No abstract

How microbial communities change over time in response to the environment is poorly understood. Previously a six-year time series of 16S rRNA V6 data from the Western English Channel demonstrated robust seasonal structure within the bacterial community, with diversity negatively correlated with day-length. Here we determine whether metagenomes and metatranscriptomes follow similar patterns. We generated 16S rRNA datasets, metagenomes (1.2 GB) and metatranscriptomes (157 MB) for eight additional time points sampled in 2008, representing three seasons (Winter, Spring, Summer) and including day and night samples. This is the first microbial ‘multi-omic’ study to combine 16S rRNA amplicon sequencing with metagenomic and metatranscriptomic profiling. Five main conclusions can be drawn from analysis of these data: 1) Archaea follow the same seasonal patterns as Bacteria, but show lower relative diversity; 2) Higher 16S rRNA diversity also reflects a higher diversity of transcripts; 3) Diversity is highest in winter and at night; 4) Community-level changes in 16S-based diversity and metagenomic profiles are better explained by seasonal patterns (with samples closest in time being most similar), while metatranscriptomic profiles are better explained by diel patterns and shifts in particular categories (i.e., functional groups) of genes; 5) Changes in key genes occur among seasons and between day and night (i.e., photosynthesis); but these samples contain large numbers of orphan genes without known homologues and it is these unknown gene sets that appear to contribute most towards defining the differences observed between times. Despite the huge diversity of these microbial communities, there are clear signs of predictable patterns and detectable stability over time. Renewed and intensified efforts are required to reveal fundamental deterministic patterns in the most complex microbial communities. Further, the presence of a substantial proportion of orphan sequences underscores the need to determine the gene products of sequences with currently unknown function.

Symbiodinium, the dinoflagellate photosymbiont of corals, is posited to become more susceptible to viral infections when heat-stressed. To investigate this hypothesis, we mined transcriptome data of a thermosensitive and a thermotolerant type C1 Symbiodinium population at ambient (27 °C) and elevated (32°C) temperatures. We uncovered hundreds of transcripts from nucleocytoplasmic large double-stranded DNA viruses (NCLDVs) and the genome of a novel positive-sense single-stranded RNA virus (+ssRNAV). In the transcriptome of the thermosensitive population only, +ssRNAV transcripts had remarkable expression levels in the top 0.03% of all transcripts at 27 °C, but at 32 °C, expression levels of +ssRNAV transcripts decreased, while expression levels of anti-viral transcripts increased. In both transcriptomes, expression of NCLDV transcripts increased at 32 °C, but thermal induction of NCLDV transcripts involved in DNA manipulation was restricted to the thermosensitive population. Our findings reveal that viruses infecting Symbiodinium are affected by heat stress and may contribute to Symbiodinium thermal sensitivity.

Recent metagenomic analyses have revealed a high diversity of viruses in the pelagic ocean and uncovered clear habitat-specific viral distribution patterns. Conversely, similar insights into the composition, host specificity and function of viruses associated with marine organisms have been limited by challenges associated with sampling and computational analysis. Here, we performed targeted viromic analysis of six coral reef invertebrate species and their surrounding seawater to deliver taxonomic and functional profiles of viruses associated with reef organisms. Sponges and corals' host species-specific viral assemblages with low sequence identity to known viral genomes. While core viral genes involved in capsid formation, tail structure and infection mechanisms were observed across all reef samples, auxiliary genes including those involved in herbicide resistance and viral pathogenesis pathways such as host immune suppression were differentially enriched in reef hosts. Utilising a novel OTU based assessment, we also show a prevalence of dsDNA viruses belonging to the Mimiviridae, Caudovirales and Phycodnaviridae in reef environments and further highlight the abundance of ssDNA viruses belonging to the Circoviridae, Parvoviridae, Bidnaviridae and Microviridae in reef invertebrates. These insights into coral reef viruses provide an important framework for future research into how viruses contribute to the health and evolution of reef organisms.

† These authors have contributed equally to this work.Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

SummaryReef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities and requires specialized methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this minireview, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double-and singlestranded DNA and RNA viruses. The identified viruses are dominated by double-stranded DNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral and other eukaryotes in close association.

SummaryOstreococcus tauri virus (OtV-1) is a large doublestranded DNA virus and a prospective member of the family Phycodnaviridae, genus Prasinovirus. OtV-1 infects the unicellular marine green alga O. tauri, the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNAencoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N-myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history.

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.