Insights into the nature of ichthyotoxins from the Chrysochromulina leadbeateri blooms in Northern Norwegian fjords.

In May-June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC-DAD-MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M + H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromsø, thereby consistent with a role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins reported from a number of dinoflagellates.

An uneven distribution of strontium in the coccolithophore Scyphosphaera apsteinii revealed by nanoscale X-ray fluorescence tomography.

Coccolithophores are biogeochemically and ecologically important phytoplankton that produce a composite calcium carbonate-based exoskeleton - the coccosphere - comprised of individual platelets, known as coccoliths. Coccoliths are stunning examples of biomineralization; their formation featuring exceptional control over both biomineral chemistry and shape. Understanding how coccoliths are formed requires information about minor element distribution and chemical environment. Here, the first high-resolution 3D synchrotron X-ray fluorescence (XRF) mapping of a coccolith is presented, showing that the lopadoliths of Scyphosphaera apsteinii display stripes of different Sr concentration. The presence of Sr stripes is unaffected by elevated Sr in the culture medium, macro-nutrient concentration, and light intensity, indicating that the observed stripiness is an expression of the fundamental coccolith formation process in this species. Current Sr fractionation models, by contrast, predict an even Sr distribution and will have to be modified to account for this stripiness. Additionally, nano-XANES analyses show that Sr resides in a Ca site in the calcite lattice in both high and low Sr stripes, confirming a central assumption of current Sr fractionation models.

Insights into Toxic Prymnesium parvum Blooms as a Cause of the Ecological Disaster on the Odra River.

In 2022, Poland and Germany experienced a prolonged and extensive mass fish kill in the Odra River. During the period from the end of July to the beginning of September 2022, a high level of incidental disease and mortality was observed in various fish species (dozens of different species were found dead). The fish mortality affected five Polish provinces (Silesia, Opole, Lower Silesia, Lubuskie, and Western Pomerania) and involved reservoir systems covering most of the river (the Odra River is 854 km long, of which 742 km are in Poland). Fatal cases were investigated using toxicological, anatomopathological, and histopathological tests. Water samples were collected to determine nutrient status in the water column, phytoplankton biomass, and community composition. High nutrient concentrations indicated high phytoplankton productivity, with favorable conditions for golden algal blooms. The harmful toxins (prymnesins secreted by Prymnesium parvum habitats) had not been found in Poland before, but it was only a matter of time, especially in the Odra River, whose waters are permanently saline and still used for navigation. The observed fish mortality resulted in a 50% decrease in the fish population in the river and affected mainly cold-blooded species. Histopathological examinations of fish showed acute damage to the most perfused organs (gills, spleen, kidneys). The disruption to hematopoietic processes and damage to the gills were due to the action of hemolytic toxins (prymnesins). An evaluation of the collected hydrological, meteorological, biological, and physico-chemical data on the observed spatio-temporal course of the catastrophe, as well as the detection of three compounds from the group of B-type prymnesins in the analyzed material (the presence of prymnesins was confirmed using an analysis of the fragmentation spectrum and the accurate tandem mass spectrometry (MS/MS) measurement, in combination with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), allowed the formulation and subsequent testing of the hypothesis for a direct link between the observed fish mortality and the presence of prymnesins in the Odra River. This article systematizes what is known about the causes of the fish kill in the Odra River in 2022, based on official government reports (one Polish and one German) and the EU technical report by the Joint Research Centre. A review and critical analysis of government findings (Polish and German) on this disaster were conducted in the context of what is known to date about similar cases of mass fish kills.

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii.

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.

Differential responses of Pleurochrysis sp. (Haptophyta) to the effect of copper and light intensity.

The effect of light, copper ions, copper oxide nanoparticles on the change in the structural, functional, cytometric, fluorescent parameters of coccolithophore Pleurochrysis sp. was investigated. The culture Pleurochrysis sp. was represented by two cell forms: (1) covered with coccoliths; and (2) not covered, the ratio of which depends from growth conditions. An increase in light from 20 to 650µEm-2 s-1 led to a decrease in the concentration of cells covered with coccoliths from 90 to 35%. With an increase in light, the decrease in the values of variable chlorophyll a fluorescence was observed, a decrease in the chlorophyll concentration was noted, and an increase in cell volumes and their granularity due to coccoliths 'overproduction' was recorded. A tolerance of Pleurochrysis sp. to the effect of copper was registered, both in the ionic form and in the form of a nanopowder. This is probably due to the morphological (presence of coccoliths) and physiological (ligand production) peculiarities of species. Copper did not affect the ratio of cells covered with coccoliths; its value was about 85%. Growth inhibition, a 2-fold decrease in the intracellular chlorophyll content, a decrease in F v /F m , and a pronounced cell coagulation were recorded at the maximum Cu2+ concentration (625µgL-1 ). The mechanical effect was registered of CuO nanoparticles on the surface of Pleurochrysis sp. coccosphere, which results in the emergence of destroyed and deformed coccoliths. A hypothesis is proposed considering the protective function of coccoliths acting as a barrier when the cells are exposed to nanoparticles and copper ions.

High-Purity Fucoxanthin Can Be Efficiently Prepared from Isochrysis zhangjiangensis by Ethanol-Based Green Method Coupled with Octadecylsilyl (ODS) Column Chromatography.

The exploitation of new economically valuable microalgae as a sustainable source of minor high-value products can effectively promote the full utilization of microalgae. The efficient preparation of minor products from microalgae remains the challenge, owing to the coexistence of various components with a similar polarity in the microalgae biomass. In this study, a novel approach based on the sustainable-oriented strategy for fucoxanthin (FX) production was proposed, which consisted of four steps, including the culture of microalga, ethanol extraction, ODS column chromatography, and ethanol precipitation. The high-purity FX (around 95%) was efficiently obtained in a total recovery efficiency of 84.28 ± 2.56%. This study reveals that I. zhangjiangensis is a potentially promising feedstock for FX production and firstly provides a potentially eco-friendly method for the scale-up preparation of FX from the microalga I. zhangjiangensis.

The variability in the structural and functional properties of coccolith base plates.

Biomineralization processes exert varying levels of control over crystallization, ranging from poorly ordered polycrystalline arrays to intricately shaped single crystals. Coccoliths, calcified scales formed by unicellular algae, are a model for a highly controlled crystallization process. The coccolith crystals nucleate next to an organic oval structure that was termed the base plate, leading to the assumption that it is responsible for the oriented nucleation of the crystals via stereochemical interactions. In recent years, several works focusing on a well-characterized model species demonstrated a fundamental role for indirect interactions that facilitate coccolith crystallization. Here, we developed the tools to extract the base plates from five different species, giving the opportunity to systematically explore the relations between base plate and coccolith properties. We used multiple imaging techniques to evaluate the structural and chemical features of the base plates under native hydrated conditions. The results show a wide range of properties, overlaid on a common rudimentary scaffold that lacks any detectable structural or chemical motifs that can explain direct nucleation control. This work emphasizes that it is the combination between the base plate and the chemical environment inside the cell that cooperatively facilitate the exquisite control over the crystallization process. STATEMENT OF SIGNIFICANCE: Biological organic scaffolds can serve as functional surfaces that guide the formation of inorganic materials. However, in many cases the specific interactions that facilitate such tight regulation are complex and not fully understood. In this work, we elucidate the architecture of such amodel biological template, an organic scale that directs the assembly of exquisite crystalline arrays of marine microalgae. By using cryo electron microscopy, we reveal the native state organization of these scales from several species. The observed similarities and differences allow us to propose that the chemical microenvironment, rather than stereochemical matching, is the pivotal regulator of the process.

A Comparative In Vitro Evaluation of the Anti-Inflammatory Effects of a Tisochrysis lutea Extract and Fucoxanthin.

In this study, we compared the effects of a Tisochrysis lutea (T. lutea) F&M-M36 methanolic extract with those of fucoxanthin (FX) at equivalent concentration, on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The T. lutea F&M-M36 methanolic extract contained 4.7 mg of FX and 6.22 mg of gallic acid equivalents of phenols per gram. HPLC analysis revealed the presence of simple phenolic acid derivatives. The T. lutea F&M-M36 extract exhibited a potent and concentration-dependent inhibitory activity against COX-2 dependent PGE2 production compared to FX alone. Compared to LPS, T. lutea F&M-M36 extract and FX reduced the expression of IL-6 and of Arg1 and enhanced that of IL-10 and of HO-1; T. lutea F&M-M36 extract also significantly abated the expression of NLRP3, enhanced mir-223 expression and reduced that of mir-146b, compared to LPS (p < 0.05). These findings indicate that T. lutea F&M-M36 methanolic extract has a peculiar anti-inflammatory activity against COX-2/PGE2 and NLRP3/mir-223 that might be attributable to the known anti-inflammatory effects of simple phenolic compounds found in the extract that may synergize with FX. Our data suggest that T. lutea F&M-M36 may serve as a source of anti-inflammatory compounds to be further evaluated in in vivo models of inflammation.

Production, Purification, and Study of the Amino Acid Composition of Microalgae Proteins.

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis, Nostoc sp., Dunaliella salina, and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera, the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

Development of a Method for Fucoxanthin Production Using the Haptophyte Marine Microalga Pavlova sp. OPMS 30543.

The natural pigment fucoxanthin has attracted global attention because of its superior antioxidant properties. The haptophyte marine microalgae Pavlova spp. are assumed to be promising industrial fucoxanthin producers as their lack of a cell wall could facilitate the commercialization of cultured cells as a whole food. This study screened promising Pavlova strains with high fucoxanthin content to develop an outdoor cultivation method for fucoxanthin production. Initial laboratory investigations of P. pinguis NBRC 102807, P. lutheri NBRC 102808, and Pavlova sp. OPMS 30543 identified OPMS 30543 as having the highest fucoxanthin content. The culture conditions were optimized for OPMS 30543. Compared with f/2 and Walne's media, the use of Daigo's IMK medium led to the highest biomass production and highest fucoxanthin accumulation. The presence of seawater elements in Daigo's IMK medium was necessary for the growth of OPMS 30543. OPMS 30543 was then cultured outdoors using acrylic pipe photobioreactors, a plastic bag, an open tank, and a raceway pond. Acrylic pipe photobioreactors with small diameters enabled the highest biomass production. Using an acrylic pipe photobioreactor with 60-mm diameter, a fucoxanthin productivity of 4.88 mg/L/day was achieved in outdoor cultivation. Thus, this study demonstrated the usefulness of Pavlova sp. OPMS 30543 for fucoxanthin production in outdoor cultivation.

Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

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

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

Surface-Enhanced Raman Scattering Microspectroscopy Enables the Direct Characterization of Biomineral-Associated Organic Material on Single Calcareous Microskeletons.

Biominerals are composite materials with inorganic and organic components. The latter provide insights into how organisms control mineralization and, if derived from micro/nannofossils, into past climates. Many calcifying organisms cannot be cultured or are extinct; the only materials available for their study are therefore complex environmental samples in which the organism of interest may only be a minor component. There is currently no method for characterizing the biomineral-associated organic material from single particles within such assemblages, so its compositional diversity is unknown. Focusing on coccoliths, we demonstrate that surface-enhanced Raman scattering microspectroscopy can be used to determine the origin and composition of fossil organic matter at the single-particle level in a heterogeneous micro/nannofossil assemblage. This approach may find applications in the study of micro/nannofossil assemblages and uncultivated species, providing evolutionary insights into the macromolecular repertoire involved in biomineralization.

Advanced Extraction of Lipids with DHA from Isochrysis galbana with Enzymatic Pre-Treatment Combined with Pressurized Liquids and Ultrasound Assisted Extractions.

Microalgal biomass is a sustainable and valuable source of lipids with omega-3 fatty acids. The efficient extraction of lipids from microalgae requires fast and alternative extraction methods, frequently combined with biomass pre-treatment by different procedures. In this work, Pressurized liquid extraction (PLE) was optimized and compared with traditional lipid extraction methods, Folch and Bligh and Dyer, and with a new Ultrasound Assisted Extraction (UAE) method for lipids from microalgae Isochrysis galbana. To further optimize PLE and UAE, enzymatic pre-treatment of microalga Isochrysis galbana was studied with commercial enzymes Viscozyme and Celluclast. No significant differences were found for lipid yields among different extraction techniques used. However, advanced extraction techniques with or without pre-treatment are a green, fast, and toxic solvent free alternative to traditional techniques. Lipid composition of Isochrysis was determined by HPLC-ELSD and included neutral and polar lipids, showing that each fraction comprised different contents in omega-3 polyunsaturated fatty acids (PUFA). The highest polar lipids content was achieved with UAE (50 °C and 15 min) and PLE (100 °C) techniques. Moreover, the highest omega-3 PUFA (33.2%), eicosapentaenoic acid (EPA) (3.3%) and docosahexaenoic acid (DHA) (12.0%) contents were achieved with the advanced technique UAE, showing the optimized method as a practical alternative to produce valuable lipids for food and nutraceutical applications.

Proteomic Profiling of Emiliania huxleyi Using a Three-Dimensional Separation Method Combined with Tandem Mass Spectrometry.

Emiliania huxleyi is one of the most abundant marine planktons, and it has a crucial feature in the carbon cycle. However, proteomic analyses of Emiliania huxleyi have not been done extensively. In this study, a three-dimensional liquid chromatography (3D-LC) system consisting of strong cation exchange, high- and low-pH reversed-phase liquid chromatography was established for in-depth proteomic profiling of Emiliania huxleyi. From tryptic proteome digest, 70 fractions were generated and analyzed using liquid chromatography-tandem mass spectrometry. In total, more than 84,000 unique peptides and 10,000 proteins groups were identified with a false discovery rate of ≤0.01. The physicochemical properties of the identified peptides were evaluated. Using ClueGO, approximately 700 gene ontology terms and 15 pathways were defined from the identified protein groups with p-value ≤0.05, covering a wide range of biological processes, cellular components, and molecular functions. Many biological processes associated with CO2 fixation, photosynthesis, biosynthesis, and metabolic process were identified. Various molecular functions relating to protein binding and enzyme activities were also found. The 3D-LC strategy is a powerful approach for comparative proteomic studies on Emiliania huxleyi to reveal changes in its protein level and related mechanism.

The potential of fatty acid isotopes to trace trophic transfer in aquatic food-webs.

Compound-specific isotope analyses (CSIA) of fatty acids (FA) constitute a promising tool for tracing energy flows in food-webs. However, past applications of FA-specific carbon isotope analyses have been restricted to a relatively coarse food-source separation and mainly quantified dietary contributions from different habitats. Our aim was to evaluate the potential of FA-CSIA to provide high-resolution data on within-system energy flows using algae and zooplankton as model organisms. First, we investigated the power of FA-CSIA to distinguish among four different algae groups, namely cyanobacteria, chlorophytes, haptophytes and diatoms. We found substantial within-group variation but also demonstrated that δ13C of several FA (e.g. 18:3ω3 or 18:4ω3) differed among taxa, resulting in group-specific isotopic fingerprints. Second, we assessed changes in FA isotope ratios with trophic transfer. Isotope fractionation was highly variable in daphnids and rotifers exposed to different food sources. Only δ13C of nutritionally valuable poly-unsaturated FA remained relatively constant, highlighting their potential as dietary tracers. The variability in fractionation was partly driven by the identity of food sources. Such systematic effects likely reflect the impact of dietary quality on consumers' metabolism and suggest that FA isotopes could be useful nutritional indicators in the field. Overall, our results reveal that the variability of FA isotope ratios provides a substantial challenge, but that FA-CSIA nevertheless have several promising applications in food-web ecology. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

In Vivo Shaping of Inorganic Functional Devices using Microalgae.

The usage of biomineralization processes performed by living microalgae to create 3D nanostructured materials are advantageous compared to conventional synthesis routes. Exploitation of in vivo shaping using living cells leads to inorganic intricate biominerals, produced with low environmental impact. Since biomineralization processes are genetically controlled, the formation of nanostructured materials is highly reproducible. The shells of microalgae, like coccoliths, are particularly of great interest. This study shows the generation of mesoporous highly structured functional materials with induced optoelectronical properties using in vivo processes of the microalga species Emiliania huxleyi. It demonstrates the metabolically driven incorporation of the lanthanide terbium into the coccoliths of E. huxleyi as a route for the synthesis of finely patterned photoluminescent particles by feeding the microalgae with this luminescent element. The resulting green luminescent particles have hierarchical ordered pores on the nano- and microscale and may act as powerful tools for many applications; they may serve as imaging probes for biomedical applications, or in microoptics. The luminescent coccoliths combine a unique hierarchical structure with a characteristic luminescence pattern, which make them superior to conventional produced Tb doted material. With this study, the possibility of the further exploitation of coccoliths as advanced functional materials for nanotechnological applications is given.

Coccolith mass and morphology of different Emiliania huxleyi morphotypes: A critical examination using Canary Islands material.

Different morphotypes of the abundant marine calcifying algal species Emiliania huxleyi are commonly linked to various degrees of E. huxleyi calcification, but few studies have been done to validate this assumption. This study investigated therefore whether E. huxleyi morphotypes can be related to coccolithophore calcification and coccolith mass. Samples from January (high productivity) and September (low productivity) 1997 at an open ocean and a coastal site near the Canary Islands were analysed using a combination of thickness measurements (Circular Polarizer Retardation estimates (CPR) method), Scanning Electron Microscope imaging, and Markov Chain Monte Carlo (MCMC) models. Mean E. huxleyi coccolith mass varied from a maximum of 2.9pg at the open ocean station in January to a minimum of 1.7pg in September at both stations. In contrast, overall calcite produced by E. huxleyi (assuming 23 coccoliths/cell) varied from a maximum of 2.6 µgL-1 at the coastal station in January to a minimum of 0.5 µgL-1 in September at the open ocean site. The relative abundance of "Overcalcified" Type A, Type A, Group B and malformed coccoliths was determined from SEM images. The mean coccolith mass of "Overcalcified" Type A was 2.0pg using the CPR-method, while mean mass of Type A and Group B coccoliths was determined using coccolith length measurements from SEM images and MCMC models relating thickness measurements to morphotype relative abundance. Type A cocccolith mass varied from a 1.6pg to 2.6pg and Group B coccolith mass varied from 1.5pg to 2.0pg. These results demonstrate that the coccolith mass of Type A, "Overcalcified" Type A, and Group B do not differ systematically and there is no systematic relationship between relative abundance of a morphotype and the overall calcite production of E. huxleyi. Therefore, morphotype appearance and relative abundance can not be uniformly used as reliable indicators of E. huxleyi calcification or calcite production.

Mechanism Analysis of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide from Isochrysis zhanjiangensis Microalgae for Suppressing Vascular Injury in Human Umbilical Vein Endothelial Cells.

Microalgae are primary producers with multiple nutrients in aquatic environments and mostly have applications in biological feed and fuel industry. There are few studies assessing the angiotensin-I-converting enzyme (ACE) inhibition potential of Isochrysis zhanjiangensis, other than its antioxidant potential. In this study, we evaluated a peptide from I. zhanjiangensis (PIZ, FEIHCC) and its vascular endothelial factors and mechanism in human umbilical vein endothelial cells (HUVEC). The results reveal that PIZ (IC50 = 61.38 µM) acts against ACE in a non-competitive binding mode. In addition, PIZ inhibits angiotensin II (Ang II)-induced vascular factor secretion and expression by blocking inflammation and apoptosis through nuclear factor κB (NF-κB), nuclear erythroid 2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), and the serine/threonine kinase (Akt) signal pathways. This study reveals that PIZ has potential to be developed as a therapeutic agent for hypertension and provides a new method of high-value utilization of I. zhanjiangensis.