The Tara Pacific expedition-A pan-ecosystemic approach of the "-omics" complexity of coral reef holobionts across the Pacific Ocean.

Coral reefs are the most diverse habitats in the marine realm. Their productivity, structural complexity, and biodiversity critically depend on ecosystem services provided by corals that are threatened because of climate change effects-in particular, ocean warming and acidification. The coral holobiont is composed of the coral animal host, endosymbiotic dinoflagellates, associated viruses, bacteria, and other microeukaryotes. In particular, the mandatory photosymbiosis with microalgae of the family Symbiodiniaceae and its consequences on the evolution, physiology, and stress resilience of the coral holobiont have yet to be fully elucidated. The functioning of the holobiont as a whole is largely unknown, although bacteria and viruses are presumed to play roles in metabolic interactions, immunity, and stress tolerance. In the context of climate change and anthropogenic threats on coral reef ecosystems, the Tara Pacific project aims to provide a baseline of the "-omics" complexity of the coral holobiont and its ecosystem across the Pacific Ocean and for various oceanographically distinct defined areas. Inspired by the previous Tara Oceans expeditions, the Tara Pacific expedition (2016-2018) has applied a pan-ecosystemic approach on coral reefs throughout the Pacific Ocean, drawing an east-west transect from Panama to Papua New Guinea and a south-north transect from Australia to Japan, sampling corals throughout 32 island systems with local replicates. Tara Pacific has developed and applied state-of-the-art technologies in very-high-throughput genetic sequencing and molecular analysis to reveal the entire microbial and chemical diversity as well as functional traits associated with coral holobionts, together with various measures on environmental forcing. This ambitious project aims at revealing a massive amount of novel biodiversity, shedding light on the complex links between genomes, transcriptomes, metabolomes, organisms, and ecosystem functions in coral reefs and providing a reference of the biological state of modern coral reefs in the Anthropocene.

Thirtieth Anniversary of the Discovery of Laxaphycins. Intriguing Peptides Keeping a Part of Their Mystery.

Lipopeptides are a class of compounds generally produced by microorganisms through hybrid biosynthetic pathways involving non-ribosomal peptide synthase and a polyketyl synthase. Cyanobacterial-produced laxaphycins are examples of this family of compounds that have expanded over the past three decades. These compounds benefit from technological advances helping in their synthesis and characterization, as well as in deciphering their biosynthesis. The present article attempts to summarize most of the articles that have been published on laxaphycins. The current knowledge on the ecological role of these complex sets of compounds will also be examined.

Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells.

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

Structure and biological evaluation of new cyclic and acyclic laxaphycin-A type peptides.

Five new laxaphycins were isolated and fully characterised from the bloom forming cyanobacteria Anabaena torulosa sampled from Moorea, French Polynesia: three acyclic laxaphycin A-type peptides, acyclolaxaphycin A (1), [des-Gly11]acyclolaxaphycin A (2) and [des-(Leu10-Gly11)]acyclolaxaphycin A (3), as well as two cyclic ones, [l-Val8]laxaphycin A (4) and [d-Val9]laxaphycin A (5). The absolute configuration of the amino acids, established using advanced Marfey's analysis for compounds 2-5, highlights a conserved stereochemistry at the Cα carbons of the peptide ring that is characteristic of this family. To the best of our knowledge, this is the first report of acyclic analogues within the laxaphycin A-type peptides. Whether these linear laxaphycins with the aliphatic ß-amino acid on the N-terminal are biosynthetic precursors or compounds obtained after enzymatic hydrolysis of the macrocycle is discussed. Biological evaluation of the new compounds together with the already known laxaphycin A shows that [l-Val8]laxaphycin A, [d-Val9]laxaphycin A and [des-Gly11]acyclolaxaphycin induce cellular toxicity whereas laxaphycin A and des-[(Leu10-Gly11)]acyclolaxaphycin A do not affect the cellular viability. An analysis of cellular death shows that the active peptides do not induce apoptosis or necrosis but instead, involve the autophagy pathway.

Structures and Activities of Tiahuramides A-C, Cyclic Depsipeptides from a Tahitian Collection of the Marine Cyanobacterium Lyngbya majuscula.

The structures of three new cyclic depsipeptides, tiahuramides A (1), B (2), and C (3), from a French Polynesian collection of the marine cyanobacterium Lyngbya majuscula are described. The planar structures of these compounds were established by a combination of mass spectrometry and 1D and 2D NMR experiments. Absolute configurations of natural and nonproteinogenic amino acids were determined through a combination of acid hydrolysis, derivitization with Marfey's reagent, and HPLC. The absolute configuration of hydroxy acids was confirmed by Mosher's method. The antibacterial activities of tiahuramides against three marine bacteria were evaluated. Compound 3 was the most active compound of the series, with an MIC of 6.7 µM on one of the three tested bacteria. The three peptides inhibit the first cell division of sea urchin fertilized eggs with IC50 values in the range from 3.9 to 11 µM. Tiahuramide B (2), the most potent compound, causes cellular alteration characteristics of apoptotic cells, blebbing, DNA condensation, and fragmentation, already at the first egg cleavage. The cytotoxic activity of compounds 1-3 was tested in SH-SY5Y human neuroblastoma cells. Compounds 2 and 3 showed an IC50 of 14 and 6.0 µM, respectively, whereas compound 1 displayed no toxicity in this cell line at 100 µM. To determine the type of cell death induced by tiahuramide C (3), SH-SY5Y cells were costained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The double staining indicated that the cytotoxicity of compound 3 in this cell line is produced by necrosis.

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis.

The laxaphyci's B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.

Additional cytotoxic pyridoacridine alkaloids from the ascidian Cystodytes violatinctus and biogenetic considerations.

The extraction and purification of the bioactive extract of Cystodytes violatinctus (Solomon Islands) led to the isolation and identification of six pyridoacridine alkaloids. The structures of four new members of this family, shermilamine F (1), dehydrokuanoniamine F (2), and arnoamines C (3) and D (4), were elucidated on the basis of NMR and MS data and by comparison with data of known compounds isolated from this genus. A general hypothetical biogenetic pathway is then proposed for pyridoacridine alkaloids that contain a fused pyrrole ring. Comparison of the biological properties of the isolated alkaloids is also discussed.

Sources of secondary metabolite variation in Dysidea avara (Porifera: Demospongiae): the importance of having good neighbors.

Several studies report temporal, geographical, and intra-individual variation in sponge metabolite yields. However, the internal and/or external factors that regulate the metabolite production remain poorly understood. Dysidea avara is a demosponge that produces sesquiterpenoids (avarol and derivatives) with interesting medical properties, which has prompted addressed studies to obtain enough amounts of these metabolites for research on drug discovery. Within this framework, specimens of Dysidea avara from a population of the Northwest Mediterranean were sampled and their secondary metabolites quantified to assess their variability and the possible relationship with external (seasonality, interactions with neighbors) and internal (reproductive stages) factors. The results show a variation of the amount of both avarol and its monoacetate derivative with time, with no clear relationship with seawater temperature. A trade-off with sponge reproduction was not found either. However, our results showed for the first time that sponges are able to increase production or accumulation of secondary metabolites in their peripheral zone depending on the nature of their neighbors. This finding could explain part of the high variability in the amount of secondary metabolites usually found in chemical ecology studies on sponges and opens new biotechnological approaches to enhance the metabolite yield in sponge cultures.

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.

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.

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.

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.

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.

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.

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.

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.

Characterization of a new anticancer agent, EAPB0203, and its main metabolites: nuclear magnetic resonance and liquid chromatography-mass spectrometry studies.

The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the (1)H and (13)C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H](+) ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H(•), CH(3)(•), or even C(7)H(7)(•) giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional (1)H NMR spectroscopies.

Temporal trends in the secondary metabolite production of the sponge Aplysina aerophoba.

Temporal changes in the production of secondary metabolites are far from being fully understood. Our study quantified, over a two-year period, the concentrations of brominated alkaloids in the ectosome and the choanosome of Aplysina aerophoba, and examined the temporal patterns of these natural products. Based on standard curves, we quantified the concentrations of aerophobin-2, aplysinamisin-1, and isofistularin-3: three of the four major peaks obtained through chemical profiling with high-performance liquid chromatography. Our results showed a striking variation in compound abundance between the outer and inner layers of the sponge. The ectosome showed high concentrations of bromocompounds during the summer months, while the choanosome followed no pattern. Additionally, we found that, from the outer layer of the sponge, aerophobin-2 and isofistularin-3 were significantly correlated with water temperature. The present study is one of the first to document quantitative seasonal variations in individual compounds over multiple years. Further studies will clarify the role of environmental, biological, and physiological factors in determining the seasonal patterns in the concentration of brominated alkaloids.

Exploring the links between natural products and bacterial assemblages in the sponge Aplysina aerophoba.

The sponge Aplysina aerophoba produces a large diversity of brominated alkaloids (BAs) and hosts a complex microbial assemblage. Although BAs are located within sponge cells, the enzymes that bind halogen elements to organic compounds have been exclusively described in algae, fungi, and bacteria. Bacterial communities within A. aerophoba could therefore be involved in the biosynthesis of these compounds. This study investigates whether changes in both the concentration of BAs and the bacterial assemblages are correlated in A. aerophoba. To do so, we quantified major natural products using high-performance liquid chromatography and analyzed bacterial assemblages using denaturing gradient gel electrophoresis on the 16S rRNA gene. We identified multiple associations between bacteria and natural products, including a strong relationship between a Chloroflexi phylotype and aplysinamisin-1 and between an unidentified bacterium and aerophobin-2 and isofistularin-3. Our results suggest that these bacteria could either be involved in the production of BAs or be directly affected by them. To our knowledge, this is one of the first reports that find a significant correlation between natural products and bacterial populations in any benthic organism. Further investigating these associations will shed light on the organization and functioning of host-endobiont systems such as Aplysina aerophoba.