vAMPirus: A versatile amplicon processing and analysis program for studying viruses.

Amplicon sequencing is an effective and increasingly applied method for studying viral communities in the environment. Here, we present vAMPirus, a user-friendly, comprehensive, and versatile DNA and RNA virus amplicon sequence analysis program, designed to support investigators in exploring virus amplicon sequencing data and running informed, reproducible analyses. vAMPirus intakes raw virus amplicon libraries and, by default, performs nucleotide- and amino acid-based analyses to produce results such as sequence abundance information, taxonomic classifications, phylogenies and community diversity metrics. The vAMPirus analytical framework leverages 16 different opensource tools and provides optional approaches that can increase the ratio of biological signal-to-noise and thereby reveal patterns that would have otherwise been masked. Here, we validate the vAMPirus analytical framework and illustrate its implementation as a general virus amplicon sequencing workflow by recapitulating findings from two previously published double-stranded DNA virus datasets. As a case study, we also apply the program to explore the diversity and distribution of a coral reef-associated RNA virus. vAMPirus is streamlined within Nextflow, offering straightforward scalability, standardization and communication of virus lineage-specific analyses. The vAMPirus framework is designed to be adaptable; community-driven analytical standards will continue to be incorporated as the field advances. vAMPirus supports researchers in revealing patterns of virus diversity and population dynamics in nature, while promoting study reproducibility and comparability.

Filamentous virus-like particles are present in coral dinoflagellates across genera and ocean basins.

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.

Endogenous viral elements reveal associations between a non-retroviral RNA virus and symbiotic dinoflagellate genomes.

Endogenous viral elements (EVEs) offer insight into the evolutionary histories and hosts of contemporary viruses. This study leveraged DNA metagenomics and genomics to detect and infer the host of a non-retroviral dinoflagellate-infecting +ssRNA virus (dinoRNAV) common in coral reefs. As part of the Tara Pacific Expedition, this study surveyed 269 newly sequenced cnidarians and their resident symbiotic dinoflagellates (Symbiodiniaceae), associated metabarcodes, and publicly available metagenomes, revealing 178 dinoRNAV EVEs, predominantly among hydrocoral-dinoflagellate metagenomes. Putative associations between Symbiodiniaceae and dinoRNAV EVEs were corroborated by the characterization of dinoRNAV-like sequences in 17 of 18 scaffold-scale and one chromosome-scale dinoflagellate genome assembly, flanked by characteristically cellular sequences and in proximity to retroelements, suggesting potential mechanisms of integration. EVEs were not detected in dinoflagellate-free (aposymbiotic) cnidarian genome assemblies, including stony corals, hydrocorals, jellyfish, or seawater. The pervasive nature of dinoRNAV EVEs within dinoflagellate genomes (especially Symbiodinium), as well as their inconsistent within-genome distribution and fragmented nature, suggest ancestral or recurrent integration of this virus with variable conservation. Broadly, these findings illustrate how +ssRNA viruses may obscure their genomes as members of nested symbioses, with implications for host evolution, exaptation, and immunity in the context of reef health and disease.

Stony coral tissue loss disease induces transcriptional signatures of in situ degradation of dysfunctional Symbiodiniaceae.

Stony coral tissue loss disease (SCTLD), one of the most pervasive and virulent coral diseases on record, affects over 22 species of reef-building coral and is decimating reefs throughout the Caribbean. To understand how different coral species and their algal symbionts (family Symbiodiniaceae) respond to this disease, we examine the gene expression profiles of colonies of five species of coral from a SCTLD transmission experiment. The included species vary in their purported susceptibilities to SCTLD, and we use this to inform gene expression analyses of both the coral animal and their Symbiodiniaceae. We identify orthologous coral genes exhibiting lineage-specific differences in expression that correlate to disease susceptibility, as well as genes that are differentially expressed in all coral species in response to SCTLD infection. We find that SCTLD infection induces increased expression of rab7, an established marker of in situ degradation of dysfunctional Symbiodiniaceae, in all coral species accompanied by genus-level shifts in Symbiodiniaceae photosystem and metabolism gene expression. Overall, our results indicate that SCTLD infection induces symbiophagy across coral species and that the severity of disease is influenced by Symbiodiniaceae identity.

Viruses of a key coral symbiont exhibit temperature-driven productivity across a reefscape.

Viruses can affect coral health by infecting their symbiotic dinoflagellate partners (Symbiodiniaceae). Yet, viral dynamics in coral colonies exposed to environmental stress have not been studied at the reef scale, particularly within individual viral lineages. We sequenced the viral major capsid protein (mcp) gene of positive-sense single-stranded RNA viruses known to infect symbiotic dinoflagellates ('dinoRNAVs') to analyze their dynamics in the reef-building coral, Porites lobata. We repeatedly sampled 54 colonies harboring Cladocopium C15 dinoflagellates, across three environmentally distinct reef zones (fringing reef, back reef, and forereef) around the island of Moorea, French Polynesia over a 3-year period and spanning a reef-wide thermal stress event. By the end of the sampling period, 28% (5/18) of corals in the fringing reef experienced partial mortality versus 78% (14/18) of corals in the forereef. Over 90% (50/54) of colonies had detectable dinoRNAV infections. Reef zone influenced the composition and richness of viral mcp amino acid types ('aminotypes'), with the fringing reef containing the highest aminotype richness. The reef-wide thermal stress event significantly increased aminotype dispersion, and this pattern was strongest in the colonies that experienced partial mortality. These findings demonstrate that dinoRNAV infections respond to environmental fluctuations experienced in situ on reefs. Further, viral productivity will likely increase as ocean temperatures continue to rise, potentially impacting the foundational symbiosis underpinning coral reef ecosystems.

Thermal stress triggers productive viral infection of a key coral reef symbiont.

Climate change-driven ocean warming is increasing the frequency and severity of bleaching events, in which corals appear whitened after losing their dinoflagellate endosymbionts (family Symbiodiniaceae). Viral infections of Symbiodiniaceae may contribute to some bleaching signs, but little empirical evidence exists to support this hypothesis. We present the first temporal analysis of a lineage of Symbiodiniaceae-infecting positive-sense single-stranded RNA viruses ("dinoRNAVs") in coral colonies, which were exposed to a 5-day heat treatment (+2.1 °C). A total of 124 dinoRNAV major capsid protein gene "aminotypes" (unique amino acid sequences) were detected from five colonies of two closely related Pocillopora-Cladocopium (coral-symbiont) combinations in the experiment; most dinoRNAV aminotypes were shared between the two coral-symbiont combinations (64%) and among multiple colonies (82%). Throughout the experiment, seventeen dinoRNAV aminotypes were found only in heat-treated fragments, and 22 aminotypes were detected at higher relative abundances in heat-treated fragments. DinoRNAVs in fragments of some colonies exhibited higher alpha diversity and dispersion under heat stress. Together, these findings provide the first empirical evidence that exposure to high temperatures triggers some dinoRNAVs to switch from a persistent to a productive infection mode within heat-stressed corals. Over extended time frames, we hypothesize that cumulative dinoRNAV production in the Pocillopora-Cladocopium system could affect colony symbiotic status, for example, by decreasing Symbiodiniaceae densities within corals. This study sets the stage for reef-scale investigations of dinoRNAV dynamics during bleaching events.

De novo transcriptome assembly of the digitate morphotype of Briareum asbestinum (Octocorallia: Alcyonacea) from the southwest shelf of Puerto Rico.

Octocorals have now become the most visually dominant metazoan benthic taxa of most Caribbean reefs, following the precipitous decline of scleractinian corals. Yet taxonomic issues because of their extensive phenotypic plasticity are still abound. Briareum asbestinum one of the iconic octocorals of the shallow Caribbean coral reefs exhibits a biform morphology, the digitate and the encrusting one. The taxonomic status of each form has not been clarified, yet. Until recently, there were few genetic resources for non-model metazoans, however, affordable high-throughput DNA sequencing has removed this hindrance. We present the first transcriptome of the digitate form of Briareum asbestinum from southwest Puerto Rico. We used paired-end sequencing (Illumina NextSeq 500), with a total yield of 159,754,702 raw reads. De novo assembly was performed utilizing a multi-assembler approach generating 371,554 biologically true, non-redundant transcripts. Open reading frame analysis identified 102,839 putative ORFs of which 78,607 were with annotations. BUSCO analysis indicated a total of 96.4% complete orthologous genes from the metazoan dataset. The assembly presented here serves as an important new genomic reference for the Briareum genus that will facilitate future population and phylogenetic studies aiming to better understand the molecular basis of phenotypic plasticity exhibited throughout the genus.

De novo transcriptome assembly of the coral Agaricia lamarcki (Lamarck's sheet coral) from mesophotic depth in southwest Puerto Rico.

The plating coral, Agaricia lamarcki is a widely distributed species inhabiting reefs across the Caribbean basin and Florida. This species is of interest since it is considered a depth-generalist, found from 10 to 70 m. Given the scope of contemporary studies on this coral's population dynamics and physiology, as well as, the potential of mesophotic reefs to be refuge habitats for deteriorated shallow water reefs, we present the first de novo transcriptome assembly of an important mesophotic coral. Using next-generation paired-end sequencing (Illumina Hiseq4000; 2 × 150 bp), we obtained a total of 82,506,058 raw reads. The novel transcriptome assembly strategy included the recently developed National Center for Genome Analysis Support de novo transcriptome assembly pipeline. Assembly produced a total of 101,322 biologically true, non-redundant transcripts with an average contig length of 959 and N50 of 1830. EvidentialGene and TransDecoder were used to identify open reading frames (ORFs) with homology insight provided by the UniProtKb and PFAM databases. ORF prediction resulted in 38,517 putative ORFs of which 12,107 ORFs were annotated as genes dealing with molecular function, 1266 with biological processes and 416 with cellular components.