Martina Bradic

Experimental evolution reveals natural selection on standing genetic variation

Evolution depends on genetic variation generated by mutation or recombination from standing genetic variation. In sexual organisms, little is known about the molecular population genetics of adaptation and reverse evolution. We carry out 50 generations of experimental reverse evolution in populations of Drosophila melanogaster, previously differentiated by forward evolution, and follow changes in the frequency of SNPs in both arms of the third chromosome. We characterize the effects of sampling finite population sizes and natural selection at the genotype level. We demonstrate that selection has occurred at several loci and further that there is no general loss or gain of allele diversity. We also observe that despite the complete convergence to ancestral levels of adaptation, allele frequencies only show partial return.

The Population Genomics of Repeated Evolution in the Blind Cavefish Astyanax mexicanus

Distinct populations of Astyanax mexicanus cavefish offer striking examples of repeatable convergence or parallelism in their independent evolutions from surface to cave phenotypes. However, the extent to which the repeatability of evolution occurred at the genetic level remains poorly understood. To address this, we first characterized the genetic diversity of 518 single-nucleotide polymorphisms (SNPs), obtained through RAD tag sequencing and distributed throughout the genome, in seven cave and three groups of surface populations. The cave populations represented two distinct lineages (old and new). Thirty-one SNPs were significantly differentiated between surface and old cave populations, two SNPs were differentiated between surface and new cave populations, and 44 SNPs were significantly differentiated in both old and new cave populations. In addition, we determined whether these SNPs map to the same locations of previously described quantitative trait loci (QTL) between surface and cave populations. A total of 25 differentiated SNPs co-map with several QTL, such as one containing a fibroblast growth factor gene (Fgf8) involved in eye development and lens size. Further, the identity of many SNPs that co-mapped with QTL was the same in independently derived cave populations. These conclusions were further confirmed by haplotype analyses of SNPs within QTL regions. Our findings indicate that the repeatability of evolution at the genetic level is substantial, suggesting that ancestral standing genetic variation significantly contributed to the population genetic variability used in adaptation to the cave environment.

Genotyping with Sequenom.

Often in evolutionary genetics research, one needs to analyze polymorphisms in populations for which cost-efficient high-throughput arrays are nonexistent, either because the species is not a model organism or because the populations have been subjected to such specific conditions that their base variation is almost unique. In this situation, custom-made genotyping assays are required. Sequenoms MassARRAY(®) genotyping platform is a powerful and flexible method for assaying up to a few thousand markers and up to thousands of individuals. It is based on distinguishing allele-specific primer extension products by mass spectrometry (MALDI-TOF). Most stages of the experimental protocol reflect adaptations of established PCR protocols to multiplexing, which allows the simultaneous amplification and detection of multiple markers per reaction.

Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis

Abstract Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.

The Tc1/mariner transposable element family shapes genetic variation and gene expression in the protist Trichomonas vaginalis

BackgroundTrichomonas vaginalis is the most prevalent non-viral sexually transmitted parasite. Although the protist is presumed to reproduce asexually, 60% of its haploid genome contains transposable elements (TEs), known contributors to genome variability. The availability of a draft genome sequence and our collection of >200 global isolates of T. vaginalis facilitate the study and analysis of TE population dynamics and their contribution to genomic variability in this protist.ResultsWe present here a pilot study of a subset of class II Tc1/mariner TEs that belong to the T. vaginalis Tvmar1 family. We report the genetic structure of 19 Tvmar1 loci, their ability to encode a full-length transposase protein, and their insertion frequencies in 94 global isolates from seven regions of the world. While most of the Tvmar1 elements studied exhibited low insertion frequencies, two of the 19 loci (locus 1 and locus 9) show high insertion frequencies of 1.00 and 0.96, respectively. The genetic structuring of the global populations identified by principal component analysis (PCA) of the Tvmar1 loci is in general agreement with published data based on genotyping, showing that Tvmar1 polymorphisms are a robust indicator of T. vaginalis genetic history. Analysis of expression of 22 genes flanking 13 Tvmar1 loci indicated significantly altered expression of six of the genes next to five Tvmar1 insertions, suggesting that the insertions have functional implications for T. vaginalis gene expression.ConclusionsOur study is the first in T. vaginalis to describe Tvmar1 population dynamics and its contribution to genetic variability of the parasite. We show that a majority of our studied Tvmar1 insertion loci exist at very low frequencies in the global population, and insertions are variable between geographical isolates. In addition, we observe that low frequency insertion is related to reduced or abolished expression of flanking genes. While low insertion frequencies might be expected, we identified two Tvmar1 insertion loci that are fixed across global populations. This observation indicates that Tvmar1 insertion may have differing impacts and fitness costs in the host genome and may play varying roles in the adaptive evolution of T. vaginalis.

Single-dose versus 7-day-dose metronidazole for the treatment of trichomoniasis in women: an open-label, randomised controlled trial

BACKGROUND Among women, trichomoniasis is the most common non-viral sexually transmitted infection worldwide, and is associated with serious reproductive morbidity, poor birth outcomes, and amplified HIV transmission. Single-dose metronidazole is the first-line treatment for trichomoniasis. However, bacterial vaginosis can alter treatment efficacy in HIV-infected women, and single-dose metronidazole treatment might not always clear infection. We compared single-dose metronidazole with a 7-day dose for the treatment of trichomoniasis among HIV-uninfected, non-pregnant women and tested whether efficacy was modified by bacterial vaginosis. METHODS In this multicentre, open-label, randomised controlled trial, participants were recruited at three sexual health clinics in the USA. We included women positive for Trichomonas vaginalis infection according to clinical screening. Participants were randomly assigned (1:1) to receive either a single dose of 2 g of metronidazole (single-dose group) or 500 mg of metronidazole twice daily for 7 days (7-day-dose group). The randomisation was done by blocks of four or six for each site. Patients and investigators were aware of treatment assignment. The primary outcome was T vaginalis infection by intention to treat, at test-of-cure 4 weeks after completion of treatment. The analysis of the primary outcome per nucleic acid amplification test or culture was also stratified by bacterial vaginosis status. This trial is registered with ClinicalTrials.gov, number NCT01018095, and with the US Food and Drug Administration, number IND118276, and is closed to accrual. FINDINGS Participants were recruited from Oct 6, 2014, to April 26, 2017. Of the 1028 patients assessed for eligibility, 623 women were randomly assigned to treatment groups (311 women in the single-dose group and 312 women in the 7-day-dose group; intention-to-treat population). Although planned enrolment had been 1664 women, the study was stopped early because of funding limitations. Patients in the 7-day-dose group were less likely to be T vaginalis positive at test-of-cure than those in the single-dose group (34 [11%] of 312 vs 58 [19%] of 311, relative risk 0·55, 95% CI 0·34-0·70; p<0·0001). Bacterial vaginosis status had no significant effect on relative risk (p=0·17). Self-reported adherence was 96% in the 7-day-dose group and 99% in the single-dose group. Side-effects were similar by group; the most common side-effect was nausea (124 [23%]), followed by headache (38 [7%]) and vomiting (19 [4%]). INTERPRETATION The 7-day-dose metronidazole should be the preferred treatment for trichomoniasis among women. FUNDING National Institutes of Health.

Genetic indicators of drug resistance in the highly repetitive genomes of Trichomonas vaginalis and other trichomonads

Background Trichomonas vaginalis, the most common non-viral sexually transmitted parasite, causes ~283 million trichomoniasis infections annually and is associated with complications during pregnancy and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but has lead to widespread drug resistance. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Results Reduced representation genome sequencings of more than 100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium decays rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in sets of genes for which no function has yet been assigned, as well as in functionally-characterized genes highly relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase (PFO)). Transcription profiling of these and other genes served as proxy for testing the functional consequences of multiple identified SNPs. Transcription profiles of lab-derived drug resistance strain as well as clinically resistant strain depict common regulation changes in carbohydrate metabolism and oxygen detoxification pathways correlated with Mz resistance. Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly-related species that causes venereal disease in cattle. Conclusions Our observation of shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggests conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.