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      Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae ( Diptera: Culicidae) from Its Sympatric Non-vector Sister Species, Aedes lilii

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          Abstract

          Introduction

          Yellow fever continues to be a problem in sub-Saharan Africa with repeated epidemics occurring. The mosquito Aedes bromeliae is a major vector of yellow fever, but it cannot be readily differentiated from its non-vector zoophilic sister species Ae. lilii using morphological characters. Genetic differences have been reported between anthropophilic Ae. bromeliae and zoophilic Ae. lilii and between forest and domestic populations. However, due to the application of different molecular markers and non-overlapping populations employed in previous studies, interpretation of species delimitation is unclear.

          Methodology/Principle Findings

          DNA sequences were generated from specimens of Ae. simpsoni s.l. from the Republic of Benin, Tanzania and Uganda for two nuclear genes apolipophorin 2 ( apoLp2) and cytochrome p450 ( CYPJ92), the ribosomal internal transcribed spacer region ( ITS) and the mitochondrial cytochrome c oxidase ( COI) barcoding region. Nuclear genes apoLp2 and CYPJ92 were unable to differentiate between species Ae. bromeliae and Ae. lilii due to ancestral lineage sorting, while ITS sequence data provided clear topological separation on a phylogeny. The standard COI barcoding region was shown to be subject to species introgression and unable to clearly distinguish the two taxa. Here we present a reliable direct PCR-based method for differentiation of the vector species Ae. bromeliae from its isomorphic, sympatric and non-biomedically important sister taxon, Ae. lilii, based on the ITS region. Using molecular species verification, we describe novel immature habitats for Ae. lilii and report both sympatric and allopatric populations. Whereas only Ae. lilii is found in the Republic of Benin and only Ae. bromeliae in Tanzania, both species are sympatric in Uganda.

          Conclusions/Significance

          Our accurate identification method will allow informed distribution and detailed ecological studies that will facilitate assessment of arboviral disease risk and development of future targeted vector control.

          Author Summary

          In Africa, epidemic outbreaks of yellow fever continue despite the availability of an effective vaccine. Effective understanding of disease epidemiology and control requires the ability to reliably identify vectors of yellow fever. The mosquito Ae. bromeliae, a competent vector of yellow fever virus, cannot be reliably morphologically differentiated from its sister species Ae. lilii, which does not bite humans and so does not transmit yellow fever. DNA sequencing of four molecular markers allowed comparisons of how they perform at distinguishing these species. We found that the mitochondrial cytochrome c oxidase ( COI) barcoding region and nuclear apolipophorin 2 ( apoLp2) and cytochrome p450 ( CYPJ92) were unable to reliably distinguish these species. Conversely, genetic variation at the internal transcribed spacer region ( ITS) was able to confirm the vector Ae. bromeliae and non-vector Ae. lilii as distinct species. Based on ITS sequence differences, we developed a robust molecular method to identify the vector Ae. bromeliae from its sister species Ae. lilii. Consequently, we find that these species use the same larval habitats including banana, cocoyam and Dracena spp. in Uganda. Whereas only Ae. lilii appears to be present in Benin and only Ae. bromeliae in Tanzania, we confirm that both species occur in Uganda. Reliable species designation will promote more detailed studies of distribution, ecology and vector status essential for disease risk assessment and mosquito control.

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          Most cited references37

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          Use of DNA barcodes to identify flowering plants.

          Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.
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            Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals

            Background The internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is regarded as one of the candidate DNA barcodes because it possesses a number of valuable characteristics, such as the availability of conserved regions for designing universal primers, the ease of its amplification, and sufficient variability to distinguish even closely related species. However, a general analysis of its ability to discriminate species in a comprehensive sample set is lacking. Methodology/Principal Findings In the current study, 50,790 plant and 12,221 animal ITS2 sequences downloaded from GenBank were evaluated according to sequence length, GC content, intra- and inter-specific divergence, and efficiency of identification. The results show that the inter-specific divergence of congeneric species in plants and animals was greater than its corresponding intra-specific variations. The success rates for using the ITS2 region to identify dicotyledons, monocotyledons, gymnosperms, ferns, mosses, and animals were 76.1%, 74.2%, 67.1%, 88.1%, 77.4%, and 91.7% at the species level, respectively. The ITS2 region unveiled a different ability to identify closely related species within different families and genera. The secondary structure of the ITS2 region could provide useful information for species identification and could be considered as a molecular morphological characteristic. Conclusions/Significance As one of the most popular phylogenetic markers for eukaryota, we propose that the ITS2 locus should be used as a universal DNA barcode for identifying plant species and as a complementary locus for CO1 to identify animal species. We have also developed a web application to facilitate ITS2-based cross-kingdom species identification (http://its2-plantidit.dnsalias.org).
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              Resurgent vector-borne diseases as a global health problem.

              D J Gubler (1998)
              Vector-borne infectious diseases are emerging or resurging as a result of changes in public health policy, insecticide and drug resistance, shift in emphasis from prevention to emergency response, demographic and societal changes, and genetic changes in pathogens. Effective prevention strategies can reverse this trend. Research on vaccines, environmentally safe insecticides, alternative approaches to vector control, and training programs for health-care workers are needed.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                7 December 2015
                December 2015
                : 9
                : 12
                : e0004250
                Affiliations
                [1 ]Computational Evolutionary Biology Group, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
                [2 ]Walter Reed Biosystematics Unit, Smithsonian Institution Museum Support Centre, Suitland, Maryland, United States of America
                [3 ]Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America
                [4 ]Uniformed Services University of Health Sciences, Bethesda, Maryland, United States of America
                [5 ]Department of Entomology, National Museum of Natural History, Smithsonian Institution, Washington, DC, United States of America
                [6 ]Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, Morogoro, Tanzania
                [7 ]Department of Arbovirology, Emerging and Re-emerging Infections, Uganda Virus Research Institute, Entebbe, Uganda
                [8 ]Agro-Eco-Health Platform for West and Central Africa, International Institute for Tropical Agriculture, Republic of Benin
                United States Army Medical Research Institute of Infectious Diseases, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CW GM YML KLB. Performed the experiments: KLB. Analyzed the data: KLB. Contributed reagents/materials/analysis tools: KLB CW YML FS RD MK JL YMH LBM MR ET. Wrote the paper: KLB CW YML.

                Article
                PNTD-D-15-01311
                10.1371/journal.pntd.0004250
                4671560
                26641858
                b3e1c3b0-d5fb-4094-92a9-63425f1452d0

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

                History
                : 26 July 2015
                : 29 October 2015
                Page count
                Figures: 6, Tables: 1, Pages: 19
                Funding
                This work was supported by an Africa Award from the Royal Society and Leverhulme Trust (AA110092) and a PhD studentship to KLB funded by the Natural Environment Research Council [NE/H525170/1,NE/1528134/1, NE/J500057/1]. This manuscript was prepared whilst YML held a National Research Council (NRC) Research Associateship Award at the Walter Reed Army Institute of Research. The material to be published reflects the views of the authors and should not be construed to represent those of the Department of the Army or the Department of Defense. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All sequence files are available from the GenBank database (accession number(s) KT998333- KT998452).

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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