Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These endogenous mavirus-like elements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.

the heterotrophic stramenopile Cafeteria roenbergensis is a globally distributed marine bacterivorous protist. This unicellular flagellate is host to the giant DNA virus CroV and the virophage mavirus. We sequenced the genomes of four cultured C. roenbergensis strains and generated 23.53 Gb of Illumina MiSeq data (99-282 × coverage per strain) and 5.09 Gb of PacBio RSII data (13-45 × coverage). Using the Canu assembler and customized curation procedures, we obtained high-quality draft genome assemblies with a total length of 34-36 Mbp per strain and contig N50 lengths of 148 kbp to 464 kbp. the C. roenbergensis genome has a GC content of ~70%, a repeat content of ~28%, and is predicted to contain approximately 7857-8483 protein-coding genes based on a combination of de novo, homologybased and transcriptome-supported annotation. These first high-quality genome assemblies of a bicosoecid fill an important gap in sequenced stramenopile representatives and enable a more detailed evolutionary analysis of heterotrophic protists.

Endogenous viral elements (EVEs) are frequently found in eukaryotic genomes, yet their integration dynamics and biological functions remain largely unknown. Unlike most other eukaryotic DNA viruses, the virophage mavirus integrates efficiently into the nuclear genome of its host, the marine heterotrophic flagellate Cafeteria burkhardae. Mavirus EVEs can reactivate upon superinfection with the lytic giant virus CroV and may act as an adaptive antiviral defense system, because mavirus increases host population survival during a coinfection with CroV. However, the prevalence of endogenous virophages in natural flagellate populations has not been explored. Here we report dozens of endogenous mavirus-like elements (EMALEs) in the nuclear genomes of four C. burkhardae strains. EMALEs were typically 20 kilobase pairs long and constituted 0.7% to 1.8% of each host genome. We analyzed 33 fully assembled EMALEs that fell into two main clusters and eight types based on GC-content, nucleotide similarity, and coding potential. Inter-strain comparison showed conservation of some EMALE insertion loci, whereas the majority of integration sites were unique to a given host strain. We also describe a group of tyrosine recombinase retrotransposons, some of which exhibited a strong preference for integration into EMALEs and represent yet another layer of parasitism in this microbial system. Our findings show that virophages are common, diverse, and dynamic genome components of the marine protist C. burkhardae, which implies important eco-evolutionary roles for these enigmatic viruses.

The heterotrophic stramenopile Cafeteria roenbergensis is a globally distributed marine bacterivorous protist. This unicellular flagellate is host to the giant DNA virus CroV and the virophage mavirus. We sequenced the genomes of four cultured C. roenbergensis strains and generated 23.53 Gb of Illumina MiSeq data (99-282 × coverage per strain) and 5.09 Gb of PacBio RSII data (13-54 × coverage). Using the Canu assembler and customized curation procedures, we obtained high-quality draft genome assemblies with a total length of 34-36 Mbp per strain and contig N50 lengths of 148 kbp to 464 kbp. The C. roenbergensis genome has a GC content of~70%, a repeat content of~28%, and is predicted to contain approximately 7857-8483 protein-coding genes based on a combination of de novo, homology-based and transcriptome-supported annotation. These first high-quality genome assemblies of a Bicosoecid fill an important gap in sequenced Stramenopile representatives and enable a more detailed evolutionary analysis of heterotrophic protists.

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