Maternal provisioning of an obligate symbiont in a sponge.

The transmission of microbes from mother to offspring is an ancient, advantageous, and widespread feature of metazoan life history. Despite this, little is known about the quantitative strategies taken to maintain symbioses across generations. The quantity of maternal microbes that is provided to each offspring through vertical transmission could theoretically be stochastic (no trend), consistent (an optimal range is allocated), or provisioned (a trade-off with fecundity). Examples currently come from animals that release free-living eggs (oviparous) and suggest that offspring are provided a consistent quantity of symbionts. The quantity of maternal microbes that is vertically transmitted in other major reproductive strategies has yet to be assessed. We used the brooding (viviparous) sponge Halichondria panicea to test whether offspring receive quantitatively similar numbers of maternal microbes. We observed that H. panicea has a maternal pool of the obligate symbiont Candidatus Halichondribacter symbioticus and that this maternal pool is provisioned proportionally to reproductive output and allometrically by offspring size. This pattern was not observed for the total bacterial community. Experimental perturbation by antibiotics could not reduce the abundance of Ca. H. symbioticus in larvae, while the total bacterial community could be reduced without affecting the ability of larvae to undergo metamorphosis. A trade-off between offspring size and number is, by definition, maternal provisioning and parallel differences in Ca. H. symbioticus abundance would suggest that this obligate symbiont is also provisioned.

Stability of a dominant sponge-symbiont in spite of antibiotic-induced microbiome disturbance.

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.

Biodiversity, environmental drivers, and sustainability of the global deep-sea sponge microbiome.

In the deep ocean symbioses between microbes and invertebrates are emerging as key drivers of ecosystem health and services. We present a large-scale analysis of microbial diversity in deep-sea sponges (Porifera) from scales of sponge individuals to ocean basins, covering 52 locations, 1077 host individuals translating into 169 sponge species (including understudied glass sponges), and 469 reference samples, collected anew during 21 ship-based expeditions. We demonstrate the impacts of the sponge microbial abundance status, geographic distance, sponge phylogeny, and the physical-biogeochemical environment as drivers of microbiome composition, in descending order of relevance. Our study further discloses that fundamental concepts of sponge microbiology apply robustly to sponges from the deep-sea across distances of >10,000 km. Deep-sea sponge microbiomes are less complex, yet more heterogeneous, than their shallow-water counterparts. Our analysis underscores the uniqueness of each deep-sea sponge ground based on which we provide critical knowledge for conservation of these vulnerable ecosystems.

Symbiont transmission in marine sponges: reproduction, development, and metamorphosis.

Marine sponges (phylum Porifera) form symbioses with diverse microbial communities that can be transmitted between generations through their developmental stages. Here, we integrate embryology and microbiology to review how symbiotic microorganisms are transmitted in this early-diverging lineage. We describe that vertical transmission is widespread but not universal, that microbes are vertically transmitted during a select developmental window, and that properties of the developmental microbiome depends on whether a species is a high or low microbial abundance sponge. Reproduction, development, and symbiosis are thus deeply rooted, but why these partnerships form remains the central and elusive tenet of these developmental symbioses.

Wounding response in Porifera (sponges) activates ancestral signaling cascades involved in animal healing, regeneration, and cancer.

Upon injury, the homeostatic balance that ensures tissue function is disrupted. Wound-induced signaling triggers the recovery of tissue integrity and offers a context to understand the molecular mechanisms for restoring tissue homeostasis upon disturbances. Marine sessile animals are particularly vulnerable to chronic wounds caused by grazers that can compromise prey's health. Yet, in comparison to other stressors like warming or acidification, we know little on how marine animals respond to grazing. Marine sponges (Phylum Porifera) are among the earliest-diverging animals and play key roles in the ecosystem; but they remain largely understudied. Here, we investigated the transcriptomic responses to injury caused by a specialist spongivorous opisthobranch (i.e., grazing treatment) or by clipping with a scalpel (i.e., mechanical damage treatment), in comparison to control sponges. We collected samples 3 h, 1 d, and 6 d post-treatment for differential gene expression analysis on RNA-seq data. Both grazing and mechanical damage activated a similar transcriptomic response, including a clotting-like cascade (e.g., with genes annotated as transglutaminases, metalloproteases, and integrins), calcium signaling, and Wnt and mitogen-activated protein kinase signaling pathways. Wound-induced gene expression signature in sponges resembles the initial steps of whole-body regeneration in other animals. Also, the set of genes responding to wounding in sponges included putative orthologs of cancer-related human genes. Further insights can be gained from taking sponge wound healing as an experimental system to understand how ancient genes and regulatory networks determine healthy animal tissues.

Individuality in the Immune Repertoire and Induced Response of the Sponge Halichondria panicea.

The animal immune system mediates host-microbe interactions from the host perspective. Pattern recognition receptors (PRRs) and the downstream signaling cascades they induce are a central part of animal innate immunity. These molecular immune mechanisms are still not fully understood, particularly in terms of baseline immunity vs induced specific responses regulated upon microbial signals. Early-divergent phyla like sponges (Porifera) can help to identify the evolutionarily conserved mechanisms of immune signaling. We characterized both the expressed immune gene repertoire and the induced response to lipopolysaccharides (LPS) in Halichondria panicea, a promising model for sponge symbioses. We exposed sponges under controlled experimental conditions to bacterial LPS and performed RNA-seq on samples taken 1h and 6h after exposure. H. panicea possesses a diverse array of putative PRRs. While part of those PRRs was constitutively expressed in all analyzed sponges, the majority was expressed individual-specific and regardless of LPS treatment or timepoint. The induced immune response by LPS involved differential regulation of genes related to signaling and recognition, more specifically GTPases and post-translational regulation mechanisms like ubiquitination and phosphorylation. We have discovered individuality in both the immune receptor repertoire and the response to LPS, which may translate into holobiont fitness and susceptibility to stress. The three different layers of immune gene control observed in this study, - namely constitutive expression, individual-specific expression, and induced genes -, draw a complex picture of the innate immune gene regulation in H. panicea. Most likely this reflects synergistic interactions among the different components of immunity in their role to control and respond to a stable microbiome, seawater bacteria, and potential pathogens.

Opisthobranch grazing results in mobilisation of spherulous cells and re-allocation of secondary metabolites in the sponge Aplysina aerophoba.

Sponges thrive in marine benthic communities due to their specific and diverse chemical arsenal against predators and competitors. Yet, some animals specifically overcome these defences and use sponges as food and home. Most research on sponge chemical ecology has characterised crude extracts and investigated defences against generalist predators like fish. Consequently, we know little about chemical dynamics in the tissue and responses to specialist grazers. Here, we studied the response of the sponge Aplysina aerophoba to grazing by the opisthobranch Tylodina perversa, in comparison to mechanical damage, at the cellular (via microscopy) and chemical level (via matrix-assisted laser desorption/ionization imaging mass spectrometry, MALDI-imaging MS). We characterised the distribution of two major brominated alkaloids in A. aerophoba, aerophobin-2 and aeroplysinin-1, and identified a generalised 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 oxidised 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.

Receptors Mediating Host-Microbiota Communication in the Metaorganism: The Invertebrate Perspective.

Multicellular organisms live in close association with a plethora of microorganism, which have a profound effect on multiple host functions. As such, the microbiota and its host form an intimate functional entity, termed the metaorganism or holobiont. But how does the metaorganism communicate? Which receptors recognize microbial signals, mediate the effect of the microbiota on host physiology or regulate microbiota composition and homeostasis? In this review we provide an overview on the function of different receptor classes in animal host-microbiota communication. We put a special focus on invertebrate hosts, including both traditional invertebrate models such as Drosophila melanogaster and Caenorhabditis elegans and "non-model" invertebrates in microbiota research. Finally, we highlight the potential of invertebrate systems in studying mechanism of host-microbiota interactions.

A Phage Protein Aids Bacterial Symbionts in Eukaryote Immune Evasion.

Phages are increasingly recognized as important members of host-associated microbiomes, with a vast genomic diversity. The new frontier is to understand how phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we use marine sponges as a model to investigate the interplay between phages, bacterial symbionts, and eukaryotic hosts. Using viral metagenomics, we find that sponges, although massively filtering seawater, harbor species-specific and even individually unique viral signatures that are taxonomically distinct from other environments. We further discover a symbiont phage-encoded ankyrin-domain-containing protein, which is widely spread in phages of many host-associated contexts including human. We confirm in macrophage infection assays that the ankyrin protein (ANKp) modulates the eukaryotic host immune response against bacteria. We predict that the role of ANKp in nature is to facilitate coexistence in the tripartite interplay between phages, symbionts, and sponges and possibly many other host-microbe associations.

Neutrality in the Metaorganism.

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.

Differential expression of immune receptors in two marine sponges upon exposure to microbial-associated molecular patterns.

The innate immune system helps animals to navigate the microbial world. The response to microbes relies on the specific recognition of microbial-associated molecular patterns (MAMPs) by immune receptors. Sponges (phylum Porifera), as early-diverging animals, provide insights into conserved mechanisms for animal-microbe crosstalk. However, experimental data is limited. We adopted an experimental approach followed by RNA-Seq and differential gene expression analysis in order to characterise the sponge immune response. Two Mediterranean species, Aplysina aerophoba and Dysidea avara, were exposed to a "cocktail" of MAMPs (lipopolysaccharide and peptidoglycan) or to sterile artificial seawater (control) and sampled 1 h, 3 h, and 5 h post-treatment for RNA-Seq. The response involved, first and foremost, a higher number of differentially-expressed genes in A. aerophoba than D. avara. Secondly, while both species constitutively express a diverse repertoire of immune receptors, they differed in their expression profiles upon MAMP challenge. The response in D. avara was mediated by increased expression of two NLR genes, whereas the response in A. aerophoba involved SRCR and GPCR genes. From the set of annotated genes we infer that both species activated apoptosis in response to MAMPs while in A. aerophoba phagocytosis was additionally stimulated. Our study assessed for the first time the transcriptomic responses of sponges to MAMPs and revealed conserved and species-specific features of poriferan immunity as well as genes potentially relevant to animal-microbe interactions.

Metaorganisms in extreme environments: do microbes play a role in organismal adaptation?

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.

Emerging Sponge Models of Animal-Microbe Symbioses.

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.

Symbiotic archaea in marine sponges show stability and host specificity in community structure and ammonia oxidation functionality.

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.

Till death do us part: stable sponge-bacteria associations under thermal and food shortage stresses.

Sporadic mass mortality events of Mediterranean sponges following periods of anomalously high temperatures or longer than usual stratification of the seawater column (i.e. low food availability) suggest that these animals are sensitive to environmental stresses. The Mediterranean sponges Ircinia fasciculata and I. oros harbor distinct, species-specific bacterial communities that are highly stable over time and space but little is known about how anomalous environmental conditions affect the structure of the resident bacterial communities. Here, we monitored the bacterial communities in I. fasciculata (largely affected by mass mortalities) and I. oros (overall unaffected) maintained in aquaria during 3 weeks under 4 treatments that mimicked realistic stress pressures: control conditions (13°C, unfiltered seawater), low food availability (13°C, 0.1 µm-filtered seawater), elevated temperatures (25°C, unfiltered seawater), and a combination of the 2 stressors (25°C, 0.1 µm-filtered seawater). Bacterial community structure was assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences and transmission electron microscopy (TEM). As I. fasciculata harbors cyanobacteria, we also measured chlorophyll a (chl a) levels in this species. Multivariate analysis revealed no significant differences in bacterial T-RFLP profiles among treatments for either host sponge species, indicating no effect of high temperatures and food shortage on symbiont community structure. In I. fasciculata, chl a content did not significantly differ among treatments although TEM micrographs revealed some cyanobacteria cells undergoing degradation when exposed to both elevated temperature and food shortage conditions. Arguably, longer-term treatments (months) could have eventually affected bacterial community structure. However, we evidenced no appreciable decay of the symbiotic community in response to medium-term (3 weeks) environmental anomalies purported to cause the recurrent sponge mortality episodes. Thus, changes in symbiont structure are not likely the proximate cause for these reported mortality events.

Host rules: spatial stability of bacterial communities associated with marine sponges (Ircinia spp.) in the Western Mediterranean Sea.

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 long-term symbiotic relationships have favored spatial stability of sponge-associated bacterial communities.

Biogeography and host fidelity of bacterial communities in Ircinia spp. from the Bahamas.

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.

Stability of sponge-associated bacteria over large seasonal shifts in temperature and irradiance.

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.