyhydroxymethylcholine (DGCC) and diacylglyceryl glucuronide (DGGA) as major lipid components. Prominent fatty acids are 14:0, 16:0, 16:1, 18:4, 20:5, 22:5 and 22:6. In MGDG and DGDG, 18:4 and 20:5 predominate, while SQDG contains high proportions of 14:0 and 16:0. DGCC is enriched in 16:0 and 20:5, DGGA in 18:1, 22:5 and 22:6, and DGTA in 22:6. Analysis of subcellular membrane fractions demonstrated an accumulation of DGCC, DGTA and DGGA in non-plastid membranes. On incubation of cells with [1- 14C] oleic acid, after 60 min of pulse, 30% of the incorporated label was found in TAG and 70% in polar lipids. DGCC (52%) and DGTA (12%) were the most strongly labelled polar lipids. Within 72 h of chase, in DGCC and in TAG the label rapidly decreased to 11 % and 17%, respectively, but in MGDG it increased in the same time up to 35% of the total. Only minor changes were observed in DGTA and no significant label was recorded in DAG during the chase-period. The shift of label from 18:1 to 18:4, 20:5 and 22:6 reflected further elongation and/or desaturation of the substrate. Very similar results obtained using [2- 14C] acetate as precursor suggested that DGCC acts as a primary acceptor of de novo-formed or exogenous fatty acids which subsequently undergo processing and redistribution. It is suggested that C 18 and C 20 fatty acids are transferred individually from the cytoplasm to the chloroplast allowing the synthesis of eukaryotic MGDG without the import of DAG. © 1997 Elsevier Science Ltd. All rights reserved