Endogenous virophages populate the genomes of a marine heterotrophic flagellate

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.

Gene expression during bacterivorous growth of a widespread marine heterotrophic flagellate

Phagocytosis is a fundamental process in marine ecosystems by which prey organisms are consumed and their biomass incorporated in food webs or remineralized. However, studies searching for the genes underlying this key ecological process in free-living phagocytizing protists are still scarce, in part due to the lack of appropriate ecological models. Our reanalysis of recent molecular datasets revealed that the cultured heterotrophic flagellate Cafeteria burkhardae is widespread in the global oceans, which prompted us to design a transcriptomics study with this species, grown with the cultured flavobacterium Dokdonia sp. We compared the gene expression between exponential and stationary phases, which were complemented with three starvation by dilution phases that appeared as intermediate states. We found distinct expression profiles in each condition and identified 2056 differentially expressed genes between exponential and stationary samples. Upregulated genes at the exponential phase were related to DNA duplication, transcription and translational machinery, protein remodeling, respiration and phagocytosis, whereas upregulated genes in the stationary phase were involved in signal transduction, cell adhesion, and lipid metabolism. We identified a few highly expressed phagocytosis genes, like peptidases and proton pumps, which could be used to target this ecologically relevant process in marine ecosystems.

Heterotrophic flagellates (Amorpha and Diaphoretiches) in phytotelmata bromeliad (Bromeliaceae)

Many plants may accumulate rainwater, forming phytotelmata, aquatic microhabitats inhabited by various organisms. The aim of this study was to conduct an inventory of heterotrophic flagellates associated with phytotelmata of the bromeliad Aechmea distichantha Lem., found in rocky cliffs on the Upper Paraná River. The bromeliads were removed manually from the rocky wall, the water was removed and cultures of organisms of each plant were mounted in Petri dishes. Sixteen species of heterotrophic flagellate were recorded, drawn and described, among them one species belonging to the Amorpha Domain and 15 species to the Diaphoretiches Domain. The groups with most species were Euglenida and Kinetoplastea. The low diversity of heterotrophic flagellates recorded in this study, compared to the plankton of lakes and reservoirs, is probably related to the fact that phytotelmata are habitats with extreme environmental conditions, thus selecting organisms tolerant to these environments.

Visualization of giant virus particles using BONCAT labeling and STED microscopy

Giant DNA viruses of the phylum Nucleocytoviricota are being increasingly recognized as important regulators of natural protist populations. However, our knowledge of their infection cycles is still very limited due to a lack of cultured virus-host systems and molecular tools to study them. Here, we apply bioorthogonal noncanonical amino acid tagging (BONCAT) to pulse label the marine heterotrophic flagellate Cafeteria burkhardae during infection with the lytic giant virus CroV. In absence of CroV, we report efficient incorporation of the L-methionine analog L-azidohomoalanine (AHA) into newly synthesized proteins of the methionine prototrophic C. burkhardae. During CroV infection, AHA was predominantly found in viral proteins, and single CroV virions were imaged with stimulated emission depletion (STED) super-resolution microscopy. CroV particles incorporated AHA with 95-100% efficiency while retaining their infectivity, which makes BONCAT/STED a powerful tool to study viral replication cycles in this ecologically relevant marine bacterivore.SignificanceGiant DNA viruses are the dominant class of protist-infecting viruses, yet the vast majority of described giant virus-protist systems remain uncultured. One of the better studied cultured systems is composed of the stramenopile Cafeteria burkhardae (previously C. roenbergensis), the giant Cafeteria roenbergensis virus (CroV) and the virophage mavirus. C. burkhardae is a widespread marine phagotrophic protist that plays an important role in regulating bacterial populations. In addition to being grazed upon by larger zooplankton, C. burkhardae populations are controlled by the lytic giant virus CroV. In turn, CroV is parasitized by the virophage mavirus that increases host population survival in the presence of CroV and forms a mutualistic symbiosis with its host. Despite being of fundamental ecological and evolutionary interest, this tripartite host-virus-virophage system suffers from a lack of molecular tools. Here, we show that CroV particles can be fluorescently labeled and imaged by super-resolution microscopy. To achieve this we established robust procedures for analyzing protist and viral populations and implemented the use of bioorthogonal noncanonical amino acid tagging (BONCAT) in a marine unicellular flagellate.

Ecological and evolutionary patterns in the enigmatic protist genus Percolomonas (Heterolobosea; Discoba) from diverse habitats

The heterotrophic flagellate Percolomonas cosmopolitus (Heterolobosea) is often observed in saline habitats worldwide that range from coastal waters to saturated brines. However, only two cultures assigned to this morphospecies have been examined using molecular methods, and their 18S rRNA gene sequences are extremely different. Further the salinity tolerances of individual strains are unknown. Thus, our knowledge on the autecology and diversity in this morphospecies is deficient. Here, we report 18S rRNA gene data on seven strains similar to P. cosmopolitus from seven geographically remote locations (New Zealand, Kenya, Korea, Poland, Russia, Spain, and the USA) with sample salinities ranging from 4‰ to 280‰, and compare morphology and salinity tolerance of the nine available strains. Percolomonas cosmopolitus-like strains show few-to-no consistent morphological differences, and form six clades separated by often extremely large 18S rDNA divergences (up to 42.4%). Some strains grew best at salinities from 75 to 125‰ and represent halophiles. All but one of these belonged to two geographically heterogeneous clusters that formed a robust monophyletic group in phylogenetic trees; this likely represents an ecologically specialized subclade of halophiles. Our results suggest that P. cosmopolitus is a cluster of several cryptic species (at least), which are unlikely to be distinguished by geography. Interestingly, the 9 Percolomonas strains formed a clade in 18S rDNA phylogenies, unlike most previous analyses based on two sequences.

Chromulinavorax destructans, a pathogenic TM6 bacterium with an unusual replication strategy targeting protist mitochondrion

Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts.Chromulinavorax destructansis an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate,Spumella elongata. Chromulinavorax destructansis characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential andC. destructansinstead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.