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      Detection and Characterization of Wolbachia Infections in Natural Populations of Aphids: Is the Hidden Diversity Fully Unraveled?

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          Abstract

          Aphids are a serious threat to agriculture, despite being a rather small group of insects. The about 4,000 species worldwide engage in highly interesting and complex relationships with their microbial fauna. One of the key symbionts in arthropods is Wolbachia, an α-Proteobacterium implicated in many important biological processes and believed to be a potential tool for biological control. Aphids were thought not to harbour Wolbachia; however, current data suggest that its presence in aphids has been missed, probably due to the low titre of the infection and/or to the high divergence of the Wolbachia strains of aphids. The goal of the present study is to map the Wolbachia infection status of natural aphids populations, along with the characterization of the detected Wolbachia strains. Out of 425 samples from Spain, Portugal, Greece, Israel and Iran, 37 were found to be infected. Our results, based mainly on 16S rRNA gene sequencing, indicate the presence of two new Wolbachia supergroups prevailing in aphids, along with some strains belonging either to supergroup B or to supergroup A.

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          Isolation of plant DNA from fesh tissue

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            Wolbachia as a bacteriocyte-associated nutritional mutualist.

            Many insects are dependent on bacterial symbionts that provide essential nutrients (ex. aphid-Buchnera and tsetse-Wiglesworthia associations), wherein the symbionts are harbored in specific cells called bacteriocytes that constitute a symbiotic organ bacteriome. Facultative and parasitic bacterial symbionts like Wolbachia have been regarded as evolutionarily distinct from such obligate nutritional mutualists. However, we discovered that, in the bedbug Cimex lectularius, Wolbachia resides in a bacteriome and appears to be an obligate nutritional mutualist. Two bacterial symbionts, a Wolbachia strain and an unnamed gamma-proteobacterium, were identified from different strains of the bedbug. The Wolbachia symbiont was detected from all of the insects examined whereas the gamma-proteobacterium was found in a part of them. The Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes, infecting the incipient symbiotic organ at an early stage of the embryogenesis. Elimination of the Wolbachia symbiont resulted in retarded growth and sterility of the host insect. These deficiencies were rescued by oral supplementation of B vitamins, confirming the essential nutritional role of the symbiont for the host. The estimated genome size of the Wolbachia symbiont was around 1.3 Mb, which was almost equivalent to the genome sizes of parasitic Wolbachia strains of other insects. These results indicate that bacteriocyte-associated nutritional mutualism can evolve from facultative and prevalent microbial associates like Wolbachia, highlighting a previously unknown aspect of the parasitism-mutualism evolutionary continuum.
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              Multilocus sequence typing system for the endosymbiont Wolbachia pipientis.

              The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                13 December 2011
                : 6
                : 12
                : e28695
                Affiliations
                [1 ]Department of Environmental and Natural Resources Management, University of Ioannina, Agrinio, Greece
                [2 ]Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Valencia, Spain
                [3 ]ISOPlexis Gene Bank, Universidade da Madeira, Funchal, Portugal
                [4 ]Department of Greenhouse Crops and Floriculture, Technological Educational Institute of Messolonghi, Messolonghi, Greece
                [5 ]Laboratório de Qualidade Agrícola, Núcleo de Fitopatologia,, Madeira, Portugal
                [6 ]Departamento de Ciências Agrárias CITA-A (Azorean Biodiversity Group), Universidade dos Açores, Angra do Heroísmo, Terceira – Azores
                [7 ]Área de Genómica y Salud, Centro Superior de Investigación en Salud Pública (CSISP), Valencia, Spain
                [8 ]Biomedical Sciences Research Center Al. Fleming, Vari, Greece
                [9 ]Department of Environmental and Natural Resources Management, University of Western Greece, Agrinio, Greece
                University of Poitiers, France
                Author notes

                Conceived and designed the experiments: KB AL MK GT. Performed the experiments: AAA DSG ED MM AP MS VD SR AFA GT. Analyzed the data: AAA DSG MM AL MK GT KB. Contributed reagents/materials/analysis tools: KB AL MK AP AFA PAVB GT. Wrote the paper: KB AAA AL MK GT.

                Article
                PONE-D-11-16968
                10.1371/journal.pone.0028695
                3236762
                22174869
                9daae74e-3693-43ec-be76-d26b3cf362fd
                Augustinos et al. 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.
                History
                : 31 August 2011
                : 14 November 2011
                Page count
                Pages: 11
                Categories
                Research Article
                Agriculture
                Agroecology
                Agro-Population Ecology
                Biology
                Evolutionary Biology
                Organismal Evolution
                Microbial Evolution
                Microbiology
                Bacteriology
                Bacterial Taxonomy
                Microbial Ecology
                Microbial Evolution
                Zoology
                Entomology

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                Uncategorized

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