High microbiome and metabolome diversification in coexisting sponges with different bio-ecological traits.

Marine Porifera host diverse microbial communities, which influence host metabolism and fitness. However, functional relationships between sponge microbiomes and metabolic signatures are poorly understood. We integrate microbiome characterization, metabolomics and microbial predicted functions of four coexisting Mediterranean sponges -Petrosia ficiformis, Chondrosia reniformis, Crambe crambe and Chondrilla nucula. Microscopy observations reveal anatomical differences in microbial densities. Microbiomes exhibit strong species-specific trends. C. crambe shares many rare amplicon sequence variants (ASV) with the surrounding seawater. This suggests important inputs of microbial diversity acquired by selective horizontal acquisition. Phylum Cyanobacteria is mainly represented in C. nucula and C. crambe. According to putative functions, the microbiome of P. ficiformis and C. reniformis are functionally heterotrophic, while C. crambe and C. nucula are autotrophic. The four species display distinct metabolic profiles at single compound level. However, at molecular class level they share a "core metabolome". Concurrently, we find global microbiome-metabolome association when considering all four sponge species. Within each species still, sets of microbe/metabolites are identified driving multi-omics congruence. Our findings suggest that diverse microbial players and metabolic profiles may promote niche diversification, but also, analogous phenotypic patterns of "symbiont evolutionary convergence" in sponge assemblages where holobionts co-exist in the same area.

Divergent morphological and microbiome strategies of two neighbor sponges to cope with low pH in Mediterranean CO2 vents.

Ocean Acidification (OA) profoundly impacts marine biochemistry, resulting in a net loss of biodiversity. Porifera are often forecasted as winner taxa, yet the strategies to cope with OA can vary and may generate diverse fitness status. In this study, microbial shifts based on the V3-V4 16S rRNA gene marker were compared across neighboring Chondrosia reniformis sponges with high microbial abundance (HMA), and Spirastrella cunctatrix with low microbial abundance (LMA) microbiomes. Sponge holobionts co-occurred in a CO2 vent system with low pH (pHT ~ 7.65), and a control site with Ambient pH (pHT ~ 8.05) off Ischia Island, representing natural analogues to study future OA, and species' responses in the face of global environmental change. Microbial diversity and composition varied in both species across sites, yet at different levels. Increased numbers of core taxa were detected in S. cunctatrix, and a more diverse and flexible core microbiome was reported in C. reniformis under OA. Vent S. cunctatrix showed morphological impairment, along with signs of putative stress-induced dysbiosis, manifested by: 1) increases in alpha diversity, 2) shifts from sponge related microbes towards seawater microbes, and 3) high dysbiosis scores. Chondrosia reniformis in lieu, showed no morphological variation, low dysbiosis scores, and experienced a reduction in alpha diversity and less number of core taxa in vent specimens. Therefore, C. reniformis is hypothesized to maintain an state of normobiosis and acclimatize to OA, thanks to a more diverse, and likely metabolically versatile microbiome. A consortium of differentially abundant microbes was identified associated to either vent or control sponges, and chiefly related to carbon, nitrogen and sulfur-metabolisms for nutrient cycling and vitamin production, as well as probiotic symbionts in C. reniformis. Diversified symbiont associates supporting functional convergence could be the key behind resilience towards OA, yet specific acclimatization traits should be further investigated.

Rhodobacteraceae dominate the core microbiome of the sea star Odontaster validus (Koehler, 1906) in two opposite geographical sectors of the Antarctic Ocean.

Microbiota plays essential roles in the health, physiology, and in adaptation of marine multi-cellular organisms to their environment. In Antarctica, marine organisms have a wide range of unique physiological functions and adaptive strategies, useful for coping with extremely cold conditions. However, the role of microbiota associated with Antarctic organisms in such adaptive strategies is underexplored. In the present study, we investigated the diversity and putative functions of the microbiome of the sea star Odontaster validus, one of the main keystone species of the Antarctic benthic ecosystems. We compared the whole-body bacterial microbiome of sea stars from different sites of the Antarctic Peninsula and Ross Sea, two areas located in two opposite geographical sectors of the Antarctic continent. The taxonomic composition of O. validus microbiomes changed both between and within the two Antarctic sectors, suggesting that environmental and biological factors acting both at large and local scales may influence microbiome diversity. Despite this, one bacterial family (Rhodobacteraceae) was shared among all sea star individuals from the two geographical sectors, representing up to 95% of the microbial core, and suggesting a key functional role of this taxon in holobiont metabolism and well-being. In addition, the genus Roseobacter belonging to this family was also present in the surrounding sediment, implying a potential horizontal acquisition of dominant bacterial core taxa via host-selection processes from the environment.

New Imidazolium Alkaloids with Broad Spectrum of Action from the Marine Bacterium Shewanella aquimarina.

The continuous outbreak of drug-resistant bacterial and viral infections imposes the need to search for new drug candidates. Natural products from marine bacteria still inspire the design of pharmaceuticals. Indeed, marine bacteria have unique metabolic flexibility to inhabit each ecological niche, thus expanding their biosynthetic ability to assemble unprecedented molecules. The One-Strain-Many-Compounds approach and tandem mass spectrometry allowed the discovery of a Shewanella aquimarina strain as a source of novel imidazolium alkaloids via molecular networking. The alkaloid mixture was shown to exert bioactivities such as: (a) antibacterial activity against antibiotic-resistant Staphylococcus aureus clinical isolates at 100 µg/mL, (b) synergistic effects with tigecycline and linezolid, (c) restoration of MRSA sensitivity to fosfomycin, and (d) interference with the biofilm formation of S. aureus 6538 and MRSA. Moreover, the mixture showed antiviral activity against viruses with and without envelopes. Indeed, it inhibited the entry of coronavirus HcoV-229E and herpes simplex viruses into human cells and inactivated poliovirus PV-1 in post-infection assay at 200 µg/mL. Finally, at the same concentration, the fraction showed anthelminthic activity against Caenorhabditis elegans, causing 99% mortality after 48 h. The broad-spectrum activities of these compounds are partially due to their biosurfactant behavior and make them promising candidates for breaking down drug-resistant infectious diseases.

Hawaiian coral holobionts reveal algal and prokaryotic host specificity, intraspecific variability in bleaching resistance, and common interspecific microbial consortia modulating thermal stress responses.

Historically, Hawai'i had few massive coral bleaching events, until two consecutive heatwaves in 2014-2015. Consequent mortality and thermal stress were observed in Kane'ohe Bay (O'ahu). The two most dominant local species exhibited a phenotypic dichotomy of either bleaching resistance or susceptibility (Montipora capitata and Porites compressa), while the third predominant species (Pocillopora acuta) was broadly susceptible to bleaching. In order to survey shifts in coral microbiomes during bleaching and recovery, 50 colonies were tagged and periodically monitored. Metabarcoding of three genetic markers (16S rRNA gene ITS1 and ITS2) followed by compositional approaches for community structure analysis, differential abundance and correlations for longitudinal data were used to temporally compare Bacteria/Archaea, Fungi and Symbiodiniaceae dynamics. P. compressa corals recovered faster than P. acuta and Montipora capitata. Prokaryotic and algal communities were majorly shaped by host species, and had no apparent pattern of temporal acclimatization. Symbiodiniaceae signatures were identified at the colony scale, and were often related to bleaching susceptibility. Bacterial compositions were practically constant between bleaching phenotypes, and more diverse in P. acuta and M. capitata. P. compressa's prokaryotic community was dominated by a single bacterium. Compositional approaches (via microbial balances) allowed the identification of fine-scale differences in the abundance of a consortium of microbes, driving changes by bleaching susceptibility and time across all hosts. The three major coral reef founder-species in Kane'ohe Bay revealed different phenotypic and microbiome responses after 2014-2015 heatwaves. It is difficult to forecast, a more successful strategy towards future scenarios of global warming. Differentially abundant microbial taxa across time and/or bleaching susceptibility were broadly shared among all hosts, suggesting that locally, the same microbes may modulate stress responses in sympatric coral species. Our study highlights the potential of investigating microbial balances to identify fine-scale microbiome changes, serving as local diagnostic tools of coral reef fitness.

DNA Barcoding Procedures for Taxonomical and Phylogenetic Studies in Marine Animals: Porifera as a Case Study.

DNA barcoding is a versatile approach that has revolutionized taxonomy and other akin topics in biology and ecology, due to its simplicity and relatively costless procedures. The method consists in the production of one or a few amplicons from informative genetic regions via Sanger sequencing. These markers are selected because they tend to evolve at a similar pace as speciation, allowing to discriminate organismal species. The applicability of this technique is here portrayed for the taxonomical identification of marine sponges (phylum: Porifera) as an exemplification.

A community perspective on the concept of marine holobionts: current status, challenges, and future directions.

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

Molecular Network and Culture Media Variation Reveal a Complex Metabolic Profile in Pantoea cf. eucrina D2 Associated with an Acidified Marine Sponge.

The Gram-negative Pantoea eucrina D2 was isolated from the marine sponge Chondrosia reniformis. Sponges were collected in a shallow volcanic vents system in Ischia island (South Italy), influenced by CO2 emissions and lowered pH. The chemical diversity of the secondary metabolites produced by this strain, under different culture conditions, was explored by a combined approach including molecular networking, pure compound isolation and NMR spectroscopy. The metabolome of Pantoea cf. eucrina D2 yielded a very complex molecular network, allowing the annotation of several metabolites, among them two biosurfactant clusters: lipoamino acids and surfactins. The production of each class of metabolites was highly dependent on the culture conditions, in particular, the production of unusual surfactins derivatives was reported for the first time from this genus; interestingly the production of these metabolites only arises by utilizing inorganic nitrogen as a sole nitrogen source. Major components of the extract obtained under standard medium culture conditions were isolated and identified as N-lipoamino acids by a combination of 1D and 2D NMR spectroscopy and HRESI-MS analysis. Assessment of the antimicrobial activity of the pure compounds towards some human pathogens, indicated a moderate activity of leucine containing N-lipoamino acids towards Staphylococcus aureus, Staphylococcus epidermidis and a clinical isolate of the emerging food pathogen Listeria monocytogenes.

Marine Terpenoids from Polar Latitudes and Their Potential Applications in Biotechnology.

Polar marine biota have adapted to thrive under one of the ocean's most inhospitable scenarios, where extremes of temperature, light photoperiod and ice disturbance, along with ecological interactions, have selected species with a unique suite of secondary metabolites. Organisms of Arctic and Antarctic oceans are prolific sources of natural products, exhibiting wide structural diversity and remarkable bioactivities for human applications. Chemical skeletons belonging to terpene families are the most commonly found compounds, whereas cytotoxic antimicrobial properties, the capacity to prevent infections, are the most widely reported activities from these environments. This review firstly summarizes the regulations on access and benefit sharing requirements for research in polar environments. Then it provides an overview of the natural product arsenal from Antarctic and Arctic marine organisms that displays promising uses for fighting human disease. Microbes, such as bacteria and fungi, and macroorganisms, such as sponges, macroalgae, ascidians, corals, bryozoans, echinoderms and mollusks, are the main focus of this review. The biological origin, the structure of terpenes and terpenoids, derivatives and their biotechnological potential are described. This survey aims to highlight the chemical diversity of marine polar life and the versatility of this group of biomolecules, in an effort to encourage further research in drug discovery.

Biotechnological Applications of Bioactive Peptides From Marine Sources.

This review is an overview on marine bioactive peptides with promising activities for the development of alternative drugs to fight human pathologies. In particular, we focus on potentially prolific producers of peptides in microorganisms, including sponge-associated bacteria and marine photoautotrophs such as microalgae and cyanobacteria. Microorganisms are still poorly explored for drug discovery, even if they are highly metabolically plastic and potentially amenable to culturing. This offers the possibility of obtaining a continuous source of bioactive compounds to satisfy the challenging demands of pharmaceutical industries. This review targets peptides because of the variety of potent biological activities demonstrated by these molecules, including antiviral, antimicrobial, antifungal, antioxidant, anticoagulant, antihypertensive, anticancer, antidiabetic, antiobesity, and calcium-binding bioactivities. Several of these peptides have already gained recognition as effective drug agents in recent years. We also focus on cutting-edge omic approaches for the discovery of novel compounds for pharmacological applications. With rapid depletion of natural resources, omic technologies may be the solution to efficiently produce a vast variety of novel peptides with unique pharmacological potential.

UV-Protective Compounds in Marine Organisms from the Southern Ocean.

Solar radiation represents a key abiotic factor in the evolution of life in the oceans. In general, marine, biota-particularly in euphotic and dysphotic zones-depends directly or indirectly on light, but ultraviolet radiation (UV-R) can damage vital molecular machineries. UV-R induces the formation of reactive oxygen species (ROS) and impairs intracellular structures and enzymatic reactions. It can also affect organismal physiologies and eventually alter trophic chains at the ecosystem level. In Antarctica, physical drivers, such as sunlight, sea-ice, seasonality and low temperature are particularly influencing as compared to other regions. The springtime ozone depletion over the Southern Ocean makes organisms be more vulnerable to UV-R. Nonetheless, Antarctic species seem to possess analogous UV photoprotection and repair mechanisms as those found in organisms from other latitudes. The lack of data on species-specific responses towards increased UV-B still limits the understanding about the ecological impact and the tolerance levels related to ozone depletion in this region. The photobiology of Antarctic biota is largely unknown, in spite of representing a highly promising reservoir in the discovery of novel cosmeceutical products. This review compiles the most relevant information on photoprotection and UV-repair processes described in organisms from the Southern Ocean, in the context of this unique marine polar environment.

Author Correction: Exploring the pathology of an epidermal disease affecting a circum-Antarctic sea star.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Exploring the pathology of an epidermal disease affecting a circum-Antarctic sea star.

Over the past decade, unusual mortality outbreaks have decimated echinoderm populations over broad geographic regions, raising awareness globally of the importance of investigating such events. Echinoderms are key components of marine benthos for top-down and bottom-up regulations of plants and animals; population declines of these individuals can have significant ecosystem-wide effects. Here we describe the first case study of an outbreak affecting Antarctic echinoderms and consisting of an ulcerative epidermal disease affecting ~10% of the population of the keystone asteroid predator Odontaster validus at Deception Island, Antarctica. This event was first detected in the Austral summer 2012-2013, coinciding with unprecedented high seawater temperatures and increased seismicity. Histological analyses revealed epidermal ulceration, inflammation, and necrosis in diseased animals. Bacterial and fungal alpha diversity was consistently lower and of different composition in lesioned versus unaffected tissues (32.87% and 16.94% shared bacterial and fungal operational taxonomic units OTUs respectively). The microbiome of healthy stars was more consistent across individuals than in diseased specimens suggesting microbial dysbiosis, especially in the lesion fronts. Because these microbes were not associated with tissue damage at the microscopic level, their contribution to the development of epidermal lesions remains unclear. Our study reveals that disease events are reaching echinoderms as far as the polar regions thereby highlighting the need to develop a greater understanding of the microbiology and physiology of marine diseases and ecosystems health, especially in the era of global warming.

Who's there? - First morphological and DNA barcoding catalogue of the shallow Hawai'ian sponge fauna.

The sponge fauna has been largely overlooked in the Archipelago of Hawai'i, notwithstanding the paramount role of this taxon in marine ecosystems. The lack of knowledge about Porifera populations inhabiting the Hawai'ian reefs limits the development of ecological studies aimed at understanding the functioning of these marine systems. Consequently, this project addresses this gap by describing the most representative sponge species in the shallow waters of the enigmatic bay of Kane'ohe Bay, in O'ahu Island. A total of 30 species (28 demosponges and two calcareous sponges) living associated to the reef structures are here reported. Six of these species are new records to the Hawai'ian Porifera catalogue and are suspected to be recent introductions to these islands. Morphological descriptions of the voucher specimens are provided, along with sequencing data of two partitions involving the mitochondrial cytochrome oxidase subunit 1 (COI) marker and a fragment covering partial (18S and 28S) and full (ITS-1, 5.8S and ITS-2) nuclear ribosomal genes. Species delimitations based on genetic distances were calculated to valitate how taxonomic assignments from DNA barcoding aligned with morphological identifications. Of the 60 sequences submitted to GenBank ~88% are the first sequencing records for the corresponding species and genetic marker. This work compiles the first catalogue combining morphological characters with DNA barcoding of Hawai'ian sponges, and contributes to the repository of public databases through the Sponge Barcoding Project initiative.

Symbiont dynamics during thermal acclimation using cnidarian-dinoflagellate model holobionts.

Warming oceans menace reef ecosystems by disrupting symbiosis between cnidarians and Symbiodinium zooxanthellae, thus triggering bleach episodes. Temperature fluctuations promote adjustments in physiological variables and symbiont composition, which can cause stress responses, but can also yield adaptation if fitter host-symbiont homeostasis are achieved. To understand such processes manipulative studies are required, but many reef-building cnidarians pose limitations to experimental prospects. We exposed Exaiptasia anemones to Gradual Thermal Stress (GTS) and Heat Shock (HS) exposures and monitored chlorophyll and symbiont dynamics to test the phenotypic plasticity of these photosynthetic holobionts. GTS enhanced chlorophyll concentrations and decreased Symbiodinium proliferation. A recovery period after GTS returned chlorophyll to lower concentrations and symbiont divisions to higher rates. HS triggered a stress response characterized by intense symbiont declines through degradation and expulsion, algal compensatory proliferation, and chlorophyll accumulation. Anemones pre-exposed to GTS displayed more acute signs of symbiont paucity after HS, demonstrating that recurrent stress does not always induce bleaching-resistance. Our study is the first documenting Symbiodinium C and D, along with the predominant Clade B1 in Exaiptasia anemones. C subclades found in outdoor specimens faded under laboratory exposures. Clade D emerged after HS treatments, and especially after GTS pre-exposure. This highlights the thermotolerance of D subclades found in E. pallida and shows that bleaching-recovery can involve shifts of background symbiont phylotypes. This study enlightens the capability of Exaiptasia anemones to acclimate to gradually increased temperatures, and explores into how thermal history influences in subsequent stress tolerance in symbiotic cnidarians.

Experimental evidence of chemical defence mechanisms in Antarctic bryozoans.

Bryozoans are among the most abundant and diverse members of the Antarctic benthos, however the role of bioactive metabolites in ecological interactions has been scarcely studied. To extend our knowledge about the chemical ecology of Antarctic bryozoans, crude ether extracts (EE) and butanol extracts (BE) obtained from two Antarctic common species (Cornucopina pectogemma and Nematoflustra flagellata), were tested for antibacterial and repellent activities. The extracts were screened for quorum quenching and antibacterial activities against four Antarctic bacterial strains (Bacillus aquimaris, Micrococcus sp., Oceanobacillus sp. and Paracoccus sp.). The Antarctic amphipod Cheirimedon femoratus and the sea star Odontaster validus were selected as sympatric predators to perform anti-predatory and substrate preference assays. No quorum quenching activity was detected in any of the extracts, while all EE exhibited growth inhibition towards at least one bacterium strain. Although the species were not repellent against the sea star, they caused repellence to the amphipods in both extracts, suggesting that defence activities against predation derive from both lipophilic and hydrophilic metabolites. In the substrate preference assays, one EE and one BE deriving from different specimens of the species C. pectogemma were active. This study reveals intraspecific variability of chemical defences and supports the fact that chemically mediated interactions are common in Antarctic bryozoans as means of protection against fouling and predation.

Mass spectrometry detection of minor new meridianins from the Antarctic colonial ascidians Aplidium falklandicum and Aplidium meridianum.

Taking into account the broad biological activities found in the meridianin indole alkaloids isolated to date, we have re-examined the organic extracts of an Antarctic collection of the tunicates Aplidium meridianum and A. falklandicum (Chordata: Ascidiacea) by HPLC in conjunction with a high-resolution mass spectrometer (HPLC-MS). A new set of analogs of meridianins A-G has been detected, and their structures are proposed on the basis of the molecular formulae identified by LC-HRMS analysis using a C18 column with a gradient of water/acetonitrile and an LTQ-FT-MS Orbitrap detector. Remarkably, dimers derived from meridianin A and from meridianin B or E were also detected. Our findings provide further evidence of the broad variability within the meridianin-like derivatives of this highly bioactive alkaloid family.

Defensive metabolites from Antarctic invertebrates: does energetic content interfere with feeding repellence?

Many bioactive products from benthic invertebrates mediating ecological interactions have proved to reduce predation, but their mechanisms of action, and their molecular identities, are usually unknown. It was suggested, yet scarcely investigated, that nutritional quality interferes with defensive metabolites. This means that antifeedants would be less effective when combined with energetically rich prey, and that higher amounts of defensive compounds would be needed for predator avoidance. We evaluated the effects of five types of repellents obtained from Antarctic invertebrates, in combination with diets of different energetic values. The compounds came from soft corals, ascidians and hexactinellid sponges; they included wax esters, alkaloids, a meroterpenoid, a steroid, and the recently described organic acid, glassponsine. Feeding repellency was tested through preference assays by preparing diets (alginate pearls) combining different energetic content and inorganic material. Experimental diets contained various concentrations of each repellent product, and were offered along with control compound-free pearls, to the Antarctic omnivore amphipod Cheirimedon femoratus. Meridianin alkaloids were the most active repellents, and wax esters were the least active when combined with foods of distinct energetic content. Our data show that levels of repellency vary for each compound, and that they perform differently when mixed with distinct assay foods. The natural products that interacted the most with energetic content were those occurring in nature at higher concentrations. The bioactivity of the remaining metabolites tested was found to depend on a threshold concentration, enough to elicit feeding repellence, independently from nutritional quality.

Occurrence of a taurine derivative in an antarctic glass sponge.

The n-butanol extract of an Antarctic hexactinellid sponge, Anoxycalyx (Scolymastra) joubini, was found to contain a taurine-conjugated anthranilic acid, never reported so far either as a natural product or by synthesis. The compound was inactive against human cancer cells in an in vitro growth inhibitory test, and also showed no antibacterial activity.

Feeding repellence in Antarctic bryozoans.

The Antarctic sea star Odontaster validus and the amphipod Cheirimedon femoratus are important predators in benthic communities. Some bryozoans are part of the diet of the asteroid and represent both potential host biosubstrata and prey for this omnivorous lysianassid amphipod. In response to such ecological pressure, bryozoans are expected to develop strategies to deter potential predators, ranging from physical to chemical mechanisms. However, the chemical ecology of Antarctic bryozoans has been scarcely studied. In this study we evaluated the presence of defenses against predation in selected species of Antarctic bryozoans. The sympatric omnivorous consumers O. validus and C. femoratus were selected to perform feeding assays with 16 ether extracts (EE) and 16 butanol extracts (BE) obtained from 16 samples that belonged to 13 different bryozoan species. Most species (9) were active (12 EE and 1 BE) in sea star bioassays. Only 1 BE displayed repellence, indicating that repellents against the sea star are mainly lipophilic. Repellence toward C. femoratus was found in all species in different extracts (10 EE and 12 BE), suggesting that defenses against the amphipod might be both lipophilic and hydrophilic. Interspecific and intraspecific variability of bioactivity was occasionally detected, suggesting possible environmental inductive responses, symbiotic associations, and/or genetic variability. Multivariate analysis revealed similarities among species in relation to bioactivities of EE and/or BE. These findings support the hypothesis that, while in some cases alternative chemical or physical mechanisms may also provide protection, repellent compounds play an important role in Antarctic bryozoans as defenses against predators.