Perturbations in natural systems generally are the combination of multiple interactions among individual stressors. However, methods to interpret the effects of interacting stressors remain challenging and are biased to identifying synergies which are prioritized in conservation. Therefore we conducted a multiple stressor experiment (no stress, single, double, triple) on the coral Pocillopora meandrina to evaluate how its microbiome changes compositionally with increasing levels of perturbation. We found that effects of nutrient enrichment, simulated predation, and increased temperature are antagonistic, rather than synergistic or additive, for a variety of microbial community diversity measures. Importantly, high temperature and scarring alone had the greatest effect on changing microbial community composition and diversity. Using differential abundance analysis, we found that the main effects of stressors increased the abundance of opportunistic taxa, and two-way interactions among stressors acted antagonistically on this increase, while three-way interactions acted synergistically. These data suggest that: (1) multiple statistical analyses should be conducted for a complete assessment of microbial community dynamics, (2) for some statistical metrics multiple stressors do not necessarily increase the disruption of microbiomes over single stressors in this coral species, and (3) the observed stressor-induced community dysbiosis is characterized by a proliferation of opportunists rather than a depletion of a proposed coral symbiont of the genus Endozoicomonas.

Filamentous viruses are hypothesized to play a role in stony coral tissue loss disease (SCTLD) through infection of the endosymbiotic dinoflagellates (Family Symbiodiniaceae) of corals. To evaluate this hypothesis, it is critical to understand the global distribution of filamentous virus infections across the genetic diversity of Symbiodiniaceae hosts. Using transmission electron microscopy, we demonstrate that filamentous virus-like particles (VLPs) are present in over 60% of Symbiodiniaceae cells (genus Cladocopium) within Pacific corals (Acropora hyacinthus, Porites c.f. lobata); these VLPs are more prevalent in Symbiodiniaceae of in situ colonies experiencing heat stress. Symbiodiniaceae expelled from A. hyacinthus also contain filamentous VLPs, and these cells are more degraded than their in hospite counterparts. Similar to VLPs reported from SCTLD-affected Caribbean reefs, VLPs range from ~150 to 1500 nm in length and 16-37 nm in diameter and appear to constitute various stages in a replication cycle. Finally, we demonstrate that SCTLD-affected corals containing filamentous VLPs are dominated by diverse Symbiodiniaceae lineages from the genera Breviolum, Cladocopium, and Durusdinium. Although this study cannot definitively confirm or refute the role of filamentous VLPs in SCTLD, it demonstrates that filamentous VLPs are not solely observed in SCTLD-affected corals or reef regions, nor are they solely associated with corals dominated by members of a particular Symbiodiniaceae genus. We hypothesize that filamentous viruses are a widespread, common group that infects Symbiodiniaceae. Genomic characterization of these viruses and empirical tests of the impacts of filamentous virus infection on Symbiodiniaceae and coral colonies should be prioritized.

Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community's functional capabilities. Here we describe PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this 'predictive metagenomic' approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available.

Island Conservation (IC) and Scripps Institution of Oceanography (SIO), UC San Diego, co-convened a meeting of practitioners and researchers to assess the Global Links for Island Marine Restoration(GLIMR). These global experts in the diverse fields of island restoration, invasive species management, and wildlife conservation, were assembled and worked for eight months to evaluate the state of the knowledge of land-sea linkages in the context of invasive species management and island restoration. The result is this white paper synthesis of the science and research recommendations and a manuscript that will be submitted to a peer-reviewed journal. The intended audiences for this endeavor include island communities, governments, conservation and sustainable development non-governmental organizations, scientists, and funders. Throughout the report, readers will find that the key takeaways, identified by the authors, are represented in blue italics. This study was made possible by the generous support from the IC Board of Directors, the Center for Marine Biodiversity and Conservation at SIO, the Nature Conservancy California Chapter, the Leo Model Foundation, and the Archie Arnold Trust.

Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as "Candidatus Aquarickettsia." Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of "Ca. Aquarickettsia" are found globally in dozens of invertebrate lineages. The coral-associated "Candidatus A. rohweri" is the first finished genome in this new clade. "Ca. A. rohweri" lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, "Ca. A. rohweri" possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that "Ca. A. rohweri" reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of "Ca. A. rohweri" in its host by providing abundant N-rich metabolites to be scavenged.

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.