Global omics study of Tetraselmis chuii reveals time-related metabolic adaptations upon oxidative stress.

Microalgae species encounter oxidative stress in their natural environments, prompting the development of species-specific adaptation mechanisms. Understanding these mechanisms can offer valuable insights for biotechnological applications in microalgal metabolic manipulation. In this study, we investigated the response of Tetraselmis chuii, an industrially important microalga, to H2O2-induced oxidative stress. Exposure to 0.5-mM H2O2 resulted in reduced cell viability, and higher concentrations led to a drastic decline. After 1 h of exposure to H2O2, photosynthetic capacity (Qy) was negatively impacted, and this reduction intensified after 6 h of continuous stress. Global multi-omics analysis revealed that T. chuii rapidly responded to H2O2-induced oxidative stress within the first hour, causing significant changes in both transcriptomic and metabolomic profiles. Among the cellular functions negatively affected were carbon and energy flow, with photosynthesis-related PSBQ having a 2.4-fold downregulation, pyruvate kinase decreased by 1.5-fold, and urea content reduced by threefold. Prolonged exposure to H2O2 incurred a high energy cost, leading to unsuccessful attempts to enhance carbon metabolism, as depicted, for example, by the upregulation of photosystems-related PETC and PETJ by more than twofold. These findings indicate that T. chuii quickly responds to oxidative stress, but extended exposure can have detrimental effects on its cellular functions. KEY POINTS: • 0.5-mM H2O2-induced oxidative stress strongly affects T. chuii • Distinct short- and long-term adaptation mechanisms are induced • Major metabolic adaptations occur within the first hour of exposure.

TetraSOD®, a Unique Marine Microalgae Ingredient, Promotes an Antioxidant and Anti-Inflammatory Status in a Metabolic Syndrome-Induced Model in Rats.

Increased oxidative stress has been linked to the pathogenic process of obesity and can trigger inflammation, which is often linked with the risk factors that make up metabolic syndrome (MetS), including obesity, insulin resistance, dyslipidaemia and hypertension. TetraSOD®, a natural marine vegan ingredient derived from the microalgae Tetraselmis chuii that is high in the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) has recently demonstrated in vitro increased activity of these key antioxidant enzymes. In the present study, the potential bioactive effects of three dietary dosages of TetraSOD® in enhancing antioxidant and anti-inflammatory mechanisms to combat the metabolic disturbances that compose MetS were assessed in rats given a cafeteria (CAF) diet. Chronic supplementation with 0.17, 1.7, and 17 mg kg-1 day-1 of TetraSOD® for 8 weeks ameliorated the abnormalities associated with MetS, including oxidative stress and inflammation, promoting endogenous antioxidant defence mechanisms in the liver (GPx and GSH), modulating oxidative stress and inflammatory markers in plasma (NOx, oxLDL and IL-10), and regulating genes involved in antioxidant, anti-inflammatory and immunomodulatory pathways in the liver, mesenteric white adipose tissue (MWAT), thymus, and spleen. Overall, TetraSOD® appears to be a potential therapeutic option for the management of MetS.

Metabolic pathways for biosynthesis and degradation of starch in Tetraselmis chui during nitrogen deprivation and recovery.

Tetraselmis chui is known to accumulate starch when subjected to stress. This phenomenon is widely studied for the purpose of industrial production and process development. Yet, knowledge about the metabolic pathways involved is still immature. Hence, in this study, transcription of 27 starch-related genes was monitored under nitrogen deprivation and resupply in 25 L tubular photobioreactors. T. chui proved to be an efficient starch producer under nitrogen deprivation, accumulating starch up to 56% of relative biomass content. The prolonged absence of nitrogen led to an overall down-regulation of the tested genes, in most instances maintained even after nitrogen replenishment when starch was actively degraded. These gene expression patterns suggest post-transcriptional regulatory mechanisms play a key role in T. chui under nutrient stress. Finally, the high productivity combined with an efficient recovery after nitrogen restitution makes this species a suitable candidate for industrial production of high-starch biomass.

Extracting protein from microalgae (Tetraselmis chuii) for proteome analysis.

Microalgae have high potential as a resource for sustainable and green protein for food or bioactive molecules. Nonetheless, despite the high protein content of microalgae (40 - 70% dry weight) progress in the characterization of their protein composition remains challenging. This is due to the highly variable chemical composition of microalgae strains and factors such as their rigid thick cell wall, polysaccharide content, protein stability, pH. The method described herein was developed to optimize protein extraction for proteome analysis of microalgae (Tetraselmis chuii) biomass. The effects on protein solubility of solvent type (organic, denaturing, and non-denaturing) combined with three customized microalgae disruption methods were investigated. The proteome targeted high quality protein extracts were for hydro-soluble proteins recovered by cell disruption using bead milling coupled to centrifugation (protein yield ≈ 13%). The developed method is inexpensive, efficient (yielding high-quality protein extracts with a low content of interfering compounds) and from an industrial perspective easily scalable and compatible with other applications. To add value to the end product we additionally propose the use of stabilizing agents to maintain protein solubility during refrigerated storage and a method targeting the fractionation of low molecular weight proteins. • An inexpensive easy-to-do 5 step protocol for microalgae protein extracts. • A protein extraction method free from dangerous or highly polluting chemicals. • Production of high yield aqueous protein extracts suitable for proteomics.

Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui.

Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.

Microalgal extracts induce larval programming and modify growth and the immune response to bioactive treatments and LCDV in Senegalese sole post-larvae.

Immunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days post-hatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast ß-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL-1) enriched for 30 min with MAe or Y (at 2 mg mL-1 SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The post-larvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total anti-protease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN post-larvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast ß-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses.

Yeast ß-glucans and microalgal extracts modulate the immune response and gut microbiome in Senegalese sole (Solea senegalensis).

One bottleneck to sustainability of fish aquaculture is the control of infectious diseases. Current trends include the preventive application of immunostimulants and prebiotics such as polysaccharides. The present study investigated how yeast ß-glucan (Y), microalgal polysaccharide-enriched extracts (MAe) and whole Phaeodactylum tricornutum cells (MA) modulated the gut microbiome and stimulated the immune system in Senegalese sole (Solea senegalensis) when administered by oral intubation. Blood, intestine and spleen samples were taken at 3 h, 24 h, 48 h and 7 days after treatment. The short-term response (within 48 h after treatment) consisted of up-regulation of il1b and irf7 expression in the gut of the Y treated group. In contrast, administration of MAe decreased expression of tnfa and the chemokine cxc10 in the gut and spleen. Both treatments down-regulated the expression of irf3 with respect to the control group. Lysozyme activity in plasma decreased at 48 h only in the MAe-treated soles. Medium-term response consisted of the up-regulation of clec and irf7 expression in the gut of the Y, MAe and MA groups and of il1b mRNAs in the spleen of the MA group compared to the control group. Microbiome analysis using 16S rDNA gene sequencing indicated that the intestine microbiome was dominated by bacteria of the Vibrio genus (>95%). All the treatments decreased the relative proportion of Vibrio in the microbiome and Y and MAe decreased and MA increased diversity. Quantitative PCR confirmed the load of bacteria of the Vibrio genus was significantly decreased and this was most pronounced in Y treated fish. These data indicate that orally administrated insoluble yeast ß-glucans acted locally in the gut modulating the immune response and controlling the Vibrio abundance. In contrast, the MAe slightly reduced the Vibrio load in the intestine and caused a transient systemic anti-inflammatory response. The results indicate that these polysaccharides are a promising source of prebiotics for the sole aquaculture industry.