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19 pages, 4 figures, 1 table, supporting information https://doi.org/10.1111/1462-2920.16249.-- Data Availability Statement: All 16 S rRNA gene amplicon reads and the sample metadata and attributes are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA): PRJNA786895; BioSample accessions: SAMN23732322- SAMN23732615, Marine sponges are known for their complex and stable microbiomes. However, the lack of a gnotobiotic sponge-model and experimental methods to manipulate both the host and the microbial symbionts currently limit our mechanistic understanding of sponge-microbial symbioses. We have used the North Atlantic sponge species Halichondria panicea to evaluate the use of antibiotics to generate gnotobiotic sponges. We further asked whether the microbiome can be reestablished via recolonization with the natural microbiome. Experiments were performed in marine gnotobiotic facilities equipped with a custom-made, sterile, flow-through aquarium system. Bacterial abundance dynamics were monitored qualitatively and quantitatively by 16 S rRNA gene amplicon sequencing and qPCR, respectively. Antibiotics induced dysbiosis by favouring an increase of opportunistic, antibiotic-resistant bacteria, resulting in more complex, but less specific bacteria-bacteria interactions than in untreated sponges. The abundance of the dominant symbiont, Candidatus Halichondribacter symbioticus, remained overall unchanged, reflecting its obligately symbiotic nature. Recolonization with the natural microbiome could not reverse antibiotic-induced dysbiosis. However, single bacterial taxa that were transferred, successfully recolonized the sponge and affected bacteria-bacteria interactions. By experimentally manipulating microbiome composition, we could show the stability of a sponge-symbiont clade despite microbiome dysbiosis. This study contributes to understanding both host-bacteria and bacteria-bacteria interactions in the sponge holobiont, This project is supported by funding of the DFG (“Origin and Function of Metaorganisms”, CRC1182-TP B01) and the Gordon and Betty Moore Foundation (“Symbiosis in Aquatic Systems Initiative”, GBMF9352) to UH. LS is further supported by the International Max Planck Research School for Evolutionary Biology, and LP receives funding by the DFG (‘IMMUBASE’, 417,981,041) and is currently supported by ‘la Caixa’ Foundation (ID 10010434) and from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grand agreement No 847648, fellowship code is 104855, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Vertical transmission of microbial symbionts is interpreted as all offspring within a clutch being provided a similar number of symbionts irrespective of reproductive output (fecundity). This interpretation, however, stems primarily from oviparous insects and, thus, has yet to consider other major reproductive strategies. We used the viviparous spongeHalichondria paniceaand its obligate symbiont “CandidatusHalichondribacter symbioticus” to test the hypothesis that offspring receive quantitatively similar numbers of its obligate symbiont. This quantitative strategy of vertical transmission was not observed. Instead, we find thatH. paniceahas a maternal pool of ‘Ca. H. symbioticus’ that is partitioned proportionally to reproductive output and allometrically by offspring size. Moreover, ‘Ca. H. symbioticus’ could not be experimentally reduced in larvae by antibiotics, while the total bacterial community could be depleted. The ability to undergo metamorphosis was unaffected by this perturbation. Together, this demonstrates that the obligate symbiont ‘Ca. H. symbioticus’ is maternally provisioned and, thus, provides an additional strategy for how microbes can be vertically transmitted.

This protocol summarizes experience and recommendations regarding DNA and RNA extractions from the sponge Halichondria panicea and qPCR to quantify bacterial abundance. We have used both bacterial and sponge DNA and RNA for subsequent qPCR. Further, bacterial 16S rRNA amplicon sequencing was performed on DNA and sponge transcriptomes were sequenced successfully from extracted RNA.

On the sea floor, prey and predator commonly engage in a chemical warfare. Here, sponges thrive due to their specific and diverse chemical arsenal. Yet, some animals use these chemically-defended organisms as food and home. Most research on sponge chemical ecology has characterized crude extracts and investigated defences against generalist predators like fish. Consequently, we know little about intraindividual chemical dynamics and responses to specialist grazers. Here, we studied the response of the sponge Aplysina aerophoba to grazing by the opistobranch Tylodina perversa, in comparison to mechanical damage, at the cellular (via microscopy) and chemical level (via matrix-assisted laser desorption/ionization imaging mass spectrometry). We characterized the distribution of two major brominated compounds in A. aerophoba, aerophobin-2 and aeroplysinin-1, and identified a generalized wounding response that was similar in both wounding treatments: (i) brominated compound-carrying cells (spherulous cells) accumulated at the wound and (ii) secondary metabolites reallocated to the sponge surface. Upon mechanical damage, the wound turned dark due to oxidized compounds, causing T. perversa deterrence. During grazing, T. perversa’s way of feeding prevented oxidation. Thus, the sponge has not evolved a specific response to this specialist predator, but rather relies on rapid regeneration and flexible allocation of constitutive defences.

This chapter covers the current knowledge on interkingdom communication in marine sponge holobionts. Sponges (Porifera) are known to contain dense and diverse microbial symbiotic communities located extracellularly in the sponge tissue. Despite their early evolutionary origin, sponges already possess a diverse array of immune genes involved in host–microbe interactions. Besides sponge–bacteria interactions, quorum sensing and quorum quenching molecules have been identified in sponge-derived bacterial isolates and genomes that mediate bacterial signaling. Thirdly, a tripartite interaction of sponge cells, bacteria, and bacteriophages has recently been discovered that provides a functional understanding of phage-mediated immune evasion by bacteria on the cellular level. This review discusses the various types of interactions (host–microbe, microbe–microbe, phage–microbe–eukaryote) in sponge holobionts, which are considered to be just the iceberg of a plethora of possible interactions awaiting discovery in sponge–microbe interactions.

Sponges—like all multicellular organisms—are holobionts, complex ecosystems comprising the host and its microbiota. The symbiosis of sponges with their microbial communities is a highly complex system, requiring interaction mechanisms and adaptation on both sides. The microbiome seems to rely on eukaryotic-like protein domains, such as ankyrins, modifications of the lipopolysaccharide structure, CRISPR-Cas, toxin-antitoxin, and restriction-modification systems, as well as secondary metabolism to communicate with the host and within the microbial community, evade phagocytosis, and defend itself against foreign DNA. Secondary metabolites produced by certain symbionts may even defend the entire holobiont against predators. On the other hand, the immune system of the sponge itself has evolved to discriminate not only between self and nonself but also between its associated microbiota and foreign microbes, such as food bacteria. Sponge holobionts are inextricably dependent on the surrounding environmental conditions due to their sessile nature. Thus, we discuss the link between environmental stress and sponge disease and dysbiosis, with a particular focus on the holobiont’s response to ongoing global change. While some species may be the “winners of climate change,” other species are adversely affected, e.g., by metabolic and immune suppression, as well as microbiome shifts resulting in loss of symbiotic functions. Hence, a much better understanding of sponge holobionts and the underlying molecular mechanisms of host-microbe interaction is required before the fate of sponge holobionts in a changing ocean can finally be validated.

Phages are increasingly recognized as important members of host associated microbial communities. While recent studies have revealed vast genomic diversity in the virosphere, the new frontier is to understand how newly discovered phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we aim to understand the tripartite interplay between phages, bacterial symbionts and marine sponges. In a viromics approach, we discover 491 novel viral clusters and show that sponges, as filter-feeding organisms, are distinct viral niches. By using a nested sampling design, we show that each sponge individual of the four species investigated harbours its own unique virome, regardless of the tissue investigated. We further discover a novel, symbiont phage-encoded ankyrin domain-containing protein which appears to be widely spread in phages of many host-associated contexts including human. The ankyrin protein (ANKp) modulates the eukaryotic immune response against bacteria as confirmed in macrophage infection assays. We predict that the role of ANKp in nature is to facilitate co-existence in the tripartite interplay between phages, symbionts and sponges and possibly in many other host-microbe associations.

Almost all animals and plants are inhabited by diverse communities of microorganisms, the microbiota, thereby forming an integrated entity, the metaorganism. Natural selection should favor hosts that shape the community composition of these microbes to promote a beneficial host-microbe symbiosis. Indeed, animal hosts often pose selective environments, which only a subset of the environmentally available microbes are able to colonize. How these microbes assemble after colonization to form the complex microbiota is less clear. Neutral models are based on the assumption that the alternatives in microbiota community composition are selectively equivalent and thus entirely shaped by random population dynamics and dispersal. Here, we use the neutral model as a null hypothesis to assess microbiata composition in host organisms, which does not rely on invoking any adaptive processes underlying microbial community assembly. We show that the overall microbiota community structure from a wide range of host organisms, in particular including previously understudied invertebrates, is in many cases consistent with neutral expectations. Our approach allows to identify individual microbes that are deviating from the neutral expectation and are therefore interesting candidates for further study. Moreover, using simulated communities, we demonstrate that transient community states may play a role in the deviations from the neutral expectation. Our findings highlight that the consideration of neutral processes and temporal changes in community composition are critical for an in-depth understanding of microbiota-host interactions., A study of microbiome compositions from many animal host species across the tree of life reveals them to be consistent with neutral expectations, suggesting that after colonization, neutral processes and dispersal play a role in shaping host-associated microbial communities.

From protists to humans, all animals and plants are inhabited by microbial organisms. There is an increasing appreciation that these resident microbes influence the fitness of their plant and animal hosts, ultimately forming a metaorganism consisting of a uni- or multicellular host and a community of associated microorganisms. Research on host-microbe interactions has become an emerging cross-disciplinary field. In both vertebrates and invertebrates a complex microbiome confers immunological, metabolic and behavioural benefits; conversely, its disturbance can contribute to the development of disease states. However, the molecular and cellular mechanisms controlling the interactions within a metaorganism are poorly understood and many key interactions between the associated organisms remain unknown. In this perspective article, we outline some of the issues in interspecies interactions and in particular address the question of how metaorganisms react and adapt to inputs from extreme environments such as deserts, the intertidal zone, oligothrophic seas, and hydrothermal vents. published

Research on sponge microbial assemblages has revealed different trends in the geographic variability and specificity of bacterial symbionts. Here, we combined replicated terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of 16S rRNA gene sequences to investigate the biogeographic and host-specific structure of bacterial communities in two congeneric and sympatric sponges: Ircinia strobilina, two color morphs of Ircinia felix and ambient seawater. Samples were collected from five islands of the Bahamas separated by 80 to 400 km. T-RFLP profiles revealed significant differences in bacterial community structure among sponge hosts and ambient bacterioplankton. Pairwise statistical comparisons of clone libraries confirmed the specificity of the bacterial assemblages to each host species and differentiated symbiont communities between color morphs of I. felix. Overall, differences in bacterial communities within each host species and morph were unrelated to location. Our results show a high degree of symbiont fidelity to host sponge across a spatial scale of up to 400 km, suggesting that host-specific rather than biogeographic factors play a primary role in structuring and maintaining sponge-bacteria relationships in Ircinia species from the Bahamas.

Sponges have a significant impact on marine benthic communities, they are of biotechnological interest owing to their production of bioactive natural compounds, and they promise to provide insights into conserved mechanisms of host–microbe interactions in basal metazoans. The natural variability of sponge-microbe associations across species and environments provides a meaningful ecological and evolutionary framework to investigate animal-microbial symbiosis through experimentation in the field and also in aquaria. In addition, next-generation sequencing technologies have shed light on the genomic repertoire of the sponge host and revealed metabolic capacities and symbiotic lifestyle features of their microbiota. However, our understanding of symbiotic mechanisms is still in its infancy. Here, we discuss the potential and limitations of the sponge-microbe symbiosis as emerging models for animal-associated microbiota.

11 páginas,5 figuras, 4 tablas., Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the northwestern (NW) Mediterranean Sea, using replicated terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host species-specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living and on symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitrite oxidation, and sulfate reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health., This research was supported by the Spanish Government projects CTM2010-17755 and CTM2010-22218, by the Catalan Government grant 2009SGR-484 for Consolidated Research Groups, and by the U.S. National Science Foundation under grant 0853089.

Este artículo contiene 13 páginas, 6 figuras, 4 tablas., Recent episodes of mass mortalities in the Mediterranean Sea have been reported for the closely related marine sponges Ircinia fasciculata and Ircinia variabilis that live in sympatry. In this context, the assessment of the genetic diversity, bottlenecks and connectivity of these sponges has become urgent in order to evaluate the potential effects of mass mortalities on their latitudinal range. Our study aims to establish (1) the genetic structure, connectivity and signs of bottlenecks across the populations of I. fasciculata and (2) the hybridization levels between I. fasciculata and I. variabilis. To accomplish the first objective, 194 individuals of I. fasciculata from 12 locations across the Mediterranean were genotyped at 14 microsatellite loci. For the second objective, mitochondrial cytochrome c oxidase subunit I sequences of 16 individuals from both species were analyzed along with genotypes at 12 microsatellite loci of 40 individuals coexisting in 3 Mediterranean populations. We detected strong genetic structure along the Mediterranean for I. fasciculata, with high levels of inbreeding in all locations and bottleneck signs in most locations. Oceanographic barriers like the Almeria-Oran front, North-Balearic front and the Ligurian-Thyrrenian barrier seem to be impeding gene flow for I. fasciculata, adding population divergence to the pattern of isolation by distance derived from the low dispersal abilities of sponge larvae. Hybridization between both species occurred in some populations that might be increasing genetic diversity and somewhat palliating the genetic loss caused by population decimation in I. fasciculata., This research was funded by the Spanish Government project MARSYMBIOMICS CTM2013-43287-P and the Catalan Government Grant 2014SGR-336 for Consolidated Research Groups.

Archaea associated with marine sponges are active and influence the nitrogen metabolism of sponges. However, we know little about their occurrence, specificity, and persistence. We aimed to elucidate the relative importance of host specificity and biogeographic background in shaping the symbiotic archaeal communities. We investigated these communities in sympatric sponges from the Mediterranean (Ircinia fasciculata and Ircinia oros, sampled in summer and winter) and from the Caribbean (Ircinia strobilina and Mycale laxissima). PCR cloning and sequencing of archaeal 16S rRNA and amoA genes showed that the archaeal community composition and structure were different from that in seawater and varied among sponge species. We found that the communities were dominated by ammonia-oxidizing archaea closely related to Nitrosopumilus. The community in M. laxissima differed from that in Ircinia spp., including the sympatric sponge I. strobilina; yet, geographical clusters within Ircinia spp. were observed. Whereas archaeal phylotypes in Ircinia spp. were persistent and belong to 'sponge-enriched' clusters, archaea in M. laxissima were closely related with those from diverse habitats (i.e. seawater and sediments). For all four sponge species, the expression of the archaeal amoA gene was confirmed. Our results indicate that host-specific processes, such as host ecological strategy and evolutionary history, control the sponge-archaeal communities.

9 páginas, 3 tablas, 3 figuras., Dispersal limitation and environmental selection are the main processes shaping free-living microbial communities, but host-related factors may also play a major role in structuring symbiotic communities. Here, we aimed to determine the effects of isolation-by-distance and host species on the spatial structure of sponge-associated bacterial communities using as a model the abundant demosponge genus Ircinia. We targeted three co-occurring Ircinia species and used terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences to explore the differentiation of their bacterial communities across a scale of hundreds of kilometres in the Western Mediterranean Sea. Multivariate analysis and nonmetric multidimensional scaling plots of T-RFLP profiles showed that bacterial communities in Ircinia sponges were structured by host species and remained stable across sampling locations, despite geographic distances (80–800 km) and diverse local conditions. While significant differences among some locations were observed in Ircinia variabilis-derived communities, no correlation between geographic distance and community similarity was consistently detected for symbiotic bacteria in any host sponge species. Our results indicate that bacterial communities are mostly shaped by host species-specific factors and suggest that evolutionary processes acting on longterm symbiotic relationships have favored spatial stability of sponge-associated bacterial communities., This research was funded by the Spanish Government projects CTM2010-17755 and CTM2010- 22218, the Catalan Government grant 2009SGR-484 for Consolidated Research Groups, the US National Science Foundation under grant 0853089, and a FI-DGR fellowship to L.P.

11 páginas, 5 figuras, 5 tablas., Spatio-temporal variability in settlement and recruitment, high mortality during the first life-history stages, and selection may determine the genetic structure of cohorts of long-lived marine invertebrates at small scales. We conducted a spatial and temporal analysis of the common Mediterranean Sea urchin Paracentrotus lividus to determine the genetic structure of cohorts at different scales. In Tossa de Mar (NW Mediterranean), recruitment was followed over 5 consecutive springs (2006–2010). In spring 2008, recruits and two-year-old individuals were collected at 6 locations along East and South Iberian coasts separated from 200 to over 1,100 km. All cohorts presented a high genetic diversity based on a fragment of mtCOI. Our results showed a marked genetic homogeneity in the temporal monitoring and a low degree of spatial structure in 2006. In 2008, coupled with an abnormality in the usual circulation patterns in the area, the genetic structure of the southern populations studied changed markedly, with arrival of many private haplotypes. This fact highlights the importance of point events in renewing the genetic makeup of populations, which can only be detected through analysis of the cohort structure coupling temporal and spatial perspectives. Introduction The last few years have seen an increase in the number of studies aimed at discerning temporal genetic structure in marine invertebrates with large dispersal abilities. Longlived larval phases theoretically ensure connectivity over, This research was funded by projects CTM2010-2218 of the Spanish Ministry of Science and Innovation and BIOCON08-187 of the Foundation of the BBVA banking institution.

11 pages, 5 figures, 1 table, supplementary information https://doi.org/10.1038/s41396-021-00900-6, Bacteriophages (phages) are ubiquitous elements in nature, but their ecology and role in animals remains little understood. Sponges represent the oldest known extant animal-microbe symbiosis and are associated with dense and diverse microbial consortia. Here we investigate the tripartite interaction between phages, bacterial symbionts, and the sponge host. We combined imaging and bioinformatics to tackle important questions on who the phage hosts are and what the replication mode and spatial distribution within the animal is. This approach led to the discovery of distinct phage-microbe infection networks in sponge versus seawater microbiomes. A new correlative in situ imaging approach (‘PhageFISH-CLEM‘) localised phages within bacterial symbiont cells, but also within phagocytotically active sponge cells. We postulate that the phagocytosis of free virions by sponge cells modulates phage-bacteria ratios and ultimately controls infection dynamics. Prediction of phage replication strategies indicated a distinct pattern, where lysogeny dominates the sponge microbiome, likely fostered by sponge host-mediated virion clearance, while lysis dominates in seawater. Collectively, this work provides new insights into phage ecology within sponges, highlighting the importance of tripartite animal-phage-bacterium interplay in holobiont functioning. We anticipate that our imaging approach will be instrumental to further understanding of viral distribution and cellular association in animal hosts, We acknowledge funding by the DFG CRC1182 to UH (TPC4.3), TL (TPC4.2). MTJ was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Wuerzburg, and a Young Investigator Award of the CRC1182. SMM was supported by the Studienstiftung des Deutschen Volkes (German Academic Scholarship Foundation). CS was supported by the grants DFG STI700/1-1 and GRK2581 (P6). MR was supported by the Spanish Government grant (RTI2018-094187-B100) and ‘Generalitat de Catalunya’ research group grant (2017SGR1011). BED was supported by the Netherlands Organisation for Scientific Research (NWO) Vidi grant 864.14.004 and by the European Research Council (ERC) Consolidator grant 865694: DiversiPHI. /.../ Open Access funding enabled and organized by Projekt DEA, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

The recognition that all macroorganisms live in symbiotic association with microbial communities has opened up a new field in biology. Animals, plants, and algae are now considered holobionts, complex ecosystems consisting of the host, the microbiota, and the interactions among them. Accordingly, ecological concepts can be applied to understand the host-derived and microbial processes that govern the dynamics of the interactive networks within the holobiont. In marine systems, holobionts are further integrated into larger and more complex communities and ecosystems, a concept referred to as “nested ecosystems.” In this review, we discuss the concept of holobionts as dynamic ecosystems that interact at multiple scales and respond to environmental change. We focus on the symbiosis of sponges with their microbial communities—a symbiosis that has resulted in one of the most diverse and complex holobionts in the marine environment. In recent years, the field of sponge microbiology has remarkably advanced in terms of curated databases, standardized protocols, and information on the functions of the microbiota. Like a Russian doll, these microbial processes are translated into sponge holobiont functions that impact the surrounding ecosystem. For example, the sponge-associated microbial metabolisms, fueled by the high filtering capacity of the sponge host, substantially affect the biogeochemical cycling of key nutrients like carbon, nitrogen, and phosphorous. Since sponge holobionts are increasingly threatened by anthropogenic stressors that jeopardize the stability of the holobiont ecosystem, we discuss the link between environmental perturbations, dysbiosis, and sponge diseases. Experimental studies suggest that the microbial community composition is tightly linked to holobiont health, but whether dysbiosis is a cause or a consequence of holobiont collapse remains unresolved. Moreover, the potential role of the microbiome in mediating the capacity for holobionts to acclimate and adapt to environmental change is unknown. Future studies should aim to identify the mechanisms underlying holobiont dynamics at multiple scales, from the microbiome to the ecosystem, and develop management strategies to preserve the key functions provided by the sponge holobiont in our present and future oceans.