Discovery of a Novel Species of Trichomonasvirus in the Human Parasite Trichomonas vaginalis Using Transcriptome Mining.

Trichomonas vaginalis is the most common non-viral cause of sexually transmitted infections globally. Infection by this protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory disease with acute and chronic consequences. Half or more isolates of this parasite are themselves infected with one or more dsRNA viruses that can exacerbate the inflammatory syndrome. At least four distinct viruses have been identified in T. vaginalis to date, constituting species Trichomonas vaginalis virus 1 through Trichomonas vaginalis virus 4 in genus Trichomonasvirus. Despite the global prevalence of these viruses, few complete coding sequences have been reported. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-Seq accessions representing at least 13 distinct T. vaginalis isolates. The results led to sequence assemblies for 27 novel trichomonasvirus strains across all four recognized species. Using a strategy of de novo sequence assembly followed by taxonomic classification, we additionally discovered six strains of a newly identified fifth species, for which we propose the name Trichomonas vaginalis virus 5, also in genus Trichomonasvirus. These additional strains exhibit high sequence identity to each other, but low sequence identity to strains of the other four species. Phylogenetic analyses corroborate the species-level designations. These results substantially increase the number of trichomonasvirus genome sequences and demonstrate the utility of mining publicly available transcriptomes for virus discovery in a critical human pathogen.

The G119S ace-1 mutation confers adaptive organophosphate resistance in a nontarget amphipod.

Organophosphate (OP) and carbamate (CM) insecticides are widely used in the United States and share the same mode of toxic action. Both classes are frequently documented in aquatic ecosystems, sometimes at levels that exceed aquatic life benchmarks. We previously identified a population of the nontarget amphipod, Hyalella azteca, thriving in an agricultural creek with high sediment levels of the OP chlorpyrifos, suggesting the population may have acquired genetic resistance to the pesticide. In the present study, we surveyed 17 populations of H. azteca in California to screen for phenotypic resistance to chlorpyrifos as well as genetic signatures of resistance in the acetylcholinesterase (ace-1) gene. We found no phenotypic chlorpyrifos resistance in populations from areas with little or no pesticide use. However, there was ~3- to 1,000-fold resistance in H. azteca populations from agricultural and/or urban areas, with resistance levels in agriculture being far higher than urban areas due to greater ongoing use of OP and CM pesticides. In every case of resistance in H. azteca, we identified a glycine-to-serine amino acid substitution (G119S) that has been shown to confer OP and CM resistance in mosquitoes and has been associated with resistance in other insects. We found that the G119S mutation was always present in a heterozygous state. Further, we provide tentative evidence of an ace-1 gene duplication in H. azteca that may play a role in chlorpyrifos resistance in some populations. The detection of a genetically based, adaptive OP and CM resistance in some of the same populations of H. azteca previously shown to harbor a genetically based adaptive pyrethroid resistance indicates that these nontarget amphipod populations have become resistant to many of the insecticides now in common use. The terrestrial application of pesticides has provided strong selective pressures to drive evolution in a nontarget, aquatic species.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

Beta vulgaris mitovirus 1 in diverse cultivars of beet and chard.

Recent results indicate that mitoviruses, which replicate persistently in host mitochondria, are not restricted to fungi, but instead are found also in plants. Beta vulgaris mitovirus 1 (BevuMV1) is an example first discovered in sugar beet cultivars. For the current study, complete coding sequences of 42 BevuMV1 strains were newly determined, derived from not only sugar beet but also fodder beet, table beet, and Swiss chard cultivars of Beta vulgaris, as well as wild sea beet. BevuMV1 is thus a common phytobiome component of this valuable crop species. Most of the new BevuMV1 sequences originated from RNA extracted from B. vulgaris seed clusters, consistent with vertical transmission of this virus. Results suggest that BevuMV1 entered the B. vulgaris lineage prior to human cultivation and also provides a marker for tracing the maternal ancestry of B. vulgaris cultivars. Especially notable is the monophyletic relationship and limited sequence divergence among BevuMV1 strains from cultivars that are thought or shown to share the "Owen" trait for cytoplasmic male sterility, which is transmitted by maternal mitochondria and has been broadly established in commercial breeding lines of B. vulgaris since the mid-20th century.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

A barnavirus sequence mined from a transcriptome of the Antarctic pearlwort Colobanthus quitensis.

Because so few viruses in the family Barnaviridae have been reported, we searched for more of them in public sequence databases. Here, we report the complete coding sequence of Colobanthus quitensis associated barnavirus 1, mined from a transcriptome of the Antarctic pearlwort Colobanthus quitensis. The 4.2-kb plus-strand sequence of this virus encompasses four main open reading frames (ORFs), as expected for barnaviruses, including ORFs for a protease-containing polyprotein, an RNA-dependent RNA polymerase whose translation appears to rely on - 1 ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic RNA. The possible derivation of this virus from a fungus associated with C. quitensis is discussed.

Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the ß1-ß2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications.

Resurrecting ancestral structural dynamics of an antiviral immune receptor: adaptive binding pocket reorganization repeatedly shifts RNA preference.

BACKGROUND: Although resurrecting ancestral proteins is a powerful tool for understanding the molecular-functional evolution of gene families, nearly all studies have examined proteins functioning in relatively stable biological processes. The extent to which more dynamic systems obey the same 'rules' governing stable processes is unclear. Here we present the first detailed investigation of the functional evolution of the RIG-like receptors (RLRs), a family of innate immune receptors that detect viral RNA in the cytoplasm. RESULTS: Using kinetic binding assays and molecular dynamics simulations of ancestral proteins, we demonstrate how a small number of adaptive protein-coding changes repeatedly shifted the RNA preference of RLRs throughout animal evolution by reorganizing the shape and electrostatic distribution across the RNA binding pocket, altering the hydrogen bond network between the RLR and its RNA target. In contrast to observations of proteins involved in metabolism and development, we find that RLR-RNA preference 'flip flopped' between two functional states, and shifts in RNA preference were not always coupled to gene duplications or speciation events. We demonstrate at least one reversion of RLR-RNA preference from a derived to an ancestral function through a novel structural mechanism, indicating multiple structural implementations of similar functions. CONCLUSIONS: Our results suggest a model in which frequent shifts in selection pressures imposed by an evolutionary arms race preclude the long-term functional optimization observed in stable biological systems. As a result, the evolutionary dynamics of immune receptors may be less constrained by structural epistasis and historical contingency.