Marine subsidy promotes spatial and dietary niche variation in an omnivore,the Keen’s mouse (Peromyscus keeni)

Marine‐derived resource subsidies can generate intrapopulation variation in the behaviors and diets of terrestrial consumers. How omnivores respond, given their multiple trophic interactions, is not well understood. We sampled mice (Peromyscus keeni) and their food sources at five sites on three islands of the Central Coast of British Columbia, Canada, to test predictions regarding variation in the spatial behavior and consumption of marine‐subsidized foods among individuals. About 50% of detections (n = 27 recaptures) occurred at traps closest to shoreline (25 m), with capture frequencies declining significantly inland (up to 200 m). Stable isotope signatures (δ ¹³C and δ ¹⁵N), particularly δ ¹⁵N, in plant foods, forest arthropod prey, and mouse feces were significantly enriched near shorelines compared with inland, while δ ¹³C patterns were more variable. Bayesian isotope mixing models applied to isotope values in mouse hair indicated that over one‐third (35–37%) of diet was comprised of beach‐dwelling arthropods, a marine‐derived food source. Males were more abundant near the shoreline than females and consumed more marine‐derived prey, regardless of reproductive status or availability of other food sources. Our results identify how multiple pathways of marine nutrient transfer can subsidize terrestrial omnivores and how subsets of recipient populations can show variation in spatial and dietary response.     

Coscinosulfate, a CDC25 phosphatase inhibitor from the sponge Coscinoderma mathewsi

The dual specificity CDC25 phosphatases dephosphorylate two inhibitory phospho-amino acids of cyclin-dependent kinases, a major family of cell cycle regulators. CDC25 inhibitors constitute new anti-mitotic agents with potential anticancer activity. While screening through a collection of natural products derived from marine organisms for CDC25A inhibitors, we purified and identified coscinosulfate 1, a sesquiterpene sulfate from the New Caledonian sponge Coscinoderma matthewsi, along with 4. The purified compound 1 displayed significant inhibitory activity towards CDC25A (IC(50): 3 microM).     

Bioactive Natural Products from Papua New Guinea Marine Sponges

The discovery of novel natural products for drug development relies heavily upon a rich biodiversity, of which the marine environment is an obvious example. Marine natural product research has spawned several drugs and many other candidates, some of which are the focus of current clinical trials. The sponge megadiversity of Papua New Guinea is a rich but underexplored source of bioactive natural products. Here, we review some of the many natural products derived from PNG sponges with an emphasis on those with interesting biological activity and, therefore, drug potential. Many bioactive natural products discussed here appear to be derived from non‐ribosomal peptide and polyketide biosynthesis pathways, strongly suggesting a microbial origin of these compounds. With this in mind, we also explore the notion of sponge‐symbiont biosynthesis of these bioactive compounds and present examples to support the working hypothesis.     

Effects of spawning Pacific salmon on terrestrial invertebrates: Insects near spawning habitat are isotopically enriched with nitrogen‐15 but display no differences in body size

When Pacific salmon (Oncorhynchus spp.) spawn and die, they deliver marine‐derived nutrient subsidies to freshwater and riparian ecosystems. These subsidies can alter the behavior, productivity, and abundance of recipient species and their habitats. Isotopes, such as nitrogen‐15 (¹⁵N), are often used to trace the destination of marine‐derived nutrients in riparian habitats. However, few studies have tested for correlations between stable isotopes and physiological responses of riparian organisms. We examined whether increases in δ ¹⁵N in terrestrial insect bodies adjacent to salmon spawning habitat translate to changes in percent nitrogen content and body size. This involved comparisons between distance from a salmon‐bearing river, marine‐derived nutrients in soils and insects, soil moisture content, and body size and nitrogen content in two common beetle families (Coleoptera: Curculionidae, Carabidae). As predicted, δ¹⁵N in riparian soils attenuated with distance from the river but was unaffected by soil moisture. This gradient was mirrored by δ¹⁵N in the herbivorous curculionid beetles, whereas carabid beetles, which feed at a higher trophic level and are more mobile, did not show discernable patterns in their δ¹⁵N content. Additionally, neither distance from the river nor body δ¹⁵N content was related to beetle body size. We also found that nitrogen‐15 was not correlated with total percent nitrogen in insect bodies, meaning that the presence of spawning salmon did not increase the percent nitrogen content of these insects. We conclude that while salmon‐derived nutrients had entered terrestrial food webs, the presence of δ¹⁵N alone did not indicate meaningful physiological changes in these insects in terms of percent nitrogen nor body size. While stable isotopes may be useful tracers of marine‐derived nutrients, they cannot necessarily be used as a proxy for physiologically important response variables.     

Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage–host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage–host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages’ host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

This study presents the BASEL collection of phages that infect the model bacterium Escherichia coli; this resource for the community is representative of natural E. coli phage diversity and has been extensively characterized phenotypically and genomically.     

Composite Sampling of a Bacillus anthracis Surrogate with Cellulose Sponge Surface Samplers from a Nonporous Surface

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm² (four separate 929 cm² areas), larger than the 645 cm² prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.     

Structural Variation among Wild and Industrial Strains of Penicillium chrysogenum

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.     

A Sponge-like Structure Involved in the Association and Transport of Maternal Products during Drosophila Oogenesis

Localization of maternally provided RNAs during oogenesis is required for formation of the antero–posterior axis of the Drosophila embryo. Here we describe a subcellular structure in nurse cells and oocytes which may function as an intracellular compartment for assembly and transport of maternal products involved in RNA localization. This structure, which we have termed “sponge body,” consists of ER-like cisternae, embedded in an amorphous electron-dense mass. It lacks a surrounding membrane and is frequently associated with mitochondria. The sponge bodies are not identical to the Golgi complexes. We suggest that the sponge bodies are homologous to the mitochondrial cloud in Xenopus oocytes, a granulo-fibrillar structure that contains RNAs involved in patterning of the embryo.     

Tolerance of Sponge Assemblages to Temperature Anomalies: Resilience and Proliferation of Sponges following the 1997–8 El-Ni?o Southern Oscillation

Coral reefs across the world are under threat from a range of stressors, and while there has been considerable focus on the impacts of these stressors on corals, far less is known about their effect on other reef organisms. The 1997–8 El-Niño Southern Oscillation (ENSO) had notable and severe impacts on coral reefs worldwide, but not all reef organisms were negatively impacted by this large-scale event. Here we describe how the sponge fauna at Bahia, Brazil was influenced by the 1997–8 ENSO event. Sponge assemblages from three contrasting reef habitats (reef tops, walls and shallow banks) at four sites were assessed annually from 1995 to 2011. The within-habitat sponge diversity did not vary significantly across the study period; however, there was a significant increase in density in all habitats. Multivariate analyses revealed no significant difference in sponge assemblage composition (ANOSIM) between pre- and post-ENSO years for any of the habitats, suggesting that neither the 1997–8 nor any subsequent smaller ENSO events have had any measurable impact on the reef sponge assemblage. Importantly, this is in marked contrast to the results previously reported for a suite of other taxa (including corals, echinoderms, bryozoans, and ascidians), which all suffered mass mortalities as a result of the ENSO event. Our results suggest that of all reef taxa, sponges have the potential to be resilient to large-scale thermal stress events and we hypothesize that sponges might be less affected by projected increases in sea surface temperature compared to other major groups of reef organisms.     

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2′–3′ phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

Isolation and Characterization of New Phenolic Compounds with Estrogen Biosynthesis-Inhibiting and Antioxidation Activities from Broussonetia papyrifera Leaves

Broussonetia papyrifera leaves (BPL) as a traditional Chinese medicine are also used in livestock feed for stimulating reproduction, adipose tissue and muscle development; however, the mechanism of their action is still unknown. Through estrogen biosynthesis-guided fractionation in human ovarian granulosa-like KGN cells, five new phenolic glycosides, broussoside A–E(1–5), along with fifteen known dietary phenolic compounds, were isolated from the n-butanol extract of BPL, and their structures were elucidated on the basis of NMR spectra analysis and chemical evidence. New compounds 3, 4, 5 and the known compounds 9 and 10 were found to potently inhibit estrogen biosynthesis in KGN cells. In addition, compounds 9, 17, 18, and 20 showed strong antioxidant activity against ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH (1, 1′-diphenyl -2-picryl-hydrazyl radical) assays. These findings suggest that BPL may improve meat quality through the regulation of estrogen biosynthesis. Furthermore, they may be useful for the discovery of potential aromatase modulators from natural products. Finally, they could be considered as a new source for natural antioxidants.     

Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process developments and economics

Large-scale, renewable supplies of chemical constituents derived from marine invertebrates have limited development of potential new natural product drugs. This paper describes the development of two in-sea aquaculture systems designed and engineered for production of large quantities of biomass for two species of marine invertebrates desired for their natural product chemical constituents. The two invertebrates and their products were: (1) the cosmopolitan, arborescent bryozoan Bugula neritina (Phylum Bryozoa) for its anticancer chemical constituent bryostatin 1; and (2) Ecteinascidia turbinate (Phylum Tunicata) the source of anticancer ecteinascidin 743. For the third invertebrate Phylum Porifera, and its representative sponge Acanthella cavernosa (desired for its anti-parasitic and anti-infective kalihinols) in-sea systems were not developed in favor of controlled environment tank aquaculture systems. For the bryozoan and tunicate, projected economics for commercial-scale in-sea production proved cost effective. This was in contrast to the controlled environment sponge culture tank system, which did not prove to be economical due to inherent slow growth and low natural product yields of the sponge in culture. A non-destructive method for "milking" natural product chemicals from sponges was tested and is described.     

Acceptability and Feasibility of Repeated Mucosal Specimen Collection in Clinical Trial Participants in Kenya

Background

Mucosal specimens are essential to evaluate compartmentalized immune responses to HIV vaccine candidates and other mucosally targeted investigational products. We studied the acceptability and feasibility of repeated mucosal sampling in East African clinical trial participants at low risk of HIV and other sexually transmitted infections.

Methods and Findings

The Kenya AIDS Vaccine Initiative (KAVI) enrolled participants into three Phase 1 trials of preventive HIV candidate vaccines in 2011–2012 at two clinical research centers in Nairobi. After informed consent to a mucosal sub-study, participants were asked to undergo collection of mucosal secretions (saliva, oral fluids, semen, cervico-vaginal and rectal), but could opt out of any collection at any visit. Specimens were collected at baseline and two additional time points. A tolerability questionnaire was administered at the final sub-study visit. Of 105 trial participants, 27 of 34 women (79%) and 62 of 71 men (87%) enrolled in the mucosal sub-study. Nearly all sub-study participants gave saliva and oral fluids at all visits. Semen was collected from about half the participating men (47–48%) at all visits. Cervico-vaginal secretions were collected by Softcup from about two thirds of women (63%) at baseline, increasing to 78% at the following visits, with similar numbers for cervical secretion collection by Merocel sponge; about half of women (52%) gave cervico-vaginal samples at all visits. Rectal secretions were collected with Merocel sponge from about a quarter of both men and women (24%) at all 3 visits, with 16% of men and 19% of women giving rectal samples at all visits.

Conclusions

Repeated mucosal sampling in clinical trial participants in Kenya is feasible, with a good proportion of participants consenting to most sampling methods with the exception of rectal samples. Experienced staff members of both sexes and trained counselors with standardized messaging may improve acceptance of rectal sampling.     

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.     

Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction

Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.     

Freshwater shrimp-sponge association from an ancient lake

Shrimp–sponge associations occur frequently in marine ecosystems, serving as model systems for the evolution of eusociality. Here, we describe the first known instance of such association in freshwater from an ancient lake in Indonesia. The shrimp Caridina spongicola forms an exclusive and probably commensal association with a yet undescribed spongillinid sponge. Phylogenetic and ecological data suggest a comparatively recent origin of both taxa. Climatic fluctuations may have facilitated speciation and occasional hybridization of the shrimp species, which is derived from a rock-dwelling ancestor. Their extremely localized occurrence in an increasingly disturbed area makes both taxa a conservation priority.     

Nepetin inhibits osteoclastogenesis by inhibiting RANKL‐induced activation of NF‐κB and MAPK signalling pathway,and autophagy

Aseptic prosthetic loosening due to wear particle–induced inflammatory osteolysis is the main cause of failure for artificial joint replacement. The inflammatory response and the production of pro‐osteoclastic factors lead to elevation of osteoclast formation and excessive activity results in extensive bone destruction around the bone‐implant interface. Here we showed that Nepetin, a natural bioactive flavonoid with proven anti‐inflammatory and anti‐proliferative properties, potently inhibited RANKL‐induced osteoclast differentiation, formation and bone resorption in vitro, and protected mice against the deleterious effects of titanium particle–induced calvarial osteolysis in vivo. Mechanistically, Nepetin attenuated RANKL‐induced activation of NF‐κB and MAPK signalling pathways and TRAF6‐dependent ubiquitination of Beclin 1 which is necessary for the induction of autophagy. In brief, our study demonstrates the potential therapeutic application of Nepetin against osteoclast‐mediated osteolytic diseases.     

Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase–like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde–2-piperidinol products, we evaluated the lysyl oxidase–like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde–2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.     

Host-specificity among abundant and rare taxa in the sponge microbiome

Microbial communities have a key role in the physiology of the sponge host, and it is therefore essential to understand the stability and specificity of sponge–symbiont associations. Host-specific bacterial associations spanning large geographic distance are widely acknowledged in sponges. However, the full spectrum of specificity remains unclear. In particular, it is not known whether closely related sponges host similar or very different microbiota over wide bathymetric and geographic gradients, and whether specific associations extend to the rare members of the sponge microbiome. Using the ultra-deep Illumina sequencing technology, we conducted a comparison of sponge bacterial communities in seven closely related Hexadella species with a well-resolved host phylogeny, as well as of a distantly related sponge Mycale. These samples spanned unprecedentedly large bathymetric (15–960 m) gradients and varying European locations. In addition, this study included a bacterial community analysis of the local background seawater for both Mycale and the widespread deep-sea taxa Hexadella cf. dedritifera. We observed a striking diversity of microbes associated with the sponges, spanning 47 bacterial phyla. The data did not reveal any Hexadella microbiota co-speciation pattern, but confirmed sponge-specific and species-specific host–bacteria associations, even within extremely low abundant taxa. Oligotyping analysis also revealed differential enrichment preferences of closely related Nitrospira members in closely related sponges species. Overall, these results demonstrate highly diverse, remarkably specific and stable sponge–bacteria associations that extend to members of the rare biosphere at a very fine phylogenetic scale, over significant geographic and bathymetric gradients.