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A scuticociliate most closely related to Philaster apodigitiformis caused mass mortalities of diadematoid sea urchins and was cultured as Philaster sp. isolate FWC2. We sequenced the metagenomic content of this isolate, which was predicted to represent ≤ 56% of the complete genome. Based on k-mer counts, the haploid genome size was predicted to be 122–136 Mbp. We assembled and annotated a 4,088 bp nuclear ribosomal operon, a 41,396 bp mitochondrial genome with 19.22% G + C content, 24 protein-coding genes, 6 tRNA genes, and 2 rRNA genes, and a protein sequence homologous to β-PKA in Philaster apodigitiformis potentially involved in host infection. 

ABSTRACT Seagrasses are a polyphyletic group of marine flowering plants that play crucial roles in nearshore ecology, yet their interactions with viruses remain largely unexplored. This study presents the construction and characterization of an infectious cDNA clone of the potexvirus turtle grass virus X (TGVX). The complete genome of this positive-sense single-stranded RNA virus was amplified from field samples ofThalassia testudinumand assembled into a pLX-based mini binary vector using a multi-fragment directional cloning strategy, resulting in the infectious clone pLX-TGVX. Agroinfection assays of potexvirus-freeT. testudinumplants resulted in systemic infections by TGVX, as confirmed by multiplex RT-PCR experiments and phenotypic changes reflecting virus-induced symptoms. Ultrastructural studies also demonstrated significant cytopathological changes resulting from TGVX infection, including chloroplast swelling, reduced thylakoid grana, and the presence of viral replication organelles and filamentous virus-like particles. The development of the TGVX infectious clone offers a novel tool for investigating the impact of this virus on seagrass health and productivity. This study demonstrates the first successful agroinfection of a marine plant with an infectious clone, creating a new avenue for studying viruses identified through sequence-based surveys and paving the way for exploring the ecological significance of viral infection in these critical marine ecosystems.IMPORTANCEThis study pioneers the construction of an infectious clone of turtle grass virus X and describes its application in the natural marine plant host,Thalassia testudinum. The creation of this infectious clone not only provides a valuable tool for marine plant virology research but also opens new avenues for exploring the influence of viral infections on the health and productivity of seagrass meadows. Given that seagrasses play a crucial role in sediment stabilization, nutrient cycling, and habitat provisioning, understanding the impact of viruses on these ecosystems is essential for their effective conservation and management. This methodological advance enables detailed studies of viral replication, virus-host interactions, and the broader ecological implications of viral infections in marine plants. 

Lysogens, bacteria with one or more viruses (prophages) integrated into their genomes, are abundant in the gut of animals. Prophages often influence bacterial traits; however, the influence of prophages on the gut microbiota–host immune axis in animals remains poorly understood. Here, we investigate the influence of the prophage SfPat onShewanella fidelis3313, a persistent member of the gut microbiome of the model marine tunicate,Ciona robusta. Establishment of a SfPat deletion mutant (ΔSfPat) reveals the influence of this prophage on bacterial physiology in vitro and during colonization of theCionagut. In vitro, deletion of SfPat reducesS. fidelis3313 motility and swimming while increasing biofilm formation. To understand the in vivo impact of these prophage-induced changes in bacterial traits, we exposed metamorphic stage 4Cionajuveniles to wildtype (WT) and ΔSfPat strains. During colonization, ΔSfPat localizes to overlapping and distinct areas of the gut compared to the WT strain. We examined the differential expression of various regulators of cyclic-di-GMP, a secondary signaling molecule that mediates biofilm formation and motility. ThepdeBgene, which encodes a bacterial phosphodiesterase known to influence biofilm formation and motility by degrading cyclic-di-GMP, is upregulated in the WT strain but not in ΔSfPat when examined in vivo. Expression of theCionagut immune effector, VCBP-C, is enhanced during colonization by ΔSfPat compared to the WT strain; however, VCBP-C binding to the WT strain does not promote the excision of SfPat in an SOS-dependent pathway. Instead, VCBP-C binding significantly reduces the expression of a phage major capsid protein. Our findings suggest that SfPat influences host perception of this important colonizing commensal and highlights the significance of investigating tripartite dynamics between prophages, bacteria, and their animal hosts to better understand the gut microbiota-host immune axis. 

Seagrasses are marine angiosperms that form highly productive and diverse ecosystems. These ecosystems, however, are declining worldwide. Plant-associated microbes affect critical functions like nutrient uptake and pathogen resistance, which has led to an interest in the seagrass microbiome. However, despite their significant role in plant ecology, viruses have only recently garnered attention in seagrass species. In this study, we produced original data and mined publicly available transcriptomes to advance our understanding of RNA viral diversity inZostera marina,Zostera muelleri,Zostera japonica, andCymodocea nodosa. InZ.marina, we present evidence for additional Zostera marina amalgavirus 1 and 2 genotypes, and a complete genome for an alphaendornavirus previously evidenced by an RNA-dependent RNA polymerase gene fragment. InZ.muelleri, we present evidence for a second complete alphaendornavirus and near complete furovirus. Both are novel, and, to the best of our knowledge, this marks the first report of a furovirus infection naturally occurring outside of cereal grasses. InZ.japonica, we discovered genome fragments that belong to a novel strain of cucumber mosaic virus, a prolific pathogen that depends largely on aphid vectoring for host-to-host transmission. Lastly, inC.nodosa, we discovered two contigs that belong to a novel virus in the familyBetaflexiviridae. These findings expand our knowledge of viral diversity in seagrasses and provide insight into seagrass viral ecology. 

Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity affects host-virus interactions. 

ABSTRACT Mass mortality of Diadematidae urchins, caused by theDiadema antillarumscuticociliatosis Philaster clade (DScPc),affected the Caribbean in spring 2022 and subsequently spread to the eastern Mediterranean, Red Sea, and western Indian Ocean. A key question around Diadematidae scuticociliatosis (DSc), the disease caused by the scuticociliate, is whether the urchin microbiome varies between scuticociliatosis-affected and grossly normal urchins. Tissue samples from both grossly normal and abnormalDiadema antillarumwere collected in the field during the initial assessment of the DSc causative agent and from an experimental challenge of DScPc culture on aquaculturedD. antillarum. Specimens were analyzed using 16S rRNA gene amplicon sequencing. Additional abnormal urchin samples were collected from the most recent outbreak site in the western Indian Ocean (Réunion Island). At reference (i.e., unaffected by DSc) sites,Kistimonasspp., Propionigeniumspp., andEndozoicomonasspp. were highly represented in amplicon libraries. DSc-affected urchin amplicon libraries had lower taxonomic richness and a greater representation of taxa related toFangia hongkongensisandPsychrobiumspp. Amplicon libraries of urchins experimentally challenged with the DSc pathogen had some shifts in microbial composition, butF. hongkongensiswas not a part of the core bacteria in DSc-challenged specimens. DSc-affectedEchinothrix diademafrom Réunion Island showed a similar high representation ofF. hongkongensisas that seen on CaribbeanD. antillarum. Our results suggest that DSc altersDiadematidaemicrobiomes and thatF. hongkongensismay be a candidate bacterial biomarker for DSc in environmental samples. The mechanism driving microbiome variation in host–pathogen interactions remains to be explored.IMPORTANCEThe mass mortality of Diadematidae urchins due toDiadema antillarumscuticociliatosis (DSc) has had significant ecological impacts, spreading from the Caribbean to the eastern Mediterranean, Red Sea, and western Indian Ocean. This study investigates whether the microbiome of urchins varies between those affected by DSc and those that are not. Using 16S rRNA gene amplicon sequencing, researchers found that DSc-affected urchins had lower taxonomic richness and a greater representation ofFangia hongkongensisandPsychrobiumspp. The findings indicate thatF. hongkongensiscould serve as a bacterial biomarker for DSc in environmental samples, providing a potential tool for early detection and management of the disease. Understanding these microbiome changes is crucial for developing strategies to mitigate the spread and impact of DSc on marine ecosystems. 

BackgroundEchinoderms play crucial roles in coral reef ecosystems, where they are significant detritivores and herbivores. The phylum is widely known for its boom and bust cycles, driven by food availability, predation pressure and mass mortalities. Hence, surveillance of potential pathogens and associates of grossly normal specimens is important to understanding their roles in ecology and mass mortality. MethodsWe performed viral surveillance in two common coral reef echinoderms,Diadema antillarumandHolothuria floridana, using metagenomics. Urchin specimens were obtained during the 2022Diadema antillarumscuticociliatosis mass mortality event from the Caribbean and grossly normalH. floridanaspecimens from a reef in Florida. Viral metagenomes were assembled and aligned against viral genomes and protein encoding regions. Metagenomic reads and previously sequenced transcriptomes were further investigated for putative viral elements by Kraken2. ResultsD. antillarumwas devoid of viruses typically seen in echinoderms, butH. floridanayielded viral taxa similar to those found in other sea cucumbers, includingPisoniviricetes(Picornaviruses),Ellioviricetes(Bunyaviruses), andMagsaviricetes(Nodaviruses). The lack of viruses detected inD. antillarummay be due to the large amount of host DNA in viral metagenomes, or because viruses are less abundant inD. antillarumtissues when compared toH. floridanatissues. Our results also suggest that RNA amplification approach may influence viral representation in viral metagenomes. While our survey was successful in describing viruses associated with both echinoderms, our results indicate that viruses are less pronounced inD. antillarumthan in other echinoderms. These results are important in context of wider investigation on the association between viruses andD. antillarummass mortalities, since the conventional method used in this study was unsuccessful. 

Abstract Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms. 

In nature, concentrations of dissolved inorganic carbon (DIC; = CO 2 + HCO 3 - + CO 3 2- ) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO 2 fixation. Such mechanisms have been well-studied in Cyanobacteria , but much remains to be learned about their activity in other phyla. Novel multi-subunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea, and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations were chosen for study. A high-CO 2 requiring, carbonic anhydrase-deficient ( yadF - cynT - ) strain of E. coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) yadF - cynT - to grow under low CO 2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO 2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO 2 , autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multi-subunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, begging the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with varied subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO 2 .