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    Risk assessment for the native anurans from an alien invasive species, American bullfrogs (Lithobates catesbeianus), in South Korea

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    Chlorophytes response to habitat complexity and human disturbance in the catchment of small and shallow aquatic systems

    Response of chlorophytes to environmental variables in field vs. forest pondsOur study demonstrated that human-originated transformation in the catchment area surrounding a small water body may influence the water conditions in terms of physical, chemical, and biological parameters as well as the ecological state of the aquatic environment in respect to green algae communities.Chlorophytes inhabiting field ponds were more abundant compared with the forest ponds. This shows that field ponds, due to the higher values of TRP and water conductivity, created favorable conditions for chlorophyte development. The high concentrations of TRP and conductivity in aquatic environments are characteristic in the case of agricultural catchments exposed to anthropogenic pressure because of the inflow from the surrounding fertilized fields42. In this type of pond, we also observed significantly higher water temperatures and pH due to the lack of trees around them compared to the forest ponds, two factors which also positively influenced the growth of chlorophytes. Both the higher light intensity and the smaller size of the field ponds cause earlier warming up than the forest ponds and give an advantage to high light tolerant species. Moreover, it is well known that an increase in temperature stimulates the release of phosphorus from the bottom sediments, so this could be another reason for the higher levels of TRP in the field ponds. Our CCA analysis showed that TRP and conductivity were the strongest determinants of the distribution of chlorophyte species in the examined water bodies. We found a large group of dominant species indicated high values of TRP (e.g. Ankistrodesmus falcatus, A. arcuatus, Monoraphidium griffithii, Pseudopediastrum boryanum, Pediastrum duplex, Scenedesmus obtusus, Scenedesmus arcuatus var. gracilis, Desmodesmus communis, Coelastrum microporum), and another group of species (e.g. Kirchneriella irregularis var. spiralis, Tetraedron minimum, Scenedesmus ecornis) that preferred high levels of conductivity.In the field ponds generally higher mean abundances of filtrators and Rotifera were observed. This could be another important factor stimulating the growth of chlorophytes and increasing their abundances by the resupply of nutrients through excretion43,44. On the other hand, the high densities of algae could be the factor that caused better zooplankton development, and therefore its abundance in field ponds was greater. Filtrating cladocerans and Rotifera also had a significant influence on the distribution of chlorophyte dominating species. However, even though the total abundance of both chlorophytes and filtering zooplankton was greater in the field ponds, CCA analysis revealed a negative relationship existing between filtrators and most dominant species of chlorophytes (e.g. Pandorina morum, Willea rectangularis, Desmodesmus armatus, Nephrochlamys willeana, Cosmarium trilobulatum). Only two chlorophyte species—Lemmermannnia tetrapedia and Tetraedron triangulare—co-occurred with cladoceran zooplankton. These latter species are very small compared to the species above and can therefore be overlooked by filtrators, which have a choice of larger and perhaps more nutritiously satisfying algae of the genus Pandorina, Crucigeniella, Cosmarium or Nephrochlamys, but still of a size suitable for zooplankton. It can also be interpreted in such a way that Crucigenia and Tetraedron are among the r-strategists that reproduce very quickly, so grazing pressure by zooplankton can stimulate their rapid development45 and thus they remain at a stable level.Specific environmental conditions prevailing in the field ponds resulted in a high number of exclusive taxa44, found only in this type of water body. Moreover, a greater diversity of the representatives of different functional groups were found here, compared to the forest ponds.Analyzing the distribution of chlorophytes in terms of phytoplankton functional groups39,40, we found that group W1 was represented by only one species, Gonium pectorale. This was especially noted in the field water bodies. This group is known to prefer small water bodies rich in organic matter from husbandry or sewage40, which suggests that the field catchment in our study migh be a supplier of these substances. It also proves that field surroundings are far more human impacted. In the field ponds we observed a higher abundance of chlorophytes belonging to the groups G (Eudorina elegans, Pandorina morum, Pandorina smithii and Volvox aureus), J (e.g. representatives of the genus Actinastrum, Chlorotetraedron, Coelastrum, Crucigenia, Desmodesmus/Scenedesmus, Golenkinia, Pediastrum, Tetraedron, Tetrastrum, Westella, Willea/Crucigeniella), W0 (genera Chlamydomonas, Chlorangiopsis, Chlamydomonadopsis, Planktococcomyxa/Coccomyxa) and X3 (Chlorella sp.), typical for shallow nutrient-rich waters (G and J), ponds with extremely high organic contents (W0), and for shallow well-mixed layers (X3), according to classification given by Padisak et al.40. Considering that nitrogen compounds had a similar level in both types of ponds it can be stated that the representatives of the above mentioned functional groups of chlorophytes associated with the field ponds were presumably dependent on higher concentrations of TRP and conductivity and not that much on nitrogen concentrations.In the forest ponds significantly higher values of water saturation were recorded compared to the field ponds. Moreover, the lack of inflow of fertilizers from the catchment area resulted in lower TRP concentrations, which along with lower water temperatures, pH and conductivity in the forest ponds may have contributed to the reduced abundance of chlorophytes compared to the field water bodies. RDA analysis showed that some dominant chlorophyte species (e.g. Closterium moniliferum, Closterium tumidulum, Cosmarium trilobulatum and Mougeotia sp.) were associated with this type of small water body. At the same time the abundance of these species was smaller in the field ponds. We also found that chlorophyte diversity (Shannon–Weaver index) was greater in the forest ponds. This suggests that water bodies located within the forested area, usually more natural ponds being less exposed to anthropogenic pressure, are characterized by greater biodiversity. Moreover, in this type of water body we found many exclusive species39, not reported from the field ponds. Interestingly, about the half of these taxa belonged to desmids, which prefer lower pH and conductivity46, conditions typical for forest ponds. This could be also a reason for the dominance of desmid species with the highest abundance/frequency, associated with forest ponds.Taking into consideration the phytoplankton functional groups39,40 our study showed that the chlorophytes associated with forest ponds prefer mesotrophic waters (from the group TD: Cladophora glomerata, Geminella turfosa, Geminella planctonica, Microspora sp., Netrium digitus, Oedogonium sp., Oocystidium ovale, Spirogyra sp. Zygnema sp. and those belonging to the group N: mainly genera Closterium, Cosmarium, Euastrum, Micrasterias, Staurastrum, Staurodesmus, Xanthidium). This explains their greater share in the less fertile forest ponds. Another group associated with the forest ponds – T (Mougeotia sp., Binuclearia lauterbornii) contains species tolerant to light deficiency, so they were able to develop well in the more shaded water bodies located in the forest catchment.Chlorophyte community structure in two types of habitats (open water vs. macrophyte-dominated zone)In our study, the type of habitat (open water and macrophyte-dominated zones) also had a significant structuring effect on chlorophytes. There were a group of species linked to the open water zone (Pandorina morum, Nephrochlamys willeana, Oocystis lacustris, Scenedesmus armatus, Scenedesmus intermedius and Desmodesmus communis), being negatively related to vegetated stations at the same time. Generally, we found here a higher mean abundance of chlorophytes compared to the macrophyte-dominated zones, possibly due to the higher values of nutrients such as NH4 and TRP, the conditions favouring the development of many algae species. The results of the CCA analysis with habitats confirmed the high importance of both nutritional factors in structuring the distribution of chlorophyte species. There was a group of species associated with a rise in the concentration of ammonium (e.g. Scenedesmus arcuatus var. gracilis, Pediastrum duplex, Closterium moniliferum, Closterium tumidulum, Cosmarium trilobulatum, Willea rectangularis) as well as with phosphates (Monoraphidium tortile, Scenedesmus ecornis, Tetradesmus lagerheimii and Tetraedron minimum). Generally, high abundance of chlorophytes in the open water area was accompanied by a small-sized fraction of zooplankton–rotifers. Therefore, rotifers had a lower impact on the distribution of chlorophytes than filtrators. The increasing numbers of cladocerans contributed to the lowering abundance of some chlorophytes, such as Monoraphidium tortile, Scenedesmus ecornis, Tetradesmus lagerheimii or Tetraedron minimum. This shows that filtrators, whose densities were significantly higher among macrophytes, were able to control the development of some chlorophyte species much more efficiently than small-bodied rotifers.The effect of habitat was also visible in the case of phytoplankton functional groups39,40. We found that representatives of the group N (e.g. Closterium, Cosmarium, Euastrum, Micrasterias, Staurastrum) had a significantly higher mean abundance in the open water zones compared to the macrophyte-dominated zones. Interestingly, according to Padisak et al.40 group N prefers less fertile (mesotrophic) conditions, which is inconsistent with our results. However, we think that their association with the open water sites could be connected rather with the place/level where they live in the water column, rather than with the trophic state of water. The above mentioned chlorophytes taxonomically belong to desmids, which are mostly benthic organisms. Their greater quantitative share in the samples from the open water areas could be an effect of the intensive water mixing in the shallow ponds due to the lack of macrophytes. Neustupa et al.47 confirm that desmids are able to form tychoplanktonic communities due to water movements. In the samples collected from the macrophyte-dominated stations the mean abundance of desmids was generally lower, probably because of the macrophyte stabilizing effect. Aquatic plants are known to reduce turbidity and stabilize bottom sediments48, so they can prevent any intensive water mixing in ponds. In the examined open water stations, we also found a higher mean abundance of chlorophytes typical for shallow nutrient-rich waters (group G: Eudorina, Pandorina, Volvox and group K: Radiococcus) and/or for ponds with extremely high organic contents (group W0: e.g. Chlamydomonas), which proves that the sites lacking macrophytes were more fertile. Additionally, clearly more representatives from the codon J and X1 (typical for waters with high trophic levels) and a greater diversity of the representatives of different functional groups were recorded in the open water area compared to the macrophyte-dominated zones.The macrophyte-dominated stations had more abundant communities of filtrators, as aquatic plants are known to provide a profitable shelter for zooplankton49. Cladoceran predominance among macrophytes may have been a force reducing green algae numbers. The chlorophytes of the investigated ponds were mostly small- or medium-size species. Their size distribution makes them a high quality food for zooplankton, particularly for cladoceran filtrators. According to RDA analysis apart from pond size, the presence of filtrators significanly reduced the abundance of several chlorophyte dominating species. The lower algae abundance among macrophytes compared to the open water zone could also be explained by competition between algae and macrophytes for light and nutrients37,50 and/or with the secretion of allelopathic substances e.g. by Ceratophyllum demersum51 inhibiting algal development. Our studies demonstrated that among chemical factors which clearly differentiated the two types of analysed habitat, TRP and NH4 significantly influenced the distribution of chlorophyte dominating species. The lower levels of these parameters in macrophyte-dominated zones suggest that the nutrient uptake by aquatic plants in the investigated water bodies was high. There are many reports on the decrease of nutrient concentrations by macrophytes30,37,52, which are consistent with our observations. Despite lower, compared to the open water zone, chlorophyte densities within the macrophyte-dominated zones there was a group of species (e.g. Mougeotia sp., Pediastrum tetras, Scenedesmus obtusus, Monoraphidium contortum) that selectively chose vegetated stands. Furthermore, we found a great number29 of exclusive chlorophyte species for macrophyte-dominated zones. Half of these taxa belong to desmids, which are often periphytic organisms associated with aquatic macrophytes53,54.Preference towards macrophyte-dominated stations was also documented for two phytoplankton functional groups (T: Mougeotia sp. and Binuclearia lauterbornii and TD: e.g., Spirogyra sp., Zygnema sp., Cladophora glomerata, Oedogonium sp.) and one group which occurred exlusively among vegetated sites (MP—Ulothrix). Interestingly, all the representatives of these groups had a similar filamentous morphological form, which suggests that many of them are of epithytic origin, coexisting within aquatic plants. Two more groups—X2 (Pseudodidymocystis/Didymocystis, Pteromonas) and W1 (Gonium pectorale) were clearly affected by the presence of macrophytes. According to Padisak et al.40, codons TD and X2 indicate mesoeutrophic conditions and their higher abundances in the macrophyte-dominated zones also proves that plants contribute to lowering the trophic levels in the examined ponds. On the other hand, the relatively high abundance of the representative of the group W1 in these habitats suggests that macrophytes could enrich ponds with organic matter during the process of their decomposition.Concluding, our results prove that different types of catchment area (field and forest) as well as different types of habitats (open water zone and macrophyte-dominated zone) create distinct, specific conditions (dependent on some physical–chemical and biological variables) for the occurrence of chlorophytes in small water bodies. We conclude that cosmopolitan chlorophytes undoubtedly respond to the level of habitat heterogeneity, contributing to the ecological assessment of small water bodies. Chlorophytes in particularl react to the level of human transformation in the ponds’ vicinities. This is why we suggest using them for water quality evaluation in ponds. This interdisciplinary research significantly broadens the knowledge, not only about the response of chlorophytes to physical–chemical parameters of water, but also about the food preferences of zooplankton for which green algae are the basic food, and vice versa about the impact of zooplankton on microalgae communities. The analyses provide valuable information on chlorophytes-zooplankton interactions and also about the relationships between chlorophytes and macrophytes. Received data emphasize the high value of field ponds, underestimated habitats particularly vulnerable to destruction in the agricultural landscape. The research will help to better understand the functioning of poorly studied small water bodies, which will contribute to the preservation of their biodiversity and protection against degradation. They will also be useful in the management of small water bodies based on the specificity of chlorophyte occurrence in various habitats and catchment type ponds. Moreover, these results are important in a broader context, as the interactions between the studied organisms and the physico-chemical parameters of water in small bodies of water are to some extent universal, so the analyses will broaden the knowledge about the functioning of larger bodies of water. More

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    Trawling the ocean virome

    Microbial biodiversity surveys have often been done in a number of generally better-studied regions3, as with the San Pedro Time Series from the San Pedro Channel off the coast of Southern California. Global surveys have also been emerging, such as the Sorcerer II Global Ocean Sampling Expedition from 2004 to 2006 launched by J. Craig Venter. There are also data and samples from the Malaspina circumnavigation, an expedition devoted to data collection on ocean biodiversity and climate change that was led by the Spanish Ministry of Science and Innovation.As microbiome researcher Shinichi Sunagawa of the ETH Zurich and colleagues point out4, sequencing technologies have advanced such that they now enable systematic and quantitative global ocean surveys. These advances, in turn, made it possible to find and assess marine double-stranded DNA virus populations. This latest work on marine RNA viruses, says Sunagawa, in which he was also involved, embeds new phylum-level findings into a “robust taxonomic framework.” In his view, this research ranks in importance with the reconstruction a few years ago of a group of bacterial genomes representing more than 35 phyla that the researchers call “the candidate phyla radiation”5. If one counts viruses in with other taxonomic groups, the finding might be the largest single expansion of established microbial taxonomy, he says. And he especially likes the definition of a new basal Orthornavirae megataxon, the proposed phylum ‘Taraviricota’. This proposed phylum is one of several findings from recently published analyses of sampling data from Tara Oceans1,2, a global expedition supported by the Tara Ocean Foundation, or Fondation Tara Océan, based in France and with many partner organizations and supporters. The foundation is a major source of global data about the ocean and ocean microbes and, as its president Étienne Bourgois says, it’s a “family project.” The family business is the French fashion house agnès b., founded by his mother Agnès Troublé.Because the family cares about the sea, they bought a 36-meter schooner from Lady Pippa Blake, widow of yachtsman and explorer Sir Peter Blake, after pirates killed him during an environmental expedition in the Amazon delta, and turned it into the expedition vessel and floating science laboratory Tara, devoted to understanding and protecting the world’s marine environment. It’s a way to continue what Peter Blake started, to continue the conversation about the ocean and do research as well, says sailor-scientist Romain Troublé, executive director of the foundation and nephew of Agnès Troublé. The boat had been previously owned by explorer Jean-Louis Étienne. The foundation has supported several expeditions with Tara including the Tara Oceans and Tara Oceans Polar Circle expeditions, as well as Tara Mission Microbiomes, which is currently underway. The equilibrium of the planet “depends on the microbiome of the ocean in the same way we depend on our own microbiome,” says Romain Troublé. Viruses are part of the larger picture of how life is supported on the planet. It’s “a great mystery of the century” to decipher the roles, behaviors and functions of the ocean microbiome, including its beneficial effects. Over the last decade, he says, the expeditions have, for example, collected plankton samples from coastal waters, coral reefs and the high seas around the world for scientists to ask questions of. Microplastics in the ocean concentrate chemical pollutants such as pesticides, and microplastics appear to be substrates for distinct microbiomes. Polystyrene and polypropylene, for example, harbor different microbial communities. “We call it the plastisphere,” he says. All sample collection, not just of microplastics, happens with a view to scientific rigor to assure data quality, says Troublé. Many institutes are part of and support the expeditions through the Tara Ocean Foundation, including AtlantECO, the French Ministry of Research, the Swiss National Science Foundation, the US National Science Foundation, the European Molecular Biology Laboratory and the French National Centre for Scientific Research.Tara Oceans was an expedition initiated by EMBL researcher Eric Karsenti, here in the foreground. He is checking a rosette of Niskin bottles that collect water, and ocean microbe samples, at various depths. Sensors capture parameters such as temperature.
    Credit: Fondation Tara OcéanIts expedition Tara Oceans was initiated by cell and marine biologist Eric Karsenti of the European Molecular Biology Laboratory. The expedition ran from 2009 to 2013 and covered 125,000 kilometers of ocean, taking ocean water and samples. It collected nearly 35,000 samples of viruses, algae and plankton and delivered more than 60 terabases of DNA and RNA sequences.The research community strives to follow FAIR data principles, the principles of findability, accessibility, interoperability and reusability, says Sunagawa. Tara Ocean’s data troves can be found, for instance, in the European Nucleotide Archive (ENA), Pangeaea, Cyverse, iVIRUS and on Genoscope. Other data-collection efforts target users with less programming experience and offer various types of data relevant to marine microbial research, he says: for example, the Ocean Gene Atlas, a portal to search for a gene or protein sequence to see, for instance, its abundance on an ocean map. The Ocean Barcode Atlas lets users explore, for example, operational taxonomic units (OTU) data and plankton communities from Tara Oceans and OTUs from Malaspina prokaryote data. Sunagawa also points to the Ocean Microbiomics Database and its high-quality genome-resolved information about the global microbiome, which has sequencing data from 2003 onwards and which includes Tara Oceans data as well as datasets such as the Hawaii Ocean Time-Series (HOT), the Bermuda Atlantic Time-series Study (BATS), with its collection of ocean data dating back to 1988, and BioGeotraces, with hydrographic and marine geochemical data from various expeditions.The recent publications on RNA viruses1,2, in which Sunagawa was also involved, have expanded the known diversity of these viruses, he says. They build on efforts by, for example, the research team that created and applied a cloud-based infrastructure called Serratus6, with which researchers can perform sequence alignment using bowtie2 for nucleotide sequences and DIAMOND2 for protein sequences in ‘ultra-high throughput’ on a petabase scale. Using Serratus, the team identified more than 130,000 previously unknown RNA viruses, both on land and in the oceans. The wealth of resources for microbial and viral data about the oceans is helpful to the research community, but “we could still improve the connectivity between various datasets though,” says Sunagawa. That would help, for example, with searching and finding data products that are derived from primary data, such as identifiers of individual genome assemblies, genes and metagenome assembled genomes, which are all presented in different online locations. But connecting data resources is a project that itself takes resources, and such projects are hard to get funding for.Going forward, it will be challenging, says Sunagawa, to update and keep up to date both past projects and ongoing projects such as the Global Ocean Ship-based Hydrographic Investigations program (GO-SHIP), which is focused on physical oceanography; the Antarctic Circumnavigation Expedition (ACE), on carbon-cycle marine biogeochemistry; Mission Microbiomes; and many more. “And ultimately, we will need to cross boundaries that currently separate biome-focused research to better understand processes at the sea–land–atmosphere interfaces.”Tara Mission Microbiomes has been underway for nearly two years and wraps up in October 2022. At press time, the schooner Tara was off the Angolan Coast. At the end of the expedition, it will have traveled a total of 70,000 km of ocean area around South America, Africa, Europe and Antarctica. Mission Microbiomes is part of the EU-funded AtlantECO and also includes 42 research organizations from 13 countries. The microbiome mission is collecting data on how climate change is affecting the marine microbiome, on how pollution, microplastics pollution in particular, affects the marine environments and on the beneficial impact of the ocean microbiome.Krill are small ocean crustaceans that mainly eat phytoplankton and are a food source for animals such as whales and seals. Krill play a crucial role in biogeochemical cycles.
    Credit: F. Aurat, Fondation Tara OcéanChris Bowler, from the Institut de Biologie de l’École Normale Supérieure, is scientific director of the Tara Oceans consortium, was scientific coordinator of the Tara Oceans expedition and was onboard in Antarctica during the Tara Mission Microbiomes expedition to collect data on the impact of icebergs on the Weddell Sea ecosystem. The project’s scientists in Tara Mission Microbiomes, he says, are studying specific processes, including the Amazon plume, the Malvinas confluence, the impact of tabular icebergs in the Weddell Sea, the Benguela upwelling and more. The data from this expedition will be similar to those from Tara Oceans but, he says, “we will have much more contextual data related to the specific processes we have been studying.” The applied techniques are all ones that have undergone much advancement since Tara Oceans, he says. They include long-read sequencing, Hi-C sequencing to capture chromatin organization on a genome-wide basis and various types of microscopy.Data and results from previous and ongoing expeditions are impressive, says Sunagawa but “we are still data-limited in our field of research.” Geographically, sampling stations are usually still separated by hundreds of kilometers, and often they are even further apart than that. This means that what is missing is both temporal and seasonal resolution, “and we keep detecting new organisms,” he says. Tara Mission Microbiomes will help to fill in some of these gaps. The mission is unlike Tara Oceans, with its focus more on coastal areas and environmental pollutants such as microplastics. Sunagawa and his group are not currently involved with Tara Mission Microbiomes, “but we look forward to seeing the first results coming out soon.”Through photosynthesis, phytoplankton deliver oxygen to the planet. They are food for zooplankton, which are food for other marine organisms. This food web and its associated decomposition are part of the ocean’s carbon pump, in which marine viruses play an important role that scientists have only begun exploring.
    Credit: M. Bardy, Fondation Tara Océan More

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    Why the ocean virome matters

    Kyoto University microbiome researcher Hiroyuki Ogata says that the recent work2,3 further connects RNA viruses and the carbon pump, which affects the Earth’s biogeochemical cycles and thus its climate. And it sheds light on the diversity, evolution and ecology of RNA viruses, which has not previously been possible through applying the techniques of traditional DNA-based metagenomics. The team found many new lineages at the phylum-level by using “highly sensitive” computational approaches.It’s possible to assess the ecosystem impact of viruses by inferring auxiliary metabolic genes (AMGs). AMGs hint at the ways RNA viruses manipulate the physiology of their hosts as they seek to maximize production of more virus through the host. As Jian explains, labs have identified a variety of AMGs that are encoded by DNA viruses and, he says, it’s “well-recognized” that AMGs probably play a role in marine ecosystems. It was unknown if AMGs could be found in RNA viruses, which the recent Science paper2 has now established, he says. Jian sees this work as providing “a very important foundational dataset” for exploring questions connected to AMGs. “In my opinion, if more long-sequence or complete marine RNA virus genomes can be obtained in the future, and they can be further connected with specific hosts, it will greatly promote the understanding of the ecological impact of RNA viruses in the oceans.”To tease out AMGs, the scientists used a variety of tools, such as viral identification software for both DNA and RNA viruses, says Wainaina. The ones for DNA viruses are available on Cyverse, and the protocols for the tools from the Sullivan lab are on protocols.io. One method for RNA viruses is in progress and will be soon available on Cyverse, he says. DNA viral identification tools include VirSorter2, a pipeline for identifying viral sequence from metagenomics data, and the protocol for using this and other tools are also on protocols.io. To identify AMGs from viral sequence that had been identified through VirSorter, the team used use DRAM-v, a software tool from the lab of microbiome researcher Kelly Wrighton at Colorado State University. Her group had created Distilled and Refined Annotation of Metabolism (DRAM), a framework to resolve metabolic information from microbial data. The companion tool DRAM-v is for viruses and can be applied to metagenomic data sets for annotating metagenomics-based assembled genomes, for example through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, and to contiguous viral sequences identified by VirSorter.The hunt for AMGs is one instance in which the team needed to determine in each case whether a sequence was likely ‘stolen’ from host cells, says Dominguez-Huerta. RNA viral genomes are less than 40 kilobases long and usually have complicated genomic organization, both in a structural genomics sense related to the physical arrangement of genes along the viral genome and in a functional sense in terms of transcription and translation: there are overlapping genes, frameshifts and more, all of which makes this kind of annotation difficult. And sometimes information in the annotation databases is wrong and indicates that a match is cellular when it is in fact viral. Thus, to find AMGs, “we don’t have a defined clean methodology automated in a pipeline yet,” he says. It remains a time-consuming task. Assigning putative function to the protein sequences encoded by AMGs also involves checking the literature and comparing different annotation sources.Dominguez-Huerta says he and the team were glad they could assemble AMG functionalities to suggest the range of ways in which RNA viruses manipulate the metabolisms of their hosts—from photosynthesis to central carbon metabolism to vacuolar digestion and RNA repair. This overview let them see how some AMGs are repeated across different viruses across the oceans. Finding AMGs in long-read sequence is what he calls a “fire test” for the lab. To avoid ‘false AMGs’ from unreliable matches, they use BLASTP, the Basic Alignment Search Tool that compares a protein query sequence to a protein database.“I am fascinated by the ability of viruses to metabolic reprogram not only their hosts but more importantly at the ecosystem level,” says Wainaina. It is probable that the AMGs the team identified “are a central cog in microbial metabolism networks.” Current and future modeling efforts will hopefully provide insights into the ecosystem roles of viruses—both DNA viruses and RNA viruses—and on a global scale both within the ocean ecosystem and beyond.Host inference is challenging, says Dominguez-Huerta, because, for example, viruses with RNA genomes do not share genetic information with their host genomic DNA the way dsDNA viruses do when they infect bacteria. That means there is no clear signal to be derived from the host genome to help one guess the possible host. But sometimes RNA viruses do integrate into host genomes, and those, likely more accidental, events were sufficient for the scientists to capture some signal to infer hosts. “We also performed statistical co-occurrence analytics using abundances to infer the hosts with certain success,” he says.Unlike dsDNA viruses, RNA viruses infect mostly eukaryotes, from protists and fungi to invertebrates and fish larvae; only a minority infect bacteria. Overall, the team has been able to capture “a picture of dsDNA viruses infecting prokaryotes and RNA viruses infecting eukaryotes in the oceans, complementing each other in their marine hosts,” says Dominguez-Huerta. The fact that the scientists can infer “that RNA viruses can steal genes from the host,” in the form of AMGs, to then reprogram host metabolism matters not only as scientists complete the picture of how viruses directly tune the activity of hosts during infection, but also in regard to how this influences biogeochemical cycles, he says. “We think that these AMGs are incorporated into the RNA virus genomes from cellular mRNA transcripts by non-homologous recombination,” he says. This gives, in his view, a new picture of RNA viruses, which, despite their small genome sizes, can squeeze in protein-coding genes. Such proteins could be sufficient to boost the production of virus particles per infected cell, perhaps increasing viral fitness in the difficult conditions of the oligotrophic open ocean and letting the viruses better propagate in the environment.More generally, says Dominguez-Huerta, capturing RNA from ocean samples is difficult, because RNA is physically fragile and degrades rapidly. When digging into metatranscriptomic data, which include the RNA from plankton and RNA from other organisms, less than 1% of this RNA is likely to be viral RNA, he says. Previously, some labs have first purified RNA from samples, enriched it for replicating RNA viruses and then applied a method called dsRNA-seq to recover dsRNA virus sequence and replicate sequences from single-stranded RNA viruses. For future ocean RNA virus projects, he says that the lab is currently working on a wet-lab method to purify RNA virus particles from seawater to solve the challenges of obtaining viral RNA for analysis. 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