Interspecific variability in phosphorus-induced lipid remodelling among marine eukaryotic phytoplankton.

The response of marine microalgal lipids to phosphorus is of central importance in phytoplankton ecology but remains poorly understood. We determined how taxonomically diverse microalgal species remodelled their lipid class profile in response to phosphorus availability and whether these changes coincided with those already known to occur in land plants and in the limited number of phytoplankton species for which data are available. The complete lipid class profile and specific lipid ratios influenced by phosphorus availability were quantified in two green microalgae and seven Chromalveolates exposed to phosphorus repletion, deprivation and replenishment. Lipid class cell quota changes in the two green microalgae resembled the currently described pattern of betaine lipids substituting for phospholipids under phosphorus depletion, whereas only two of the studied Chromalveolates showed this pattern. Sulpholipids counterbalanced phosphatidylglycerol only in Picochlorum atomus. In all other species, both lipids decreased simultaneously under phosphorus deprivation, although sulpholipids declined more slowly. Phosphorus deprivation always induced a decrease in digalactosyl-diacylglycerol. However, the ratio of digalactosyl-diacylglycerol to total phospholipids increased in eight species and remained unchanged in Isochrysis galbana. Marine phytoplankton seems to have evolved a diversified mechanism for remodelling its lipid class profile under the influence of phosphorus, with cryptophytes and particularly haptophytes exhibiting previously unobserved lipid responses to phosphorus.

Polar Lipids Analysis of Cultured Phytoplankton Reveals Significant Inter-taxa Changes, Low Influence of Growth Stage, and Usefulness in Chemotaxonomy.

The high lipid diversity of microalgae has been used to taxonomically differentiate phytoplankton taxa at the class level. However, important lipids such as phospholipids (PL) and betaine lipids (BL) with potential chemotaxonomy application in phytoplankton ecology have been scarcely studied. The chemotaxonomy value of PL and BL depends on their intraspecific extent of variation as microalgae respond to external changing factors. To determine such effects, lipid class changes occurring at different growth stages in 15 microalgae from ten different classes were analyzed. BL occurred in 14 species and were the less affected lipids by growth stage with diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGTA) showing the highest stability. PL were more influenced by growth stage with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidyletanolamine (PE) declining towards older culture stages in some species. Glycolipids were the more common lipids, and no evident age-related variability pattern could be associated to taxonomic diversity. Selecting BL and PL as descriptor variables optimally distinguished microalgae taxonomic variability at all growth stages. Principal coordinate analysis arranged species through a main tendency from diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGCC) containing species (mainly dinoflagellates and haptophytes) to DGTA or PC containing species (mainly cryptophytes). Two diatom classes with similar fatty acid profiles could be distinguished from their respective content in DGTA (Bacillariophyceae) or DGCC (Mediophyceae). In green lineage classes (Trebouxiophyceae, Porphyridophyceae, and Chlorodendrophyceae), PC was a better descriptor than BL. BL and PL explained a higher proportion of microalgae taxonomic variation than did fatty acids and played a complementary role as lipid markers.

Common and Species-Specific Effects of Phosphate on Marine Microalgae Fatty Acids Shape Their Function in Phytoplankton Trophic Ecology.

The use of fatty acids (FA) to infer structure of phytoplankton assemblages and as indicators of microalgae nutritional value is acquiring relevance in modern phytoplankton ecology and new advances concerning factors influencing FA variability among microalgae are demanded. In this regard, the relationship between phosphorus and FA remains particularly little studied in marine phytoplankton. In the present study, we focus on phosphate effects on FA from a diversified set of marine microalgae and provide new insights into the applicability of FA in phytoplankton trophic ecology. Phosphate deprivation mainly induced monounsaturated FA production in eight out of nine microalgae and their changes were species-specific, with palmitoleic acid exhibiting extreme variation and discriminating between haptophyte classes. The important phosphate-induced and interspecific variability found for oleic acid was perceived as a concern for the current application of this FA as a trophic position indicator in grazers. Chloroplast C-16 and C-18 polyunsaturated FA were more affected by phosphate than C-20 and C-22 highly unsaturated FA (HUFA). The relative stability of stearidonic acid to phosphate in cryptophytes and haptophytes pinpointed this FA as a suited marker for both microalgae groups. Taken all species together, phosphate deprivation and taxonomy accounted for 20.8 and 50.7% of total FA variation, respectively. HUFA were minimally affected by phosphate indicating their suitability as indicators of phytoplankton trophic value. The asymptotic relationship between HUFA and phosphorus cell content suggested mineral composition (phosphorus) could be more important than HUFA content as attribute of marine microalgae nutritional value at the species level.

Assessment of Full-Fat Tenebrio molitor as Feed Ingredient for Solea senegalensis: Effects on Growth Performance and Lipid Profile.

Tenebrio molitor (TM) is considered as one of the most promising protein sources for replacing fish meal in aquafeeds, among other things because it is rich in protein, a good source of micronutrients and has a low carbon footprint and land use. However, the main drawback of TM is its fatty acid profile, in particular its low content of n-3 PUFA. This study evaluates the effects of partially replacing plant or marine-derived with full-fat TM meal at two different levels on growth performance and lipid profiles of Senegalese sole (Solea senegalensis). For this purpose, a control diet (CTRL) and four experimental isoproteic (53%) and isolipidic (16%) diets were formulated containing 5 and 10% TM meal replacing mostly fish meal (FM5 and FM10), or 10 and 15% TM meal replacing mostly plant meal (PP10 and PP15). Fish (215 g) were fed at 1% of their body weight for 98 days. The final body weight of fish fed the experimental diets containing TM meal was not different from that of fish fed the CTRL diet (289 g). However, the inclusion of TM meal resulted in a gradual improvement in growth rate and feed efficiency in both cases (replacement of fish or plant meals), and significant differences in specific growth rate (SGR) were observed between fish fed the CTRL diet (SGR = 0.30% day-1) and those fed diets with the highest TM meal content (PP15; SGR = 0.35% day-1). The experimental groups did not show any differences in the protein content of the muscle (19.6% w/w). However, significant differences were observed in the total lipid content of the muscle, with the FM10, PP10, and PP15 groups having the lowest muscle lipid contents (2.2% ww). These fish also showed the lowest neutral lipid content in muscle (6.6% dw), but no differences were observed in the total phospholipid content (2.6% dw). Regarding the fatty acid profile, fish fed FM10, PP10 and PP15 had lower levels of linoleic acid (18:2n-6) and higher levels of oleic acid (18:1n-9) in liver and muscle compared to fish fed CTRL. However, no differences were found between fish fed CTRL and TM-based diets for docosahexaenoic acid (22:6n-3) and total n-3 PUFA in liver and muscle. In conclusion, our study demonstrated that full-fat TM inclusion up to 15% in S. senegalensis diets had no negative effects or even some positive effects on fish survival, growth performance, nutrient utilization and flesh quality.

Sperm production and quality in brill Scophthalmus rhombus L.: relation to circulating sex steroid levels.

The aims of the present study were to characterize sperm quality and to quantify seasonal changes in sexual hormone (testosterone [T], 11-ketotestosterone [11-KT] and 17,20ß-dihydroxypregn-4-en-3-one [17,20ß-P]) levels in male brill (Scophthalmus rhombus) plasma, as well as to test a more intensive sampling strategy to establish relationships between sex steroid levels and sperm production parameters. Sperm concentration ranged from 0.5 to 3.1 × 10(9) spermatozoa mL(-1), and changes in sperm quality parameters depending on sampling date were observed. Plasma sexual steroid levels remained high and changed in parallel during the spawning season and afterwards decreased to very low levels in summer. The analysis of annual changes of 11-KT and T ratios suggests that 11-KT can be the main circulating androgen for stimulating spermatogenesis in S. rhombus and that T could be involved in the beginning of spermatogenesis through the positive feedback on brain-pituitary-gonad axis. Finally, daily 11-KT and T levels showed similar patterns of variation in males sampled, whereas 17,20ß-P amounts showed somewhat opposite trends. These differences could be related with the different role of androgens and progestin during the spermatogenesis.

De novo transcriptome reconstruction in aquacultured early life stages of the cephalopod Octopus vulgaris.

Cephalopods have been considered enigmatic animals that have attracted the attention of scientists from different areas of expertise. However, there are still many questions to elucidate the way of life of these invertebrates. The aim of this study is to construct a reference transcriptome in Octopus vulgaris early life stages to enrich existing databases and provide a new dataset that can be reused by other researchers in the field. For that, samples from different developmental stages were combined including embryos, newly-hatched paralarvae, and paralarvae of 10, 20 and 40 days post-hatching. Additionally, different dietary and rearing conditions and pathogenic infections were tested. At least three biological replicates were analysed per condition and submitted to RNA-seq analysis. All sequencing reads from experimental conditions were combined in a single dataset to generate a reference transcriptome assembly that was functionally annotated. The number of reads aligned to this reference was counted to estimate the transcript abundance in each sample. This dataset compiled a complete reference for future transcriptomic studies in O. vulgaris.

Dietary Phospholipids Enhance Growth Performance and Modulate Cold Tolerance in Meagre (Argyrosomus regius) Juveniles.

Meagre (Argyrosomus regius) is a fast-growing species currently produced in aquaculture. This species is highly sensitive to low environmental temperatures which results in high mortality events during production cycles. In this study, the effects of dietary phospholipids (PLs) on growth and cold tolerance were evaluated. For this purpose, control (CTRL) and PL-enriched diets (three-fold higher levels than CTRL) were supplied to meagre juveniles (12.9 ± 2.5 g) for 60 days, and growth was determined using a longitudinal approach. Weight gaining and SGR reduction were significantly different between dietary treatments. Animals fed with the PL-enriched diet were 4.1% heavier and grew 3.2% faster than those fed with the CTRL diet. Survival was higher than 98% in both groups. After finishing the growth trial, animals were submitted to two cold challenges and cold tolerance was evaluated as temperature at death (Tdeath), risk to death and lethal doses (LD) 50 and 90 using the cumulative degree cooling hours 6 h (CD6H). Tdeath ranged between 7.54 and 7.91 °C without statistical differences between dietary treatments. However, risk to death was significantly smaller (0.91-fold lower) and LD50 and LD90 were higher in animals fed with the PL-enriched than those supplied the CTRL diet. To assess the fatty acid (FA) composition of liver and brain in animals fed both diets after a cold challenge, FA profiles were determined in juveniles maintained at 14 °C and challenged at 7 °C. FA amounts increased in the liver of animals challenged at 7 °C. In contrast, several FAs reduced their levels in the PL-enriched diet with respect to CTRL indicating that these animals were able to mobilize efficiently lipids from this organ mitigating the negative effects of lipid accumulation during the cold challenge. In brain, the PL-enriched diet increased DHA level during the cold shock indicating a role in maintaining of brain functions. These results open a new research line that could improve the cold tolerance of meagre through dietary supplementation before winter.

Assessment of Growth, Lipid Metabolism and Gene Expression Responses in Senegalese Sole Larvae Fed With Low Dietary Phospholipid Levels.

Phospholipids (PL) are essential molecules for larval growth and development. In this study, growth, lipid metabolism and gene expression responses associated with different dietary PL levels in pelagic sole larvae were evaluated. In a first trial, the long-term effects on growth and survival of two experimental microdiets (MD) containing high (High-PL) or low (Low-PL) PL levels were tested and compared to a diet based on live prey (rotifers). The MD were supplied from 3 to 10 days post-hatch (dph) and Artemia from day 8 to 29 dph. High-PL fed larvae had higher dry mass (1.2-fold) than Low-PL fed larvae at 8 dph and both MD were smaller (2.9-fold) than larvae fed live preys. However, a compensatory growth (33% between 8 and 20 dph) occurred when MD were substituted by Artemia and by the end of the trial no significant differences in mass or survival occurred between the dietary treatments. In a second trial, growth, lipid metabolism and gene expression profiles of larvae fed with MD up to 8 dph were analyzed. Growth data confirmed that mass of larvae fed with High-PL was higher (1.3-fold) than the those fed Low-PL and they had lower levels of triacylglycerol (2.8-fold), cholesterol (1.2-fold) and cetoleic acid (1.7-fold). Histological analysis indicated an excess of lipid vacuoles in larvae fed with Low-PL and the expression analysis revealed a coordinated response to enhance lipid mobilization since the expression of genes involved in PL intermediate synthesis, PL remodeling as well as eight apolipoprotein was up-regulated. The down-regulation of apolipoprotein apob2 in larvae fed with Low-PL indicated a specific regulation by PL levels. The present work provides insight into the responses associated with dietary PL in early fish larvae, which will be of use for future studies aimed as designing effective larval sole diets.

Phylogeny and expression patterns of two apolipoprotein E genes in the flatfish Senegalese sole.

The apolipoprotein E (ApoE) is a key component of several lipoproteins involved in lipid homeostasis. In this study, two cDNA sequences encoding ApoE (referred to as apoEa and apoEb) were characterized in the flatfish Solea senegalensis. The predicted peptides contained conserved structural blocks related with their capacity for lipid binding and lipoprotein receptor interaction. At genomic level, both genes contained five exons and four introns and they were organized into two tandem arrays with apoA-IV gene copies. The phylogenetic analysis clearly separated them into two well-supported clusters that matched with their organization in the genome of teleosts. Whole-mount in situ hybridization located the apoEa signal in the yolk syncytial layer (YSL) of lecitothrophic larval stages (0dph) and in the anterior intestine of exotrophic larvae and benthic fish. In the case of apoEb, hybridization signals were located in the YSL, tail bud, eyes and mouth at 0dph and in the otic vesicle, hindbrain, eyes, pharynx, mouth, heart and intestine at 1dph. In exotrophic larvae, apoEb was ubiquitously expressed in several tissues such as taste buds, brain, mouth, nostril, gills, intestine, liver and around the neuromasts and eyes. Quantification of mRNA levels in pools of whole larvae confirmed distinct expression patterns with a significant reduction of apoEa and an increase of apoEb mRNA levels throughout larval development. Moreover, only apoEa transcripts increased in response to food supply suggesting that this paralog mostly participates in the absorption and transport of dietary lipids and the apoEb in the redistribution of endogenous lipids as well as in neural tissue regeneration.

Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in Integrated Multi-Trophic Aquaculture.

Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration are ecosystem services provided by salt marshes. Additionally, salt ponds offer coastal breeding and a nursery habitat for fishes and they provide abundant invertebrates, such as amphipods, which are potentially useful as a resource in aquaculture. Fishmeal and fish oil are necessary food resources to support aquaculture of carnivorous species due to their omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Currently, aquaculture depends on limited fisheries and feed with elevated n-3 LC-PUFA levels, but the development of more sustainable food sources is necessary. Amphipods appear to be a potential high quality alternative feed resource for aquaculture. Hence, a nutritional study was carried out for several main amphipod species-Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosa-in terrestrial ponds in the South of Spain. These species showed high protein content (up to 40%), high n-3 PUFA and phospholipid levels, and high levels of phophatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerols (TAG), the latter being significantly high for M. acherusicum. M. gryllotalpa and M. acherusicum showed the highest proportion of lipids (19.15% and 18.35%, respectively). Isoleucine, glycine and alanine were the dominant amino acids in all species. In addition, amphipods collected from ponds showed low levels of heavy metals. Furthermore, the biochemical profiles of the five species of amphipods have been compared with other studied alternative prey. Therefore, pond amphipods are good candidates to be used as feed, and are proposed as a new sustainable economic resource to be used in aquaculture. G. insensibilis may be the best for intensive culture as an alternative feed resource because it shows: (1) adequate n-3 PUFA and PL composition; (2) high levels of glycine, alanine, tyrosine, isoleucine and lysine; (3) high natural densities; (4) large body size (≥1 cm), and (5) high concentration of calcium. Moreover, a combined culture of amphipods and fishes in these marsh ponds seems a promising and environmentally sustainable way to develop Integrate Multi-Trophic Aquaculture (IMTA) in these ecosystems.

Aspects of phosphorus physiology associated with phosphate-induced polar lipid remodelling in marine microalgae.

Marine microalgae exhibit a diversified phosphorus physiology and have also been recently found to show high inter-taxa variability in their phosphate induced-polar lipids' remodelling. Identification of phosphorus physiology aspects that are more related to lipid remodelling can contribute to better understanding of such intricate phytoplankton lipid metabolism. Therefore, some aspects of phosphorus physiology related to its uptake, storage and use were evaluated in a taxonomically diversified group of nine marine microalgae that was arranged into three subgroups, each of them including species showing similar polar lipid responses to phosphate. Luxury phosphate uptake (PU) was the physiological aspect best associated to microalgal polar lipid metabolism as it was maximal in species (Picochlorum atomus, Tetraselmis suecica and Nannochloropsis gaditana) that were able to counterbalance between phospholipids (PL) and betaine lipids (BL). Cryptophytes (Rhodomonas baltica, Chroomonas placoidea), characterized by their constitutive BL and flexible PL contents in response to phosphate, had almost no luxury PU and showed higher phosphorus cell quota (QP) under phosphate deprivation. Haptophyes (Isochrysis galbana, Diacronema vlkianum), with constitutive BL contents and permanently minimal PL contents, showed the lowest QP when deprived of phosphate while their luxury PU was below that for green microalgae. Induction of alkaline phosphatase activity following phosphate depletion was maximal in diatoms (Phaeodactylum tricornutum, Chaetoceros gracilis) and I. galbana but it was unrelated to lipid remodelling. Despite strong influence of taxonomy, polar lipid remodelling accounted for 38.8% of total variation when microalgae were ordinated using their physiological responses to phosphorus as descriptive variables.

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.

Towards Integrated Multi-Trophic Aquaculture: Lessons from Caprellids (Crustacea: Amphipoda).

The search for alternative live feed organisms and the progression of Integrative Multi-Trophic Aquaculture (IMTA) are currently being highly prioritised in EU strategies. Caprellids could potentially be an important exploitable resource in aquaculture due to their high levels of beneficial polyunsaturated fatty acids, fast growing nature and widespread distribution. Furthermore, since they are mainly detritivorous, they could be excellent candidates for integration into IMTA systems, potentially benefitting from uneaten feed pellets and faeces released by cultured fish in fish farms and sea-cage structures. Despite this, there is a lack of experimental studies to: (i) test inexpensive diets for caprellids, such as detritus, (ii) develop sustainable caprellid culture techniques and (iii) include caprellids in IMTA systems. The main aim of this study was to determine whether detritus (D) in the form of fish faeces provided an adequate diet for caprellids in comparison to other traditional diets, such as Artemia nauplii (A) or phytoplankton (P). Adult survival rate was shown to be significantly higher for caprellids fed with D. Conversely, hatchlings had the highest survival rate with A, although the juvenile growth rate and number of moults was similar in the three diets. With regard to lipid composition, caprellids fed with A had higher concentrations of Triacylglycerols (TAG) and Phosphatidylcholine (PC) while those fed with P or D were richer in polyunsaturated fatty acids, especially 22:6(n-3) (DHA). Interestingly, caprellids fed with D were also a rich source of 18:2(n-6) (LA), considered to be an essential fatty acid in vertebrates. It was found that detritus based mainly on fish faeces and uneaten feed pellets can be considered an adequate feed for adult caprellids, providing a source of both omega-3 (DHA) and omega-6 (LA) fatty acids. Hatchlings however seem to require an additional input of TAG and PC during juvenile stages to properly grow.

A sensory and nutritional comparison of mussels (Mytilus sp.) produced in NW Iberia and in the Armona offshore production area (Algarve, Portugal).

A biometric, nutritional and sensory analysis of raw and cooked mussels comparing Mytilus sp. from the north-west coast of Portugal and Spain (Minho and Galicia, respectively) and the new offshore production site of Armona (Algarve, south Portugal) was carried out. In addition, multiple factorial analysis was performed to explore potential relationships between sensory attributes and nutritional content properties between the different mussels. Results showed that, at similar times of sale, biometrics of mussels from Armona and Vigo were similar and bigger than the remaining. Nonetheless, despite some similarities in proximate composition, mussels presented differences in lipid classes, fatty acid content and free amino acids profiles. These differences were not fully reflected in the sensory assessment by the panel, which were able to distinguish different production sites in raw specimens but displayed problems in discrimination these in cooked mussels. Some nutritional components were related to specific sensory sensations.

Effect of varying dietary levels of LC-PUFA and vegetable oil sources on performance and fatty acids of Senegalese sole post larvae: puzzling results suggest complete biosynthesis pathway from C18 PUFA to DHA.

Lipid nutrition of marine fish larvae has focused on supplying essential fatty acids (EFA) at high levels to meet requirements for survival, growth and development. However, some deleterious effects have been reported suggesting that excessive supply of EFA might result in insufficient supply of energy substrates, particularly in species with lower EFA requirements such as Senegalese sole (Solea senegalensis). This study addressed how the balance between EFA and non-EFA (better energy sources) affects larval performance, body composition and metabolism and retention of DHA, by formulating enrichment emulsions containing two different vegetable oil sources (olive oil or soybean oil) and three DHA levels. DHA positively affected growth and survival, independent of oil source, confirming that for sole post-larvae it is advantageous to base enrichments on vegetable oils supplying higher levels of energy, and supplement these with a DHA-rich oil. In addition, body DHA levels were generally comparable considering the large differences in their dietary supply, suggesting that the previously reported ∆4 fatty acyl desaturase (fad) operates in vivo and that DHA was synthesized at physiologically significant rates through a mechanism involving transcriptional up-regulation of ∆4fad, which was significantly up-regulated in the low DHA treatments. Furthermore, data suggested that DHA biosynthesis may be regulated by an interaction between dietary n-3 and n-6 PUFA, as well as by levels of LC-PUFA, and this may, under certain nutritional conditions, lead to DHA production from C18 precursors. The molecular basis of putative fatty acyl ∆5 and ∆6 desaturation activities remains to be fully determined as thorough searches have found only a single (∆4) Fads2-type transcript. Therefore, further studies are required but this might represent a unique activity described within vertebrate fads.

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-ß-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.