Decoupling of strain- and intrastrain-level interactions of microbiomes in a sponge holobiont.

Holobionts are highly organized assemblages of eukaryotic hosts, cellular microbial symbionts, and viruses, whose interactions and evolution involve complex biological processes. It is largely unknown which specific determinants drive similarity or individuality in genetic diversity between holobionts. Here, we combine short- and long-read sequencing and DNA-proximity-linkage technologies to investigate intraspecific diversity of the microbiomes, including host-resolved viruses, in individuals of a model marine sponge. We find strong impacts of the sponge host and the cellular hosts of viruses on strain-level organization of the holobiont, whereas substantial overlap in nucleotide diversity between holobionts suggests frequent exchanges of microbial cells and viruses at intrastrain level in the local sponge population. Immune-evasive arms races likely restricted virus-host co-evolution at the intrastrain level, generated holobiont-specific genome variations, and linked virus-host genetics through recombination. Our work shows that a decoupling of strain- and intrastrain-level interactions is a key factor in the genetic diversification of holobionts.

Viral ecogenomics across the Porifera.

BACKGROUND: Viruses directly affect the most important biological processes in the ocean via their regulation of prokaryotic and eukaryotic populations. Marine sponges form stable symbiotic partnerships with a wide diversity of microorganisms and this high symbiont complexity makes them an ideal model for studying viral ecology. Here, we used morphological and molecular approaches to illuminate the diversity and function of viruses inhabiting nine sponge species from the Great Barrier Reef and seven from the Red Sea. RESULTS: Viromic sequencing revealed host-specific and site-specific patterns in the viral assemblages, with all sponge species dominated by the bacteriophage order Caudovirales but also containing variable representation from the nucleocytoplasmic large DNA virus families Mimiviridae, Marseilleviridae, Phycodnaviridae, Ascoviridae, Iridoviridae, Asfarviridae and Poxviridae. Whilst core viral functions related to replication, infection and structure were largely consistent across the sponge viromes, functional profiles varied significantly between species and sites largely due to differential representation of putative auxiliary metabolic genes (AMGs) and accessory genes, including those associated with herbicide resistance, heavy metal resistance and nylon degradation. Furthermore, putative AMGs varied with the composition and abundance of the sponge-associated microbiome. For instance, genes associated with antimicrobial activity were enriched in low microbial abundance sponges, genes associated with nitrogen metabolism were enriched in high microbial abundance sponges and genes related to cellulose biosynthesis were enriched in species that host photosynthetic symbionts. CONCLUSIONS: Our results highlight the diverse functional roles that viruses can play in marine sponges and are consistent with our current understanding of sponge ecology. Differential representation of putative viral AMGs and accessory genes across sponge species illustrate the diverse suite of beneficial roles viruses can play in the functional ecology of these complex reef holobionts. Video Abstract.

Marine virus predation by non-host organisms.

Viruses are the most abundant biological entities in marine environments, however, despite its potential ecological implications, little is known about virus removal by ambient non-host organisms. Here, we examined the effects of a variety of non-host organisms on the removal of viruses. The marine algal virus PgV-07T (infective to Phaeocystis globosa) can be discriminated from bacteriophages using flow cytometry, facilitating its use as a representative model system. Of all the non-host organisms tested, anemones, polychaete larvae, sea squirts, crabs, cockles, oysters and sponges significantly reduced viral abundance. The latter four species reduced viral abundance the most, by 90, 43, 12 and 98% over 24 h, respectively. Breadcrumb sponges instantly removed viruses at high rates (176 mL h-1 g tissue dry wt-1) which continued over an extended period of time. The variety of non-host organisms capable of reducing viral abundance highlights that viral loss by ambient organisms is an overlooked avenue of viral ecology. Moreover, our finding that temperate sponges have the huge potential for constant and effective removal of viruses from the water column demonstrates that natural viral loss has, thus far, been underestimated.

dsRNA-seq Reveals Novel RNA Virus and Virus-Like Putative Complete Genome Sequences from Hymeniacidon sp. Sponge.

Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genome sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing, called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences, including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.

Potential Interactions between Clade SUP05 Sulfur-Oxidizing Bacteria and Phages in Hydrothermal Vent Sponges.

In deep-sea hydrothermal vent environments, sulfur-oxidizing bacteria belonging to the clade SUP05 are crucial symbionts of invertebrate animals. Marine viruses, as the most abundant biological entities in the ocean, play essential roles in regulating the sulfur metabolism of the SUP05 bacteria. To date, vent sponge-associated SUP05 and their phages have not been well documented. The current study analyzed microbiomes of Haplosclerida sponges from hydrothermal vents in the Okinawa Trough and recovered the dominant SUP05 genome, designated VS-SUP05. Phylogenetic analysis showed that VS-SUP05 was closely related to endosymbiotic SUP05 strains from mussels living in deep-sea hydrothermal vent fields. Homology and metabolic pathway comparisons against free-living and symbiotic SUP05 strains revealed that the VS-SUP05 genome shared many features with the deep-sea mussel symbionts. Supporting a potentially symbiotic lifestyle, the VS-SUP05 genome contained genes involved in the synthesis of essential amino acids and cofactors that are desired by the host. Analysis of sponge-associated viral sequences revealed putative VS-SUP05 phages, all of which were double-stranded viruses belonging to the families Myoviridae, Siphoviridae, Podoviridae, and Microviridae Among the phage sequences, one contig contained metabolic genes (iscR, iscS, and iscU) involved in iron-sulfur cluster formation. Interestingly, genome sequence comparison revealed horizontal transfer of the iscS gene among phages, VS-SUP05, and other symbiotic SUP05 strains, indicating an interaction between marine phages and SUP05 symbionts. Overall, our findings confirm the presence of SUP05 bacteria and their phages in sponges from deep-sea vents and imply a beneficial interaction that allows adaptation of the host sponge to the hydrothermal vent environment.IMPORTANCE Chemosynthetic SUP05 bacteria dominate the microbial communities of deep-sea hydrothermal vents around the world, SUP05 bacteria utilize reduced chemical compounds in vent fluids and commonly form symbioses with invertebrate organisms. This symbiotic relationship could be key to adapting to such unique and extreme environments. Viruses are the most abundant biological entities on the planet and have been identified in hydrothermal vent environments. However, their interactions with the symbiotic microbes of the SUP05 clade, along with their role in the symbiotic system, remain unclear. Here, using metagenomic sequence-based analyses, we determined that bacteriophages may support metabolism in SUP05 bacteria and play a role in the sponge-associated symbiosis system in hydrothermal vent environments.

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.

Thermal stress modifies the marine sponge virome.

Marine sponges can form stable partnerships with a wide diversity of microbes and viruses, and this high intraspecies symbiont specificity makes them ideal models for exploring how host-associated viromes respond to changing environmental conditions. Here we exposed the abundant Great Barrier Reef sponge Rhopaloiedes odorabile to elevated seawater temperature for 48 h and utilised a metaviromic approach to assess the response of the associated viral community. An increase in endogenous retro-transcribing viruses within the Caulimorviridae and Retroviridae families was detected within the first 12 h of exposure to 32 °C, and a 30-fold increase in retro-transcribing viruses was evident after 48 h at 32 °C. Thermally stressed sponges also exhibited a complete loss of ssDNA viruses which were prevalent in field samples and sponges from the control temperature treatment. Despite these viromic changes, functional analysis failed to detect any loss or gain of auxiliary metabolic genes, indicating that viral communities are not providing a direct competitive advantage to their host under thermal stress. In contrast, endogenous sponge retro-transcribing viruses appear to be replicating under thermal stress, and consistent with retroviral infections in other organisms, may be contributing to the previously described rapid decline in host health evident at elevated temperature.

MicroRNA and Nonsense Transcripts as Putative Viral Evasion Mechanisms.

Viral proteins encode numerous antiviral activities to modify the host immunity. In this article, we hypothesize that viral genomes and gene transcripts interfere with host gene expression using passive mechanisms to deregulate host microRNA (miRNA) activity. We postulate that various RNA viruses mimic or block binding between a host miRNA and its target transcript, a phenomenon mediated by the miRNA seed site at the 5' end of miRNA. Virus-encoded miRNA seed sponges (vSSs) can potentially bind to host miRNA seed sites and prevent interaction with their native targets thereby relieving native miRNA suppression. In contrast, virus-encoded miRNA seed mimics (vSMs) may mediate considerable downregulation of host miRNA activity. We analyzed genomes from diverse RNA viruses for vSS and vSM signatures and found an abundance of these motifs indicating that this may be a mechanism of deceiving host immunity. Employing respiratory syncytial virus and measles virus as models, we reveal that regions surrounding vSS or vSM motifs have features characteristics of pre-miRNA templates and show that RSV viral transcripts are processed into small RNAs that may behave as vSS or vSM effectors. These data suggest that complex molecular interactions likely occur at the host-virus interface. Identifying the mechanisms in the network of interactions between the host and viral transcripts can help uncover ways to improve vaccine efficacy, therapeutics, and potentially mitigate the adverse events that may be associated with some vaccines.

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

Evaluation of sewage source and fate on southeast Florida coastal reefs.

Water, sponge and coral samples were collected from stations impacted by a variety of pollution sources and screened for human enteric viruses as conservative markers for human sewage. While human enteroviruses and adenoviruses were not detected, noroviruses (NoV; human genogroups I and II) were detected in 31% of samples (especially in sponge tissue). Stations near inlets were the only ones to show multiple sample types positive for NoV. Fecal indicator bacteria and enteric viruses were further evaluated at multiple inlet stations on an outgoing tide. Greatest indicator concentrations and highest prevalence of viruses were found at the mouth of the inlet and offshore in the inlet plume. Results suggest that inlets moving large volumes of water into the coastal zone with tides may be an important source of fecal contaminants. Efforts to reduce run-off or unintended release of water into the Intracoastal Waterway may lower contaminants entering sensitive coastal areas.

Exploring the role of microorganisms in the disease-like syndrome affecting the sponge Ianthella basta.

A disease-like syndrome is currently affecting a large percentage of the Ianthella basta populations from the Great Barrier Reef and central Torres Strait. Symptoms of the syndrome include discolored, necrotic spots leading to tissue degradation, exposure of the skeletal fibers, and disruption of the choanocyte chambers. To ascertain the role of microbes in the disease process, a comprehensive comparison of bacteria, viruses, fungi, and other eukaryotes was performed in healthy and diseased sponges using multiple techniques. A low diversity of microbes was observed in both healthy and diseased sponge communities, with all sponges dominated by an Alphaproteobacteria, a Gammaproteobacteria, and a group I crenarchaeota. Bacterial cultivation, community analysis by denaturing gradient gel electrophoresis (Bacteria and Eukarya), sequencing of 16S rRNA clone libraries (Bacteria and Archaea), and direct visual assessment by electron microscopy failed to reveal any putative pathogens. In addition, infection assays could not establish the syndrome in healthy sponges even after direct physical contact with affected tissue. These results suggest that microbes are not responsible for the formation of brown spot lesions and necrosis in I. basta.

Mimivirus and Mimiviridae: giant viruses with an increasing number of potential hosts, including corals and sponges.

Mimivirus, a giant DNA virus (i.e. "girus") infecting species of the genus Acanthamoeba, was first identified in 2003. With a particle size of 0.7microm in diameter, and a genome size of 1.2Mb encoding more than 900 proteins, it is the most complex virus described to date. Beyond its unusual size, the Mimivirus genome was found to contain the first viral homologues of many genes thought to be the trademark of cellular organisms, such as central components of the translation apparatus. These findings revived the debate on the origin of DNA viruses, and the role they might have played in the emergence of eukaryotes. Published and ongoing studies on Mimivirus continue to lead to unexpected findings concerning a variety of aspects, such as the structure of its particle, unique features of its replication cycle, or the distribution and abundance of Mimivirus relatives in the oceans. Following a summary of these recent findings, we present preliminary results suggesting that octocorals might have come in close contact with an ancestor of Mimivirus, and that modern sponges might be host to a yet unidentified, even larger, member of the Mimiviridae.

Sponge disease: a global threat?

Sponges are the most simple and primitive metazoans, yet they have various biological and ecological properties that make them an influential component of coral-reef ecosystems. Marine sponges provide refuge for many small invertebrates and are critical to benthic-pelagic coupling across a wide range of habitats. Reports of sponge disease have increased dramatically in recent years with sponge populations decimated throughout the Mediterranean and Caribbean. Reports also suggest an increased prevalence of sponge disease in Papua New Guinea, the Great Barrier Reef and in the reefs of Cozumel, Mexico. These epidemics can have severe impacts on the survival of sponge populations, the ecology of the reef and the fate of associated marine invertebrates. Despite the ecological and commercial importance of sponges, the understanding of sponge disease is limited. There has generally been a failure to isolate and identify the causative agents of sponge disease, with only one case confirming Koch's postulates and identifying a novel Alphaproteobacteria strain as the primary pathogen. Other potential disease agents include fungi, viruses, cyanobacteria and bacterial strains within the Bacillus and Pseudomonas genera. There is some evidence for correlations between sponge disease and environmental factors such as climate change and urban/agricultural runoff. This review summarizes the occurrence of sponge disease, describes the syndromes identified thus far, explores potential linkages with environmental change and proposes a strategy for future research towards better management of sponge disease outbreaks.

Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR.

A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100 kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5' untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3 viruses/ml to 83 viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters.