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      Wolbachia Variants Induce Differential Protection to Viruses in Drosophila melanogaster: A Phenotypic and Phylogenomic Analysis

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          Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups — wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ∼21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.

          Author Summary

          Wolbachia are bacterial symbionts that infect many arthropods and can protect insects from viral infection. Here we show that different variants of Wolbachia from Drosophila melanogaster (wMel) are phenotypically heterogeneous: they differ in the level of protection they confer and the titres they reach in their host. The Wolbachia with higher titres have higher antiviral protection but can also exert a cost on their host. Based on the observed phenotypes, we divided the wMel variants into two groups and demonstrated that the division is reflected in their phylogeny. Moreover, we discovered the genetic difference between two otherwise almost identical wMel variants, wMelPop and wMelCS, that may explain why one is highly pathogenic while the other produces benign infections. Our study helps to explain the prevalence of the different wMel variants in wild Drosophila populations and sheds light on the factors shaping it. In particular, the recent replacement of some wMel variants caused a decrease in anti-viral resistance and probably reduced the cost of the symbiont for the host. Finally, our work helps to understand the interaction of wMel with its natural host and inform Wolbachia use in the control of diseases transmitted by insects.

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          Most cited references 110

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          The Sequence Alignment/Map format and SAMtools

          Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: Contact:
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            Fast and accurate short read alignment with Burrows–Wheeler transform

            Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: Contact:
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              A new mathematical model for relative quantification in real-time RT-PCR.

               M. Pfaffl (2001)
              Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

                Author and article information

                Role: Editor
                PLoS Genet
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                December 2013
                December 2013
                12 December 2013
                : 9
                : 12
                [1 ]Instituto Gulbenkian de Ciência, Oeiras, Portugal
                [2 ]Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
                [3 ]Department of Genetics, University of Cambridge, Cambridge, United Kingdom
                Fred Hutchinson Cancer Research Center, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EC MSPM FMJ LT. Performed the experiments: EC MSPM SSE JM. Analyzed the data: EC LAW FMJ LT. Wrote the paper: EC FMJ LT.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 22
                This work was supported by Fundação para a Ciência e Tecnologia ( grants PTDC/BIA-MIC/108327/2008 and PTDC/SAU-MII/105655/2008 and the Wellcome Trust ( grant 094664/Z/10/Z. EC is supported by the PhD fellowship from Fundação para a Ciência e Tecnologia SFRH/BD/51625/2011. FMJ is supported by a Royal Society University Research Fellowship ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article



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