Molecular Response to Toxic Diatom-Derived Aldehydes in the Sea Urchin Paracentrotus lividus

Diatoms are dominant photosynthetic organisms in the world’s oceans and represent a major food source for zooplankton and benthic filter-feeders. However, their beneficial role in sustaining marine food webs has been challenged after the discovery that they produce secondary metabolites, such as polyunsaturated aldehydes (PUAs), which negatively affect the reproductive success of many invertebrates. Here, we report the effects of two common diatom PUAs, heptadienal and octadienal, which have never been tested before at the molecular level, using the sea urchin, Paracentrotus lividus, as a model organism. We show that both PUAs are able to induce teratogenesis (i.e., malformations), as already reported for decadienal, the better-studied PUA of this group. Moreover, post-recovery experiments show that embryos can recover after treatment with all three PUAs, indicating that negative effects depend both on PUA concentrations and the exposure time of the embryos to these metabolites. We also identify the time range during which PUAs exert the greatest effect on sea urchin embryogenesis. Finally, we report the expression levels of thirty one genes (having a key role in a broad range of functional responses, such as stress, development, differentiation, skeletogenesis and detoxification processes) in order to identify the common targets affected by PUAs and their correlation with morphological abnormalities. This study opens new perspectives for understanding how marine organisms afford protection from environmental toxicants through an integrated network of genes.     

Polyunsaturated Aldehydes from Large Phytoplankton of the Atlantic Ocean Surface (42°N to 33°S)

Polyunsaturated aldehydes (PUAs) are organic compounds mainly produced by diatoms, after cell wounding. These compounds are increasingly reported as teratogenic for species of grazers and deleterious for phytoplanktonic species, but there is still scarce information regarding concentration ranges and the composition of PUAs in the open ocean. In this study, we analyzed the spatial distribution and the type of aldehydes produced by the large-sized (>10 μm) phytoplankton in the Atlantic Ocean surface. Analyses were conducted on PUAs released after mechanical disruption of the phytoplankton cells, referred to here as potential PUAs (pPUAs). Results show the ubiquitous presence of pPUA in the open ocean, including upwelling areas, as well as oligotrophic gyres. Total pPUA concentrations ranged from zero to 4.18 pmol from cells in 1 L. Identified PUAs were heptadienal, octadienal and decadienal, with heptadienal being the most common (79% of total stations). PUA amount and composition across the Atlantic Ocean was mainly related to the nitrogen:phosphorus ratio, suggesting nutrient-driven mechanisms of PUA production. Extending the range of trophic conditions considered by adding data reported for productive coastal waters, we found a pattern of PUA variation in relation to trophic status.     

The relevance of marine chemical ecology to plankton and ecosystem function: an emerging field

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.     

Potential Polyunsaturated Aldehydes in the Strait of Gibraltar under Two Tidal Regimes

Diatoms, a major component of the large-sized phytoplankton, are able to produce and release polyunsaturated aldehydes after cell disruption (potential PUAs or pPUA). These organisms are dominant in the large phytoplankton fraction (>10 µm) in the Strait of Gibraltar, the only connection between the Mediterranean Sea and the Atlantic Ocean. In this area, the hydrodynamics exerts a strong control on the composition and physiological state of the phytoplankton. This environment offers a great opportunity to analyze and compare the little known distribution of larger sized PUA producers in nature and, moreover, to study how environmental variables could affect the ranges and potential distribution of these compounds. Our results showed that, at both tidal regimes studied (Spring and Neap tides), diatoms in the Strait of Gibraltar are able to produce three aldehydes: Heptadienal, Octadienal and Decadienal, with a significant dominance of Decadienal production. The PUA released by mechanical cell disruption of large-sized collected cells (pPUA) ranged from 0.01 to 12.3 pmol from cells in 1 L, and from 0.1 to 9.8 fmol cell⁻¹. Tidal regime affected the abundance, distribution and the level of physiological stress of diatoms in the Strait. During Spring tides, diatoms were more abundant, usually grouped nearer the coastal basin and showed less physiological stress than during Neap tides. Our results suggest a significant general increase in the pPUA productivity with increasing physiological stress for the cell also significantly associated to low nitrate availability.     

Biological Activity of trans-2-Hexenal Against Bradysia odoriphaga (Diptera: Sciaridae) at Different Developmental Stages

trans-2-Hexenal, one of the C6 green leaf volatiles, is potentially useful for the control of Bradysia odoriphaga Yang et Zhang. In this study, the biological activity of trans-2-hexenal on B. odoriphaga was assessed in the laboratory. trans-2-Hexenal was observed to kill B. odoriphaga in different developmental stages at a relatively low concentration under fumigation. The respiration rate in the male treatment group decreased from 131.44 to 4.07 nmol/g·min with a prolonged fumigation time, while the respiration rate in females decreased from 128.82 to 24.20 nmol/g·min. Male adults exhibited a more sensitive electroantennogram response at 0.05–500 μl/ml at the dose of 10.0 μl than female adults. Moreover, trans-2-hexenal had a repellent effect on adults based on the results with a Y-tube olfactometer at 10.0 μl, as shown by the deterrent rate of male and female adults with 96.67% and 98.33%, respectively. The results showed that trans-2-hexenal had good biological activity in different developmental stages of B. odoriphaga, which could reduce the need for, and risks associated with, the use of traditional insecticides and enable nonharmful management.     

The Negative Relationship between Fouling Organisms and the Content of Eicosapentaenoic Acid and Docosahexaenoic Acid in Cultivated Pacific Oysters,Crassostrea gigas

Bivalves serve as an important aquaculture product, as they are the source of essential fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in our diet. However, their cultivation in the wild can be affected by fouling organisms that, in turn, affect their EPA and DHA content. The effects of fouling organisms on the EPA and DHA contents of cultivated bivalves have not been well documented. We examined the effects of fouling organisms on the EPA and DHA contents and condition index of cultured oysters, Crassostrea gigas, in an aquaculture system. We sampled two-year-old oysters from five sites in Shizugawa Bay, Japan, in August 2014. Most of the fouling organisms were sponges, macroalgae, and Mytilus galloprovincialis. A significant negative relationship existed between the DHA content in C. gigas and the presence of sponges and macroalgae. A lower C. gigas EPA content corresponded to a higher M. galloprovincialis fouling mass and a lower C. gigas condition index. This can be explained by dietary competition between C. gigas and M. galloprovincialis for diatoms, which were the main producer of EPA in our study sites. Our findings indicate that fouling organisms likely reduce the EPA and DHA content in cultivated oysters. Therefore, our results suggest that the current efforts to remove fouling organisms from oyster clusters is an effective strategy to enhance the content of EPA and DHA in oysters.     

Marine Benthic Diatoms Contain Compounds Able to Induce Leukemia Cell Death and Modulate Blood Platelet Activity

In spite of the high abundance and species diversity of diatoms, only a few bioactive compounds from them have been described. The present study reveals a high number of mammalian cell death inducing substances in biofilm-associated diatoms sampled from the intertidal zone. Extracts from the genera Melosira, Amphora, Phaeodactylum and Nitzschia were all found to induce leukemia cell death, with either classical apoptotic or autophagic features. Several extracts also contained inhibitors of thrombin-induced blood platelet activation. Some of this activity was caused by a high content of adenosine in the diatoms, ranging from 0.07 to 0.31 μg/mg dry weight. However, most of the bioactivity was adenosine deaminase-resistant. An adenosine deaminase-resistant active fraction from one of the extracts was partially purified and shown to induce apoptosis with a distinct phenotype. The results show that benthic diatoms typically found in the intertidal zone may represent a richer source of interesting bioactive compounds than hitherto recognized.     

Cross-linking of arabinoxylans via 8-8-coupled diferulates as demonstrated by isolation and identification of diarabinosyl 8-8(cyclic)-dehydrodiferulate from maize bran

Dehydrodiferulates are likely the most important arabinoxylan cross-links in cereals and grasses in general. However, association of dehydrodiferulates and arabinoxylans has only been authenticated for 5-5- and 8-O-4-dehydrodiferulates to date. In the present study, a saccharide ester of 8-8(cyclic)-dehydrodiferulate was isolated from maize bran insoluble fibre following mild acidic hydrolysis by using Sephadex LH-20 chromatography, gel chromatography on Bio-Gel P-2, and RP-HPLC. Mass spectrometry, one- and two-dimensional NMR and analysis of the carbohydrate and phenolic constituents following further hydrolysis identified the isolated compound as the di-5-O-l-arabinosyl ester of 8-8(cyclic)-dehydrodiferulic acid. From this finding it is apparent that 8-8(cyclic)-dehydrodiferulate exists as such in the plant cell wall and acts as an arabinoxylan cross-link. In addition, a fraction was isolated that contained two saccharide esters of 8-O-4-dehydrodiferulates. This fraction was comprised of two compounds, both built from 8-O-4-dehydrodiferulate, a 5-linked arabinofuranose and a 5-linked xylopyranosyl-(1→2)-arabinofuranose unit. These compounds show that, in addition to the 5-O-(trans-feruloyl)-l-arabinofuranosyl sidechain, the more complex β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranosyl sidechains are involved in the formation of 8-O-4-dehydrodiferulates.     

The Effect of Dissolved Polyunsaturated Aldehydes on Microzooplankton Growth Rates in the Chesapeake Bay and Atlantic Coastal Waters

Allelopathy is wide spread among marine phytoplankton, including diatoms, which can produce cytotoxic secondary metabolites such as polyunsaturated aldehydes (PUA). Most studies on diatom-produced PUA have been dedicated to their inhibitory effects on reproduction and development of marine invertebrates. However, little information exists on their impact on key herbivores in the ocean, microzooplankton. This study examined the effects of dissolved 2E,4E-octadienal and 2E,4E-heptadienal on the growth rates of natural ciliate and dinoflagellate populations in the Chesapeake Bay and the coastal Atlantic waters. The overall effect of PUA on microzooplankton growth was negative, especially at the higher concentrations, but there were pronounced differences in response among common planktonic species. For example, the growth of Codonella sp., Leegaardiella sol, Prorodon sp., and Gyrodinium spirale was impaired at 2 nM, whereas Strombidium conicum, Cyclotrichium gigas, and Gymnodinium sp. were not affected even at 20 nM. These results indicate that PUA can induce changes in microzooplankton dynamics and species composition.     

1-O-Alkylglycerol Ethers from the Marine Sponge Guitarra abbotti and Their Cytotoxic Activity

The cytotoxicity-bioassay-guided fractionation of the ethanol extract from the marine sponge Guitarra abbotti, whose 1-O-alkyl-sn-glycerol ethers (AGEs) have not been investigated so far, led to the isolation of a complex lipid fraction containing, along with previously known compounds, six new lipids of the AGE type. The composition of the AGE fraction as well as the structures of 6 new and 22 previously known compounds were established using ¹H and ¹³C NMR, GC/MS, and chemical conversion methods. The new AGEs were identified as: 1-O-(Z-docos-15-enyl)-sn-glycerol (1), 1-O-(Z-docos-17-enyl)-sn-glycerol (2), 1-O-(Z-tricos-15-enyl)-sn-glycerol (3), 1-O-(Z-tricos-16-enyl)-sn-glycerol (4), 1-O-(Z-tricos-17-enyl)-sn-glycerol (5), and 1-O-(Z-tetracos-15-enyl)-sn-glycerol (6). The isolated AGEs show weak cytotoxic activity in THP-1, HL-60, HeLa, DLD-1, SNU C4, SK-MEL-28, and MDA-MB-231 human cancer cells. A further cytotoxicity analysis in JB6 P⁺ Cl41 cells bearing mutated MAP kinase genes revealed that ERK2 and JNK1 play a cytoprotective role in the cellular response to the AGE-induced cytotoxic effects.     

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.     

Silaffins in Silica Biomineralization and Biomimetic Silica Precipitation

Biomineralization processes leading to complex solid structures of inorganic material in biological systems are constantly gaining attention in biotechnology and biomedical research. An outstanding example for biomineral morphogenesis is the formation of highly elaborate, nano-patterned silica shells by diatoms. Among the organic macromolecules that have been closely linked to the tightly controlled precipitation of silica in diatoms, silaffins play an extraordinary role. These peptides typically occur as complex posttranslationally modified variants and are directly involved in the silica deposition process in diatoms. However, even in vitro silaffin-based peptides alone, with and without posttranslational modifications, can efficiently mediate biomimetic silica precipitation leading to silica material with different properties as well as with encapsulated cargo molecules of a large size range. In this review, the biomineralization process of silica in diatoms is summarized with a specific focus on silaffins and their in vitro silica precipitation properties. Applications in the area of bio- and nanotechnology as well as in diagnostics and therapy are discussed.     

bZIP Transcription Factor UvATF21 Mediates Vegetative Growth,Conidiation, Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes. Several bZIP proteins have previously been identified in Ustilaginoidea virens. However, the biological roles of these bZIP proteins in this pathogen are still unknown. Here, one of these bZIP protein coding genes, UvATF21, was functionally characterized. Targeted deletion of UvATF21 resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth. The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses. Interestingly, deletion of UvATF21 exhibited different performances to cell wall integrity stress. These results indicated that UvATF21 played crucial roles in vegetative growth, conidiation, stress response, and full virulence in U. virens.     

Cytotoxic Effect of Natural trans-Resveratrol Obtained from Elicited Vitis vinifera Cell Cultures on Three Cancer Cell Lines

trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose- and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G₀/G₁ phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a down-regulation of Bcl-2.     

CS5931, a Novel Polypeptide in Ciona savignyi,Represses Angiogenesis via Inhibiting Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs)

CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell) in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF) production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9) both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.     

Oxylipin Diversity in the Diatom Family Leptocylindraceae Reveals DHA Derivatives in Marine Diatoms

Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C₂₀ and C₂₂ non-volatile oxylipins in five species of the family Leptocylindraceae, which constitute a basal clade in the diatom phylogeny. We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species.     

Degradation of Carotenoids in Apricot (Prunus armeniaca L.) During Drying Process

Carotenoids are natural compounds whose nutritional importance comes from the provitamin A activity of some of them and their protection against several serious human disorders. The degradation of carotenoids was investigated during apricot drying by microwave and convective hot-air at 60 and 70 °C. Seven carotenoids were identified: antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, 13-cis-β-carotene, all-trans-β-carotene and 9-cis-β-carotene; among these, all-trans-β-carotene was found to be about 50 % of total carotenoids. First-order kinetic models were found to better describe all-trans-β-carotene reduction during drying, with a degradation rate constant (k₁) that increased two folds when temperatures increased by 10 °C, in both methods. No differences were found in k₁ between apricots dried by hot air at 70 °C (k₁?=?0.0340 h^?1) and by microwave at 60 °C. The evolution of total carotenoids (117.1 mg/kg on dry basis) during drying highlighted a wider decrease (about 50 %) when microwave heating was employed, for both set temperatures. Antheraxantin was found to be the carotenoid most susceptible to heat, disappearing at 6 h during both trials with microwave as well as during convective hot-air at 70 °C. For this reason, antheraxanthin could be a useful marker for the evaluation of thermal damage due to the drying process. Also the degree of isomerization of all-trans-β-carotene could be a useful marker for the evaluation of the drying process.     

Constitutive expression of <Emphasis Type="Italic">REL1</Emphasis> confers the rice response to drought stress and abscisic acid

Leaf rolling is one of the most significant symptoms of drought stress in plant. Previously, we identified a dominant negative mutant, termed rolled and erect 1 (hereafter referred to rel1-D), regulating leaf rolling and erectness in rice. However, the role of REL1 in drought response is still poorly understood. Here, our results indicated that rel1-D displayed higher tolerance to drought relative to wild type, and the activity of superoxide dismutase (SOD) and drought responsive genes were significantly up-regulated in rel1-D. Moreover, our results revealed that rel1-D was hypersensitive to ABA and the expression of ABA associated genes was significantly increased in rel1-D, suggesting that REL1 likely coordinates ABA to regulate drought response. Using the RNA-seq approach, we identified a large group of differentially expressed genes that regulate stimuli and stresses response. Consistently, we also found that constitutive expression of REL1 alters the expression of biotic and abiotic stress responsive genes by the isobaric tags for relative and absolute quantification (iTRAQ) analysis. Integrative analysis demonstrated that 8 genes/proteins identified by both RNA-seq and iTRAQ would be the potential targets in term of the REL1-mediated leaf morphology. Together, we proposed that leaf rolling and drought tolerance of rel1-D under normal condition might be caused by the endogenously perturbed homeostasis derived from continuous stressful dynamics.     

Ecotoxicological Impact of the Marine Toxin Palytoxin on the Micro-Crustacean Artemia franciscana

Palytoxin (PLTX) is a highly toxic polyether identified in various marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. In addition to adverse effects in humans, negative impacts on different marine organisms have been often described during Ostreopsis blooms and the concomitant presence of PLTX and its analogues. Considering the increasing frequency of Ostreopsis blooms due to global warming, PLTX was investigated for its effects on Artemia franciscana, a crustacean commonly used as a model organism for ecotoxicological studies. At concentrations comparable to those detected in culture media of O. cf. ovata (1.0–10.0 nM), PLTX significantly reduced cysts hatching and induced significant mortality of the organisms, both at larval and adult stages. Adults appeared to be the most sensitive developmental stage to PLTX: significant mortality was recorded after only 12 h of exposure to PLTX concentrations > 1.0 nM, with a 50% lethal concentration (LC₅₀) of 2.3 nM (95% confidence interval = 1.2–4.7 nM). The toxic effects of PLTX toward A. franciscana adults seem to involve oxidative stress induction. Indeed, the toxin significantly increased ROS levels and altered the activity of the major antioxidant enzymes, in particular catalase and peroxidase, and marginally glutathione-S-transferase and superoxide dismutase. On the whole, these results indicate that environmentally relevant concentrations of PLTX could have a negative effect on Artemia franciscana population, suggesting its potential ecotoxicological impact at the marine level.