Metagenomic Analysis of Viral Communities in (Hado)Pelagic Sediments

Citation: Yoshida M, Takaki Y, Eitoku M, Nunoura T, Takai K ( Metagenomic Analysis of Viral Communities in (Hado)Pelagic Sediments Mitsuhiro Yoshida 0 1 Yoshihiro Takaki 0 1 Masamitsu Eitoku 0 1 Takuro Nunoura 0 1 Ken Takai 0 1 Jianming Qiu, University of Kansas Medical Center, United States of America 0 Current address: Department of Environmental Medicine, Kochi Medical School, Kochi University , Nankoku, Kochi , Japan 1 Japan Agency for Marine-Earth Science and Technology (JAMSTEC) , Yokosuka, Kanagawa , Japan In this study, we analyzed viral metagenomes (viromes) in the sedimentary habitats of three geographically and geologically distinct (hado)pelagic environments in the northwest Pacific; the Izu-Ogasawara Trench (water depth = 9,760 m) (OG), the Challenger Deep in the Mariana Trench (10,325 m) (MA), and the forearc basin off the Shimokita Peninsula (1,181 m) (SH). Virus abundance ranged from 106 to 1011 viruses/cm3 of sediments (down to 30 cm below the seafloor [cmbsf]). We recovered viral DNA assemblages (viromes) from the (hado)pelagic sediment samples and obtained a total of 37,458, 39,882, and 70,882 sequence reads by 454 GS FLX Titanium pyrosequencing from the virome libraries of the OG, MA, and SH (hado)pelagic sediments, respectively. Only 24230% of the sequence reads from each virome library exhibited significant similarities to the sequences deposited in the public nr protein database (E-value ,1023 in BLAST). Among the sequences identified as potential viral genes based on the BLAST search, 95299% of the sequence reads in each library were related to genes from single-stranded DNA (ssDNA) viral families, including Microviridae, Circoviridae, and Geminiviridae. A relatively high abundance of sequences related to the genetic markers (major capsid protein [VP1] and replication protein [Rep]) of two ssDNA viral groups were also detected in these libraries, thereby revealing a high genotypic diversity of their viruses (833 genotypes for VP1 and 2,551 genotypes for Rep). A majority of the viral genes predicted from each library were classified into three ssDNA viral protein categories: Rep, VP1, and minor capsid protein. The deep-sea sedimentary viromes were distinct from the viromes obtained from the oceanic and fresh waters and marine eukaryotes, and thus, deep-sea sediments harbor novel viromes, including previously unidentified ssDNA viruses. - Viruses represent the most abundant number of biological components by far in aquatic ecosystems [1], and viral ecology in environments such as oceanic surface waters, coastal, and fresh waters have been intensively investigated [2]. Viral activity in aquatic environments is known to regulate the dynamics and mortality of the host microbial community [39]. The lytic processes of the host microbial cells infected by marine viruses, termed the viral shunt, supply organic matter to dissolved carbon and nutrient pools [1012]. Furthermore, viruses have been noted as natural genetic vectors for horizontal gene transfer events [13,14]. Despite their ecological and evolutionary importance, our current knowledge of marine viruses is restricted to the euphotic zone of the habitat, which represents only a limited portion of the oceanic biosphere [15]. Viral ecology in sedimentary environments has been poorly studied, although the seafloor sediments cover almost two-thirds of the Earths surface and serve as highly vital and dynamic interface habitats in global ocean biogeochemical cycles [16]. Deep-sea sediments (down to 10 cm below the seafloor [cmbsf]) harbor a great number of viral particles (.107 viruses/cm3 sediment) and high virus productivity associated with large prokaryotic biomasses ranging from 106 to 108 cells/cm3 sediment [17,18]. These observations suggest that viral infections have a large impact on deep-sea sedimentary microbial communities and that the benthic prokaryotic biomass is sustained by the viral shunt, which is estimated to provide 35% of organic carbon for the total benthic microbial production [18]. However, the genetic composition and diversity of viral communities in deep-sea sediments have not yet been reported. A comprehensive metagenomic approach to environmental viral populations (viromes) can provide insight into the genetic diversity and previously unidentified constituents of the viral communities of various ecosystems [1926]. Two different wholegenome amplification methods have been used for virome analysis. One is known as the linker-amplified shotgun library (LASL) method [27] and is only applicable to double-stranded DNA (dsDNA). The LASL method has been applied in several virome studies, such as of surface seawater [27], human feces [28], and fermented foods [29]. These virome studies have suggested that a large proportion of the DNA viruses infect prokaryotic hosts, while most RNA viruses analyzed by reverse transcription infect eukaryotes [30]. The other method, known as multiple displacement amplification (MDA) with phi29 polymerase [31], can amplify both the dsDNA and single-stranded DNA (ssDNA) of the viral genomes, although this method is known to have a considerable bias for the preferential amplification of small circular genomes (129 kb) from ssDNA viruses [32,33]. Using this method, the distribution and diversity of ssDNA viruses (including both phages and eukaryotic viruses) have been investigated in various environments, such as marine waters [19,34], modern microbialites [35], coral [36], temperate freshwater lakes [37], the Antarctic lake [21], reclaimed water [38], the human gut [39,40], and rice paddy soil [33]. However, the host ranges and ecological impacts of these ssDNA viruses are still largely uncertain [41]. In this study, we used 454 pyrosequencing to conduct a virome analysis of deep-sea shallow subseafloor sediments (down to 40 cmbsf) in three distinct (hado)pelagic environments of the northwest Pacific: the hadopelagic sediments in the Izu-Ogasawara Trench (water depth = 9,760 m), the hadopelagic sediments in the Challenger Deep of the Mariana Trench (water depth = 10,325 m), and the pelagic sediments off Shimokita Peninsula (water depth = 1,181 m). To our knowledge, this study is the first to describe the characteristics of viromes in deep-sea sediments and identify novel ssDNA viruses that are distinct from viral genotypes previously known to occur in ocean environments. Materials and Methods Ethics Statement The sampling in the Mariana Trench during the JAMSTEC KR08-05 cruise was approved by the U.S. Government. No specific permits were required for the other field studies described here and sampling locations are not privately-owned or protected. The field studies did not involve endangered or protected species. Sediment Samples Sediment cores from the Izu-Ogasawara Trench (29u099 N, 142u49 E; 9,760 m water depth) (Fig. 1) and the Challenger Deep in the Mariana Trench (11u229 N, 142u429 E; 10,332 m water depth) (Fig. 1) were obtained with (...truncated)