Transcriptome Sequencing of the Diatom Asterionellopsis thurstonii and In Silico Identification of Enzymes Potentially Involved in the Synthesis of Bioactive Molecules.

Microalgae produce a plethora of primary and secondary metabolites with possible applications in several market sectors, including cosmetics, human nutrition, aquaculture, biodiesel production and treatment/prevention of human diseases. Diatoms, in particular, are the most diversified microalgal group, many species of which are known to have anti-cancer, anti-oxidant, anti-diabetes, anti-inflammatory and immunomodulatory properties. Compounds responsible for these activities are often still unknown. The aim of this study was to de novo sequence the full transcriptome of two strains of the diatom Asterionellopsis thurstonii, sampled from two different locations and cultured in both control and phosphate starvation conditions. We used an RNA-sequencing approach to in silico identify transcripts potentially involved in the synthesis/degradation of compounds with anti-cancer and immunomodulatory properties. We identified transcript coding for L-asparaginase I, polyketide cyclase/dehydrase, bifunctional polyketide phosphatase/kinase, 1-deoxy-D-xylulose-5-phosphate synthase (fragment), inositol polyphosphate 5-phosphatase INPP5B/F, catechol O-Methyltransferase, digalactosyldiacylglycerol synthase (DGD1), 1,2-diacylglycerol-3-beta-galactosyltransferase and glycerolphosphodiester phosphodiesterase. Differential expression analysis also allowed to identify in which culturing condition these enzymes are more expressed. Overall, these data give new insights on the annotation of diatom genes, enzymatic pathways involved in the generation of bioactive molecules and possible exploitation of Asterionellopsis thurstonii.

Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation.

Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ∼15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60-100%) and HMs removal (21-100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities. Most promising yields (∼100%) were obtained towards naphthalene, pyrene and lead. We propose these novel bacterial strains and related genetic repertoire to be further exploited for effective bioremediation of marine environments contaminated with both PAHs and HMs.

De Novo Transcriptome of the Flagellate Isochrysis galbana Identifies Genes Involved in the Metabolism of Antiproliferative Metabolites.

Haptophytes are important primary producers in the oceans, and among the phylum Haptophyta, the flagellate Isochrysis galbana has been found to be rich in high-value compounds, such as lipids, carotenoids and highly branched polysaccharides. In the present work, I. galbana was cultured and collected at both stationary and exponential growth phases. A transcriptomic approach was used to analyze the possible activation of metabolic pathways responsible for bioactive compound synthesis at the gene level. Differential expression analysis of samples collected at the exponential versus stationary growth phase allowed the identification of genes involved in the glycerophospholipid metabolic process, the sterol biosynthetic process, ADP-ribose diphosphatase activity and others. I. galbana raw extracts and fractions were tested on specific human cancer cells for possible antiproliferative activity. The most active fractions, without affecting normal cells, were fractions enriched in nucleosides (fraction B) and triglycerides (fraction E) for algae collected in the exponential growth phase and fraction E for stationary phase samples. Overall, transcriptomic and bioactivity data confirmed the activation of metabolic pathways involved in the synthesis of bioactive compounds giving new insights on possible Isochrysis applications in the anticancer sector.

First identification and characterization of detoxifying plastic-degrading DBP hydrolases in the marine diatom Cylindrotheca closterium.

Diatoms are photosynthetic organisms with potential biotechnological applications in the bioremediation sector, having shown the capacity to reduce environmental concentrations of different pollutants. The diatom Cylindrotheca closterium is known to degrade di-n-butyl phthalate (DBP), one of the most abundant phthalate esters in aquatic environments and a known endocrine-disrupting chemical. In this study, we present for the first time the in silico identification of two putative DBP hydrolases (provisionally called DBPH1 and DBPH2) in the transcriptome of C. closterium. We modeled the structure of both DBPH1-2 and their proposed interactions with the substrate to gain insights into their mechanism of action. Finally, we analyzed the expression levels of the two putative hydrolases upon exposure of C. closterium to different concentrations of DBP (5 and 10 mg/l) for 24 and 48 h. The data showed a DBP concentration-dependent increase in expression levels of both dbph1 and 2 genes, further highlighting their potential involvement in phthalates degradation. This is the first identification of phthalate-degrading enzymes in microalgae, providing new insights into the possible use of diatoms in bioremediation strategies targeting phthalates.

A Treasure of Bioactive Compounds from the Deep Sea.

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.

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction.

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.

Metagenome-assembled genome (MAG) of Oceancaulis alexandrii NP7 isolated from Mediterranean Sea polluted marine sediments and its bioremediation potential.

Oceanicaulis alexandrii strain NP7 is a marine bacterium which belongs to the Hyphomonadaceae family and was isolated from sediments highly contaminated with metals and polycyclic aromatic hydrocarbons released for decades by industrial activities in the Gulf of Naples (Mediterranean Sea). Here, we report the partial genome sequence and annotation of O. alexandrii strain NP7 that contains a chromosome of 2,954,327 bp and encodes for 2914 predicted coding sequences (CDSs) and 44 RNA-encoding genes. Although the presence of some CDSs for genes involved in hydrocarbon degradation processes (e.g., alkB) have already been described in the literature associated with the Oceanicaulis, this is the first time that more than 100 genes involved in metal detoxification processes and hydrocarbon degradation are reported belonging to this genus. The presence of a heterogeneous set of genes involved in stress response, hydrocarbon degradation, heavy metal resistance, and detoxification suggests a possible role for O. alexandrii NP7 in the bioremediation of these highly contaminated marine sediments.

Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols.

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.

Bacteria, Fungi and Microalgae for the Bioremediation of Marine Sediments Contaminated by Petroleum Hydrocarbons in the Omics Era.

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.

A Metataxonomic Approach Reveals Diversified Bacterial Communities in Antarctic Sponges.

Marine sponges commonly host a repertoire of bacterial-associated organisms, which significantly contribute to their health and survival by producing several anti-predatory molecules. Many of these compounds are produced by sponge-associated bacteria and represent an incredible source of novel bioactive metabolites with biotechnological relevance. Although most investigations are focused on tropical and temperate species, to date, few studies have described the composition of microbiota hosted by Antarctic sponges and the secondary metabolites that they produce. The investigation was conducted on four sponges collected from two different sites in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018. Collected species were characterized as Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemigellius pilosus and Microxina sarai by morphological analysis of spicules and amplification of four molecular markers. Metataxonomic analysis of these four Antarctic sponges revealed a considerable abundance of Amplicon Sequence Variants (ASVs) belonging to the phyla Proteobacteria, Bacteroidetes, Actinobacteria and Verrucomicrobia. In particular, M. (Oxymycale) acerata, displayed several genera of great interest, such as Endozoicomonas, Rubritalea, Ulvibacter, Fulvivirga and Colwellia. On the other hand, the sponges H. pilosus and H. (Rhizoniera) dancoi hosted bacteria belonging to the genera Pseudhongella, Roseobacter and Bdellovibrio, whereas M. sarai was the sole species showing some strains affiliated to the genus Polaribacter. Considering that most of the bacteria identified in the present study are known to produce valuable secondary metabolites, the four Antarctic sponges could be proposed as potential tools for the discovery of novel pharmacologically active compounds.

Unlocking the Health Potential of Microalgae as Sustainable Sources of Bioactive Compounds.

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.

Highly Contaminated Marine Sediments Can Host Rare Bacterial Taxa Potentially Useful for Bioremediation.

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 <1%) by As and aliphatic hydrocarbons concentrations and by sediment redox potential. These results suggest a differential response of bacterial taxa to environmental features and chemical contamination and those different bacterial groups may be inhibited or promoted by different contaminants. This hypothesis was confirmed by culturing and isolating 80 bacterial strains using media highly enriched in PAHs, only nine of which were contextually resistant to high HM concentrations. Such resistant isolates represented novel Gammaproteobacteria strains affiliated to 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.

Genome Sequence of an Alkaliphilus Species Isolated from Historically Contaminated Sediments of the Gulf of Naples (Mediterranean Sea).

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.

First De Novo Transcriptome of the Copepod Rhincalanus gigas from Antarctic Waters.

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.

RNA-Seq and differential gene expression analysis in Temora stylifera copepod females with contrasting non-feeding nauplii survival rates: an environmental transcriptomics study.

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.

Chemical Defense in Marine Organisms.

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Degradation of Hydrocarbons and Heavy Metal Reduction by Marine Bacteria in Highly Contaminated Sediments.

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.

De Novo Transcriptome Assembly and Gene Expression Profiling of the Copepod Calanus helgolandicus Feeding on the PUA-Producing Diatom Skeletonema marinoi.

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 µM, 15 µM, and 20 µM. 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.

Diatom-Derived Polyunsaturated Aldehydes Activate Similar Cell Death Genes in Two Different Systems: Sea Urchin Embryos and Human Cells.

Programmed cell death, such as apoptosis and autophagy, are key processes that are activated early on during development, leading to remodelling in embryos and homeostasis in adult organisms. Genomic conservation of death factors has been largely investigated in the animal and plant kingdoms. In this study, we analysed, for the first time, the expression profile of 11 genes involved in apoptosis (extrinsic and intrinsic pathways) and autophagy in sea urchin Paracentrotus lividus embryos exposed to antiproliferative polyunsaturated aldehydes (PUAs), and we compared these results with those obtained on the human cell line A549 treated with the same molecules. We found that sea urchins and human cells activated, at the gene level, a similar cell death response to these compounds. Despite the evolutionary distance between sea urchins and humans, we observed that the activation of apoptotic and autophagic genes in response to cytotoxic compounds is a conserved process. These results give first insight on death mechanisms of P. lividus death mechanisms, also providing additional information for the use of this marine organism as a useful in vitro model for the study of cell death signalling pathways activated in response to chemical compounds.

De novo Transcriptome of the Non-saxitoxin Producing Alexandrium tamutum Reveals New Insights on Harmful Dinoflagellates.

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 "polyketide synthase"). 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.