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Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.

Most benthic invertebrates, including ecosystem engineers such as corals, sponges and bivalves, have a motile planktonic larval phase and rely on specific chemical cues to identify a suitable substrate to settle. Crustose coralline algae (CCA) can induce settlement and metamorphosis responses in many invertebrates including corals. We tested the respective coral settlement capacity of multiple CCA species in a choice experiment and investigated the composition of their microbiomes. Our findings revealed that coral larval settlement was drastically influenced by CCA genera and also suggest that bacterial communities on the CCA surface can potentially serve as a driver of coral larval settlement. The composition of the bacterial communities on the surface of the least attractive CCA genus, Neogoniolithon fosliei, was markedly different from the other genera, Porolithon gardineri and Titanoderma prototypum and was significantly enriched in Vibrio and Flammeovirgaceae. The activity of CCA-associated bacterial communities may contribute to some of the variability observed in settlement responses between CCA species. Specific bacterial ASVs assigned to the Neptuniibacter, Methylotrophic Group 3 and Cellvibrionaceae were positively correlated with coral settlement. Conversely, ASVs assigned as Vibrio and Flammeovirga were negatively correlated with coral settlement. This study identifies putative bacterial taxa involved in coral settlement, which is an essential step to understand the chemical cues involved in this process and to predict the ability of corals to recolonize damaged reefs following disturbances.

With each cell division, phytoplankton create new space for primary colonization by marine bacteria. Although this surface microenvironment is available to all planktonic bacterial colonizers, we show the assembly of bacterial consortia on a cosmopolitan marine diatom to be highly specific and reproducible. While phytoplankton–bacteria interactions play fundamental roles in marine ecosystems, namely primary production and the carbon cycle, the ecological paradigm behind epiphytic microbiome assembly remains poorly understood. In a replicated and repeated primary colonization experiment, we exposed the axenic diatom Thalassiosira rotula to several complex and compositionally different bacterial inocula derived from phytoplankton species of varying degrees of relatedness to the axenic Thalassiosira host or natural seawater. This revealed a convergent assembly of diverse and compositionally different bacterial inocula, containing up to 2071 operational taxonomic units (OTUs), towards a stable and reproducible core community. Four of these OTUs already accounted for a cumulative abundance of 60%. This core community was dominated by Rhodobacteraceae (30.5%), Alteromonadaceae (27.7%), and Oceanospirillales (18.5%) which was qualitatively and quantitatively most similar to its conspecific original. These findings reject a lottery assembly model of bacterial colonization and suggest selective microhabitat filtering. This is likely due to diatom host traits such as surface properties and different levels of specialization resulting in reciprocal stable-state associations.

34 pages, 4 figures.-- Distributed under Creative Commons CC-BY 4.0, 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, This paper is based on the results of a foresight workshop funded by the EuroMarine network, Sorbonne University, and the UMRs 8227 and 7144 of the Roscoff Biological Station. Ezequiel M. Marzinelli was funded by an Australian Research Council Discovery Project (DP180104041), and José Pintado Valverde was funded by the Galician Innovation Agency (IN607A 2017/4). The work of Simon M. Dittami ad Catherine Leblanc was funded by the ANR project IDEALG (ANR-10-TBR-04). Claire M.M. Gachon, Catherine Leblanc, and SimonMDittami received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 624575 (ALFF). The work of Fabrice Not was funded by the ANR project IMPEKAB (ANR-15-CE02-001). Ulisse Cardini was funded by the Research Council of Lithuania project INBALANCE (09.3.3-LMT-K-712-01-0069). Johan Decelle was supported by the CNRS and the ATIP-Avenir program, the LabEx GRAL (ANR-10-LABX-49-01) and Pôle Dittami et CBS from the University of Grenoble Alpes. Paco Cardenas received support from the European Union's Horizon 2020 research and innovation program through the SponGES project (grant agreement No. 679849). Elena Kazamia was funded by a Marie Curie Individual Fellowship (Horizon 2020, IRONCOMM). Aschwin H Engelen was supported by Portuguese national funds from FCT - Foundation for Science and Technology through projects UID/Multi/04326/2019 and UIDB/04326/2020

Only few naturally occurring cyclic imines have been fully structurally elucidated or synthesized to date. The configuration at the C-4 carbon plays a pivotal role in the neurotoxicity of many of these metabolites, for example, gymnodomines (GYMs) and spirolides (SPXs). However, the stereochemistry at this position is not accessible by nuclear Overhauser effect&mdash, nuclear magnetic resonance spectroscopy (NOE-NMR) due to unconstrained rotation of the single carbon bond between C-4 and C-5. Consequently, the relative configuration of GYMs and SPXs at C-4 and its role in protein binding remains elusive. Here, we determined the stereochemical configuration at carbon C-4 in the butenolide ring of spirolide- and gymnodimine-phycotoxins by comparison of measured 13C NMR shifts with values obtained in silico using force field, semiempirical and density functional theory methods. This comparison demonstrated that modeled data support S configuration at C-4 for all studied SPXs and GYMs, suggesting a biosynthetically conserved relative configuration at carbon C-4 among these toxins.

Siderophores are metal chelators produced by microorganisms to facilitate binding and uptake of iron. The isolation and characterization of siderophores are impeded by typically low siderophore yields and the complexity of siderophore-containing extracts generated with traditional purification methods. We investigated titanium dioxide nanoparticle solid-phase extraction (TiO2 NP SPE) as a technique to selectively concentrate and purify siderophores from complex matrices for subsequent LC-MS detection and identification. TiO2 NP SPE showed a high binding capacity (15.7 ± 0.2 μmol mg−1 TiO2) for the model siderophore desferrioxamine B (DFOB) and proved robust to pH changes and the presence of EDTA. These are significant advances in comparison to immobilized metal affinity chromatography (IMAC). The TiO2 NP SPE was highly selective and recovered 77.6 ± 6.2% of DFOB spiked to a compositionally complex bacterial culture supernatant. The simple clean-up procedure removed the majority of contaminants and allowed direct detection of siderophores from the LC-MS base peak chromatogram. The ‘untargeted’ purification and analysis of an untreated supernatant of iron-deprived bacterial culture allowed for the direct identification of two known and three novel ferrioxamines. Thus, TiO2 NP SPE in combination with LC-MS offers great potential as a discovery platform for the purification and subsequent quantification or identification of novel siderophores of microbial origin.

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe

This article is an outcome of the EuroMarine Foresight Workshop on “Chemical ecology of marine interactions: the chemical language that shapes future marine health”, hosted by the University of Essex. Financial support for this event was provided by EuroMarine.-- 8 pages, 4 figures, 1 panel, “Infochemicals” (information‐conveying chemicals) dominate much of the underwater communication in biological systems. They influence the movement and behavior of organisms, the ecological interactions between and across populations, and the trophic structure of marine food webs. However, relative to their terrestrial equivalents, the wider ecological and economic importance of marine infochemicals remains understudied and a concerted, cross‐disciplinary effort is needed to reveal the full potential of marine chemical ecology. We highlight current challenges with specific examples and suggest how research on the chemical ecology of marine organisms could provide opportunities for implementing new management solutions for future “blue growth” (the sustainable use of ocean resources) and maintaining healthy marine ecosystems, M Saha acknowledges funding from the German Research Foundation (DFG) cluster of excellence “Future Ocean” for research grant CP1215, and the DFG under grant number SA 2571/2‐1. TW and GP acknowledge DFG for funding through CRC1127 ChemBioSys. EB received support from the CTM2014‐53818‐R (OstreoRisk) project funded by the Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER), and by the project CoCliME an ERA4CS Network (ERA‐NET) initiated by JPI Climate, and funded by EPA (IE), ANR (FR), BMBF (DE), UEFISCDI (RO), RCN (NO), and FORMAS (SE), with co‐funding by the European Union (grant agreement number 690462)

It is well known that bacteria and fungi have evolved sophisticated systems for acquiring the abundant but biologically inaccessible trace element iron. These systems are based on high affinity Fe(III)-specific binding compounds called siderophores which function to acquire, transport, and process this essential metal ion. Many hundreds of siderophores are now known and their numbers continue to grow. Extensive studies of their isolation, structure, transport, and molecular genetics have been undertaken in the last three decades and have been comprehensively reviewed many times. In this review we focus on a unique subset of siderophores that has only been recognized in the last 20 years, namely those whose iron complexes display photoactivity. This photoactivity, which typically results in the photooxidation of the siderophore ligand with concomitant reduction of Fe(III) to Fe(II), seemingly upsets the siderophore paradigm of forming and transporting only extremely stable Fe(III) complexes into microbial cells. Here we review their structure, synthesis, photochemistry, photoproduct coordination chemistry and explore the potential biological and ecological consequences of this photoactivity.

Host-microbe interactions play crucial roles in marine ecosystems, but 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 describe and understand these complex systems. It posits that a host and its associated microbiota, living together in a long-lasting relationship, form the holobiont, and have to be studied together, as a coherent biological and functional unit, in order to understand the biology, ecology and evolution of the organisms. 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 with comparisons to terrestrial science whenever appropriate. A deeper understanding of such complex systems, however, will require further technological and conceptual advances. The most significant challenge will be to bridge functional research on simple and tractable model systems and global approaches. This will require scientists to work together as an (inter)active community in order to address, for instance, ecological and evolutionary questions and the roles of holobionts in biogeochemical cycles., PeerJ Preprints, Computer Science Preprints., 2019

Recent research suggests that corals including cold-water corals harbor a diverse community of bacteria that are not only pathogens but also potential mutualists. Here we review data on bacterial community composition and diversity on the main cold-water corals framework builder species: Lophelia pertusa and Madrepora oculata. Sampling strategies such as box core, video grabs and remotely operated vehicle did not reveal strong differences between bacterial community composition as long as samples were used that looked ‘not contaminated’. However, there were strong differences of bacterial diversity between the two coral species. An analysis of bacterial community composition by pyrosequencing of L. pertusa and M. oculata revealed for the Mediterranean Sea the presence of the potential mutualists already found in the Atlantic indicating a species-specific core microbiome. The data also suggest some biogeographical differences between the Mediterranean Sea and the North Atlantic for both coral species, however, this depends on the phylogenetic levels applied. In addition, there was also indication for a shared microbiome between the Mediterranean Sea and the Atlantic. Therefore species-specific bacterial associations seem to exist, whereas the biogeographical variability can be seen as adaptation to specific environmental conditions.

Most benthic marine invertebrates have complex life cycles that include motile larvae and more or less immobile adult stages. The colonization of surfaces by marine invertebrate larvae is a fundamental process that governs distribution patterns and biodiversity of marine invertebrates. The transition between these life stages is linked to the choice for appropriate habitats via cues for larval settlement and metamorphosis. While marine biofilms have long been recognized as a cue source for larval settlement of phylogenetically diverse invertebrates, the list of chemically identified settlement compounds is surprisingly short. In this chapter, we review the current approach to identify larval settlement cues and discuss the difficulties in differentiating between settlement cues associated with animals or plants and those originating from prokaryotic microbes associated with living surfaces. We further argue for the need to integrate larval ecology, environmental microbiology, analytical chemistry, and surface physics to measure the natural distribution and abundance of larval settlement cues and judge their ecological relevance and plausibility.

Seaweeds (macroalgae) form a diverse and ubiquitous group of photosynthetic organisms that play an essential role in aquatic ecosystems. These ecosystem engineers contribute significantly to global primary production and are the major habitat formers on rocky shores in temperate waters, providing food and shelter for aquatic life. Like other eukaryotic organisms, macroalgae harbor a rich diversity of associated microorganisms with functions related to host health and defense. In particular, epiphytic bacterial communities have been reported as essential for normal morphological development of the algal host, and bacteria with antifouling properties are thought to protect chemically undefended macroalgae from detrimental, secondary colonization by other microscopic and macroscopic epibiota. This tight relationship suggests that macroalgae and epiphytic bacteria interact as a unified functional entity or holobiont, analogous to the previously suggested relationship in corals. Moreover, given that the impact of diseases in marine ecosystems is apparently increasing, understanding the role of bacteria as saprophytes and pathogens in seaweed communities may have important implications for marine management strategies. This review reports on the recent advances in the understanding of macroalgal-bacterial interactions with reference to the diversity and functional role of epiphytic bacteria in maintaining algal health, highlighting the holobiont concept.

Larval settlement of the infaunal spionid polychaetes Polydora cornuta and Streblospio benedicti is mediated by sediment-associated microorganisms. To investigate if larval preference for certain sediment is guided by individual sediment-associated bacteria, 13 bacterial isolates (5 phyla) obtained from the natural habitat of adult polychaetes (Wadden Sea, Germany) in 2008 were screened in still-water, no-choice settlement assays. Two isolates (α-Proteobacterium-Strain DF11 and Flavobacterium-Strain 54) significantly triggered larval settlement in comparison with sterile sediment. In still-water, multiple-choice settlement assays comprising natural and sterile sediment and sediment re-inoculated with isolates DF11 and 54, significant preferences for natural sediment and sediment containing bacterial isolates at 108 cells g−1 were observed. Larval settlement was influenced by bacterial abundance in sediment but the correlation was not strictly positive; thus, maximum larval settlement in response to single bacterial species may occur at certain optimum densities. Non-viable or suspended bacteria and water-soluble bacterial products did not induce larval settlement, suggesting that sediment-associated bacterial settlement cues for P. cornuta and S. benedicti were either produced in situ and/or consisted of heat-labile bacterial products.

Invasion success of introduced species is often attributed to a lack of natural enemies as stated by the enemy release hypothesis (ERH). The ERH intuitively makes sense for specialized enemies, but it is less evident why invaders in their new area escape attacks by generalist enemies. A recent hypothesis explains low herbivore damage on invasive plants with plant defense chemicals that are evolutionarily novel to native herbivores. Support for this novel weapon hypothesis (NWH) is so far based on circumstantial evidence. To corroborate the NWH, there is a need for direct evidence through explicit characterizations of the novel chemicals and their effects on native consumers. This study evaluated the NWH using the highly invasive red alga Bonnemaisonia hamifera. In pairwise feeding experiments, preferences between B. hamifera and native competitors were assessed for four common generalist herbivores in the invaded area. Through a bioassay-guided fractionation, we identified the deterrent compound and verified its effect in an experiment with the synthesized compound at natural concentrations. The results showed that native herbivores strongly preferred native algae to B. hamifera. The resistance against herbivores could be tracked down to the algal metabolite 1,1,3,3-tetrabromo-2-heptanone, a compound not known from native algae in the invaded area. The importance of the chemical defense was further underlined by the feeding preference of herbivores for individuals with a depleted content of 1,1,3,3-tetrabromo-2-heptanone. This study thus provides the first conclusive example of a highly successful invader where low consumption in the new range can be directly attributed to a specific chemical defense against evolutionarily naive native generalists. In conclusion, our results support the notion that novel chemical weapons against naive herbivores can provide a mechanistic explanation for plant invasion success.

The need for animals and plants to control microbial colonization is important in the marine environment with its high densities of microscopic propagules and seawater that provides an ideal medium for their dispersal. In contrast to the traditional emphasis on antagonistic interactions of marine organisms with microbes, emerging studies lend support to the notion that health and performance of many marine organisms are functionally regulated and assisted by associated microbes, an ecological concept defined as a holobiont. While antimicrobial activities of marine secondary metabolites have been studied in great depth ex-situ, we are beginning to understand how some of these compounds function in an ecological context to maintain the performance of marine holobionts. The present article reviews two decades of our research on the red seaweed Delisea pulchra by addressing: the defense chemistry of this seaweed; chemically-mediated interactions between the seaweed and its natural enemies; and the negative influence of elevated seawater temperature on these interactions. Our understanding of these defense compounds and the functional roles they play for D. pulchra extends from molecular interactions with bacterial cell signaling molecules, to ecosystem-scale consequences of chemically-controlled disease and herbivory. Delisea pulchra produces halogenated furanones that antagonize the same receptor as acylated homoserine lactones (AHL)-a group of widespread intercellular communication signals among bacteria. Halogenated furanones compete with and inhibit bacterial cell-to-cell communication, and thus interfere with important bacterial communication-regulated processes, such as biofilm formation. In a predictable pattern that occurs at the ecological level of entire populations, environmental stress interferes with the production of halogenated furanones, causing downstream processes that ultimately result in disease of the algal holobiont.

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti- Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas , Phaeobacter , and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis . The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida , Pseudoalteromonas tunicata , and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata , reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.

Disease is emerging as an important impact of global climate change, due to the effects of environmental change on host organisms and their pathogens. Climate-mediated disease can have severe consequences in natural systems, particularly when ecosystem engineers, such as habitat-formers or top predators are affected, as any impacts can cascade throughout entire food webs. In temperate marine ecosystems, seaweeds are the dominant habitat-formers on rocky reefs. We investigated a putative bleaching disease affecting Delisea pulchra, a chemically defended seaweed that occurs within a global warming 'hot-spot' and assessed how patterns of this phenomenon were influenced by ocean temperature, solar radiation, algal chemical defences and microbial pathogens. Warmer waters were consistently and positively correlated with higher frequencies of bleaching in seaweed populations, but patterns of bleaching were not consistently influenced by light levels. Bleached thalli had low levels of antibacterial chemical defences relative to healthy conspecifics and this was observed across entire thalli of partially bleached algae. Microbial communities associated with bleached algae were distinct from those on the surfaces of healthy seaweeds. Direct testing of the importance of algal chemical defences, done here for the first time in the field, demonstrated that they protected the seaweed from bleaching. Treatment of algal thalli with antibiotics reduced the severity of bleaching in experimental algae, especially at high water temperatures. These results indicate that bleaching in D. pulchra is the result of temperature-mediated bacterial infections and highlight the potential for warming to influence disease dynamics by stressing hosts. Understanding the complex ways in which global change may affect important organisms such as habitat-forming seaweeds, is essential for the management and conservation of natural resources. © 2011 Blackwell Publishing Ltd.

Marine macroalgae are constantly exposed to epibacterial colonizers. The epiphytic bacterial patterns and their temporal and spatial variability on host algae are poorly understood. To investigate the interaction between marine macroalgae and epiphytic bacteria, this study tested if the composition of epibacterial communities on different macroalgae was specific and persisted under varying biotic and abiotic environmental conditions over a 2-year observation time frame. Epibacterial communities on the co-occurring macroalgae Fucus vesiculosus, Gracilaria vermiculophylla and Ulva intestinalis were repeatedly sampled in summer and winter of 2007 and 2008. The epibacterial community composition was analysed by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene libraries. Epibacterial community profiles did not only differ significantly at each sampling interval among algal species, but also showed consistent seasonal differences on each algal species at a bacterial phylum level. These compositional patterns re-occurred at the same season of two consecutive years. Within replicates of the same algal species, the composition of bacterial phyla was subject to shifts at the bacterial species level, both within the same season but at different years and between different seasons. However, 7–16% of sequences were identified as species specific to the host alga. These findings demonstrate that marine macroalgae harbour species-specific and temporally adapted epiphytic bacterial biofilms on their surfaces. Since several algal host-specific bacteria were highly similar to other bacteria known to either avoid subsequent colonization by eukaryotic larvae or to exhibit potent antibacterial activities, algal host-specific bacterial associations are expected to play an important role for marine macroalgae.

This study investigated whether surface-associated compounds isolated from the macroalga Fucus vesiculosus had the potential to mediate microbial and/or macrobial epibiosis similar to that on the natural alga. To selectively yield thallus-associated compounds and avoid contamination by intracellular algal compounds, cell lysis was monitored by surface microscopy of algal cells and chemical profiling of algal surface extracts by coupled gas chromatography mass spectroscopy. The optimized extraction resulted in polar and non-polar algal surface extracts. The non-polar surface extract was immobilized in hydrogel, the polar surface extract was homogeneously perfused through the gel to ensure a temporally constant delivery of polar extract components. During a 7 day field trial, bacterial biofilms were formed on control gels and gels featuring polar and/or non-polar extract components. PERMANOVA revealed that bacterial community profiles on controls and on gels featuring polar or non-polar extract were significantly different from the profile on F. vesiculosus, while the profile on the gels bearing both polar and non-polar extracts was not. Moreover, the polar surface extracts inhibited the settlement of barnacle cyprids. Considering the pronounced effects of bacterial biofilms on invertebrate larval settlement, these results suggest that algal surface chemistry affects macrofouling not only directly but also indirectly, via its control of biofilm formation and composition.

Hermit crabs with poor fitting shells are chemically attracted to dying gastropods and conspecifics where a shell may become available. For land hermit crabs, the shell cue is a volatile compound found in the haemolymph. Based on this knowledge, we tested the hypothesis that shell investigation behavior in aquatic hermit crabs, the ancestral predecessors of terrestrial hermit crabs, is also triggered by volatile cues. Volatile compounds from haemolymph of Clibanarius vittatus and Pagurus pollicaris and brachyuran decapod crustaceans were purged from a water–haemolymph solution, trapped in seawater and tested for induction of shell investigation behavior with juvenile C. vittatus. Only volatiles from C. vittatus haemolymph stimulated shell investigation. Volatile compounds were isolated from haemolymph by headspace solid-phase microextraction (SPME) and analyzed by coupled gas chromatography–mass spectrometry (GC–MS). Two prominent compounds were identified, 3-decanol, which was unique to C. vittatus haemolymph, and 2-ethyl-1-hexanol, which was present in the haemolymph of all 4 crustacean species. In shell investigation bioassays, 3-decanol from C. vittatus haemolymph stimulated shell investigation behavior, while 2-ethyl-1-hexanol did not. In bioassays with synthetic 1-, 2-, 4-, and 5-decanol, shell investigation behavior was evoked by 1-decanol, 5-decanol and 3-undecanol. There was no response to 2- and 4-decanol. The response of C. vittatus to volatile shell cues supports the hypothesis that volatile cue detection evolved prior to the occupation of terrestrial niches by crustaceans.

To test whether macroalgae affect microbial colonizers in close proximity in a phylum-specific fashion, the community richness of planktonic bacteria and fungi was analyzed with selective oligonucleotide probes targeting the Cytophaga/Flavobacterium/Bacteroides (CFB), Alphaproteobacteria and Roseobacter group and the ITS1 region of marine fungi. Naturally occuring planktonic microorganisms were incubated in the presence of macroalgae or in seawater previously conditioned with macroalgal metabolites. The red algae Ceramium rubrum and Mastocarpus stellatus as well as seawater conditioned with these algae reduced the community composition of bacteria to a greater extent than the brown alga Laminaria digitata, indicating that metabolites differed among macroalgae or that the susceptibility of planktonic bacteria towards alga-derived antimicrobials correlated with their phylogenetic affiliation. The most affected phylotypes belonged to the CFB and the Roseobacter clade. The planktonic fungal community was only affected in the presence of macroalgae and not in algal-conditioned water, but with a specificity different from that observed for bacteria. The macroalgae L. digitata and M. stellatus exhibited more pronounced antifungal effects than C. rubrum. This study demonstrates macroalgal defenses against epiphytic microorganisms based on natural delivery mechanisms of allelochemicals utilizing a culture-independent approach, thus minimizing the ecological bias inherent to culture-dependent studies based on few microbial isolates.

Considering that a variety of infaunal polychaetes release halogenated metabolites with potent bactericidal effects into the surrounding sediment, we tested the hypothesis that polychaete- derived halometabolites may either pose a direct negative effect on larvae or alter the bacterial com- munity in surface sediments, and thus indirectly influence larval settlement. Commercially available halogenated compounds identical or similar to the dominant halometabolites found in Streblospio benedicti and Capitella sp. I served as experimental proxies in single- and multiple-choice settlement assays with larvae of S. benedicti. In multiple-choice assays, the hypothesis of a direct influence of halogenated proxies on larval settlement was verified with 1-chlorononane at concentrations of 3.5 µg g -1 sediment. A similar effect was not observed with 2,6-dibromophenol. The no-choice assays did not show a direct influence of halometabolites on polychaete larvae. On the other hand, the hypothesis of an indirect influence of halogenated proxies on larval settlement via modification of the sediment microbial assemblage was clearly rejected. However, an analysis of similarity of the PCR-amplified bacterial genes coding for 16S rRNA corresponding to distinct phylotypes revealed a significant change in bacterial community richness between untreated and natural sediment spiked and incubated with halogenated proxies for 7 d. The change was characterized by the absence of up to 13 phylotypes and the appearance of up to 11 previously undetected phylotypes. The shift in bacterial community richness of sediments exposed to the halogenated proxies was not recognized by larvae, indicating that the halogenated proxies elicited no differential response in larvae of S. benedicti.

Microbially-derived diseases cause economic losses in aquaculture on global scale. Since many marine invertebrates and plants have evolved potent defence mechanisms against microbial epibionts, such as bacteria, viruses and fungi, there is increasing interest in these organisms as a source for natural antifouling and antibacterial substances. In this context, marine macroalgae are considered as a promising source for new antimicrobial agents. In this study, we investigated antibacterial effects of hexane and methanol extracts of the macroalgae Mastocarpus stellatus, Laminaria digitata and Ceramium rubrum on 12 marine and 7 prominent fish pathogenic bacteria at ecologically realistic concentrations of macroalgal secondary metabolites. Antibacterial activity was determined with a highly sensitive growth inhibition assay that records the fluorescence intensity of stained bacterial DNA. The assay allowed the detection of bacterial growth inhibition at and below the tissue level concentration of algal metabolites. Tests were carried out with algae collected in November 2006 and in March 2007 to investigate seasonal variation of antibacterial activities. Methanol extracts of C. rubrum at 10 mg dry weight per ml and hexane extracts of L. digitata at 31 mg dry weight per ml evoked strong antibacterial activities and inhibited almost all tested bacteria. There was no clear distinction between antibacterial effects of algae collected in November 2006 and March 2007. The growth inhibition of bacteria by algal extracts was frequently higher than 100%. This result indicated that bacteria must have been lysed during the exposure to algal extract components. Therefore, the extracts presumably evoked not only a bacteriostatic but also bacteriolytic mode of action. The assays revealed different susceptibilities of the bacterial phyla under investigation to algal extracts. Gram-positive marine Bacillaceae were generally more susceptible than Gram-negative marine Vibrionaceae. Listonella anguillarum, Pseudomonas anguilliseptica and Aeromonas salmonicida were the most susceptible fish pathogenic bacteria.

A variety of macroalgae (Ceramium rubrum, Corallina officinalis, Palmaria palmata, Mastocarpus stellatus, Fucus vesiculosus, Cladophora rupestris, Ulva sp.) were investigated by scanning electron microscopy to visualize epiphytic colonizers. The macroalgae differed in terms of their epiphytic coverage of bacteria, fungi and diatoms. Macroalgae, largely devoid of epiphytic diatoms, were hypothesized to employ effective antifouling means to reduce epiphytic coverage, whilst heavily fouled macroalgae were proposed to lack antifouling strategies. To test these hypotheses from an allelochemical perspective with regard to fouling diatoms, dichloromethane-methanol (1:1) crude extracts of macroalgae were concentrated in dimethylsulfoxide and investigated in diatom attachment and proliferation assays using four benthic diatoms (Nitzschia sp., Navicula phyllepta, Navicula arenaria and Amphora sp.). Algal extracts exhibited a distinct pattern of activity against the test diatoms, suggesting a targeted and selective effect of macroalgal metabolites on individual fouling diatoms. The main outcome of this study was that visual inspection and quantitative categorization of epiphytic colonizers on macroalgal thalli could not be used to predict reliably whether macroalgae employed a chemical defense mechanism.

Many macroalgae employ chemical means to suppress epibiosis by micro- and macroorganisms. Previous studies have focused on the effects of tissue extracts of entire algae on a few bacterial isolates, thereby missing not only ecologically relevant bacteria but also natural delivery mechanisms of algal antimicrobial agents. In this study, we investigated the potential antimicrobial effects of waterborne macroalgal metabolites utilizing a culture-independent approach to compare the bacterial community richness in seawater in the presence and absence of macroalgae. The methodology comprised the collection of planktonic bacteria in algal culture water on membrane filters followed by filter PCR and denaturant gradient gel electrophoresis (DGGE) of 16S rRNA gene sequences of harvested bacteria with universal primers. Similarity analysis distinguished two groups of macroalgae under investigation, one of which showed >55% difference, and the other

The relationship between monobacterial films and the preference of harpacticoid copepods for such films was investigated using still water multiple-choice assays with natural biofilm and sterile conditions as controls. Adult Schizopera sp. were most attracted by a heterogeneous natural biofilm, followed by monospecies-biofilms of Rhodovulum sp., Vibrio proteolyticus, and Flexibacter sp. The preferred bacterial films stemmed from different phylogenetic and physiological groups. The results indicated that the harpacticoid Schizopera sp. was effectively and differentially attracted by bacterial films. Since bacteria constitute a substantial portion of the organic carbon available at the sea bottom as nutritive sources for harpacticoid copepods, we subsequently examined the influence of 9 bacterial strains and a natural biofilm as a nutrient source on the growth and reproductive performance of ontogenetic stages (nauplii and copepodids) of Schizopera sp. The food value of bacterial strains was assayed in terms of life table data that provided growth parameters. All variables were affected by the type of food offered. A diet on Rhodovulum sp. resulted in optimal growth performance of nauplii and copepodids demonstrating that bacteria can be used as a sole diet to support postembryonic development. The present study is the first to link behavioral preferences to bacterial biofilms with life history parameters when cultivating harpacticoid copepods on the same bacterial strains as the only diet. This study revealed a discrepancy between the biofilm favored (natural biofilm) and the one leading to maximal reproductive performance (monobacterial film of Rhodovulum sp. MB253) as indicated by major life table data as net reproductive rate (Ro), mean generation time (Tm), and capacity for increase (rc).

The majority of marine invertebrates produce dispersive larvae which, in order to complete their life cycles, must attach and metamorphose into benthic forms. This process, collectively referred to as settlement, is often guided by habitat-specific cues. While the sources of such cues are well known, the links between their biological activity, chemical identity, presence and quantification in situ are largely missing. Previous work on coral larval settlement in vitro has shown widespread induction by crustose coralline algae (CCA) and in particular their associated bacteria. However, we found that bacterial biofilms on CCA did not initiate ecologically realistic settlement responses in larvae of 11 hard coral species from Australia, Guam, Singapore and Japan. We instead found that algal chemical cues induce identical behavioral responses of larvae as per live CCA. We identified two classes of CCA cell wall-associated compounds – glycoglycerolipids and polysaccharides – as the main constituents of settlement inducing fractions. These algae-derived fractions induce settlement and metamorphosis at equivalent concentrations as present in CCA, both in small scale laboratory assays and under flow-through conditions, suggesting their ability to act in an ecologically relevant fashion to steer larval settlement of corals. Both compound classes were readily detected in natural samples.

Most marine invertebrates have dispersive larvae and relatively immobile adults. These developmental stages are linked by a settlement event, which is often mediated by specific cues in bacterial biofilms. While larvae distinguish between biofilms from different environments, it remains unknown if they receive information from all, only a few or even just a single bacterial species in natural biofilms. Here we asked how specific is larval settlement to the bacterial community structure and/or taxonomically distinguishable groups of bacteria in epiphytic marine biofilms? We used novel multivariate statistical approaches to investigate if larval settlement of two sea urchins correlated with the microbial community composition. Larval settlement of Heliocidaris erythrogramma revealed a strong correlation with the community composition, highlighted by canonical analysis of principle components, a constrained ordination technique. Using this technique, the importance of operational taxonomic units (OTUs) within communities relative to larval settlement was investigated. Larval settlement not only correlated, both positively and negatively, with the epiphytic bacterial community composition but also with the relative abundance of few OTUs within these communities. In contrast, no such correlation was observed for the other urchin, Holopneustes purpurascens, whose larvae likely respond to bacterial biofilms in a more general way and specifically respond to a defined settlement cue of algal origin.

Do patchy distribution patterns of infaunal polychaetes result from active site selection of larvae influenced by sedimentassociated microbial cues? This hypothesis was tested with still-water laboratory settlement assays revealing the acceptance or rejection of polychaete larvae to qualitatively different sediments. Laboratory brood cultures of the spionid polychaetes Polydora cornuta and Streblospio benedicti yielded a sufficient number of larvae with planktotrophic development for bioassays. High settlement rates (75–95%) of test larvae were observed in response to natural sediment. Sterilization of natural sediment significantly decreased settlement of P. cornuta (25–55%) while combustion of sediment significantly decreased the settlement rate in both species (5–50%). Differences in settlement responses to sediments treated by sterilization or combustion most likely resulted from a variety of factors such as modified sediment fabric, grain size distribution and quantity of adsorbed organic matter. To experimentally address the potential role of microorganisms and microbial metabolites as mediators of larval settlement, ashed sediment was inoculated with viable microorganisms obtained from natural sediment. In both polychaete species, this treatment significantly increased larval settlement in comparison to the control of ashed sediment indicating that larval settlement was at least partially mediated by the presence of microorganisms associated with sediment.

The abundance and diversity of microorganisms on the surface of the tropical green macroalga Caulerpa racemosa var. turbinata and the effect of algal surface and waterborne compounds on fouling organisms were investigated both in laboratory and field experiments. As shown via electron microscopic enumeration, the abundance of epibiotic bacteria and diatoms on algal frond surfaces was not significantly different from the reference biofilms harvested from stones in the C. racemosa habitat. The analysis of Terminal Restriction Fragment Length Polymorphism of DNA from algal surface-associated bacterial communities revealed that despite a similar abundance of these bacteria, the community profile on algal frond surfaces differed significantly from that of inanimate, undefended substrates. These results suggest that the alga regulate the occurrence of certain bacterial ribotypes. This result was in accordance with the fact that different bacterial communities formed on the artificial substrata (i.e. Petri dishes) placed in the C. racemosa habitat and alga-free control sites. Neither C. racemosa conditioned seawater (CCW) nor hexane surface extracts affected the growth of bacterial isolates from biofilms. However, only CCW exhibited a toxic effect on the larvae of the fouling polychaete Hydroides elegans, and evoked abnormal larval development in a concentration-dependent fashion. At sublethal concentrations, the

Epi- and endozoic bacterial communities associated with four bryozoan species from the Jade (North Sea, Germany) were investigated by the combined application of molecular tools and electron microscopic visualisation of zooids. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments of associated bacteria displayed specific bacterial community profiles in the examined species Aspidelectra melolontha , Conopeum reticulum , Electra monostachys and Electra pilosa . Actual bacterial epibiosis was only observed on C. reticulum whilst the other bryozoans under investigation were largely free of microbial epibionts. These observations indicated that bryozoan-associated bacteria identified by molecular methods originated from internal cavities of bryozoan zooids. Cluster analysis of DGGE band patterns revealed species-specific bacterial communities in A. melolontha , E. monostachys and E. pilosa . Bacteria associated with C. reticulum were seemingly influenced by site-specific parameters. A comparison of bacterial community profiles between reference and invertebrate surfaces allowed for an interpretation of conspicuous group-specific differences. Operational taxonomic units (OTUs) obtained from a single set of bryozoan replicates that were absent on the inorganic reference samples (mussel shells) were hypothesized to be favourable endobionts. Contrary, OTUs present in the references but absent in bryozoan samples could be assumed to stem from bryozoan-specific defenses against ubiquitous bacterial colonizers. Although there was no experimental evidence for a mutual relationship between prokaryotes and their eukaryotic bryozoan hosts, this study demonstrated that in three out of four bryozoans under investigation associated bacterial communities were characteristically shaped by host attributes.

Extracellular polymers (EPS) produced by diatoms have been demonstrated as potent mediators of larval settlement in Hydroides elegans. Few studies have addressed the importance of environmental parameters (e.g. temperature and salinity) on quantity and quality of diatom EPS as larval settlement cues. The growth rates of the benthic pennate diatoms Achnanthes sp. and Nitzschia constricta reached a maximum and a minimum level at high and low temperature and salinity, respectively. A positive correlation was observed between the growth rate of diatoms and their production of EPS. In order to decouple the quantitative effect of diatom films from the potential qualitative effect of diatom EPS on larval settlement, the same amount of diatom EPS obtained from different growth conditions were immobilized in hydrogels and investigated in larval settlement assays. The diatom EPS obtained from high temperature treatments were more inductive to larval settlement than the low-temperature treatments, irrespective of the salinity setting. The qualitative differences of the diatom EPS samples under investigation were further analyzed by bioassay-guided gel-chromatography. The bioactive EPS fraction of Achnanthes sp. mainly consisted of large macro- molecules, while the bioactive EPS fraction of N. constricta consisted of both large and small macro- molecules. Due to their sensitivity to changes in environmental parameters, diatoms may serve as small scale proxies of substratum suitability for larval settlement of benthic marine invertebrates, analogous to the reported role of bacteria in marine biofilms.