Diverse RNA viruses discovered in multiple seagrass species.

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 in Zostera marina, Zostera muelleri, Zostera japonica, and Cymodocea nodosa. In Z. 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. In Z. 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. In Z. 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, in C. nodosa, we discovered two contigs that belong to a novel virus in the family Betaflexiviridae. These findings expand our knowledge of viral diversity in seagrasses and provide insight into seagrass viral ecology.

2024 taxonomy update for the family Circoviridae.

Circovirids have a circular single-stranded DNA genome packed into a small icosahedral capsid. They are classified within two genera, Circovirus and Cyclovirus, in the family Circoviridae (phylum Cressdnaviricota, class Arfiviricetes, order Cirlivirales). Over the last five years, a number of new circovirids have been identified, and, as a result, 54 new species have been created for their classification based on the previously established species demarcation criterion, namely, that viruses classified into different species share less than 80% genome-wide pairwise sequence identity. Of note, one of the newly created species includes a circovirus that was identified in human hepatocytes and suspected of causing liver damage. Furthermore, to comply with binomial species nomenclature, all new and previously recognized species have been (re)named in binomial format with a freeform epithet. Here, we provide a summary of the properties of circovirid genomes and their classification as of June 2024 (65 species in the genus Circovirus and 90 species in the genus Cyclovirus). Finally, we provide reference datasets of the nucleotide and amino acid sequences representing each of the officially recognized circovirid species to facilitate further classification of newly discovered members of the Circoviridae.

Coral-reef ecology: Expanding urchin disease threatens ecosystem balance.

Sea urchins are critically important herbivores on coral reefs. A new study shows that a disease that decimated sea urchins in the Caribbean in 2022 has spread to the Red Sea, further threatening coral ecosystems.

Limited potexvirus diversity in eastern Gulf of Mexico seagrass meadows.

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, and 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.

Evaluation of in vitro treatments against the causative agent of Diadema antillarum scuticociliatosis (DaSc).

In the 1980s, a mass die-off of the long-spined sea urchin Diadema antillarum occurred on Florida and Caribbean coral reefs. D. antillarum populations largely did not recover, and in 2022, remaining populations experienced another mass mortality event. A ciliate most similar to Philaster apodigitiformis was identified as the causative agent of the 2022 event, which was named D. antillarum scuticociliatosis (DaSc). Here, we investigated possible treatments for this pathogen. We tested the efficacy of 10 compounds at final concentrations of 100, 50, 25, 12.5, 6.25, and 3.13 µM, or a 10-fold serial dilution series, against ciliates cultured from an infected D. antillarum specimen. Of the tested compounds, 8 induced 100% ciliate mortality at some dose after 24 h. The most effective (defined as those requiring the lowest dose to induce 100% ciliate mortality) were quinacrine and tomatine (both effective at 12.5 µM), followed by furaltadone and plumbagin (25 µM), bithionol sulfoxide and 2'4' dihydroxychalcone (50 µM), and oxyclozanide and carnidazole (100 µM). Toltrazuril and a commercially available anticiliate product containing naphthoquinones were not effective at any dose tested. Shortened (15 min) time trials were performed using ciliate cultures reared in natural seawater to better reflect natural environmental conditions, and revealed that 2 of the compounds (quinacrine and tomatine) induced 100% ciliate mortality at 100 µM, with tomatine also effective at 50 µM. This study identified several treatments effective against the causative agent of DaSc in vitro, but their toxicity and utility in vivo remain unknown.

Transglobal spread of an ecologically relevant sea urchin parasite.

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.

A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea.

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

Evaluation of DNA metabarcoding for identifying fish eggs: a case study on the West Florida Shelf.

A critical factor in fisheries management is the protection of spawning sites for ecologically and economically important fish species. DNA barcoding (i.e., amplification and sequencing of the mitochondrial cytochrome c oxidase I (COI) gene) of fish eggs has emerged as a powerful technique for identifying spawning sites. However, DNA barcoding of individual fish eggs is time-consuming and expensive. In an attempt to reduce costs and effort for long-term fisheries monitoring programs, here we used DNA metabarcoding, in which DNA is extracted and amplified from a composited sample containing all the fish eggs collected at a given site, to identify fish eggs from 49 stations on the West Florida Shelf. A total of 37 taxa were recovered from 4,719 fish eggs. Egg distributions on the West Florida Shelf corresponded with the known habitat types occupied by these taxa, which included burrower, coastal pelagic, epipelagic, mesopelagic, demersal, deep demersal, commensal, and reef-associated taxa. Metabarcoding of fish eggs was faster and far less expensive than barcoding individual eggs; however, this method cannot provide absolute taxon proportions due to variable copy numbers of mitochondrial DNA in different taxa, different numbers of cells within eggs depending on developmental stage, and PCR amplification biases. In addition, some samples yielded sequences from more taxa than the number of eggs present, demonstrating the presence of contaminating DNA and requiring the application of a threshold proportion of sequences required for counting a taxon as present. Finally, we review the advantages and disadvantages of using metabarcoding vs. individual fish egg barcoding for long-term monitoring programs.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Freshwater macrophytes harbor viruses representing all five major phyla of the RNA viral kingdom Orthornavirae.

Research on aquatic plant viruses is lagging behind that of their terrestrial counterparts. To address this knowledge gap, here we identified viruses associated with freshwater macrophytes, a taxonomically diverse group of aquatic phototrophs that are visible with the naked eye. We surveyed pooled macrophyte samples collected at four spring sites in Florida, USA through next generation sequencing of RNA extracted from purified viral particles. Sequencing efforts resulted in the detection of 156 freshwater macrophyte associated (FMA) viral contigs, 37 of which approximate complete genomes or segments. FMA viral contigs represent putative members from all five major phyla of the RNA viral kingdom Orthornavirae. Similar to viral types found in land plants, viral sequences identified in macrophytes were dominated by positive-sense RNA viruses. Over half of the FMA viral contigs were most similar to viruses reported from diverse hosts in aquatic environments, including phototrophs, invertebrates, and fungi. The detection of FMA viruses from orders dominated by plant viruses, namely Patatavirales and Tymovirales, indicate that members of these orders may thrive in aquatic hosts. PCR assays confirmed the presence of putative FMA plant viruses in asymptomatic vascular plants, indicating that viruses with persistent lifestyles are widespread in macrophytes. The detection of potato virus Y and oat blue dwarf virus in submerged macrophytes suggests that terrestrial plant viruses infect underwater plants and highlights a potential terrestrial-freshwater plant virus continuum. Defining the virome of unexplored macrophytes will improve our understanding of virus evolution in terrestrial and aquatic primary producers and reveal the potential ecological impacts of viral infection in macrophytes.

Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single-stranded DNA phage.

Advances in metagenomics have revealed the ubiquity of single-stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here, we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At ≥95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 × 105 viruses ml-1 . These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans.

Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments.

In nature, concentrations of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO2 fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit 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-CO2-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia 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) ΔyadF ΔcynT to grow under low-CO2 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 CO2 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 CO2, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising 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 various 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 CO2.

Spatial and Temporal Dynamics of Prokaryotic and Viral Community Assemblages in a Lotic System (Manatee Springs, Florida).

Flow from high-magnitude springs fed by the Floridan aquifer system contributes hundreds of liters of water per second to rivers, creating unique lotic systems. Despite their importance as freshwater sources and their contributions to the state's major rivers, little is known about the composition and spatiotemporal variability of prokaryotic and viral communities of these spring systems or their influence on downstream river sites. At four time points throughout a year, we determined the abundance and diversity of prokaryotic and viral communities at three sites within the first-magnitude Manatee Springs system (the spring head where water emerges from the aquifer, a mixed region where the spring run ends, and a downstream site in the Suwannee River). The abundance of prokaryotes and virus-like particles increased 100-fold from the spring head to the river and few members from the head communities persisted in the river at low abundance, suggesting the springs play a minor role in seeding downstream communities. Prokaryotic and viral communities within Manatee Springs clustered by site, with seasonal variability likely driven by flow. As water flowed through the system, microbial community composition was affected by changes in physiochemical parameters and community coalescence. Evidence of species sorting and mass effects could be seen in the assemblages. Greater temporal fluctuations were observed in prokaryotic and viral community composition with increasing distance from the spring outflow, reflecting the relative stability of the groundwater environment, and comparisons to springs from prior work reaffirmed that distinct first-magnitude springs support unique communities. IMPORTANCE Prokaryotic and viral communities are central to food webs and biogeochemical processes in aquatic environments, where they help maintain ecosystem health. The Floridan aquifer system (FAS), which is the primary drinking water source for millions of people in the southeastern United States, contributes large amounts of freshwater to major river systems in Florida through its springs. However, there is a paucity of information regarding the spatiotemporal dynamics of microbial communities in these essential flowing freshwater systems. This work explored the prokaryotic and viral communities in a first-magnitude spring system fed by the FAS that discharges millions of liters of water per day into the Suwannee River. This study examined microbial community composition through space and time as well as the environmental parameters and metacommunity assembly mechanisms that shape these communities, providing a foundational understanding for monitoring future changes.

Relationships among microbial indicators of fecal pollution, microbial source tracking markers, and pathogens in Costa Rican coastal waters.

Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms' concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users' health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches.

Genome Sequence of Vibrio natriegens Phage vB_VnaS-AQKL99.

Vibrio natriegens is a naturally occurring marine bacterium that is emerging as a microbiological model system. Here, we describe Aquatic Killer 99 (AQKL99), a novel phage that infects Vibrio natriegens 14048. The genome of the phage is 58,464 bp long, has a GC content of 45.9%, and contains 51 protein-coding genes.

Prokaryotic and Viral Community Composition of Freshwater Springs in Florida, USA.

Aquifers, which are essential underground freshwater reservoirs worldwide, are understudied ecosystems that harbor diverse forms of microbial life. This study investigated the abundance and composition of prokaryotic and viral communities in the outflow of five springs across northern Florida, USA, as a proxy of microbial communities found in one of the most productive aquifers in the world, the Floridan aquifer. The average abundances of virus-like particles and prokaryotic cells were slightly lower than those reported from other groundwater systems, ranging from 9.6 × 103 ml-1 to 1.1 × 105 ml-1 and 2.2 × 103 ml-1 to 3.4 × 104 ml-1, respectively. Despite all of the springs being fed by the Floridan aquifer, sequencing of 16S rRNA genes and viral metagenomes (viromes) revealed unique communities in each spring, suggesting that groundwater microbial communities are influenced by land usage in recharge zones. The prokaryotic communities were dominated by Bacteria, and though the most abundant phyla (Proteobacteria, Cyanobacteria, and Bacteroidetes) were found in relatively high abundance across springs, variation was seen at finer taxonomic resolution. The viral sequences were most similar to those described from other aquatic environments. Sequencing resulted in the completion of 58 novel viral genomes representing members of the order Caudovirales as well as prokaryotic and eukaryotic single-stranded DNA (ssDNA) viruses. Sequences similar to those of ssDNA viruses were detected at all spring sites and dominated the identifiable sequences at one spring site, showing that these small viruses merit further investigation in groundwater systems.IMPORTANCE Aquifer systems may hold up to 40% of the total microbial biomass on Earth. However, little is known about the composition of microbial communities within these critical freshwater ecosystems. Here, we took advantage of Florida's first-magnitude springs (the highest spring classification based on water discharge), each discharging at least 246 million liters of water each day from the Floridan aquifer system (FAS), to investigate prokaryotic and viral communities from the aquifer. The FAS serves as a major source of potable water in the Southeastern United States, providing water for large cities and citizens in three states. Unfortunately, the health of the FAS and its associated springs has declined in the past few decades due to nutrient loading, increased urbanization and agricultural activity in aquifer recharge zones, and saltwater intrusion. This is the first study to describe the prokaryotic and viral communities in Florida's first-magnitude springs, providing a baseline against which to compare future ecosystem change.

Environmental DNA reveals seasonal shifts and potential interactions in a marine community.

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015-2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies.

Prevalence of a vertically transmitted single-stranded DNA virus in spinybacked orbweavers (Gasteracantha cancriformis) from Florida, USA.

Spiders (order Araneae, class Arachnida) are an important group of predatory arthropods in terrestrial ecosystems that have been recently identified as an untapped reservoir of single-stranded (ss)DNA viruses. Specifically, spiders harbour a diversity of ssDNA viruses encoding a replication-associated protein (Rep) within a circular genome. However, little is known about the ecology of novel circular Rep-encoding ssDNA (CRESS DNA) viruses. Here we investigated two CRESS DNA viruses recently identified in spinybacked orbweavers (Gasteracantha cancriformis), namely spinybacked orbweaver circular virus (SpOrbCV) 1 and 2. SpOrbCV-1 was detected in the majority (> 65 %) of spider specimens from all life stages, including eggs, spiderlings and adults, demonstrating that this virus is active within spinybacked orbweavers. In contrast, SpOrbCV-2 was only detected in adults at a lower (36 %) prevalence. Since we also detected SpOrbCV-2 in other spider species and this virus has been reported from a dragonfly, we suggest that SpOrbCV-2 is accumulated in these predators through common insect prey. The prevalence of SpOrbCV-1 in collected specimens allowed us to design assays to characterize this virus, which represents a new group of CRESS DNA viruses, the 'circularisviruses'. To our knowledge, SpOrbCV-1 is the first example of a vertically transmitted virus in spiders, which may explain its high prevalence in spinybacked orbweavers. Since vertically transmitted viruses infecting insects (class Insecta) can manipulate their host's behaviour and physiology, future studies should investigate the ecological role of vertically transmitted viruses in spiders.