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Background Copepods are fundamental components of pelagic food webs, but reports on how molecular responses link to reproductive success in natural populations are still scarce. We present a de novo transcriptome assembly and differential expression (DE) analysis in Temora stylifera females collected in the Gulf of Naples, Mediterranean Sea, where this copepod dominates the zooplankton community. High-Throughput RNA-Sequencing and DE analysis were performed from adult females collected on consecutive weeks (May 23rd and 30th 2017), because opposite naupliar survival rates were observed. We aimed at detecting key genes that may have influenced copepod reproductive potential in natural populations and whose expression was potentially affected by phytoplankton-derived oxylipins, lipoxygenase-derived products strongly impacting copepod naupliar survival. Results On the two sampling dates, temperature, salinity, pH and oxygen remained stable, while variations in phytoplankton cell concentration, oxylipin concentration and oxylipin-per-diatom-cell production were observed. T. stylifera naupliar survival was 25% on May 23rd and 93% on May 30th. De novo assembly generated 268,665 transcripts (isoforms) and 120,749 unique ‘Trinity predicted genes’ (unigenes), of which 50% were functionally annotated. Out of the 331 transcript isoforms differentially expressed between the two sampling dates, 119 sequences were functionally annotated (58 up- and 61 down-regulated). Among predicted genes (unigenes), 144 sequences were differentially expressed and 31 (6 up-regulated and 25 down-regulated) were functionally annotated. Most of the significantly down-regulated unigenes and isoforms were A5 Putative Odorant Binding Protein (Obp). Other differentially expressed sequences (isoforms and unigenes) related to developmental metabolic processes, protein ubiquitination, response to stress, oxidation-reduction reactions and hydrolase activities. DE analysis was validated through Real Time-quantitative PCR of 9 unigenes and 3 isoforms. Conclusions Differential expression of sequences involved in signal detection and transduction, cell differentiation and development offered a functional interpretation to the maternally-mediated low naupliar survival rates observed in samples collected on May 23rd. Down-regulation of A5 Obp along with higher quantities of oxylipins-per-litre and oxylipins-per-diatom-cell observed on May 23rd could suggest oxylipin-mediated impairment of naupliar survival in natural populations of T. stylifera. Our results may help identify biomarker genes explaining variations in copepod reproductive responses at a molecular level.

The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays—two phenotypic and one enzymatic—for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.

The deep-sea environment is a unique, challenging extreme habitat where species have had to adapt to the absence of light, low levels of oxygen, high pressure and little food. In order to survive such harsh conditions, these organisms have evolved different biochemical and physiological features that often have no other equivalent in terrestrial habitats. Recent analyses have highlighted how the deep sea is one of the most diverse and species-rich habitats on the planet but less explored compared to more accessible sites. Because of their adaptation to this extreme environment, deep-sea species have the potential to produce novel secondary metabolites with potent biological activities. Recent advances in sampling and novel techniques in microorganism culturing and chemical isolation have promoted the discovery of bioactive agents from deep-sea organisms. However, reports of natural products derived from deep-sea species are still scarce, probably because of the difficulty in accessing deep-sea samples, sampling costs and the difficulty in culturing deep-sea organisms. In this review, we give an overview of the potential treasure represented by metabolites produced by deep marine species and their bioactivities for the treatment and prevention of various human pathologies.

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.

Oxylipins are important signal transduction lipoxygenase-derived products of fatty acids that regulate a variety of physiological and pathological processes in plants and animals. In marine diatoms, these molecules can be highly bioactive, impacting zooplankton grazers, bacteria and other phytoplankton. However, the ultimate cause for oxylipin production in diatoms is still poorly understood, from an evolutionary perspective. Here we analysed production of particulate linear oxygenated fatty acids (LOFAs, previously named non-volatile oxylipins) from natural phytoplankton collected weekly for 1 year. We demonstrate for the first time that diatoms are the main LOFA producers in natural phytoplankton assemblages. Interestingly, LOFA-per-cell production decreased with increasing diatom density and was not due to major changes in diatom community composition. An inverse relation was confirmed at a global scale by analysing diatom lipoxygenase unigenes and metagenomes from Tara Oceans datasets. A network analysis suggested that different LOFAs could contribute to modulate co-variations of different diatom taxa. Overall, we offer new insights in diatom chemical ecology, possibly explaining the evolution of oxylipin synthesis in diatoms.

Petroleum hydrocarbons (PHCs) are one of the most widespread and heterogeneous organic contaminants affecting marine ecosystems. The contamination of marine sediments or coastal areas by PHCs represents a major threat for the ecosystem and human health, calling for urgent, effective, and sustainable remediation solutions. Aside from some physical and chemical treatments that have been established over the years for marine sediment reclamation, bioremediation approaches based on the use of microorganisms are gaining increasing attention for their eco-compatibility, and lower costs. In this work, we review current knowledge concerning the bioremediation of PHCs in marine systems, presenting a synthesis of the most effective microbial taxa (i.e., bacteria, fungi, and microalgae) identified so far for hydrocarbon removal. We also discuss the challenges offered by innovative molecular approaches for the design of effective reclamation strategies based on these three microbial components of marine sediments contaminated by hydrocarbons.

Here, we report the draft genome sequence of a metagenome-assembled genome (MAG) of a new Alkaliphilus bacterium, NP8, of the Clostridiaceae family. This bacterium was isolated from polluted sediment collected from an abandoned industrial site located in the Gulf of Naples (Mediterranean Sea) as part of a microbial consortium.

Coastal areas impacted by high anthropogenic pressures typically display sediment contamination by polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs). Microbial-based bioremediation represents a promising strategy for sediment reclamation, yet it frequently fails due to poor knowledge of the diversity and dynamics of the autochthonous microbial assemblages and to the inhibition of the target microbes in the contaminated matrix. In the present study, we used an integrated approach including a detailed environmental characterization, high-throughput sequencing and culturing to identify autochthonous bacteria with bioremediation potential in the sediments of Bagnoli-Coroglio (Gulf of Naples, Mediterranean Sea), a coastal area highly contaminated by PAHs, aliphatic hydrocarbons and HMs. The analysis of the benthic prokaryotic diversity showed that the distribution of the dominant taxon (Gammaproteobacteria) was mainly influenced by PAHs, As, and Cd concentrations. The other abundant taxa (including Alphaproteobacteria, Deltaproteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, NB1-j, Desulfobacterota, and Myxococcota) were mainly driven by sediment grain size and by Cu and Cr concentrations, while the rare taxa (i.e., each contributing Vibrio, Pseudoalteromonas, and Agarivorans, which were only scarcely represented in their original assemblages. These findings suggest that rare but culturable bacterial strains resistant/tolerant to high levels of mixed contaminants can be promising candidates useful for the reclamation by bioaugmentation strategies of marine sediments that are highly contaminated with PAHs and HMs.

Microalgae are known to produce a plethora of compounds derived from the primary and secondary metabolism. Different studies have shown that these compounds may have allelopathic, antimicrobial, and antipredator activities. In addition, in vitro and in vivo screenings have shown that several compounds have interesting bioactivities (such as antioxidant, anti-inflammatory, anticancer, and antimicrobial) for the possible prevention and treatment of human pathologies. Additionally, the enzymatic pathways responsible for the synthesis of these compounds, and the targets and mechanisms of their action have also been investigated for a few species. However, further research is necessary for their full exploitation and possible pharmaceutical and other industrial applications. Here, we review the current knowledge on the chemical characteristics, biological activities, mechanism of action, and the enzymes involved in the synthesis of microalgal metabolites with potential benefits for human health.

Simple Summary Compared to more accessible sites, organisms inhabiting Antarctic waters have been poorly investigated. This study provides the first molecular resource (transcriptome from whole individual) for the eucalanoid copepod Rhincalanus gigas, one of the predominant zooplankton species of Antarctic waters. Sequence analyses identified possible adaptation strategies adopted by the organism to cope with cold environments. Among those, we identified in R. gigas transcriptome three predicted genes encoding for antifreeze proteins and gene duplication within the glutathione metabolism pathway. This new molecular resource, provided here, will be useful to study the physiology, ecology, and biology of R. gigas and it increases the information available for polar environments. Abstract Antarctic waters are the largest almost untapped diversified resource of our planet. Molecular resources for Antarctic organisms are very limited and mostly represented by sequences used for species genotyping. In this study, we present the first transcriptome for the copepod Rhincalanus gigas, one of the predominant zooplankton species of Antarctic waters. This transcriptome represents also the first molecular resource for an eucalanoid copepod. The transcriptome is of high quality and completeness. The presence of three predicted genes encoding antifreeze proteins and gene duplication within the glutathione metabolism pathway are suggested as possible adaptations to cope with this harsh environment. The R. gigas transcriptome represents a powerful new resource for investigating the molecular basis associated with polar biological processes and ecology.

Marine organisms are constantly exposed to variations in physical parameters (e [...]

Investigations on the ability of bacteria to enhance removal of hydrocarbons and reduce heavy metal toxicity in sediments are necessary to design more effective bioremediation strategies. In this study, five bacterial strains, Halomonas sp. SZN1, Alcanivorax sp. SZN2, Pseudoalteromonas sp. SZN3, Epibacterium sp. SZN4, and Virgibacillus sp. SZN7, were isolated from polluted sediments from an abandoned industrial site in the Gulf of Naples, Mediterranean Sea, and tested for their bioremediation efficiency on sediment samples collected from the same site. These bacteria were added as consortia or as individual cultures into polluted sediments to assess biodegradation efficiency of polycyclic aromatic hydrocarbons and heavy metal immobilisation capacity. Our results indicate that these bacteria were able to remove polycyclic aromatic hydrocarbons, with a removal rate up to ca. 80% for dibenzo-anthracene. In addition, these bacteria reduced arsenic, lead, and cadmium mobility by promoting their partitioning into less mobile and bioavailable fractions. Microbial consortia generally showed higher performance toward pollutants as compared with pure isolates, suggesting potential synergistic interactions able to enhance bioremediation capacity. Overall, our findings suggest that highly polluted sediments select for bacteria efficient at reducing the toxicity of hazardous compounds, paving the way for scaled-up bioremediation trials.

Many dinoflagellates species, especially of the Alexandrium genus, produce a series of toxins with tremendous impacts on human and environmental health, and tourism economies. Alexandrium tamutum was discovered for the first time in the Gulf of Naples, and it is not known to produce saxitoxins. However, a clone of A. tamutum from the same Gulf showed copepod reproduction impairment and antiproliferative activity. In this study, the full transcriptome of the dinoflagellate A. tamutum is presented in both control and phosphate starvation conditions. RNA-seq approach was used for in silico identification of transcripts that can be involved in the synthesis of toxic compounds. Phosphate starvation was selected because it is known to induce toxin production for other Alexandrium spp. Results showed the presence of three transcripts related to saxitoxin synthesis (sxtA, sxtG and sxtU), and others potentially related to the synthesis of additional toxic compounds (e.g., 44 transcripts annotated as &ldquo, polyketide synthase&rdquo, ). These data suggest that even if this A. tamutum clone does not produce saxitoxins, it has the potential to produce toxic metabolites, in line with the previously observed activity. These data give new insights into toxic microalgae, toxin production and their potential applications for the treatment of human pathologies.

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 &micro, M, 15 &micro, M, and 20 &micro, M. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

Apoptosis and autophagy are fundamental mechanisms of programed cell death activated during protostome and deuterostome embryonic development, contributing to the creation and remodeling of different anatomical structures. Programed cell death has been investigated at morphological and biochemical levels, but there is a lack of information concerning gene expression of death factors during deuterostome embryonic development. In this study, we analyze the expression patterns of 13 genes involved in autophagy, extrinsic and intrinsic apoptosis during blastula, gastrula, and pluteus stages of the sea urchin Paracentrotus lividus embryonic development. Results suggested the occurrence of all death mechanisms investigated, highlighting the simultaneous involvement of apoptosis and autophagy during embryonic development. In particular, gastrula was the developmental stage where the majority of death genes were highly expressed. During gastrulation apoptotic processes are fundamental for tissue remodeling, such as cavity formation and removal of inner ectodermal cells. This is the first report that identifies a panel of cell death genes in the P. lividus genome and analyzes their expression variations during ontogenesis.

Microalgae have been shown to be excellent producers of lipids, pigments, carbohydrates, and a plethora of secondary metabolites with possible applications in the pharmacological, nutraceutical, and cosmeceutical sectors. Recently, various microalgal raw extracts have been found to have anti-inflammatory properties. In this study, we performed the fractionation of raw extracts of the diatom Cylindrotheca closterium, previously shown to have anti-inflammatory properties, obtaining five fractions. Fractions C and D were found to significantly inhibit tumor necrosis factor alpha (TNF-⍺) release in LPS-stimulated human monocyte THP-1 cells. A dereplication analysis of these two fractions allowed the identification of their main components. Our data suggest that lysophosphatidylcholines and a breakdown product of chlorophyll, pheophorbide a, were probably responsible for the observed anti-inflammatory activity. Pheophorbide a is known to have anti-inflammatory properties. We tested and confirmed the anti-inflammatory activity of 1-palmitoyl-sn-glycero-3-phosphocholine, the most abundant lysophosphatidylcholine found in fraction C. This study demonstrated the importance of proper dereplication of bioactive extracts and fractions before isolation of compounds is commenced.

There is an urgent need to discover new drug entities due to the increased incidence of severe diseases as cancer and neurodegenerative pathologies, and reducing efficacy of existing antibiotics. Recently, there is a renewed interest in exploring the marine habitat for new pharmaceuticals also thanks to the advancement in cultivation technologies and in molecular biology techniques. Microorganisms represent a still poorly explored resource for drug discovery. The possibility of obtaining a continuous source of bioactives from marine microorganisms, more amenable to culturing compared to macro-organisms, may be able to meet the challenging demands of pharmaceutical industries. This would enable a more environmentally-friendly approach to drug discovery and overcome the over-utilization of marine resources and the use of destructive collection practices. The importance of the topic is underlined by the number of EU projects funded aimed at improving the exploitation of marine organisms for drug discovery.

Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus, these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

As people across the world live longer, chronic illness and diminished well-being are becoming major global public health challenges. Marine biotechnology may help overcome some of these challenges by developing new products and know-how derived from marine organisms. While some products from marine organisms such as microalgae, sponges, and fish have already found biotechnological applications, jellyfish have received little attention as a potential source of bioactive compounds. Nevertheless, recent studies have highlighted that scyphomedusae (Cnidaria, Scyphozoa) synthesise at least three main categories of compounds that may find biotechnological applications: collagen, fatty acids and components of crude venom. We review what is known about these compounds in scyphomedusae and their current biotechnological applications, which falls mainly into four categories of products: nutraceuticals, cosmeceuticals, biomedicals, and biomaterials. By defining the state of the art of biotechnological applications in scyphomedusae, we intend to promote the use of these bioactive compounds to increase the health and well-being of future societies.

Due to the unique biodiversity and the physical-chemical properties of their environment, marine microorganisms have evolved defense and signaling compounds that often have no equivalent in terrestrial habitats. The aim of this study was to screen extracts of the dinoflagellate Amphidinium carterae for possible bioactivities (i.e., anticancer, anti-inflammatory, anti-diabetes, antibacterial and antifungal properties) and identify bioactive compounds. Anticancer activity was evaluated on human lung adenocarcinoma (A549), human skin melanoma (A2058), human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MCF7) and human pancreas carcinoma (MiaPaca-2) cell lines. Antimicrobial activities were evaluated against Gram-positive bacteria (Staphylococcus aureus MRSA and MSSA), Gram-negative bacteria (i.e., Escherichia coli and Klebsiella pneumoniae), Mycobacterium tuberculosis and the fungus Aspergillus fumigatus. The results indicated moderate biological activities against all the cancer cells lines and microorganisms tested. Bioassay-guided fractionation assisted by HRMS analysis allowed the detection of one new and two known amphidinols that are potentially responsible for the antifungal and cytotoxic activities observed. Further isolation, purification and structural elucidation led to a new amphidinol, named amphidinol 22. The planar structure of the new compound was determined by analysis of its HRMS and 1D and 2D NMR spectra. Its biological activity was evaluated, and it displayed both anticancer and antifungal activities.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments.

Diatoms are key phytoplankton species in the world oceans and essential in the transfer of energy through marine food chains. Their beneficial role has been questioned after the discovery that some diatom species produce teratogenic compounds such as polyunsaturated aldehydes and other oxylipins that induce abortions, poor larval development and high offspring mortality in many invertebrates. To test whether maternal and post-embryonic diatom diets affected development in the copepod Paracartia latisetosa , we carried out experiments in which the PUA-producing diatom Skeletonema marinoi (SKE) was fed to this copepod species. Controls were run with the dinoflagellate, Prorocentrum minimum (PRO), which does not produce any of these compounds. Four treatments were tested: both mothers and progeny always fed on either SKE or PRO, and those in which mothers received either PRO or SKE, and neonates were switched to the other diet. Our results show that a SKE/SKE diet induced lower egg production and egg viability, as well as slower embryonic development than the control diet. Naupliar development was negatively affected with the diatom diet which blocked the metamorphosis of larvae at N3, 4 days after hatching, compared to the control diet that allowed for normal naupliar development in 10 days. Nauplii reared on PRO and spawned by females fed on SKE were able to complete development, but only after 11 days, whereas larvae reared on SKE spawned by females fed on PRO were arrested at the N5 developmental stage, 12 days after hatching. Apoptotic TUNEL-positive nauplii were detected with the SKE diet, indicating imminent death. Significant differences were also recorded by comparing biomass of equivalent naupliar stages obtained for each treatment, and the overall mean biomass female − 1 day − 1 for each diet. Growth inhibition due to oxylipin-producing diatoms may have important consequences on prey–predator relationships and on the biomass flux through marine food chains.

The chemical ecology of marine diatoms has been the subject of several studies in the last decades, due to the discovery of oxylipins with multiple simultaneous functions including roles in chemical defence (antipredator, allelopathic and antibacterial compounds) and/or cell-to-cell signalling. Diatoms represent a fundamental compartment of marine ecosystems because they contribute to about 45% of global primary production even if they represent only 1% of the Earth’s photosynthetic biomass. The discovery that they produce several toxic metabolites deriving from the oxidation of polyunsaturated fatty acids, known as oxylipins, has changed our perspectives about secondary metabolites shaping plant–plant and plant–animal interactions in the oceans. More recently, their possible biotechnological potential has been evaluated, with promising results on their potential as anticancer compounds. Here, we focus on some recent findings in this field obtained in the last decade, investigating the role of diatom oxylipins in cell-to-cell communication and their negative impact on marine biota. Moreover, we also explore and discuss the possible biotechnological applications of diatom oxylipins.

Amphidinolides are cytotoxic macrolides produced by symbiotic unicellular microalgae of the genus Amphidinium. Here we describe the identification of four related molecules belonging to this macrolide family isolated from the invertebrate Stragulum bicolor. The new molecules, named amphidinolide PX1-PX3 and stragulin A (1&ndash, 4), show an unprecedented carbon skeleton whose complete stereochemistry has been determined by spectroscopic and computational methods. Differences in the structures of these molecules modulate their biological activity in a panel of tumor cell lines, but the opened derivative stragulin (4) shows a very potent and specific cytotoxic activity (IC50 0.18 &micro, M) against the aggressive human melanoma cell A2058.

Diatoms, ubiquitous primary producers in marine systems, synthesize oxylipins, which impair copepod fitness through a maternal effect. While oxylipins do not directly affect the adults, these chemicals alter basic cellular and developmental processes of copepod embryos, thereby negatively affecting egg hatching and naupliar survival. Inspecting the effects of oxylipins on copepod reproductive success is extremely challenging, because wide variations in their synthesis potentials among diatom genera, species, populations and strains have been detected. In parallel, distinct copepod species and populations can be differently sensitive to oxylipin-producing diatoms. Lately, application of chemical and molecular methods allowed to finely characterize the oxylipin profiles of single diatom strains and to highlight detoxification responses of copepods through the relative expression of selected genes of interest. Thus far, integrative experimental approaches encompassing physiological, chemical and molecular data were mostly applied to laboratory setups, whereas few in situ experiments were presented in this perspective. Moreover, only a restricted number of papers tested the effects of oxylipins on the plankton community as a whole. This review synthesizes latest advances in conceptual and methodological approaches in diatom–copepod interactions and aims at discussing possible re-shaping of the plankton community in response to oxylipin-producing diatom occurrence.

Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world’s ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

Marine microalgae are considered a potentially new and valuable source of biologically active compounds for applications in several biotechnology sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. Considering the increasing rate of antibiotic-resistance bacteria and infections by fungi, 46 microalgae have been screened in this study for possible antibacterial and antifungal activities. Two different extraction methods have been used in order to increase the probability of finding positive hits. In particular, we screened microalgae in both control and nutrient stress conditions. We also tested different strains for 7 species in order to study potentially different bioactivities due to strain diversity. Results showed that extracts of two diatoms, Skeletonema costatum and Chaetoceros pseudocurvisetus, had anti-tuberculosis activity and were active only when cultured in the control and phosphate-starvation conditions, while the nitrogen starvation condition showed no activity. In addition, we tested both the organic and water extracts and found that only the organic extracts for both diatoms were active. The organic extracts of these two diatom species were not toxic on normal human cell lines.

Enzymes are essential components of biological reactions and play important roles in the scaling and optimization of many industrial processes. Due to the growing commercial demand for new and more efficient enzymes to help further optimize these processes, many studies are now focusing their attention on more renewable and environmentally sustainable sources for the production of these enzymes. Microalgae are very promising from this perspective since they can be cultivated in photobioreactors, allowing the production of high biomass levels in a cost-efficient manner. This is reflected in the increased number of publications in this area, especially in the use of microalgae as a source of novel enzymes. In particular, various microalgal enzymes with different industrial applications (e.g., lipids and biofuel production, healthcare, and bioremediation) have been studied to date, and the modification of enzymatic sequences involved in lipid and carotenoid production has resulted in promising results. However, the entire biosynthetic pathways/systems leading to synthesis of potentially important bioactive compounds have in many cases yet to be fully characterized (e.g., for the synthesis of polyketides). Nonetheless, with recent advances in microalgal genomics and transcriptomic approaches, it is becoming easier to identify sequences encoding targeted enzymes, increasing the likelihood of the identification, heterologous expression, and characterization of these enzymes of interest. This review provides an overview of the state of the art in marine and freshwater microalgal enzymes with potential biotechnological applications and provides future perspectives for this field.

Numerous biological activities such as grazer defense and intraspecific signaling have been described for diatom oxylipins, fatty acid derived secondary metabolites produced by some diatom species. As the function and importance of these compounds are still controversial, the production of a subclass of these molecules, nonvolatile oxylipins, was studied during an induced bloom of Skeletonema marinoi (Samo et Zingone) in a mesocosm setup. Reproductive parameters of one of the main grazers, Calanus finmarchicus, were also examined during the bloom. Oxylipins detected during the bloom were the same as those previously described for S. marinoi and were detected predominantly in the mesocosm inoculated with this diatom. Reproductive success of C. finmarchicus remained unaffected during the course of the bloom. This may have been due to a dilution effect by the availability of alternative suitable prey or to the limited exposure of the copepods to the oxylipins generated during the short bloom. Follow up laboratory studies showed that oxylipin composition changed both when the S. marinoi clone used for inoculation was grown in the laboratory and in comparison to the well-studied Adriatic clone of S. marina These results highlight the necessity of quantitatively measuring oxylipin concentrations during diatom blooms at sea to be able to correctly evaluate their ecological significance. (C) 2013 Elsevier B.V. All rights reserved.

Effects of the toxic Gulf of Mexico dinoflagellate Karenia brevis on its copepod grazers are equivocal, in that this dinoflagellate is ingested by various copepods, but may be toxic to, and/or nutritionally inadequate for grazers. Recent studies have shown that when the Mediterranean copepod Calanus helgolandicus fed on K. brevis, egg production rates and egg viability declined steadily over time, reaching almost zero levels after 5 days of feeding ( Turner et al., 2012 ). The K. brevis strain used in that study was chemically analyzed and shown not to contain any brevetoxins, but may have contained other harmful metabolites. Here we present gene expression analyses by reverse transcription-quantitative PCR (RT-qPCR) in C. helgolandicus after different exposure times (3, 5 and 8 days) to the same non-brevetoxin-containing K. brevis strain examined previously. The analyzed genes have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) and apoptosis regulation in other organisms. After 3 days of feeding on K. brevis, gene expression levels in copepods did not show significant changes. However, after 5 days of exposure, expression of stress-response genes was significantly reduced. After prolonged exposure for 8 days, expression of genes for 5 of 6 aldehyde dehydrogenases was strongly reduced, but expression of other genes was not coherently reduced, or increased. Microscopic observations revealed that after only a single day of feeding on K. brevis, freshly-spawned eggs were morphologically similar to those produced by copepods feeding on control diets, but after 3 days of feeding on K. brevis, eggs exhibited altered membrane cell morphology and apoptotic features such as granulation and degeneration of the cytoplasm matrix, compared to the unaltered eggs produced by copepods feeding on control diets. The changes in gene expression levels in copepods feeding on K. brevis were similar to those noted previously for copepods feeding upon diatoms that contained oxylipins, which interfered with copepod reproductive success. Changes in gene expression in copepods feeding upon toxic algal diets may offer important early warning signals of copepod stress responses to these diets.

The influence of six monoalgal diets was tested on the reproductive success of the copepod Acartia tonsa over a 15-day period in order to define the most favorable diet for the optimization of this copepod species to be used in aquaculture and in ecotoxicology applications. The cryptophytes Rhinomonas reticulata and Rhodomonas baltica induced highest egg production rates (mean = 24.4 eggs female− 1 day− 1and 21.9 eggs female− 1 day− 1) and hatching success (mean = 76% and 86.1%) over the 15-day period, respectively. Lowest egg production rates were recorded with both diatoms Phaeodactylum tricornutum (mean = 8.8 eggs female− 1 day− 1) and Skeletonema marinoi (mean = 8.4 eggs female− 1 day− 1). These two diatoms also had detrimental effects on hatching success (mean = 44.1% and 46.5%, respectively) and adult survival. No adults survived for longer than 13 days with a diet of P. tricornutum. Moreover, nauplii produced by females that had fed on diatoms for > 10 d, were positively stained for the apoptotic fluorescent marker TUNEL, indicating imminent death. The prasinophyta Tetraselmis suecica induced low fecundity (mean = 13.4 eggs female− 1 day− 1) and hatching success (mean = 62%), and after 15 days only 37.5% of the adults survived. Isochrysis galbana induced low egg production per female (

Diabetes is a chronic degenerative metabolic disease with high morbidity and mortality rates caused by its complications. In recent years, there has been a growing interest in looking for new bioactive compounds to treat this disease, including metabolites of marine origin. Several aquatic organisms have been screened to evaluate their possible anti-diabetes activities, such as bacteria, microalgae, macroalgae, seagrasses, sponges, corals, sea anemones, fish, salmon skin, a shark fusion protein as well as fish and shellfish wastes. Both in vitro and in vivo screenings have been used to test anti-hyperglycemic and anti-diabetic activities of marine organisms. This review summarizes recent discoveries in anti-diabetes properties of several marine organisms as well as marine wastes, existing patents and possible future research directions in this field.

Published version. Source at https://doi.org/10.3389/fmars.2016.00068 Marine microalgae are considered a potentially new and valuable source of biologically active molecules for applications in the food industry as well as in the pharmaceutical, nutraceutical, and cosmetic sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. In this study, 21 diatoms, 7 dinoflagellates, and 4 flagellate species were grown in three different culturing conditions and the corresponding extracts were tested for possible antioxidant, anti-inflammatory, anticancer, anti-diabetes, antibacterial, and anti-biofilm activities. In addition, for three diatoms we also tested two different clones to disclose diversity in clone bioactivity. Six diatom species displayed specific anti-inflammatory, anticancer (blocking human melanoma cell proliferation), and anti-biofilm (against the bacteria Staphylococcus epidermidis) activities whereas, none of the other microalgae were bioactive against the conditions tested for. Furthermore, none of the 6 diatom species tested were toxic on normal human cells. Culturing conditions (i.e., nutrient starvation conditions) greatly influenced bioactivity of the majority of the clones/species tested. This study denotes the potential of diatoms as sources of promising bioactives for the treatment of human pathologies.

There may be biogeographic aspects of interactions between toxic phytoplankton and their zooplankton grazers. Grazers that naturally co-occur with a given species of toxic phytoplankton may have evolved mechanisms to counteract adverse effects of phytoplankton toxins. Conversely, grazers that have no co-evolutionary experience with a toxic phytoplankter may be more susceptible to its effects. This may also be true for toxic metabolites other than neurotoxins that adversely affect copepod reproduction and development such as the well-described diatom oxylipins. In order to test this hypothesis, we investigated interactions in terms of feeding, reproductive success and survival of a copepod feeding on a potentially toxic dinoflagellate species with which the copepod does not naturally co-occur: the red tide dinoflagellate Karenia brevis from the Gulf of Mexico and the copepod Calanus helgolandicus from the Mediterranean. We additionally compared interactions of these two allopatric plankters with various co-occurring sympatric ones, including the copepod Temora stylifera and a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense, both from the Mediterranean, and as a control, the non-toxic dinoflagellate Prorocentrum minimum. Both Mediterranean copepods fed, reproduced and survived on diets of K. brevis from the Gulf of Mexico, but neither copepod did as well on this diet as on diets of A. tamarense from the Mediterranean or the control diet of P. minimum. Of the two copepods, T. stylifera fared worse on a diet of K. brevis than did C. helgolandicus. Egg hatching success for both copepods feeding on K. brevis was sharply diminished over the seven days of the experiments. This was surprising, since we subsequently discovered that our culture of K. brevis contained no brevetoxins. However, our culture appeared to contain another as yet unidentified compound. Similarly, we have previously demonstrated that our culture of A. tamarense, which contained no saxitoxins or aldehydes, did contain another unidentified compound that reduced egg hatching success by inhibiting sperm from fertilizing eggs. Thus, although diets of the dinoflagellate from the Gulf of Mexico (K. brevis) caused adverse effects on both of the Mediterranean copepods, T. stylifera was also adversely affected by the Mediterranean dinoflagellate (A. tamarense). The copepod T. stylifera which co-occurs in the Gulf of Mexico with the dinoflagellate K. brevis was far more adversely affected by feeding on a sole diet of this dinoflagellate than was the Mediterranean copepod (C. helgolandicus) which never naturally co-occurs with this dinoflagellate. Thus, any putative biogeographic effects of copepods feeding on allopatric versus sympatric dinoflagellates did not clearly emerge from our data. However, since both species of dinoflagellates appear to contain additional undescribed chemicals other than the neurotoxins for which they are best known, and these compounds are potentially related to the adverse effects we recorded, it appears that understanding of the biogeography must await further defining the biochemistry of some harmful algae:grazer interactions.

In this study, we applied different fluorescent techniques to analyse the induction of apoptosis in the copepod Acartia tonsa nauplii exposed to low concentrations of nickel chloride (NiCl2). Newly hatched and later naupliar stages were exposed to increasing concentrations of NiCl2 (0.016, 0.025 and 0.063 mg·L−1) for 7 days and then stained with annexin V–FITC, a vital fluorescent probe commonly used to visualise apoptotic cells in live samples. Nauplii were also stained with TUNEL, a non-vital fluorescent probe used to detect apoptosis in fixed copepods. Moreover, we used for the first time, two-photon fluorescence (2PF) microscopy and higher harmonic generation microscopy (second SHG and third THG harmonic generation) to study apoptosis induction in A. tonsa nauplii without the use of fluorescent probes. 2PF and THG intensity increased in samples exposed to higher Ni concentrations, with respect to the control, whereas SHG signals were similar in all treated samples and visualised muscles. Future perspe...

Diatom oxylipins have been observed to deleteriously impact copepod reproductive success. However, field studies have revealed very variable and case-dependent results. Therefore, the plasticity of diatom oxylipin metabolism was studied among four clones of the marine diatom Skeletonema marinoi Sarno et Zingone. Diatom oxylipin metabolism was studied by two lipoxygenase (LOX) activity assays carried out at different pH values and by oxylipin quantification. The four clones showed no major metabolic differences in terms of protein content or growth rate. However, two of the clones produced significantly higher levels of oxylipins than the other two. LOX activity measurements also indicated clonal variability in fatty acid oxidative metabolism. The presence of clone-specific differences in oxylipin metabolism may play a role in shaping diatom population dynamics by conferring selective advantages to certain clones.

In this study we develop gene expression tools in Calanus helgolandicus to study the effects of toxic diatom diets on copepod fitness and survival. We demonstrate that when adult females are fed on the control dinoflagellate Prorocentrum minimum and the flagellate Rhodomonas baltica, which are not known to produce toxic oxylipins, there are no significant changes in alpha- and beta-tubulin (microtubule subunits) gene expression levels. By contrast, the oxylipin-producing diatom Skeletonema marinoi influences tubulin expression levels which were markedly down-regulated. We scored a panel of putative reference genes (ACT, EFA, GAPDH, 18S, S7, S20, ATPs, UBI and IST) and found that two (S20 and S7) were highly stable in the tested conditions and can be used for further experiments. To normalize reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR) data we also used the third best reference gene, GAPDH, considering the possibility that the two ribosomal proteins could be co-regulated. This pilot study will pave the way for further investigations on which genes are affected by diatom diets and clarify when and if a stress response or a detoxification mechanism becomes visible in C. helgolandicus. Moreover, the RT-qPCR analysis presented here may also be useful to study the effects of other diets and/or environmental factors such as salinity, temperature, pollution and other toxic compounds, on gene expression levels in this copepod species.

A pilot re-circulating system was used for the cultivation of two Mediterranean calanoid copepods: Temora stylifera and Centropages typicus. The system automatically concentrated the naupliar and copepodite stages. Temora stylifera was fed the flagellate Rhodomonas baltica or Prorocentrum minimum, whereas C. typicus was fed with a mixture of R. baltica or P. minimum and Tetraselmis suecica. Both copepods also received Isochrysis galbana. After 21 days, the T. stylifera population increased 26-fold, reaching a density of 38 000 individuals, mostly represented by nauplii (88%). The maximum density recorded was 380 ind. L−1, with a production of 370 nauplii L−1. On average, the egg hatching success for this copepod during the rearing period was 54%, with the highest viability in April and May (>75%). The C. typicus population increased more than 10-fold after 7 weeks of rearing, reaching a density of 123 000 individuals, mainly represented by nauplii (>90%). The highest naupliar production was 100 ind. L−1, with a mean egg hatching success of 68%. This system may be useful to produce nauplii and copepodite stages to be used as live, alternative or complementary food for fish larvae or to provide a ready source of organisms for physiological and bioassay studies.

Development and sex differentiation in the copepod Temora stylifera was studied in the presence of maternal and larval diets of the diatoms Thalassiosira rotula and Skeletonema marinoi, either provided alone or supplemented with the control dinoflagellate Prorocentrum minimum. Both diatoms had deleterious effects on growth compared to the control when used as pure diets, inducing very low or even zero survival from hatching to adulthood. This effect was deleted when the diet was supplemented with a good food (P. minimum) only in the case of T. rotula. By contrast, with a maternal or larval diet of S. marinoi, nauplii did not pass metamorphosis even when this alga was mixed with P. minimum. Arrested development was not due to lack of feeding since early and late nauplii (NII and NV) ingested all three algae at similar rates when used as single diets, and did not show any preference when the algae were offered as mixtures. Since mortality rates with a mixed diet of S. marinoi + P. minimum were similar or even higher than those obtained with a single diet of S. marinoi, we suggest that this diatom is more toxic than nutritionally deficient for T. stlyfera development. No males were produced in cohorts reared on pure diatom diets or with a mixture of S. marinoi + P. minimum, and intermediate male:female sex ratios were obtained with the mixed T. rotula + P. minimum diet. Possibly some diatoms produce compounds such as oxylipins or new molecules that alter sex differentiation in T. stylifera.

The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans-decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19-docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid (gamma-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 microM with heptadienal, 16.13 microM with octadienal, 11.47 microM with octatrienal and 5.26 microM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 microM for EPA and 181 microM for stearidonic acid). Sub-lethal concentrations of decadienal (1.32-5.26 microM) delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

Egg production rates and/or hatching success in the copepods Acartia clausi, Calanus helgolandicus and Temora longicornis were negatively affected by a late spring (May-June 2003) phytoplankton bloom in the North Adriatic Sea, dominated mainly by the large diatom Cerataulina pelagica. Highest total concentrations of 3.3- 104 cells-ml(-1) were located in the vicinity of the Po River, which also corresponded to the area where the highest numbers of phaeophorbides were measured (0.779, 0.528 and 0.419 mu g.l(-1), respectively, compared to an average of the remaining stations of 0.183 +/- 0.049 SD), suggesting some grazing on the bloom. Phytoplankton biomass in terms of carbon was dominated by diatoms, representing on average 42% of total phytoplankton carbon and more than 80% at several stations. Cerataulina pelagica, Cyclotella spp., Chaetoceros spp. and small unidentified centric diatoms dominated the diatom community numerically but C. pelagica was by far the dominant diatom in terms of carbon due to its large cell size. This species represented more than 60% of the diatom biomass at nine of the 14 stations sampled, and was absent only at one station, which was the most offshore station sampled during the cruise. Although polyunsaturated aldehydes (PUAs) were not detected, other oxylipins which are hydroxy and keto derivatives of eicosapentaenoic and docosahexaenoic acids that affect copepod reproduction were found in these samples. Hence, we can attribute the negative impact of diatoms not only to PUAs, as previously believed, but also to these compounds. This is the first direct evidence of the presence of oxylipins other than PUAs in marine blooms dominated by diatoms.

Several marine diatoms produce polyunsaturated aldehydes (PUAs) that have been shown to be toxic to a wide variety of model organisms, from bacteria to invertebrates. However, very little information is available on their effect on phytoplankton. Here, we expand previous studies to six species of marine phytoplankton, belonging to different taxonomic groups that are well represented in marine plankton. The effect of three PUAs, 2 E ,4 E -decadienal, 2 E ,4 E -octadienal and 2 E ,4 E -heptadienal, was assessed on growth, cell membrane permeability, flow cytometric properties and morphology. A concentration-dependent reduction in the growth rate was observed for all cultures exposed to PUAs with longer-chained aldehydes having stronger effects on growth than shorter-chained aldehydes. Clear differences were observed among the different species. The prymnesiophyte Isochrysis galbana was the most sensitive species to PUA exposure with a lower threshold for an observed effect triggered by mean concentrations of 0.10 μmol L −1 for 2 E ,4 E -decadienal, 1.86 μmol L −1 for 2 E ,4 E -octadienal and 3.06 μmol L −1 for 2 E ,4 E -heptadienal, and a 50% growth inhibition (EC 50 ) with respect to the control at 0.99, 2.25 and 5.90 μmol L −1 for the three PUAs, respectively. Alternatively, the chlorophyte Tetraselmis suecica and the diatom Skeletonema marinoi (formerly S. costatum ) were the most resistant species with 50% growth inhibition occurring at concentrations at least two to three times higher than I. galbana . In all species, the three PUAs caused changes in flow cytometric measures of cell size and cell granulosity and increased membrane permeability, assessed using the viability stain SYTOX Green. For example, after 48 h 51.6 ± 2.6% of I. galbana cells and 15.0 ± 1.8% of S. marinoi cells were not viable. Chromatin fragmentation was observed in the dinoflagellate Amphidinium carterae while clear DNA degradation was observed in the chlorophyte Dunaliella tertiolecta . Concentrations used are in a significant range for affecting growth and performance of phytoplankton living in close vicinity of PUA-producing algae. Thus, PUAs may act as allelochemicals by mediating interactions among planktonic organisms.

Background The dense phytoplankton blooms that characterize productive regions and seasons in the oceans are dominated, from high to low latitudes and from coast line to open ocean, by comparatively few, often cosmopolitan species of diatoms. These key dominant species may undergo dramatic changes due to global climate change. Results In order to identify molecular stress-indicators for the ubiquitous diatom species Skeletonema marinoi, we tested stress-related genes in different environmental conditions (i.e. nutrient starvation/depletion, CO2-enrichment and combined effects of these stressors) using RT-qPCR. The data show that these stressors impact algal growth rate, inducing early aging and profound changes in expression levels of the genes of interest. Conclusions Most analyzed genes (e.g. antioxidant-related and aldehyde dehydrogenases) were strongly down-regulated which may indicate a strategy to avoid unnecessary over-investment in their respective proteins. By contrast, key genes were activated (e.g. HSPs, GOX) which may allow the diatom species to better cope with adverse conditions. We propose the use of this panel of genes as early bio-indicators of environmental stress factors in a changing ocean. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1574-5) contains supplementary material, which is available to authorized users.

Diatoms are a hugely diverse microalgal class, which possesses unique biological features and complex metabolic pathways and may activate sophisticated mechanisms to respond to environmental changes. Abiotic stress factors may limit growth rate of diatoms, but may also trigger intracellular signaling pathways that cause cells to undergo programmed cell death (PCD). Here we investigate the gene expression of different target genes related to cell death, namely programmed cell death 4 (PDCD4), tumor susceptibility gene 101 (TSG101), developmental and cell death (DCD) domain, death specific protein (DSP) and metacaspase (MC), using RT-qPCR in the cosmopolitan coastal centric diatom species Skeletonema marinoi, which contributes significantly to phytoplankton blooms in temperate waters. To this end, we undertook a detailed study of the best reference genes to analyze gene expression in S. marinoi under different experimental conditions (i.e. in different growth phases or under silica starvation). Results showed that DSP gene expression had a clear and constant increase along the S. marinoi growth curve reaching its maximum during the senescent phase. On the contrary, PDCD4, DCD, TSG101 and MC did not show any significant variation. These findings indicate that the DSP gene is a possible PCD marker induced by aging in this diatom species. In contrast, levels of DSP transcripts induced by silica starvation were relatively low compared to those induced by cell aging suggesting differential activation and/or regulation of the PCD machinery in response to different stressful conditions. Our study also expands the list of reference genes available for the diatom S. marinoi for normalization of RT-qPCR data of cells cultivated under different growth phases or under silica starvation.

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes Dil and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed. (C) 2003 Wiley-Liss, Inc.

Ecologically-relevant marine diatoms produce a plethora of bioactive oxylipins deriving from fatty acid oxidation, including aldehydes, hydroxy-fatty acids, epoxy-hydroxy-fatty acids, and oxo-acids. These secondary metabolites have been related to the negative effect of diatoms on copepod reproduction, causing low hatching success and teratogenesis in the offspring during periods of intense diatom blooms. The common intermediates in the formation of oxylipins are fatty acid hydroperoxides. The quantitative measurement of these intermediates can fundamentally contribute to understanding the function and role of lipoxygenase metabolites in diatom-copepod interactions. Here, we describe the successful adaptation of the ferrous oxidation-xylenol orange 2 (FOX2) assay to diatom samples, which showed several advantages over other spectrophotometric and polarographic methods tested in the present work. Using this method we assessed fatty acid hydroperoxide levels in three diatom species: Skeletonema marinoi, Thalassiosira rotula, and Chaetoceros affinis, and discuss results in light of the literature data on their detrimental effects on copepod reproduction.

The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol, and four widely-used Silica Membrane kits, GenElute TM Mammalian Total RNA Miniprep Kit, RNAqueous Micro Kit, RNeasy Micro Kit and Aurum TM Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

Eggs and embryos of the sea urchin Paracentrotus lividus were used as a model to study the effect at the cellular level of potential anti-mitotic compounds extracted from the diatom Thalassiosira rotula. Eggs and embryos incubated in a water-soluble diatom extract, corresponding to 5 × 106 and 107 cells ml−1, were totally blocked (i.e. cell division was blocked) at the one-cell stage. At lower concentrations (2.5 and 1.25 × 106 cells ml−1), the first mitotic division was inhibited in 32 ± 26% and 25 ± 3.5% of the zygotes, respectively, demonstrating the dose-dependent effect of diatom extracts on sea urchin development. Immunofluorescence dyes, specific for DNA and α-tubulin subunits, were used to stain nuclei and microtubules in sea urchin embryos during various phases of development. Images with the confocal laser scanning microscope showed that tubulin was not organised in filaments at the sperm aster and cortex levels, and that the pronuclei were not fused in embryos incubated soon after fertilisation with water-soluble diatom extracts corresponding to 107 cells ml−1. At lower diatom-extract concentrations (4 × 106 cells ml −1), fusion of the pronuclei occurred but the mitotic spindle was not formed. Microtubules were clearly de-polymerised and the chromatin appeared globular and compacted at the centre of the cell. A similar structure was observed for sea urchin embryos incubated with 0.1 mM colchicine, a potent anti-mitotic compound. When sea urchin embryos were incubated in water-soluble diatom extracts at different times prior to the first mitotic division, microtubules appeared de-polymerised at each step, from pronuclear fusion to telophase, and cell division was blocked. At the histological level, embryos incubated with 4 × 106 cells ml−1 diatom extract showed nuclear fragmentation without cytokinesis. The possible use of sea urchin embryos as a bioassay to test for other unknown compounds with cytotoxic activity in phytoplankton species is discussed.