Fig 1.
A schematic of the major lineages of the newly identified sea-lice-associated RNA viruses in the tree of RNA viruses.
The approximate phylogenetic positions of the sea-lice-associated RNA viruses are indicated by sea-lice icons and are based on analyses of the RdRp sequences for the viruses identified in this study. Levi, Leviviricetes; Narna, Narnaviridae; Ourmia, Ourlivirales; Mito, Mitoviridae; Partiti-Hypo, Partitiviridae, Hypoviridae, Picobirnaviridae, Amalgaviridae; Astro, Astroviridae; Poty, Potyviridae; Picorna, Picornavirales; Solemo, Solemoviridae; Nido, Nidovirales; Toli, Tolivirales; Martelli, Martellivirales; Noda, Nodaviridae; Flavi, Flaviviridae; Reo, Reoviridae; Cysto, Cystoviridae; Ghabri, Ghabrivirales; Bunya, Bunyavirales; Mononega, Mononegavirales. The colored branches indicate phyla recognized by the ICTV [28], and the phylogenetic relationships among them are based on Wolf et al. and Koonin et al. [29,30].
Fig 2.
Phylogenetic placement of sea-lice viruses for the phyla Lenarviricota and Pisuviricota.
Phylogenetic placement of sea-lice viruses within major taxonomic groups based on RdRp sequences for the phyla Lenarviricota (Narnaviridae related) and Pisuviricota (Hypoviridae related, Solemoviridae related, Picornavirales related, and Partitiviridae related). Branches representing members of established viral groups (i.e., families and genera) are collapsed, and branches representing unclassified arthropod-associated RNA viruses are collapsed and indicated with a butterfly icon. Viruses identified in the present study are indicated by red branches. Viruses discovered in different species of sea lice are color-coded by the host they are associated with: Caligus clemensi (blue), Caligus rogercresseyi (green), and Lepeophtheirus salmonis (red); those identified in different life stages of C. rogercresseyi are indicated by filled circles (planktonic stages) or stars (parasitic stages) at the end of each branch. Bootstrap branch support greater than 0.7, 0.8, and 0.9 are shown by empty, grey, and black circles, respectively. Each scale bar indicates 0.5 amino-acid substitutions per site. The maximum-likelihood trees are inferred based on the RdRp coding regions of the viruses with 1000 ultrafast bootstrap replicates using IQ-tree 2 [31]. The best substitution model is selected according to the BIC scores by ModelFinder [32] integrated within IQ-tree 2.
Fig 3.
Phylogenetic placement of sea-lice viruses for the phylum Kitrinoviricota.
Phylogenetic placement of sea-lice viruses within major taxonomic groups belonging to the phylum Kitrinoviricota. The figure legend is the same as for Fig 2, except the fish icons, which represent fish-associated viruses that are related to the viruses identified in sea lice.
Fig 4.
Phylogenetic placement of sea-lice viruses for the phyla Duplornaviricota and Negarnaviricota.
Phylogenetic placement of sea-lice viruses within major taxonomic groups belonging to the phyla Duplornaviricota and Negarnaviricota. The figure legend is the same as that for Fig 2.
Fig 5.
Virus-derived small RNAs (viRNAs) and EVE-derived PIWI-interacting RNAs (piRNAs) found in Caligus rogercresseyi and Lepeophtheirus salmonis.
The small RNAs were mapped to sequences of RNA viruses discovered from the transcriptomes of C. rogercresseyi, in order to identify canonical viRNAs. Each bar plot represents the size distribution and 5’ base composition of small RNAs that matched sequences of viruses discovered in C. rogercresseyi. The x-axis indicates the size of small RNAs with each axis beginning at 17 nt and ending at 29 nt. Bars above and below the x-axis indicate small RNAs mapping to the positive and negative senses of the virus, respectively. The y-axes, which are hidden to simplify the figure, indicate the number of aligned small RNAs for each size category (i.e., 17–29 nt). Bars are color-coded based on the frequency of 5-prime base of the mapped sRNAs (green: A, blue: C, yellow: G, pink: U). CARO, Caligus rogercresseyi. LESA, Lepeophtheirus salmonis. Blue stars indicate the viruses present in our phylogenetic trees. Red asterisks show the viruses that have fewer (< 100) detected sRNAs but display typical viRNA or piRNA patterns.
Fig 6.
The number and genomic location of viRNAs derived from the selected viruses in C. rogercresseyi.
Our analysis showed that viRNAs were typically generated from the entire viral sequences in C. rogercresseyi, while the number of the produced viRNAs varied markedly among genomic sites. N, nucleoprotein; P, phosphoprotein; M, matrix protein; G, glycoprotein; L, large protein that encompassing RdRp. The hypothetical protein is denoted by a question mark.
Fig 7.
Genome sizes and structures of previously unknown viruses found in sea lice.
The genome maps are arranged by phylogenetic group and by name within each group. Each viral genome is represented by a single line with the length of each line corresponding to genome size. The predicted open reading frames are shown as individual boxes on each genome map. Coding sequences are color-coded based on the functional genes they correspond to, while sequences encoding proteins with unknown functions are colored in grey.
Fig 8.
Abundance of viral sequences in different life stages of sea lice (C. rogercresseyi).
The estimated abundance is based on polyA-tail enriched libraries; hence, includes viral genomes and transcripts with polyA-tails. Transcripts per million (TPM) are shown in parentheses for Nauplius I-II (110); Copepodid (133); Chalimus I-II (319); Chalimus III-IV (597); Female (567); Male (398).
Fig 9.
Relative abundance of viruses in each taxonomic group in C. rogercresseyi.
Bars are color-coded according to the assigned taxonomic group. Relative abundance, estimated as the fraction of TPM for each taxonomic group, varied among C. rogercresseyi collected at different life stages and between males and females.