Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

Estimated US infant exposures to 3-MCPD esters and glycidyl esters from consumption of infant formula.

A dietary exposure assessment was conducted for 3-monochloropropane-1,2-diol (3-MCPD) esters (3-MCPDE) and glycidyl esters (GE) in infant formulas available for consumption in the United States. 3-MCPDE and GE are food contaminants generated during the deodorisation of refined edible oils, which are used in infant formulas and other foods. 3-MCPDE and GE are of potential toxicological concern because these compounds are metabolised to free 3-MCPD and free glycidol in rodents and may have the same metabolic fate in humans. Free 3-MCPD and free glycidol have been found to cause adverse effects in rodents. Dietary exposures to 3-MCPDE and GE from consumption of infant formulas are of particular interest because formulas are the sole or primary food source for some infants. In this analysis, US Food and Drug Administration data on 3-MCPDE and GE concentrations (as 3-MCPD and glycidol equivalents, respectively) in a small convenience sample of infant formulas were used to estimate exposures from consumption of formula by infants 0-6 months of age. 3-MCPDE and GE exposures based on mean concentrations in all formulas were estimated at 7-10 and 2 µg/kg bw/day, respectively. Estimated mean exposures from consumption of formulas produced by individual manufacturers ranged from 1 to 14 µg/kg bw/day for 3-MCPDE and from 1 to 3 µg/kg for GE.

Occurrence of 3-monochloropropanediol esters and glycidyl esters in commercial infant formulas in the United States.

This work presents occurrence data for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD) and glycidol in 98 infant formula samples purchased in the United States. These contaminants are considered potentially carcinogenic and/or genotoxic, making their presence in refined oils and foods a potential health risk. Recently, attention has focused on methodology to quantify MCPD and glycidyl esters in infant formula for risk-assessment purposes. Occurrence data for 3-MCPD and glycidyl esters were produced using a procedure for extracting fat from infant formula and an LC-MS/MS method for analysing fat extracts for intact esters. Infant formulas were produced by seven manufacturers, five of which use palm oil and/or palm olein in their formulations. In formulas containing palm/palm olein, concentrations for bound 3-MCPD and glycidol ranged from 0.021 to 0.92 mg kg-1 (ppm) and from < LOQ to 0.40 mg kg-1 (ppm), respectively. Formulas not containing palm/palm olein, bound 3-MCPD and glycidol concentrations ranged from 0.072 to 0.16 mg kg-1 (ppm) and from 0.005 to 0.15 mg kg-1 (ppm), respectively. Although formulas from manufacturers A and G did not contain palm/palm olein, formulas from manufacturer E (containing palm olein) had the lowest concentrations of bound 3-MCPD and glycidol, demonstrating the effectiveness of industrial mitigation strategies.

Extraction and Liquid Chromatography-Tandem Mass Spectrometry Detection of 3-Monochloropropanediol Esters and Glycidyl Esters in Infant Formula.

A method was developed for the extraction of fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD) and glycidol from infant formula, followed by quantitative analysis of the extracts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These process-induced chemical contaminants are found in refined vegetable oils, and studies have shown that they are potentially carcinogenic and/or genotoxic, making their presence in edible oils (and processed foods containing these oils) a potential health risk. The extraction procedure involves a liquid-liquid extraction, where powdered infant formula is dissolved in water and extracted with ethyl acetate. Following shaking, centrifugation, and drying of the organic phase, the resulting fat extract is cleaned-up using solid-phase extraction and analyzed by LC-MS/MS. Method performance was confirmed by verifying the percent recovery of each 3-MCPD and glycidyl ester in a homemade powdered infant formula reference material. Average ester recoveries in the reference material ranged from 84.9 to 109.0% (0.6-9.5% RSD). The method was also validated by fortifying three varieties of commercial infant formulas with a 3-MCPD and glycidyl ester solution. Average recoveries of the esters across all concentrations and varieties of infant formula ranged from 88.7 to 107.5% (1.0-9.5% RSD). Based on the validation results, this method is suitable for producing 3-MCPD and glycidyl ester occurrence data in all commercially available varieties of infant formula.

Monte Carlo Strategies for Selecting Parameter Values in Simulation Experiments.

Simulation experiments are used widely throughout evolutionary biology and bioinformatics to compare models, promote methods, and test hypotheses. The biggest practical constraint on simulation experiments is the computational demand, particularly as the number of parameters increases. Given the extraordinary success of Monte Carlo methods for conducting inference in phylogenetics, and indeed throughout the sciences, we investigate ways in which Monte Carlo framework can be used to carry out simulation experiments more efficiently. The key idea is to sample parameter values for the experiments, rather than iterate through them exhaustively. Exhaustive analyses become completely infeasible when the number of parameters gets too large, whereas sampled approaches can fare better in higher dimensions. We illustrate the framework with applications to phylogenetics and genetic archaeology.

Kinetics and mechanism of styrene epoxidation by chlorite: role of chlorine dioxide.

An investigation of the kinetics and mechanism for epoxidation of styrene and para-substituted styrenes by chlorite at 25 °C in the pH range of 5-6 is described. The proposed mechanism in water and water/acetonitrile includes seven oxidation states of chlorine (-I, 0, I, II, III, IV, and V) to account for the observed kinetics and product distributions. The model provides an unusually detailed quantitative mechanism for the complex reactions that occur in mixtures of chlorine species and organic substrates, particularly when the strong oxidant chlorite is employed. Kinetic control of the reaction is achieved by the addition of chlorine dioxide to the reaction mixture, thereby eliminating a substantial induction period observed when chlorite is used alone. The epoxidation agent is identified as chlorine dioxide, which is continually formed by the reaction of chlorite with hypochlorous acid that results from ClO produced by the epoxidation reaction. The overall stoichiometry is the result of two competing chain reactions in which the reactive intermediate ClO reacts with either chlorine dioxide or chlorite ion to produce hypochlorous acid and chlorate or chloride, respectively. At high chlorite ion concentrations, HOCl is rapidly eliminated by reaction with chlorite, minimizing side reactions between HOCl and Cl2 with the starting material. Epoxide selectivity (>90% under optimal conditions) is accurately predicted by the kinetic model. The model rate constant for direct reaction of styrene with ClO2(aq) to produce epoxide is (1.16 ± 0.07) × 10(-2) M(-1) s(-1) for 60:40 water/acetonitrile with 0.20 M acetate buffer. Rate constants for para substituted styrenes (R = -SO3(-), -OMe, -Me, -Cl, -H, and -NO2) with ClO2 were determined. The results support the radical addition/elimination mechanism originally proposed by Kolar and Lindgren to account for the formation of styrene oxide in the reaction of styrene with chlorine dioxide.

Evaluating phylogenetic congruence in the post-genomic era.

Congruence is a broadly applied notion in evolutionary biology used to justify multigene phylogeny or phylogenomics, as well as in studies of coevolution, lateral gene transfer, and as evidence for common descent. Existing methods for identifying incongruence or heterogeneity using character data were designed for data sets that are both small and expected to be rarely incongruent. At the same time, methods that assess incongruence using comparison of trees test a null hypothesis of uncorrelated tree structures, which may be inappropriate for phylogenomic studies. As such, they are ill-suited for the growing number of available genome sequences, most of which are from prokaryotes and viruses, either for phylogenomic analysis or for studies of the evolutionary forces and events that have shaped these genomes. Specifically, many existing methods scale poorly with large numbers of genes, cannot accommodate high levels of incongruence, and do not adequately model patterns of missing taxa for different markers. We propose the development of novel incongruence assessment methods suitable for the analysis of the molecular evolution of the vast majority of life and support the investigation of homogeneity of evolutionary process in cases where markers do not share identical tree structures.

Let them fall where they may: congruence analysis in massive phylogenetically messy data sets.

Interest in congruence in phylogenetic data has largely focused on issues affecting multicellular organisms, and animals in particular, in which the level of incongruence is expected to be relatively low. In addition, assessment methods developed in the past have been designed for reasonably small numbers of loci and scale poorly for larger data sets. However, there are currently over a thousand complete genome sequences available and of interest to evolutionary biologists, and these sequences are predominantly from microbial organisms, whose molecular evolution is much less frequently tree-like than that of multicellular life forms. As such, the level of incongruence in these data is expected to be high. We present a congruence method that accommodates both very large numbers of genes and high degrees of incongruence. Our method uses clustering algorithms to identify subsets of genes based on similarity of phylogenetic signal. It involves only a single phylogenetic analysis per gene, and therefore, computation time scales nearly linearly with the number of genes in the data set. We show that our method performs very well with sets of sequence alignments simulated under a wide variety of conditions. In addition, we present an analysis of core genes of prokaryotes, often assumed to have been largely vertically inherited, in which we identify two highly incongruent classes of genes. This result is consistent with the complexity hypothesis.

A multi-locus time-calibrated phylogeny of the brown algae (Heterokonta, Ochrophyta, Phaeophyceae): Investigating the evolutionary nature of the "brown algal crown radiation".

The most conspicuous feature in previous phaeophycean phylogenies is a large polytomy known as the brown algal crown radiation (BACR). The BACR encompasses 10 out of the 17 currently recognized brown algal orders. A recent study has been able to resolve a few nodes of the BACR, suggesting that it may be a soft polytomy caused by a lack of signal in molecular markers. The present work aims to refine relationships within the BACR and investigate the nature and timeframe of the diversification in question using a dual approach. A multi-marker phylogeny of the brown algae was built from 10 mitochondrial, plastid and nuclear loci (>10,000 nt) of 72 phaeophycean taxa, resulting in trees with well-resolved inter-ordinal relationships within the BACR. Using Bayesian relaxed molecular clock analysis, it is shown that the BACR is likely to represent a gradual diversification spanning most of the Lower Cretaceous rather than a sudden radiation. Non-molecular characters classically used in ordinal delimitation were mapped on the molecular topology to study their evolutionary history.

Network analyses structure genetic diversity in independent genetic worlds.

DNA flows between chromosomes and mobile elements, following rules that are poorly understood. This limited knowledge is partly explained by the limits of current approaches to study the structure and evolution of genetic diversity. Network analyses of 119,381 homologous DNA families, sampled from 111 cellular genomes and from 165,529 phage, plasmid, and environmental virome sequences, offer challenging insights. Our results support a disconnected yet highly structured network of genetic diversity, revealing the existence of multiple "genetic worlds." These divides define multiple isolated groups of DNA vehicles drawing on distinct gene pools. Mathematical studies of the centralities of these worlds' subnetworks demonstrate that plasmids, not viruses, were key vectors of genetic exchange between bacterial chromosomes, both recently and in the past. Furthermore, network methodology introduces new ways of quantifying current sampling of genetic diversity.

Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic "supergroups".

Nearly all of eukaryotic diversity has been classified into 6 suprakingdom-level groups (supergroups) based on molecular and morphological/cell-biological evidence; these are Opisthokonta, Amoebozoa, Archaeplastida, Rhizaria, Chromalveolata, and Excavata. However, molecular phylogeny has not provided clear evidence that either Chromalveolata or Excavata is monophyletic, nor has it resolved the relationships among the supergroups. To establish the affinities of Excavata, which contains parasites of global importance and organisms regarded previously as primitive eukaryotes, we conducted a phylogenomic analysis of a dataset of 143 proteins and 48 taxa, including 19 excavates. Previous phylogenomic studies have not included all major subgroups of Excavata, and thus have not definitively addressed their interrelationships. The enigmatic flagellate Andalucia is sister to typical jakobids. Jakobids (including Andalucia), Euglenozoa and Heterolobosea form a major clade that we name Discoba. Analyses of the complete dataset group Discoba with the mitochondrion-lacking excavates or "metamonads" (diplomonads, parabasalids, and Preaxostyla), but not with the final excavate group, Malawimonas. This separation likely results from a long-branch attraction artifact. Gradual removal of rapidly-evolving taxa from the dataset leads to moderate bootstrap support (69%) for the monophyly of all Excavata, and 90% support once all metamonads are removed. Most importantly, Excavata robustly emerges between unikonts (Amoebozoa + Opisthokonta) and "megagrouping" of Archaeplastida, Rhizaria, and chromalveolates. Our analyses indicate that Excavata forms a monophyletic suprakingdom-level group that is one of the 3 primary divisions within eukaryotes, along with unikonts and a megagroup of Archaeplastida, Rhizaria, and the chromalveolate lineages.

Phylogenomic analyses support the monophyly of Taphrinomycotina, including Schizosaccharomyces fission yeasts.

Several morphologically dissimilar ascomycete fungi including Schizosaccharomyces, Taphrina, Saitoella, Pneumocystis, and Neolecta have been grouped into the taxon Taphrinomycotina (Archiascomycota or Archiascomycotina), originally based on rRNA phylogeny. These analyses lack statistically significant support for the monophyly of this grouping, and although confirmed by more recent multigene analyses, this topology is contradicted by mitochondrial phylogenies. To resolve this inconsistency, we have assembled phylogenomic mitochondrial and nuclear data sets from four distantly related taphrinomycotina taxa: Schizosaccharomyces pombe, Pneumocystis carinii, Saitoella complicata, and Taphrina deformans. Our phylogenomic analyses based on nuclear data (113 proteins) conclusively support the monophyly of Taphrinomycotina, diverging as a sister group to Saccharomycotina + Pezizomycotina. However, despite the improved taxon sampling, Taphrinomycotina continue to be paraphyletic with the mitochondrial data set (13 proteins): Schizosaccharomyces species associate with budding yeasts (Saccharomycotina) and the other Taphrinomycotina group as a sister group to Saccharomycotina + Pezizomycotina. Yet, as Schizosaccharomyces and Saccharomycotina species are fast evolving, the mitochondrial phylogeny may be influenced by a long-branch attraction (LBA) artifact. After removal of fast-evolving sequence positions from the mitochondrial data set, we recover the monophyly of Taphrinomycotina. Our combined results suggest that Taphrinomycotina is a legitimate taxon, that this group of species diverges as a sister group to Saccharomycotina + Pezizomycotina, and that phylogenetic positioning of yeasts and fission yeasts with mitochondrial data is plagued by a strong LBA artifact.

Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: implications for the evolutionary history of the double karyomastigont of diplomonads.

BACKGROUND: Fornicata is a relatively recently established group of protists that includes the diplokaryotic diplomonads (which have two similar nuclei per cell), and the monokaryotic enteromonads, retortamonads and Carpediemonas, with the more typical one nucleus per cell. The monophyly of the group was confirmed by molecular phylogenetic studies, but neither the internal phylogeny nor its position on the eukaryotic tree has been clearly resolved. RESULTS: Here we have introduced data for three genes (SSU rRNA, alpha-tubulin and HSP90) with a wide taxonomic sampling of Fornicata, including ten isolates of enteromonads, representing the genera Trimitus and Enteromonas, and a new undescribed enteromonad genus. The diplomonad sequences formed two main clades in individual gene and combined gene analyses, with Giardia (and Octomitus) on one side of the basal divergence and Spironucleus, Hexamita and Trepomonas on the other. Contrary to earlier evolutionary scenarios, none of the studied enteromonads appeared basal to diplokaryotic diplomonads. Instead, the enteromonad isolates were all robustly situated within the second of the two diplomonad clades. Furthermore, our analyses suggested that enteromonads do not constitute a monophyletic group, and enteromonad monophyly was statistically rejected in 'approximately unbiased' tests of the combined gene data. CONCLUSION: We suggest that all higher taxa intended to unite multiple enteromonad genera be abandoned, that Trimitus and Enteromonas be considered as part of Hexamitinae, and that the term 'enteromonads' be used in a strictly utilitarian sense. Our result suggests either that the diplokaryotic condition characteristic of diplomonads arose several times independently, or that the monokaryotic cell of enteromonads originated several times independently by secondary reduction from the diplokaryotic state. Both scenarios are evolutionarily complex. More comparative data on the similarity of the genomes of the two nuclei of diplomonads will be necessary to resolve which evolutionary scenario is more probable.

Testing congruence in phylogenomic analysis.

Phylogenomic analyses of large sets of genes or proteins have the potential to revolutionize our understanding of the tree of life. However, problems arise because estimated phylogenies from individual loci often differ because of different histories, systematic bias, or stochastic error. We have developed Concaterpillar, a hierarchical clustering method based on likelihood-ratio testing that identifies congruent loci for phylogenomic analysis. Concaterpillar also includes a test for shared relative evolutionary rates between genes indicating whether they should be analyzed separately or by concatenation. In simulation studies, the performance of this method is excellent when a multiple comparison correction is applied. We analyzed a phylogenomic data set of 60 translational protein sequences from the major supergroups of eukaryotes and identified three congruent subsets of proteins. Analysis of the largest set indicates improved congruence relative to the full data set and produced a phylogeny with stronger support for five eukaryote supergroups including the Opisthokonts, the Plantae, the stramenopiles + Apicomplexa (chromalveolates), the Amoebozoa, and the Excavata. In contrast, the phylogeny of the second largest set indicates a close relationship between stramenopiles and red algae, to the exclusion of alveolates, suggesting gene transfer from the red algal secondary symbiont to the ancestral stramenopile host nucleus during the origin of their chloroplast. Investigating phylogenomic data sets for conflicting signals has the potential to both improve phylogenetic accuracy and inform our understanding of genome evolution.

Invasion and persistence of a selfish gene in the Cnidaria.

BACKGROUND: Homing endonuclease genes (HEGs) are superfluous, but are capable of invading populations that mix alleles by biasing their inheritance patterns through gene conversion. One model suggests that their long-term persistence is achieved through recurrent invasion. This circumvents evolutionary degeneration, but requires reasonable rates of transfer between species to maintain purifying selection. Although HEGs are found in a variety of microbes, we found the previous discovery of this type of selfish genetic element in the mitochondria of a sea anemone surprising. METHODS/PRINCIPAL FINDINGS: We surveyed 29 species of Cnidaria for the presence of the COXI HEG. Statistical analyses provided evidence for HEG invasion. We also found that 96 individuals of Metridium senile, from five different locations in the UK, had identical HEG sequences. This lack of sequence divergence illustrates the stable nature of Anthozoan mitochondria. Our data suggests this HEG conforms to the recurrent invasion model of evolution. CONCLUSIONS: Ordinarily such low rates of HEG transfer would likely be insufficient to enable major invasion. However, the slow rate of Anthozoan mitochondrial change lengthens greatly the time to HEG degeneration: this significantly extends the periodicity of the HEG life-cycle. We suggest that a combination of very low substitution rates and rare transfers facilitated metazoan HEG invasion.

Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms.

To generate data for comparative analyses of zygomycete mitochondrial gene expression, we sequenced mtDNAs of three distantly related zygomycetes, Rhizopus oryzae, Mortierella verticillata and Smittium culisetae. They all contain the standard fungal mitochondrial gene set, plus rnpB, the gene encoding the RNA subunit of the mitochondrial RNase P (mtP-RNA) and rps3, encoding ribosomal protein S3 (the latter lacking in R.oryzae). The mtP-RNAs of R.oryzae and of additional zygomycete relatives have the most eubacteria-like RNA structures among fungi. Precise mapping of the 5' and 3' termini of the R.oryzae and M.verticillata mtP-RNAs confirms their expression and processing at the exact sites predicted by secondary structure modeling. The 3' RNA processing of zygomycete mitochondrial mRNAs, SSU-rRNA and mtP-RNA occurs at the C-rich sequence motifs similar to those identified in fission yeast and basidiomycete mtDNAs. The C-rich motifs are included in the mature transcripts, and are likely generated by exonucleolytic trimming of RNA 3' termini. Zygomycete mtDNAs feature a variety of insertion elements: (i) mtDNAs of R.oryzae and M.verticillata were subject to invasions by double hairpin elements; (ii) genes of all three species contain numerous mobile group I introns, including one that is closest to an intron that invaded angiosperm mtDNAs; and (iii) at least one additional case of a mobile element, characterized by a homing endonuclease insertion between partially duplicated genes [Paquin,B., Laforest,M.J., Forget,L., Roewer,I., Wang,Z., Longcore,J. and Lang,B.F. (1997) Curr. Genet., 31, 380-395]. The combined mtDNA-encoded proteins contain insufficient phylogenetic signal to demonstrate monophyly of zygomycetes.

Phylogenetic reconstruction and lateral gene transfer.

Lateral gene transfer (LGT) is often seen as a form of noise, obscuring the phylogenetic signal with which we might hope to reconstruct the evolution of a group of organisms, or indeed the history of all life (the Tree of Life). Such reconstruction might still be possible if the subset of genes conserved among all genomes in a group (or common to all genomes) comprise a core that is relatively refractory to LGT. Several papers designed to test this notion have recently appeared, and here we re-analyze one, which claims that the core of single-copy orthologs shared by all sequenced genomes of the gammaproteobacteria is essentially free of LGT. This conclusion is unfortunately premature, and it is very hard to determine what fraction of this core has been affected by LGT. We discuss other difficulties with the core concept and suggest that, although the core idea must remain part of our understanding of phylogenetic relationships, it should not be the sole basis for defining such relationships, because these are not exclusively tree-like. We suggest instead a more complex but more natural framework for classification, which we call the Synthesis of Life.

Mitochondrial 3' tRNA editing in the jakobid Seculamonas ecuadoriensis: a novel mechanism and implications for tRNA processing.

The jakobid flagellates are bacteriovorus protists with mitochondrial genomes that are the most ancestral identified to date, in that they most resemble the genomes of the alpha-proteobacterial ancestors of the mitochondrion. Because of the bacterial character of jakobid mitochondrial genomes, it was expected that mechanisms for gene expression and RNA structures would be bacterial in nature. However, sequencing of the mitochondrial genome of the jakobid Seculamonas ecuadoriensis revealed several apparent mismatches in the acceptor stems of two predicted tRNAs. To investigate this observation, we determined the cDNA sequences of these tRNAs by RT-PCR. Our results show that the last three positions of the 3' extremity, plus the discriminator position of seryl and glutamyl tRNAs, are altered posttranscriptionally, restoring orthodox base-pairing and replacing the discriminator with an adenosine residue, in an editing process that resembles that of the metazoan Lithobius forficatus. However, the most 5' of the edited nucleotides is occasionally left unedited, indicating that the editing mechanism proceeds initially by exonucleolytic degradation, followed by repair of the degraded region. This 3' tRNA editing mechanism is likely distinct from that of L. forficatus, despite the apparent similarities between the two systems.