Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts

Abstract

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters.

Fig. 1. Morphology and evolutionary relationships of newly discovered Bathycoccus viruses.

A Transmission electron micrographs of BII-V1, BII-V2 and BII-V3 (scale bar, 50 nm). The capsid diameters (n = 6 virions) measured 138 ± 2 nm (BII-V1), 150 ± 5 nm (BII-V2) and 152 ± 11 nm (BII-V3). B Maximum Likelihood (ML) phylogenetic reconstruction of green algal viruses inferred from a concatenated alignment of 22 core proteins shared among the viruses (7,001 positions) under the LG + G + F model. Node support was calculated from 1000 bootstrap (BS) replicates, with all branches acquired support values of 100% (white dots). Viruses infecting Chlorella were used as an outgroup and the branch connecting the prasinoviruses to the outgroup was truncated for display purpose. The new Bathycoccus viruses isolated against Bathycoccus Clade II (sensu [12]) isolate RCC716 (named as species Bathycoccus calidus herein, see below) are in bold. Colours reflect different host species within each genus. Letters alongside vertical lines (a and b) correspond to Bathycoccus viral clades. C Venn diagram of the shared and unique protein-encoding genes in the genome sequences of the new Bathycoccus viruses.

Fig. 2. Distribution of functions and orthologous protein families across genome-sequenced prasinoviruses.

A Functional category distributions across 21 genome-sequenced prasinoviruses and chloroviruses based on EggNOG categorisation. Viruses are clustered by similarity in their distribution of the functional categories on the y-axis and the frequency of each category across the viral genomes determines clustering along the x-axis ordering. Genes with homology to proteins in the EggNOG database but could not be assigned a function are in the “function unknown” category. B Orthogroups presence/absence patterns ordered along the x-axis by ranking according to the total number of genes in the orthogroup. For inclusion, the orthogroup was required to include protein sequences from at least two different viral genomes. Viruses are ordered along the vertical by their presence/absence pattern reconstructed by hierarchical clustering (topology on the left). Top histogram: frequency of each orthogroup in sequenced prasinoviruses. C Genes in each virus (number) not assigned to any orthogroup, with viruses in the same vertical order as B.

Fig. 3. Temporal studies reveal differential infection dynamics.

Flow cytometric enumeration of host cell abundance and viral particle abundance over experimental time courses. A Non-infected Bathycoccus Clade BII RCC716 control cultures (grey circles) as well as RCC716 cells in cultures inoculated with BII-V1 (red circles), BII-V2 (orange diamonds) and BII-V3 (yellow triangles). B symbols are as in A but represent the abundance of viral particles rather than hosts. Points and error bars represent mean values and standard deviation for biological triplicates. Horizontal grey dashed lines represent the cell or viral concentration at T₀. Non-shaded and shaded areas represent light and dark periods, respectively. These experiments indicate BII-Vs are lytic viruses, although note that an intermediate state of chronic infection, without host lysis or integration into the host genome, has been described for O. tauri viruses [89].

Fig. 4. Global distribution and diversity of Bathycoccus species and viruses.

A Map of Tara Oceans stations (modified from Sunagawa et al. [46]) from which metagenomic reads were recruited using the B. prasinos (BI), Bathycoccus RCC716 (BII), BII-V1, -V2 and -V3, as well as BpV1 and BpV2 genomes. Most stations contained reads from both the host and respective virus (solid colours) while in some only hosts reads were detected (muted colours). All stations in which any of the Bathycoccus viruses were detected also contained reads from the respective hosts. B Relative contribution of each Bathycoccus species (solid lines; BI, light green; BII dark green) based on [24] at Tara Oceans stations where we also detected viruses. Also shown are the relative contributions of viruses, computed as the sum of reads competitively recruited to genome-sequenced BpVs versus BII-Vs. C Relative contribution of reads competitively mapped against all Bathycoccus viruses. The colour code for stations is the same in all panels. SRF surface water, SSCM sub-surface chlorophyll maximum. D Phylogenetic reconstruction of prasinovirus PolB genes in GenBank nr and metagenomic data. ML reconstruction using the model LG + G + I based on a multiple sequence alignment of amino acids. Branches with BS values ≥90% (1000 replicates) are indicated (black dots). A total of 159 viral PolB sequences from Tara Oceans data were incorporated from assemblies with >300,000 contigs [46] along with 21 from viruses with known hosts and sequenced. Coloured blocks represent taxonomic classification of lineages based on supported clades (BS ≥ 90%) that include viruses with known hosts (only these are named on the tree). All labels and accessions are shown in Fig. S5.

Fig. 5. Cell morphologies and molecular signatures of novel species Bathycoccus calidus, formerly Bathycoccus RCC716, as compared to Bathycoccus prasinos.

A Transmission electron micrographs of B. calidus isolate RCC716, reveal cell characteristics including, n nucleus, c chloroplast, m mitochondria, and arrows external scales. B The second internal transcribed spacer (ITS2) of Bathycoccus calidus (i.e., Bathycoccus Clade BII, KY563784) has ‘universal’ helices 1-4 and an additional helix (between helices 3 and 4), characteristic for the family Bathycoccaceae (i.e., Bathycoccus and Ostreococcus genera [103]). Helices of B. calidus were compared with homologous ITS2 helices from B. prasinos (Clade BI, JX625115) by highlighting compensatory base changes (CBCs), hemi-CBCs, base pairing/dissociation events (dotted lines), as well as hypothetical insertions/deletions (indels). A few regions, which were divergent and showed no intermediate evolutionary stages, were labelled as ‘not comparable’ (n.c.). Note that while the two Clade BII isolates have identical ITS sequences, among 13 Bathycoccus environmental rRNA/ITS sequences annotated as Bathycoccus Clade BII [12], four from tropical Pacific and Atlantic (KY382370, KY382373, KY382374, KY368637) present polymorphisms (green shading) at one nucleotide (helix 2, bp 16 reverse, G instead of A) as well as one deletion (helix 4, bp 13, bp not present). Tracing base pair evolution in the stem regions (i.e., helices) by CBCs and hemi-CBCs revealed several molecular signatures for B. calidus in helices 2 and 4, i.e., without homoplasies in the Bathycoccaceae [19], most of which evolved as hemi-CBCs. Signatures for B. calidus are indicated by orange shading. Base pairs 16 and 17 of helix 4 (corresponding to bp 18 and 19 in B. prasinos) were selected for the taxonomic diagnosis. A single-stranded nucleotide position in the conserved linker between the additional helix and helix 4 revealed another distinctive character of B. calidus.