Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

Fatty acids and sterols of selected hyphochytriomycetes and chytridiomycetes

The fatty acids and sterols of eight Chytridiomycetes and two Hyphochytriomycetes, and fatty acids of the OomycetePythium gracile, were analyzed by gas-liquid chromatography. In addition to the fatty acids anticipated for fungi, the two Hyphochytriomycetes (Hyphochytrium catenoides andRhizidiomyces apophysatus) and four of the Chytridiomycetes (Catenaria anguillulae, Blastocladiella emersonii, Monoblepharella sp., andAllomyces macrogynus) contained arachidonic acid as a major fatty acid of the polar lipid fraction, and this fatty acid was detected as a minor component ofRhizophlyctis rosea andSpizellomyces punctatum. Eicosapentaenoic acid constituted 4.6% of the polar lipid fatty acids inMonoblepharella sp., and trace amounts were detected in several other species. Both the gamma (ω-6) and alpha (ω-3) isomers of linolenic acid were detected in all of the species analyzed. Cholesterol was the predominant (≥73%) sterol ofB. emersonii, R. rosea, A. macrogynus, andChytridium confervae, and a minor (<12%) component ofC. anguillulae, andH. catenoides. The major sterols of the other species included lanosterol (C. anguillulae, 45%), stigmasta-5,22-dien-3β-ol (H. catenoides, 51%), 24-ethyl-cholesterol (S. punctatum, 38%;H. catenoides, 17%;Monoblepharella sp., 70%; andR. apophysatus, 84%), 24-methyl-cholesterol (H. catenoides, 23%;R. apophysatus, 14%;S. punctatum, 53%), and 24-methylene cholesterol (Rhizophydium sphaerotheca, 51%). Neither ergosterol nor fucosterol was detected in any of the species studied.     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.     

The fatty acid and sterol composition of two marine dinoflagellates

The fatty acid, sterol and chlorophyll pigment compositions of the marine dinoflagellates Gymnodinium wilczeki and Prorocentrum cordatum are reported. The fatty acids of both algae show a typical dinoflagellate distribution pattern with a predominance of C₁₈, C₂₀ and C₂₂ unsaturated components. The acid 18:5ω3 is present at high concentration in these two dinoflagellates. G. wilczeki contains a high proportion (93.4%) of 4-methyl-5α-stanols including 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), dinostanol and 4,23,24-trimethyl-5α-cholest-7-en-3β-ol reported for the first time in dinoflagellates. The role of this sterol in the biosynthesis of 5α-stanols in dinoflagellates is discussed. P. cordatum contains high concentrations of a number of δ ²⁴⁽²⁸⁾-sterols with dinosterol, 24-methylcholesta-5,24(28)-dien-3β-ol, 23,24-dimethylcholesta-5,22E-dien-3β-ol, 4,24-dimethyl-5α-cholest-24(28)-en-3β-ol and a sterol identified as either 4,23,24-trimethyl- or 4-methyl-24-ethyl-5α-cholest-24(28)-en-3β-ol present as the five major components. The role of marine dinoflagellates in the input of both 4-methyl- and 4-desmethyl-5α-stanols to marine sediments is discussed.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Long-chain polyenoics and the essential dietary fatty acid requirement of the waxmoth, Galleria mellonella

The essential fatty acid requirement for normal pupal-adult ecdysis in Galleria mellonella was studied using non-axenic casein-based semisynthetic diets with or without various 99% pure fatty acids. The abilities of linoleic and linolenic acids to alleviate faulty adult emergence differed markedly, linolenic acid being 10-fold more potent than linoleic acid. One other ω6 polyunsaturated fatty acid, C20:2ω6, resembled its analogue, linoleic acid (18:2ω6), in efficacy at high dosage, but three others, C18:3ω6, C20: ω6 and C20:4ω6 (arachidonic acid), were without effect. Of five ω3 polyunsatures tested, C22:3ω3 and C20:3ω3 were as effective as linolenic acid (C18:3ω3), their shorter-chained analogue. Docosahexaenoic acid (C22:6ω3) was totally ineffective, but eicosapentaenoic acid (C20:5ω3), though supporting no perfect emergences, produced some active adults having wing malformations only, and was therefore considered partially active. It is suggested that a C18 polyunsaturate is physiologically required by G. mellonella and can be derived from various dietary longer-chained analogues by simple carbon chain shortening so long as there are no additional double bonds carboxylwards of an active di- or trienoic sequence. The partial activity of C20:5ω3 suggests there may additionally be a physiological requirement for this or a related long-chain polyunsaturate. The possibility of multiple essential fatty acid requirements in Lepidoptera in general is discussed.     

Assessment of Microzooplankton Grazing on <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis> Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods

Delaware’s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day⁻¹ at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. An erratum to this article can be found at     

Fatty acid composition of ten marine algae from australian waters

Detailed fatty acid analyses are reported for ten benthic algae from Australian waters of which the data for Cladophora fascicularis (Chlorophyta); Polysiphonia pungens, Porphyra sp., Centroceras clavatum (Rhodophyta); Hormosira banksii, Ralfsia sp., and Dictyota dichomota (Phaeophyta) are presented for the first time. The analyses report the exact molecular structure of component acids which is essential for taxonomic and food chain studies. The acid 16:4ω3 could be taxonomically distinguishing for species of the Chlorophyta. The occurrence of cis-vaccenic acid (18:1ω7) in the algae reported here suggests a distribution in marine benthic algae which is wider than hitherto realised, with particular taxonomic importance for Chlorophyta species in which it occurs in high levels. Corallina officinalis was found to contain the non-methylene interrupted acids 20:2 and 22:2.     

Sterols and fatty acids of three harmful algae previously assigned as Chattonella

Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n-9, 18:4n-3 and 20:5n-3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n-3, 18:5n-3 and 22:6n-3. C. toxicum contained the 24beta-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24alpha and 24beta, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

Improvement of lipid production in the marine strains Alexandrium minutum and Heterosigma akashiwo by utilizing abiotic parameters

Two different strains of microalgae, one raphidophyte and one dinoflagellate, were tested under different abiotic conditions with the goal of enhancing lipid production. Whereas aeration was crucial for biomass production, nitrogen deficiency and temperature were found to be the main abiotic parameters inducing the high-level cellular accumulation of neutral lipids. Net neutral lipid production and especially triacylglycerol (TAG) per cell were higher in microalgae (>200% in Alexandrium minutum, and 30% in Heterosigma akashiwo) under treatment conditions (25°C; 330 μM NaNO₃) than under control conditions (20°C; 880 μM NaNO₃). For both algal species, oil production (free fatty acids plus TAG fraction) was also higher under treatment conditions (57 mg L⁻¹ in A. minutum and 323 mg L⁻¹ in H. akashiwo). Despite the increased production and accumulation of lipids in microalgae, the different conditions did not significantly change the fatty acids profiles of the species analyzed. These profiles consisted of saturated fatty acids (SAFA) and polyunsaturated fatty acids (PUFA) in significant proportions. However, during the stationary phase, the concentrations per cell of some PUFAs, especially arachidonic acid (C20:4n6), were higher in treated than in control algae. These results suggest that the adjustment of abiotic parameters is a suitable and one of the cheapest alternatives to obtain sufficient quantities of microalgal biomass, with high oil content and minimal changes in the fatty acid profile of the strains under consideration.     

Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae)

Heterosigma akashiwo and Chattonella species (Raphidophyceae) are difficult to detect and quantify in environmental samples because of their pleomorphic and fragile cell nature. In this study, we developed a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the enumeration of these algal taxa using a new DNA-binding dye, EvaGreen. Species-specific qRT PCR primers to H. akashiwo, Chattonella antiqua, Chattonella marina, Chattonella ovata, and Chattonella subsalsa were designed to target the ITS2 rRNA gene intergenic region. Primer specificities were tested via BLAST searches. In addition, specificity was verified using empirical tests, including competitive PCR. The qRT PCR assay analyzing C _t value and the log of cell number showed a significant linear relationship (r ²?≥?0.997). When light microscopy was used to monitor the population dynamics of targeted Raphidophyceae from Lake Shihwa, H. akashiwo was detected in ten samples and no Chattonella spp. were detected (70 samples collected from May, 2007 to January, 2008). In contrast, when the qRT-PCR assay was used, H. akashiwo was detected in 41 samples. C. antiqua, C. marina, and C. ovata were detected in eight samples. Most of the samples analyzed using qRT-PCR assays showed higher algal numbers than did those assayed via microscopy, suggesting that the enumeration of Raphidophyceae via classic microscopic methods most likely underestimates true algal concentration.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

The sterol and fatty acid compositions of seven tropical seagrasses from North Queensland,Australia

The sterol and fatty acid compositions of fresh leaves of the seagrasses Cymodocea serrulata, Enhalus acoroides, Halodule uninervis, Halophila ovalis, H. ovata, H. spinulosa and Thalassia hemprichii are reported. The major fatty acids were palmitic acid, linoleic acid and linolenic acid as expected. H. ovalis and H. ovata were characterized by the relatively high abundance (ca 5%) of the acid hexadeca-7,10,13-trienoic acid (16:3<7 > ). The sterol compositions were typical of higher plants, with sitosterol and stigmasterol accounting for 60–90% of the observed sterols. 28-Isofucosterol was a major sterol (20–30%) only in the Halophila spp. Cluster analysis of the sterol composition data clearly separated the Halophila spp. from the other seagrasses and enabled the distinction of Enhalus sp. from Cymodocea, Halodule and Thalassia spp. The seagrass species were clearly separated into five chemical groups using the combined fatty acid and sterol composition data and the need for a reappraisal of the taxonomic position of Halophila was indicated.     

Heterologous expression of Brassica juncea microsomal ω-3 desaturase gene (BjFad3) improves the nutritionally desirable ω-6:ω-3 fatty acid ratio in rice bran oil

Rice bran oil (RBO), being naturally rich in antioxidants, is currently regarded as one of the health-beneficial edible oils. However, the RBO has essential linoleic acid (ω-6, C18:2) and α-linolenic acid (ω-3, C18:3) in nutritionally disproportionate level (~25:1), contrary to the WHO/FAO’s recommendation of ~5:1. Among few naturally occurring C18:3 enriched oil-seeds, Brassica juncea (Indian mustard) has almost equal proportion of ω-6 and ω-3 fatty acids in its oil due to the activity of microsomal ω-3 desaturase (Fad3), which converts C18:2–C18:3. Therefore, the full length Fad3 coding DNA sequence (CDS) was isolated from the developing seeds of B. juncea, functionally characterized and heterologously expressed for the nutritional enhancement of RBO. Sequence analysis revealed that the 1,134 bp long BjFad3 CDS corresponds to a polypeptide of 377 amino acids, which is highly (85–95 %) homologous to other known Fad3 enzymes of plant kingdom. The BjFad3 gene was initially characterized in transgenic tobacco to establish its linoleate desaturase activity. Thereafter, rice bran-specific expression of the BjFad3 was carried out to alter the fatty acid profile of RBO. Several independent transgenic lines of tobacco and rice plants were developed by Agrobacterium-mediated transformation. Standard molecular biological techniques were used to confirm the transgene integration in the respective genomes and subsequent in planta expression. The BjFad3 transgene expression correlated to the significant increase in C18:3 fatty acid content (up to tenfold) in both tobacco seed oil and RBO, and thereby improving the nutritionally desirable ω-6:ω-3 ratio (~2:1) in one of the transgenic rice lines.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

Lipids of the antarctic sea ice diatom Nitzschia cylindrus

The sterol and neutral, glyco- and phospholipid fatty acid profiles of the sea ice diatom Nitzschia cylindrus, isolated from McMurdo Sound, Antarctica, are reported. Two sterols were detected, trans-22-dehydrocholesterol (66% of total sterols) and cholesterol (34%); no sterols containing alkyl groups at the C₂₄ position were present. The major fatty acids in N. cylindrus, 16:1Δ9c, 14:0, 16:0, 20:5Δ5,8,11,14,17 and 20:4Δ5,8,11,14, were typical of previous reports of diatom fatty acids. A number of long-chain monounsaturated fatty acids were also detected, with higher relative proportions present in the phospholipid fraction. GC-MS analysis of the dimethyldisulphide adducts of these monounsaturated components showed that 24: 1Δ13c, 24:1Δ15c, 26:1Δ15c and 26:1Δ17c were the major components. The distribution of these fatty acids suggests that chain elongation of monounsaturated fatty acids was occurring in N. cylindrus. The proposed chain lengthening occurring for N. cylindrus represents, to our knowledge, the first report of possible chain lengthening of monounsaturated fatty acids in microscopic algae. These features, the presence of long-chain monounsaturated fatty acids and the sterol profile, may allow the input of this alga into benthic marine sediments or food webs to be monitored.     

The biochemical composition of krill, Euphausia superba Dana,from South Georgia

Krill, Euphausia superba Dana, sampled from waters around South Georgia in 1978 contained 10–11% protein, 2–6% lipid, 0.3–0.6% carbohydrate, 2% chitin, and 3–4% mineral ash (all mean values, % fresh weight).Lipid content varied greatly with sexual maturity: males contained 2–4% lipid, immature animals 4% (both increasing to 5–6% later in the season) and mature females 5–6% (although some contained as much as 9%). Gravid females lost ≈60% of their lipid at spawning, and a biochemical estimate of fecundity gave values in the range 7000 to 15 000.The major lipid classes were phospholipid, triacylglyceropl, and free sterol, and there was 1–2% wax ester. The mean pigment content was 21.5 μg/g fresh weight. Triacylglycerol fatty acids were low in polyenoic acids and contained up to 4% phytanic acid; compositions were variable and showed the probable influence of dietary input. Phospholipids were rich in polyenoic acids, especially 20 : 5ω3 and 22 : 6ω3, and usually had 18 : 1ω7 > 18 : 1ω9. Ovarian triacylglycerol contained almost no polyenoic acids, but ovarian phospholipid was fairly unsaturated.These results are discussed in relation to the Antarctic environment, and compared with the results of previous work on caridean decapods.