Antioxidant micropeptins from a Microcoleus autumnalis-dominated benthic cyanobacterial mat from Western Ireland.

Exploring the chemical diversity present in cyanobacterial mats increasingly frequent in fresh and marine waters is imperative for both evaluating risks associated with these diverse biofilms and their potential for biodiscovery. During a project aimed at the study of the (eco)toxicity of benthic cyanobacteria blooming in some lakes of the West of Ireland, three previously undescribed ahp-cyclodepsipeptides micropeptin LOF941 (1), micropeptin LOF925 (2) and micropeptin LOF953 (3) were isolated from the Microcoleus autumnalis-dominated benthic cyanobacterial biofilm collected from the shore of Lough O'Flynn, Co. Roscommon, Ireland. Their structures remain consistent in their amino acid sequence with the presence of an unusual methionine, and differ by their exocyclic side chains. The planar structures of the previously undescribed micropeptins were elucidated by 1D and 2D NMR and HRESIMS analyses, and their 3D configurations assessed by ROESY NMR and Marfey's analyses. The three isolated compounds showed no cytotoxic effects and all three compounds were shown to exhibit antioxidant properties, with 1 showing the highest bioactivity. Additionally, several micropeptin analogues are proposed from the methanolic fraction of the biofilm extract by UHPLC-HRESIMS/MS analysis and molecular networking. Notably, the known cyanotoxins anatoxin-a and dihydroanatoxin-a were annotated in the molecular network therefore raising issues about the toxicity of this cyanobacterial mat.

Highly Concentrated Linear Guanidine Amides from the Marine Sipunculid Phascolosoma granulatum.

The chemical diversity of annelids, particularly those belonging to the class Sipuncula, remains largely unexplored. However, as part of a Marine Biodiscovery program in Ireland, the peanut worm Phascolosoma granulatum emerged as a promising source of unique metabolites. The purification of the MeOH/CH2Cl2 extract of this species led to the isolation of six new linear guanidine amides, named phascolosomines A-F (1-6). NMR analysis allowed for the elucidation of their structures, all of which feature a terminal guanidine, central amide linkage, and a terminal isobutyl group. Notably, these guanidine amides were present in unusually high concentrations, comprising ∼3% of the dry mass of the organism. The primary concentration of the phascolosomines in the viscera is similar to that previously identified in linear amides from sipunculid worms and marine fireworms. The compounds from sipunculid worms have been hypothesized to be toxins, while those from fireworms are reported to be defensive irritants. However, screening of the newly isolated compounds for inhibitory bioactivity showed no significant inhibition in any of the assays conducted.

Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types.

Blooms of the dinoflagellate Ostreopsis cf. ovata are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PlTX). Unlike for PlTX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PlTX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and -d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PlTX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PlTX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms.

Additional Sarasinosides from the Marine Sponge Melophlus sarasinorum Collected from the Bismarck Sea.

In our continuing efforts to describe the biological and chemical diversity of sponges from Kimbe Bay, Papua New Guinea, the known 30-norlanostane saponin sarasinoside C1 (1) was identified along with six new analogues named sarasinosides C4, C5, C6, C7, C8, and C9 (2-7) from the sponge Melophlus sarasinorum. The structures of the new compounds were elucidated by analysis of 1D and 2D NMR and HRMS data, as well as comparison with literature data. All new compounds are characterized by the same tetraose moiety, ß-d-Xylp-(1→6)-ß-d-GlcNAcp-(1→2)-[ß-d-GalNAcp-(1→4)]-ß-d-Xylp, as described previously for sarasinoside C1, but differed in their aglycone moieties. When comparing NMR data of sarasinoside C8 with those of known analogues, a misassignment was identified in the configuration of the C-8/C-9 diol for the previously described sarasinoside R (8), and it has been corrected here using a combination of ROESY analysis and molecular modeling.

Production of the dinoflagellate Amphidoma languida in a large scale photobioreactor and structure elucidation of its main metabolite AZA-39.

Shellfish contamination with azaspiracids (AZA) is a major and recurrent problem for the Irish shellfish industry. Amphidoma languida, a small thecate dinoflagellate of the family Amphidomataceae, is widely distributed in Irish coastal waters and is one of the identified source species of azaspiracids. Irish and North Sea strains of Am. languida have been found to produce as major metabolites AZA-38 and -39 whose structures have only been provisionally elucidated by mass spectrometry and their toxic potential is currently unknown. In order to provide pure AZA-38 and -39 for subsequent structural and toxicological analyses, we present the first successful large-scale culture of Am. languida. A 180 L in house prototype bioreactor was used for culture growth and harvesting in semi-continuous mode for two months. Two different runs of the photobiorector with different light and pH setting showed the highest toxin yield at higher light intensity and slightly higher pH. AZA-38 and -39 cell quota were measured throughout the complete growth cycle with AZA-39 cell quota increasing in proportion to AZA-38 at late stationary to senescence phase. Over two experiments a total of 700 L of culture was harvested yielding 0.45 mg of pure AZA-39. The structure of AZA-39 was elucidated through NMR data analyses, which led to a revision of the structure proposed previously by mass spectrometry. While the spirotetrahydrofuran/tetrahydrofuran of rings A and B has been confirmed by NMR for AZA-39, a methyl is still present in position C-14 and the carboxylic acid chain is different from the structure proposed initially.

Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen.

AIM: A promising approach for the development of next-generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens. METHODS AND RESULTS: Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and/or Listeria monocytogenes without causing growth inhibition. Gas chromatograph/mass spectroscopy analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, which are required for MRSA and MSSA biofilm formation, respectively. CONCLUSIONS: This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case, repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.

Differences in carbonate chemistry up-regulation of long-lived reef-building corals.

With climate projections questioning the future survival of stony corals and their dominance as tropical reef builders, it is critical to understand the adaptive capacity of corals to ongoing climate change. Biological mediation of the carbonate chemistry of the coral calcifying fluid is a fundamental component for assessing the response of corals to global threats. The Tara Pacific expedition (2016-2018) provided an opportunity to investigate calcification patterns in extant corals throughout the Pacific Ocean. Cores from colonies of the massive Porites and Diploastrea genera were collected from different environments to assess calcification parameters of long-lived reef-building corals. At the basin scale of the Pacific Ocean, we show that both genera systematically up-regulate their calcifying fluid pH and dissolved inorganic carbon to achieve efficient skeletal precipitation. However, while Porites corals increase the aragonite saturation state of the calcifying fluid (Ωcf) at higher temperatures to enhance their calcification capacity, Diploastrea show a steady homeostatic Ωcf across the Pacific temperature gradient. Thus, the extent to which Diploastrea responds to ocean warming and/or acidification is unclear, and it deserves further attention whether this is beneficial or detrimental to future survival of this coral genus.

Telomere DNA length regulation is influenced by seasonal temperature differences in short-lived but not in long-lived reef-building corals.

Telomeres are environment-sensitive regulators of health and aging. Here,we present telomere DNA length analysis of two reef-building coral genera revealing that the long- and short-term water thermal regime is a key driver of between-colony variation across the Pacific Ocean. Notably, there are differences between the two studied genera. The telomere DNA lengths of the short-lived, more stress-sensitive Pocillopora spp. colonies were largely determined by seasonal temperature variation, whereas those of the long-lived, more stress-resistant Porites spp. colonies were insensitive to seasonal patterns, but rather influenced by past thermal anomalies. These results reveal marked differences in telomere DNA length regulation between two evolutionary distant coral genera exhibiting specific life-history traits. We propose that environmentally regulated mechanisms of telomere maintenance are linked to organismal performances, a matter of paramount importance considering the effects of climate change on health.

Diversity of the Pacific Ocean coral reef microbiome.

Coral reefs are among the most diverse ecosystems on Earth. They support high biodiversity of multicellular organisms that strongly rely on associated microorganisms for health and nutrition. However, the extent of the coral reef microbiome diversity and its distribution at the oceanic basin-scale remains to be explored. Here, we systematically sampled 3 coral morphotypes, 2 fish species, and planktonic communities in 99 reefs from 32 islands across the Pacific Ocean, to assess reef microbiome composition and biogeography. We show a very large richness of reef microorganisms compared to other environments, which extrapolated to all fishes and corals of the Pacific, approximates the current estimated total prokaryotic diversity for the entire Earth. Microbial communities vary among and within the 3 animal biomes (coral, fish, plankton), and geographically. For corals, the cross-ocean patterns of diversity are different from those known for other multicellular organisms. Within each coral morphotype, community composition is always determined by geographic distance first, both at the island and across ocean scale, and then by environment. Our unprecedented sampling effort of coral reef microbiomes, as part of the Tara Pacific expedition, provides new insight into the global microbial diversity, the factors driving their distribution, and the biocomplexity of reef ecosystems.

Ecology of Endozoicomonadaceae in three coral genera across the Pacific Ocean.

Health and resilience of the coral holobiont depend on diverse bacterial communities often dominated by key marine symbionts of the Endozoicomonadaceae family. The factors controlling their distribution and their functional diversity remain, however, poorly known. Here, we study the ecology of Endozoicomonadaceae at an ocean basin-scale by sampling specimens from three coral genera (Pocillopora, Porites, Millepora) on 99 reefs from 32 islands across the Pacific Ocean. The analysis of 2447 metabarcoding and 270 metagenomic samples reveals that each coral genus harbored a distinct new species of Endozoicomonadaceae. These species are composed of nine lineages that have distinct biogeographic patterns. The most common one, found in Pocillopora, appears to be a globally distributed symbiont with distinct metabolic capabilities, including the synthesis of amino acids and vitamins not produced by the host. The other lineages are structured partly by the host genetic lineage in Pocillopora and mainly by the geographic location in Porites. Millepora is more rarely associated to Endozoicomonadaceae. Our results show that different coral genera exhibit distinct strategies of host-Endozoicomonadaceae associations that are defined at the bacteria lineage level.

Open science resources from the Tara Pacific expedition across coral reef and surface ocean ecosystems.

The Tara Pacific expedition (2016-2018) sampled coral ecosystems around 32 islands in the Pacific Ocean and the ocean surface waters at 249 locations, resulting in the collection of nearly 58 000 samples. The expedition was designed to systematically study warm-water coral reefs and included the collection of corals, fish, plankton, and seawater samples for advanced biogeochemical, molecular, and imaging analysis. Here we provide a complete description of the sampling methodology, and we explain how to explore and access the different datasets generated by the expedition. Environmental context data were obtained from taxonomic registries, gazetteers, almanacs, climatologies, operational biogeochemical models, and satellite observations. The quality of the different environmental measures has been validated not only by various quality control steps, but also through a global analysis allowing the comparison with known environmental large-scale structures. Such publicly released datasets open the perspective to address a wide range of scientific questions.

Host transcriptomic plasticity and photosymbiotic fidelity underpin Pocillopora acclimatization across thermal regimes in the Pacific Ocean.

Heat waves are causing declines in coral reefs globally. Coral thermal responses depend on multiple, interacting drivers, such as past thermal exposure, endosymbiont community composition, and host genotype. This makes the understanding of their relative roles in adaptive and/or plastic responses crucial for anticipating impacts of future warming. Here, we extracted DNA and RNA from 102 Pocillopora colonies collected from 32 sites on 11 islands across the Pacific Ocean to characterize host-photosymbiont fidelity and to investigate patterns of gene expression across a historical thermal gradient. We report high host-photosymbiont fidelity and show that coral and microalgal gene expression respond to different drivers. Differences in photosymbiotic association had only weak impacts on host gene expression, which was more strongly correlated with the historical thermal environment, whereas, photosymbiont gene expression was largely determined by microalgal lineage. Overall, our results reveal a three-tiered strategy of thermal acclimatization in Pocillopora underpinned by host-photosymbiont specificity, host transcriptomic plasticity, and differential photosymbiotic association under extreme warming.

Integrative omics framework for characterization of coral reef ecosystems from the Tara Pacific expedition.

Coral reef science is a fast-growing field propelled by the need to better understand coral health and resilience to devise strategies to slow reef loss resulting from environmental stresses. Key to coral resilience are the symbiotic interactions established within a complex holobiont, i.e. the multipartite assemblages comprising the coral host organism, endosymbiotic dinoflagellates, bacteria, archaea, fungi, and viruses. Tara Pacific is an ambitious project built upon the experience of previous Tara Oceans expeditions, and leveraging state-of-the-art sequencing technologies and analyses to dissect the biodiversity and biocomplexity of the coral holobiont screened across most archipelagos spread throughout the entire Pacific Ocean. Here we detail the Tara Pacific workflow for multi-omics data generation, from sample handling to nucleotide sequence data generation and deposition. This unique multidimensional framework also includes a large amount of concomitant metadata collected side-by-side that provide new assessments of coral reef biodiversity including micro-biodiversity and shape future investigations of coral reef dynamics and their fate in the Anthropocene.

Endogenous viral elements reveal associations between a non-retroviral RNA virus and symbiotic dinoflagellate genomes.

Endogenous viral elements (EVEs) offer insight into the evolutionary histories and hosts of contemporary viruses. This study leveraged DNA metagenomics and genomics to detect and infer the host of a non-retroviral dinoflagellate-infecting +ssRNA virus (dinoRNAV) common in coral reefs. As part of the Tara Pacific Expedition, this study surveyed 269 newly sequenced cnidarians and their resident symbiotic dinoflagellates (Symbiodiniaceae), associated metabarcodes, and publicly available metagenomes, revealing 178 dinoRNAV EVEs, predominantly among hydrocoral-dinoflagellate metagenomes. Putative associations between Symbiodiniaceae and dinoRNAV EVEs were corroborated by the characterization of dinoRNAV-like sequences in 17 of 18 scaffold-scale and one chromosome-scale dinoflagellate genome assembly, flanked by characteristically cellular sequences and in proximity to retroelements, suggesting potential mechanisms of integration. EVEs were not detected in dinoflagellate-free (aposymbiotic) cnidarian genome assemblies, including stony corals, hydrocorals, jellyfish, or seawater. The pervasive nature of dinoRNAV EVEs within dinoflagellate genomes (especially Symbiodinium), as well as their inconsistent within-genome distribution and fragmented nature, suggest ancestral or recurrent integration of this virus with variable conservation. Broadly, these findings illustrate how +ssRNA viruses may obscure their genomes as members of nested symbioses, with implications for host evolution, exaptation, and immunity in the context of reef health and disease.

Pervasive tandem duplications and convergent evolution shape coral genomes.

BACKGROUND: Over the last decade, several coral genomes have been sequenced allowing a better understanding of these symbiotic organisms threatened by climate change. Scleractinian corals are reef builders and are central to coral reef ecosystems, providing habitat to a great diversity of species. RESULTS: In the frame of the Tara Pacific expedition, we assemble two coral genomes, Porites lobata and Pocillopora cf. effusa, with vastly improved contiguity that allows us to study the functional organization of these genomes. We annotate their gene catalog and report a relatively higher gene number than that found in other public coral genome sequences, 43,000 and 32,000 genes, respectively. This finding is explained by a high number of tandemly duplicated genes, accounting for almost a third of the predicted genes. We show that these duplicated genes originate from multiple and distinct duplication events throughout the coral lineage. They contribute to the amplification of gene families, mostly related to the immune system and disease resistance, which we suggest to be functionally linked to coral host resilience. CONCLUSIONS: At large, we show the importance of duplicated genes to inform the biology of reef-building corals and provide novel avenues to understand and screen for differences in stress resilience.

In-Silico Approaches for the Screening and Discovery of Broad-Spectrum Marine Natural Product Antiviral Agents Against Coronaviruses.

The urgent need for SARS-CoV-2 controls has led to a reassessment of approaches to identify and develop natural product inhibitors of zoonotic, highly virulent, and rapidly emerging viruses. There are yet no clinically approved broad-spectrum antivirals available for beta-coronaviruses. Discovery pipelines for pan-virus medications against a broad range of betacoronaviruses are therefore a priority. A variety of marine natural product (MNP) small molecules have shown inhibitory activity against viral species. Access to large data caches of small molecule structural information is vital to finding new pharmaceuticals. Increasingly, molecular docking simulations are being used to narrow the space of possibilities and generate drug leads. Combining in-silico methods, augmented by metaheuristic optimization and machine learning (ML) allows the generation of hits from within a virtual MNP library to narrow screens for novel targets against coronaviruses. In this review article, we explore current insights and techniques that can be leveraged to generate broad-spectrum antivirals against betacoronaviruses using in-silico optimization and ML. ML approaches are capable of simultaneously evaluating different features for predicting inhibitory activity. Many also provide a semi-quantitative measure of feature relevance and can guide in selecting a subset of features relevant for inhibition of SARS-CoV-2.

Rapid Biotic and Abiotic Transformation of Toxins produced by Ostreopsis. cf. ovata.

The dinoflagellate Ostreopsis cf. ovata produces several families of toxic polyketides. Despite only a few field measurements of these phycotoxins in seawater and aerosols, they are believed to be responsible for dermatitis and the toxic inhalations reported during blooms of this species. Therefore, the stability of these compounds in seawater is essential to understanding the causes of these symptoms, however, this has never been assessed. In the current study, the optimization of a solid phase extraction (SPE) procedure was first performed to ensure the most efficient extraction of all phycotoxins known to be produced by this strain, including the recently described liguriatoxins. The SPE cartridge SDBL® under non acidified conditions offered the best option. The stability of the ovatoxins and the liguriatoxins under biotic and abiotic stress was assessed by exposing the spent medium of a culture of Ostreopsis cf. ovata to its bacterial consortium and natural sunlight. A rapid biotic transformation was detected for both families of compounds. When exposed to bacteria, the half-lives of the ovatoxins were reached before 10 h and at 36 h, 97% of these toxins had been transformed. The half-lives of the liguriatoxins were 10 h under these conditions. Photolysis (abiotic degradation) of the ovatoxins (T1/2 < 36 h) was faster than for the liguriatoxins (T1/2 > 62 h). Although none of the catabolites of these phycotoxins were thoroughly identified, an untargeted metabolomics approach combined with molecular networking highlighted the presence of several compounds exhibiting structural similarities with the ovatoxins. Additional work should confirm the preliminary findings on these potential ovatoxins' catabolites and their biological properties. The rapid transformation of O. cf. ovata's phycotoxins introduces questions concerning their presence in seawater and their dispersion in the sea spray aerosols. The compounds involved in the toxic inhalations and dermatitis often experienced by beachgoers may stem from the catabolites of these toxins or even unrelated and as yet unidentified compounds.

Application of SS-CS-HR-AAS measurements for the detection of Ag nanoparticles in marine invertebrates.

The present study describes the development of a fit-for-purpose analytical procedure for the detection of Ag NPs in different marine organisms by Solid Sampling Continuous Source High Resolution Atomic Absorption Spectrometry (SS-CS-HR-AAS). The detection is based on the observation of the Ag absorption peak and its atomization delay tad which is different for ionic Ag and Ag NPs. The temperature program was optimized in order to achieve the maximum difference between the t ad (Δtad ). The method was first developed using biota CRMs spiked with different Ag NPs standard solutions or Ag+ , at the same concentration. Then, laboratory exposure experiments were performed on mussels and marine sponges. The results showed that the developed methodology is suitable for the detection of Ag NPs for both groups of organisms, showing Δtad up to 3.1 s. The developed method is therefore a promising tool to assess the presence of AgNPs in marine invertebrates.

Toxicity of palytoxin, purified ovatoxin-a, ovatoxin-d and extracts of Ostreopsis cf. ovata on the Caco-2 intestinal barrier model.

Human intoxications in the Mediterranean Sea have been linked to blooms of the dinoflagellate Ostreopsis cf. ovata, producer of palytoxin (PlTX)-like toxins called ovatoxins (OVTXs). Exposure routes include only inhalation and contact, although PlTX-poisoning by seafood has been described in tropical regions. To address the impact of OVTXs on the intestinal barrier, dinoflagellate extracts, purified OVTX-a and -d and PlTX were tested on differentiated Caco-2 cells. Viability, inflammatory response and barrier integrity were recorded after 24 h treatment. OVTX-a and -d were not cytotoxic up to 20 ng/mL but increased IL-8 release, although to a lesser extent compared to PlTX. While PlTX and OVTX-a (at 0.5 and 5 ng/mL respectively) affected intestinal barrier integrity, OVTX-d up to 5 ng/mL did not. Overall, OVTX-d was shown to be less toxic than OVTX-a and PlTX. Therefore, oral exposure to OVTX-a and -d could provoked lower acute toxicity than PlTX.