DGLA from the Microalga Lobosphaera Incsa P127 Modulates Inflammatory Response, Inhibits iNOS Expression and Alleviates NO Secretion in RAW264.7 Murine Macrophages.

Microalgae have been considered as a renewable source of nutritional, cosmetic and pharmaceutical compounds. The ability to produce health-beneficial long-chain polyunsaturated fatty acids (LC-PUFA) is of high interest. LC-PUFA and their metabolic lipid mediators, modulate key inflammatory pathways in numerous models. In particular, the metabolism of arachidonic acid under inflammatory challenge influences the immune reactivity of macrophages. However, less is known about another omega-6 LC-PUFA, dihomo-γ-linolenic acid (DGLA), which exhibits potent anti-inflammatory activities, which contrast with its delta-5 desaturase product, arachidonic acid (ARA). In this work, we examined whether administrating DGLA would modulate the inflammatory response in the RAW264.7 murine macrophage cell line. DGLA was applied for 24 h in the forms of carboxylic (free) acid, ethyl ester, and ethyl esters obtained from the DGLA-accumulating delta-5 desaturase mutant strain P127 of the green microalga Lobosphaera incisa. DGLA induced a dose-dependent increase in the RAW264.7 cells' basal secretion of the prostaglandin PGE1. Upon bacterial lipopolysaccharide (LPS) stimuli, the enhanced production of pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1ß (IL-1ß), was affected little by DGLA, while interleukin 6 (IL-6), nitric oxide, and total reactive oxygen species (ROS) decreased significantly. DGLA administered at 100 µM in all forms attenuated the LPS-induced expression of the key inflammatory genes in a concerted manner, in particular iNOS, IL-6, and LxR, in the form of free acid. PGE1 was the major prostaglandin detected in DGLA-supplemented culture supernatants, whose production prevailed over ARA-derived PGE2 and PGD2, which were less affected by LPS-stimulation compared with the vehicle control. An overall pattern of change indicated DGLA's induced alleviation of the inflammatory state. Finally, our results indicate that microalgae-derived, DGLA-enriched ethyl esters (30%) exhibited similar activities to DGLA ethyl esters, strengthening the potential of this microalga as a potent source of this rare anti-inflammatory fatty acid.

Dietary Supplementation with Omega-6 LC-PUFA-Rich Microalgae Regulates Mucosal Immune Response and Promotes Microbial Diversity in the Zebrafish Gut.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism - cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine - interleukin 10 (il-10), immune defense - lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants - catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1%-1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

Metabolomic foundation for differential responses of lipid metabolism to nitrogen and phosphorus deprivation in an arachidonic acid-producing green microalga.

The green oleaginous microalga Lobosphaera incisa accumulates storage lipids triacylglycerols (TAG) enriched in the long-chain polyunsaturated fatty acid arachidonic acid under nitrogen (N) deprivation. In contrast, under phosphorous (P) deprivation, the production of the monounsaturated oleic acid prevails. We compared physiological responses, ultrastructural, and metabolic consequences of L. incisa acclimation to N and P deficiency to provide novel insights into the key determinants of ARA accumulation. Differential responses to nutrient deprivation on growth performance, carbon-to-nitrogen stoichiometry, membrane lipid composition and TAG accumulation were demonstrated. Ultrastructural analyses suggested a dynamic role for vacuoles in sustaining cell homeostasis under conditions of different nutrient availability and their involvement in autophagy in L. incisa. Paralleling ARA-rich TAG accumulation in lipid droplets, N deprivation triggered intensive chloroplast dismantling and promoted catabolic processes. Metabolome analysis revealed depletion of amino acids and pyrimidines, and repression of numerous biosynthetic hubs to favour TAG biosynthesis under N deprivation. Under P deprivation, despite the relatively low growth penalties, the presence of the endogenous P reserves and the characteristic lipid remodelling, metabolic signatures of energy deficiency were revealed. Metabolome adjustments to P deprivation included depletion in ATP and phosphorylated nucleotides, increased levels of TCA-cycle intermediates and osmoprotectants. We conclude that characteristic cellular and metabolome adjustments tailor the adaptive responses of L. incisa to N and P deprivation modulating its LC-PUFA production.

Dietary Supplementation With ω6 LC-PUFA-Rich Algae Modulates Zebrafish Immune Function and Improves Resistance to Streptococcal Infection.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Dietary arachidonic acid affects immune function and fatty acid composition in cultured rabbitfish Siganus rivulatus.

The marbled spinefoot rabbitfish (Siganus rivulatus) is an economically valuable fish species that has potential for commercial production in aquaculture. To overcome challenges in its sustainable production, a formulated diet is required for imparting health and robustness. This study evaluates the effect of dietary supplementation with arachidonic acid (ARA; 20:4n-6) on growth, survival, immune function and fatty acid composition of red blood cells (RBCs) in rabbitfish. We conducted two feeding trials using juvenile fish (to evaluate growth and survival) and adults (to evaluate immune function and fatty acid incorporation). Fish were fed diets supplemented with three different levels of ARA (in % of total fatty acids): 0.6 (unsupplemented control), 2.6 (moderate) and 4.7 (high). The fish fed with moderate ARA levels exhibited improved (p < 0.05) growth over the control and the high ARA level groups. During an outbreak of Streptococcus iniae, fish fed with moderate ARA survived significantly (p < 0.05) better (89%) than the control and the high ARA groups (59% and 48%, respectively). Moderate ARA supplementation resulted in elevated lysozyme and complement levels in the plasma of rabbitfish. A significant increase in the total serum immunoglobulin levels was observed in both the medium and the high ARA level groups; however, a decrease in antiprotease activity was recorded in the supplemented groups as compared to the control. Fatty acid analysis in fish red blood cells revealed a significant (p < 0.05) increase in the proportion of ARA of total fatty acids in the groups fed with the medium and the high ARA level diets (9.5% and 11.2%, respectively, compared to 7.1% in the control). Concomitantly, there was a decrease in the proportion of eicosapentaenoic acid (EPA; 20:5n-3), dihomo-γ linolenic acid (DGLA; 20:3n-6) and several 18-carbon unsaturated fatty acids in these groups. In conclusion, ARA in rabbitfish feeds improved growth, survival as well as innate and acquired humoral immune functions. Thus ARA supplementation in the diet of this species could be a valuable step towards establishing the commercial culture of rabbitfish.

The complete mitochondrial genome sequence of the green microalga Lobosphaera (Parietochloris) incisa reveals a new type of palindromic repetitive repeat.

BACKGROUND: Lobosphaera incisa, formerly known as Myrmecia incisa and then Parietochloris incisa, is an oleaginous unicellular green alga belonging to the class Trebouxiophyceae (Chlorophyta). It is the richest known plant source of arachidonic acid, an ω-6 poly-unsaturated fatty acid valued by the pharmaceutical and baby-food industries. It is therefore an organism of high biotechnological interest, and we recently reported the sequence of its chloroplast genome. RESULTS: We now report the complete sequence of the mitochondrial genome of L. incisa from high-throughput Illumina short-read sequencing. The circular chromosome of 69,997 bp is predicted to encode a total of 64 genes, some harboring specific self-splicing group I and group II introns. Overall, the gene content is highly similar to that of the mitochondrial genomes of other Trebouxiophyceae, with 34 protein-coding, 3 rRNA, and 27 tRNA genes. Genes are distributed in two clusters located on different DNA strands, a bipartite arrangement that suggests expression from two divergent promoters yielding polycistronic primary transcripts. The L. incisa mitochondrial genome contains families of intergenic dispersed DNA repeat sequences that are not shared with other known mitochondrial genomes of Trebouxiophyceae. The most peculiar feature of the genome is a repetitive palindromic repeat, the LIMP (L. Incisa Mitochondrial Palindrome), found 19 times in the genome. It is formed by repetitions of an AACCA pentanucleotide, followed by an invariant 7-nt loop and a complementary repeat of the TGGTT motif. Analysis of the genome sequencing reads indicates that the LIMP can be a substrate for large-scale genomic rearrangements. We speculate that LIMPs can act as origins of replication. Deep sequencing of the L. incisa transcriptome also suggests that the LIMPs with long stems are sites of transcript processing. The genome also contains five copies of a related palindromic repeat, the HyLIMP, with a 10-nt motif related to that of the LIMP. CONCLUSIONS: The mitochondrial genome of L. incisa encodes a unique type of repetitive palindromic repeat sequence, the LIMP, which can mediate genome rearrangements and play a role in mitochondrial gene expression. Experimental studies are needed to confirm and further characterize the functional role(s) of the LIMP.

Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (Chlorophyceae).

Cytoplasmic oil globules of Haematococcus pluvialis (Chlorophyceae) were isolated and analyzed for pigments, lipids and proteins. Astaxanthin appeared to be the only pigment deposited in the globules. Triacyglycerols were the main lipids (more than 90% of total fatty acids) in both the cell-free extract and in the oil globules. Lipid profile analysis of the oil globules showed that relative to the cell-free extract, they were enriched with extraplastidial lipids. A fatty acids profile revealed that the major fatty acids in the isolated globules were oleic acid (18:1) and linoleic acid (18:2). Protein extracts from the globules revealed seven enriched protein bands, all of which were possible globule-associated proteins. A major 33-kDa globule protein was partially sequenced by MS/MS analysis, and degenerate DNA primers were prepared and utilized to clone its encoding gene from cDNA extracted from cells grown in a nitrogen depleted medium under high light. The sequence of this 275-amino acid protein, termed the Haematococcus Oil Globule Protein (HOGP), revealed partial homology with a Chlamydomonas reinhardtii oil globule protein and with undefined proteins from other green algae. The HOGP transcript was barely detectable in vegetative cells, but its level increased by more than 100 fold within 12 h of exposure to nitrogen depletion/high light conditions, which induced oil accumulation. HOGP is the first oil-globule-associated protein to be identified in H. pluvialis, and it is a member of a novel gene family that may be unique to green microalgae.

LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology.

Microalgae offer potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFAs). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and the market for these products is continually growing. An appropriate ratio of LC-PUFA of the ω-3 and ω-6 groups is vital for "healthy" nutrition, and adequate dietary intake has strong health benefits in humans. Microalgae of diverse classes are primary natural producers of LC-PUFA. This mini-review presents an introductory overview of LC-PUFA-related health benefits in humans, describes LC-PUFA occurrence in diverse microalgal classes, depicts the major pathways of their biosynthesis in microalgae, and discusses the prospects for microalgal LC-PUFA production.

Identification and characterization of Delta12, Delta6, and Delta5 Desaturases from the green microalga Parietochloris incisa.

The freshwater microalga Parietochloris incisa accumulates, under nitrogen starvation, large amounts of triacylglycerols containing approximately 60% of the omega6 very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. Based on sequence homology, we isolated three cDNA sequences from P. incisa, designated PiDesD12, PiDesD6, PiDesD5. The deduced amino acid sequences of the three genes contained three conserved histidine motifs; the front-end desaturases, PiDes6 and PiDes5, contained a fused N-terminal cytochrome b5 domain. By functional characterization in the yeast Saccharomyces cerevisiae, we confirmed that PiDesD6, PiDesD5 cDNA encode membrane bound desaturases with Delta6, and Delta5 activity, respectively. Both PiDes6 and PiDes5 can indiscriminately desaturate both omega6 and omega3 substrates. A phylogenetic analysis showed that the three genes were homologous to the corresponding desaturases from green microalgae and lower plants that were functionally characterized. Quantitative real-time PCR revealed the concerted expression pattern of all three genes in P. incisa cells subjected to nitrogen starvation, featuring maximum expression level on day 3 of starvation, corresponding to the sharpest increase in the share of arachidonic acid.

Effect of dietary fatty acid composition on fatty acid profiles of polar and neutral lipid tissue fractions in zebra finches, Taeniopygia guttata.

The growing awareness that the fatty acid (FA) composition of the diets of birds, and ultimately their tissues, influence physiological performance variables, such as aerobic capacity, thermosensitivity, digestive efficiency, etc., underscores the need to understand how differences in dietary fatty acid composition actually translate into differences in the fatty acid composition of specific tissues. We quantified the fatty acid profiles of polar and neutral lipid fractions of several tissues in zebra finches (Taeniopygia guttata) and compared these profiles among birds fed either a control diet of only hulled millet, or one of two experimental diets of hulled millet supplemented with either 8% (by mass) sunflower seed oil (omega6-enriched diet) or linseed oil (omega3-enriched diet). We found that different lipid fractions vary widely in their diversity and complexity of FA composition, with neutral lipids being much less structurally diverse than those of polar lipids, for example, and that the fatty acid compositions of different organs exhibited different propensities to be altered by the diet, with brain and cardiac tissues having lower levels of flexibility than skeletal muscle and liver. We also present evidence suggesting that adipose tissue may be used to sequester essential FAs when they occur in the diet at levels that exceed immediate requirements. We conclude that the fatty acid composition of adipose tissue may not be a particularly useful indicator of the dietary FA composition of birds, and suggest that future studies investigating the relationships between the FA profiles of bird tissues and bird diets and/or physiological performance variables examine multiple tissues and distinguish between neutral and polar lipid fractions.