Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species
Identifieur interne : 000018 ( Istex/Corpus ); précédent : 000017; suivant : 000019Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species
Auteurs : S. Epis ; D. Sassera ; T. Beninati ; N. Lo ; L. Beati ; J. Piesman ; L. Rinaldi ; K. D. Mccoy ; A. Torina ; L. Sacchi ; E. Clementi ; M. Genchi ; S. Magnino ; C. BandiSource :
- Parasitology [ 0031-1820 ] ; 2008.
Abstract
The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.
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DOI: 10.1017/S0031182007004052
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Epis</name><affiliation><mods:affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation>These authors contributed equally to this work.</mods:affiliation></affiliation></author><author><name sortKey="Sassera, D" sort="Sassera, D" uniqKey="Sassera D" first="D." last="Sassera">D. Sassera</name><affiliation><mods:affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation>These authors contributed equally to this work.</mods:affiliation></affiliation></author><author><name sortKey="Beninati, T" sort="Beninati, T" uniqKey="Beninati T" first="T." last="Beninati">T. Beninati</name><affiliation><mods:affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</mods:affiliation></affiliation><affiliation><mods:affiliation>§Present address: Faculty of Veterinary Science, The University of Sydney, New South Wales, Australia.</mods:affiliation></affiliation></author><author><name sortKey="Lo, N" sort="Lo, N" uniqKey="Lo N" first="N." last="Lo">N. Lo</name><affiliation><mods:affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</mods:affiliation></affiliation></author><author><name sortKey="Beati, L" sort="Beati, L" uniqKey="Beati L" first="L." last="Beati">L. Beati</name><affiliation><mods:affiliation>Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA, USA</mods:affiliation></affiliation></author><author><name sortKey="Piesman, J" sort="Piesman, J" uniqKey="Piesman J" first="J." last="Piesman">J. Piesman</name><affiliation><mods:affiliation>Division of Vector Borne Infectious Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA</mods:affiliation></affiliation></author><author><name sortKey="Rinaldi, L" sort="Rinaldi, L" uniqKey="Rinaldi L" first="L." last="Rinaldi">L. Rinaldi</name><affiliation><mods:affiliation>Dipartimento di Patologia e Sanità Animale, Universita degli Studi di Napoli Federico II, Napoli, Italy</mods:affiliation></affiliation></author><author><name sortKey="Mccoy, K D" sort="Mccoy, K D" uniqKey="Mccoy K" first="K. D." last="Mccoy">K. D. Mccoy</name><affiliation><mods:affiliation>Génétique et Evolution des Maladies Infectieuses, UMR CNRS-IRD 2724, IRD, Montpellier, France</mods:affiliation></affiliation></author><author><name sortKey="Torina, A" sort="Torina, A" uniqKey="Torina A" first="A." last="Torina">A. Torina</name><affiliation><mods:affiliation>Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy</mods:affiliation></affiliation></author><author><name sortKey="Sacchi, L" sort="Sacchi, L" uniqKey="Sacchi L" first="L." last="Sacchi">L. Sacchi</name><affiliation><mods:affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Clementi, E" sort="Clementi, E" uniqKey="Clementi E" first="E." last="Clementi">E. Clementi</name><affiliation><mods:affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Genchi, M" sort="Genchi, M" uniqKey="Genchi M" first="M." last="Genchi">M. Genchi</name><affiliation><mods:affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Magnino, S" sort="Magnino, S" uniqKey="Magnino S" first="S." last="Magnino">S. Magnino</name><affiliation><mods:affiliation>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Pavia, Italy</mods:affiliation></affiliation></author><author><name sortKey="Bandi, C" sort="Bandi, C" uniqKey="Bandi C" first="C." last="Bandi">C. Bandi</name><affiliation><mods:affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: claudio.bandi@unimi.it</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Parasitology</title><title level="j" type="abbrev">Parasitology</title><idno type="ISSN">0031-1820</idno><idno type="eISSN">1469-8161</idno><imprint><publisher>Cambridge University Press</publisher><pubPlace>Cambridge, UK</pubPlace><date type="published" when="2008">2008</date><biblScope unit="volume">135</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="485">485</biblScope><biblScope unit="page" to="494">494</biblScope></imprint><idno type="ISSN">0031-1820</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0031-1820</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.</div></front></TEI><istex><corpusName>cambridge</corpusName><author><json:item><name>S. EPIS</name><affiliations><json:string>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</json:string><json:string>These authors contributed equally to this work.</json:string></affiliations></json:item><json:item><name>D. SASSERA</name><affiliations><json:string>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</json:string><json:string>These authors contributed equally to this work.</json:string></affiliations></json:item><json:item><name>T. BENINATI</name><affiliations><json:string>School of Biological Sciences, The University of Sydney, New South Wales, Australia</json:string><json:string>§Present address: Faculty of Veterinary Science, The University of Sydney, New South Wales, Australia.</json:string></affiliations></json:item><json:item><name>N. LO</name><affiliations><json:string>School of Biological Sciences, The University of Sydney, New South Wales, Australia</json:string></affiliations></json:item><json:item><name>L. BEATI</name><affiliations><json:string>Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA, USA</json:string></affiliations></json:item><json:item><name>J. PIESMAN</name><affiliations><json:string>Division of Vector Borne Infectious Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA</json:string></affiliations></json:item><json:item><name>L. RINALDI</name><affiliations><json:string>Dipartimento di Patologia e Sanità Animale, Universita degli Studi di Napoli Federico II, Napoli, Italy</json:string></affiliations></json:item><json:item><name>K. D. McCOY</name><affiliations><json:string>Génétique et Evolution des Maladies Infectieuses, UMR CNRS-IRD 2724, IRD, Montpellier, France</json:string></affiliations></json:item><json:item><name>A. TORINA</name><affiliations><json:string>Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy</json:string></affiliations></json:item><json:item><name>L. SACCHI</name><affiliations><json:string>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</json:string></affiliations></json:item><json:item><name>E. CLEMENTI</name><affiliations><json:string>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</json:string></affiliations></json:item><json:item><name>M. GENCHI</name><affiliations><json:string>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</json:string></affiliations></json:item><json:item><name>S. MAGNINO</name><affiliations><json:string>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Pavia, Italy</json:string></affiliations></json:item><json:item><name>C. BANDI</name><affiliations><json:string>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</json:string><json:string>E-mail: claudio.bandi@unimi.it</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Midichloria mitochondrii</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Ixodes ricinus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>hard ticks</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>intra-mitochondrial</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PCR screening</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>phylogeny</value></json:item></subject><articleId><json:string>00405</json:string></articleId><arkIstex>ark:/67375/6GQ-BFLRDC9S-T</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.</abstract><qualityIndicators><score>9.448</score><pdfWordCount>5399</pdfWordCount><pdfCharCount>36225</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>10</pdfPageCount><pdfPageSize>595 x 842 pts (A4)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>204</abstractWordCount><abstractCharCount>1357</abstractCharCount><keywordCount>6</keywordCount></qualityIndicators><title>Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse 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work.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species</title><author xml:id="author-0000"><persName><forename type="first">S.</forename><surname>EPIS</surname></persName><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation><affiliation>These authors contributed equally to this work.</affiliation></author><author xml:id="author-0001"><persName><forename type="first">D.</forename><surname>SASSERA</surname></persName><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation><affiliation>These authors contributed equally to this work.</affiliation></author><author xml:id="author-0002"><persName><forename type="first">T.</forename><surname>BENINATI</surname></persName><affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</affiliation><affiliation>§Present address: Faculty of Veterinary Science, The University of Sydney, New South Wales, Australia.</affiliation></author><author xml:id="author-0003"><persName><forename type="first">N.</forename><surname>LO</surname></persName><affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</affiliation></author><author xml:id="author-0004"><persName><forename type="first">L.</forename><surname>BEATI</surname></persName><affiliation>Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA, USA</affiliation></author><author xml:id="author-0005"><persName><forename type="first">J.</forename><surname>PIESMAN</surname></persName><affiliation>Division of Vector Borne Infectious Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA</affiliation></author><author xml:id="author-0006"><persName><forename type="first">L.</forename><surname>RINALDI</surname></persName><affiliation>Dipartimento di Patologia e Sanità Animale, Universita degli Studi di Napoli Federico II, Napoli, Italy</affiliation></author><author xml:id="author-0007"><persName><forename type="first">K. D.</forename><surname>McCOY</surname></persName><affiliation>Génétique et Evolution des Maladies Infectieuses, UMR CNRS-IRD 2724, IRD, Montpellier, France</affiliation></author><author xml:id="author-0008"><persName><forename type="first">A.</forename><surname>TORINA</surname></persName><affiliation>Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy</affiliation></author><author xml:id="author-0009"><persName><forename type="first">L.</forename><surname>SACCHI</surname></persName><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation></author><author xml:id="author-0010"><persName><forename type="first">E.</forename><surname>CLEMENTI</surname></persName><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation></author><author xml:id="author-0011"><persName><forename type="first">M.</forename><surname>GENCHI</surname></persName><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation></author><author xml:id="author-0012"><persName><forename type="first">S.</forename><surname>MAGNINO</surname></persName><affiliation>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Pavia, Italy</affiliation></author><author xml:id="author-0013"><persName><forename type="first">C.</forename><surname>BANDI</surname></persName><email>claudio.bandi@unimi.it</email><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation></author><idno type="istex">00EF801AD23CA06DA52754AFF1D5F6433D9AFB1A</idno><idno type="ark">ark:/67375/6GQ-BFLRDC9S-T</idno><idno type="DOI">10.1017/S0031182007004052</idno><idno type="PII">S0031182007004052</idno><idno type="article-id">00405</idno></analytic><monogr><title level="j">Parasitology</title><title level="j" type="abbrev">Parasitology</title><idno type="pISSN">0031-1820</idno><idno type="eISSN">1469-8161</idno><idno type="publisher-id">PAR</idno><imprint><publisher>Cambridge University Press</publisher><pubPlace>Cambridge, UK</pubPlace><date type="published" when="2008"></date><biblScope unit="volume">135</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="485">485</biblScope><biblScope unit="page" to="494">494</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2008</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en" style="normal"><p>The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.</p></abstract><textClass><keywords scheme="keyword"><list><head></head><item><term>Midichloria mitochondrii</term></item><item><term>Ixodes ricinus</term></item><item><term>hard ticks</term></item><item><term>intra-mitochondrial</term></item><item><term>PCR screening</term></item><item><term>phylogeny</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2008">Published</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus cambridge not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//NLM//DTD Journal Publishing DTD v2.2 20060430//EN" URI="journalpublishing.dtd" name="istex:docType"></istex:docType><istex:document><article article-type="research-article" dtd-version="2.2" xml:lang="EN"><front><journal-meta><journal-id journal-id-type="publisher-id">PAR</journal-id><journal-title>Parasitology</journal-title><abbrev-journal-title>Parasitology</abbrev-journal-title><issn pub-type="ppub">0031-1820</issn><issn pub-type="epub">1469-8161</issn><publisher><publisher-name>Cambridge University Press</publisher-name><publisher-loc>Cambridge, UK</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.1017/S0031182007004052</article-id><article-id pub-id-type="pii">S0031182007004052</article-id><article-id pub-id-type="publisher-id">00405</article-id><title-group><article-title><italic>Midichloria mitochondrii</italic> is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species</article-title><alt-title alt-title-type="left-running">S. Epis and others</alt-title><alt-title alt-title-type="right-running"><italic>Midichloria mitochondrii</italic> in hard ticks (Ixodidae)</alt-title></title-group><contrib-group><contrib><name name-style="western"><surname>EPIS</surname><given-names>S.</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="fn" rid="fn001">†</xref></contrib><contrib><name name-style="western"><surname>SASSERA</surname><given-names>D.</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="fn" rid="fn001">†</xref></contrib><contrib><name name-style="western"><surname>BENINATI</surname><given-names>T.</given-names></name><xref ref-type="aff" rid="aff002"><sup>2</sup></xref><xref ref-type="aff" rid="aff010">§</xref></contrib><contrib><name name-style="western"><surname>LO</surname><given-names>N.</given-names></name><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib><name name-style="western"><surname>BEATI</surname><given-names>L.</given-names></name><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib><name name-style="western"><surname>PIESMAN</surname><given-names>J.</given-names></name><xref ref-type="aff" rid="aff004"><sup>4</sup></xref></contrib><contrib><name name-style="western"><surname>RINALDI</surname><given-names>L.</given-names></name><xref ref-type="aff" rid="aff005"><sup>5</sup></xref></contrib><contrib><name name-style="western"><surname>McCOY</surname><given-names>K. D.</given-names></name><xref ref-type="aff" rid="aff006"><sup>6</sup></xref></contrib><contrib><name name-style="western"><surname>TORINA</surname><given-names>A.</given-names></name><xref ref-type="aff" rid="aff007"><sup>7</sup></xref></contrib><contrib><name name-style="western"><surname>SACCHI</surname><given-names>L.</given-names></name><xref ref-type="aff" rid="aff008"><sup>8</sup></xref></contrib><contrib><name name-style="western"><surname>CLEMENTI</surname><given-names>E.</given-names></name><xref ref-type="aff" rid="aff008"><sup>8</sup></xref></contrib><contrib><name name-style="western"><surname>GENCHI</surname><given-names>M.</given-names></name><xref ref-type="aff" rid="aff008"><sup>8</sup></xref></contrib><contrib><name name-style="western"><surname>MAGNINO</surname><given-names>S.</given-names></name><xref ref-type="aff" rid="aff009"><sup>9</sup></xref></contrib><contrib><name name-style="western"><surname>BANDI</surname><given-names>C.</given-names></name><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="corresp" rid="cor001">*</xref></contrib></contrib-group><aff id="aff001"><label><sup>1</sup></label><addr-line>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria</addr-line>, <institution>Università degli Studi di Milano</institution>, <addr-line>Milano</addr-line>, <country>Italy</country></aff><aff id="aff002"><label><sup>2</sup></label><addr-line>School of Biological Sciences</addr-line>, <institution>The University of Sydney</institution>, <addr-line>New South Wales</addr-line>, <country>Australia</country></aff><aff id="aff003"><label><sup>3</sup></label><addr-line>Institute of Arthropodology and Parasitology</addr-line>, <institution>Georgia Southern University</institution>, <addr-line>Statesboro</addr-line>, <addr-line>GA</addr-line>, <country>USA</country></aff><aff id="aff004"><label><sup>4</sup></label><addr-line>Division of Vector Borne Infectious Diseases</addr-line>, <addr-line>Coordinating Center for Infectious Diseases</addr-line>, <institution>Centers for Disease Control and Prevention</institution>, <addr-line>Fort Collins</addr-line>, <addr-line>CO</addr-line>, <country>USA</country></aff><aff id="aff005"><label><sup>5</sup></label><addr-line>Dipartimento di Patologia e Sanità Animale</addr-line>, <institution>Universita degli Studi di Napoli Federico II</institution>, <addr-line>Napoli</addr-line>, <country>Italy</country></aff><aff id="aff006"><label><sup>6</sup></label><addr-line>Génétique et Evolution des Maladies Infectieuses</addr-line>, <institution>UMR</institution> <addr-line>CNRS-IRD 2724</addr-line>, <addr-line>IRD</addr-line>, <addr-line>Montpellier</addr-line>, <country>France</country></aff><aff id="aff007"><label><sup>7</sup></label><institution>Istituto Zooprofilattico Sperimentale della Sicilia</institution>, <addr-line>Palermo</addr-line>, <country>Italy</country></aff><aff id="aff008"><label><sup>8</sup></label><addr-line>Dipartimento di Biologia Animale</addr-line>, <institution>Università di Pavia</institution>, <addr-line>Piazza Botta 9</addr-line>, <addr-line>Pavia</addr-line>, <country>Italy</country></aff><aff id="aff009"><label><sup>9</sup></label><institution>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna</institution>, <addr-line>Sezione di Pavia</addr-line>, <country>Italy</country></aff><author-notes><corresp id="cor001"><label>*</label>Corresponding author: <addr-line>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria</addr-line>, <institution>Università degli Studi di Milano</institution>, <addr-line>Milano</addr-line>, <country>Italy</country>. Tel: <phone>+39 02 50318093</phone>. Fax: <fax>+39 02 50318095</fax>. E-mail: <email xlink:href="claudio.bandi@unimi.it">claudio.bandi@unimi.it</email></corresp><fn fn-type="other" id="fn001"><label>†</label><p>These authors contributed equally to this work.</p></fn><fn fn-type="present-address" id="aff010"><label>§</label><p>Present address: Faculty of Veterinary Science, The University of Sydney, New South Wales, Australia.</p></fn></author-notes><pub-date><month>04</month><year>2008</year></pub-date><volume>135</volume><issue>4</issue><fpage>485</fpage><lpage>494</lpage><history><date date-type="received"><day>18</day><month>07</month><year>2007</year></date><date date-type="rev-recd"><day>23</day><month>11</month><year>2007</year></date><date date-type="accepted"><day>24</day><month>11</month><year>2007</year></date><date><day>21</day><month>01</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © Cambridge University Press 2008</copyright-statement><copyright-year>2008</copyright-year><copyright-holder>Cambridge University Press</copyright-holder></permissions><abstract abstract-type="normal"><title>SUMMARY</title><p>The hard tick <italic>Ixodes ricinus</italic> (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named <italic>Midichloria mitochondrii</italic>. The objectives of this work were (i) to screen ixodid ticks for <italic>Midichloria</italic>-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for <italic>Midichloria</italic>-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to <italic>M. mitochondrii</italic>. Samples of the tick species <italic>Rhipicephalus bursa</italic>, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of <italic>Midichloria</italic> symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.</p></abstract><kwd-group kwd-group-type=""><kwd><italic>Midichloria mitochondrii</italic></kwd><kwd><italic>Ixodes ricinus</italic></kwd><kwd>hard ticks</kwd><kwd>intra-mitochondrial</kwd><kwd>PCR screening</kwd><kwd>phylogeny</kwd></kwd-group><counts><page-count count="10"></page-count></counts><custom-meta-wrap><custom-meta><meta-name>pdf</meta-name><meta-value>S0031182007004052a.pdf</meta-value></custom-meta><custom-meta><meta-name>dispart</meta-name><meta-value>Original Articles</meta-value></custom-meta></custom-meta-wrap></article-meta></front><body><sec id="sec001" sec-type="intro"><title>INTRODUCTION</title><p>Ticks are obligate haematophagous mites parasitizing vertebrates. They act as vectors of a number of microbial agents that cause diseases in humans and animals. These pathogens include <italic>Borrelia</italic> spp., <italic>Coxiella burnetii</italic>, <italic>Francisella tularensis</italic>, as well as intracellular bacteria from the order Rickettsiales. Other tick-associated bacteria do not appear to be present in the salivary glands or to cause infections in humans and animals; some of these bacteria are transmitted to the progeny through the egg cytoplasm (transovarial transmission). These vertically transmitted microrganisms are usually referred to as symbionts, but their interaction with the host tick is generally not fully understood (for a general introduction to ticks, see Bowman and Nuttall, <xref ref-type="bibr" rid="ref006">2004</xref>).</p><p>The hard tick <italic>Ixodes ricinus</italic> (family Ixodidae) harbours an endosymbiont with the peculiar capacity to invade the mitochondria and multiply therein (first observation by Lewis, <xref ref-type="bibr" rid="ref014">1979</xref>). These mitochondrial bacteria found in <italic>I. ricinus</italic> have been identified based on 16S rRNA gene sequence data (Beninati <italic>et al.</italic> <xref ref-type="bibr" rid="ref004">2004</xref>), investigated at the ultrastructural level (Sacchi <italic>et al.</italic> <xref ref-type="bibr" rid="ref019">2004</xref>), and described as a new genus and species, i.e. <italic>Midichloria mitochondrii</italic> (Sassera <italic>et al.</italic> <xref ref-type="bibr" rid="ref020">2006</xref>). The biological role of <italic>M. mitochondrii</italic> in <italic>I. ricinus</italic> is still unknown. During oogenesis, the proportion of mitochondria invaded by <italic>M. mitochondrii</italic> is not negligible (e.g. see photos in Sacchi <italic>et al.</italic> <xref ref-type="bibr" rid="ref019">2004</xref>), and this suggests that the bacterium could have a detrimental effect on the host tick. However, the fact that <italic>M. mitochondrii</italic> is apparently at fixation in females of <italic>I. ricinus</italic> (100% prevalence according to Lo <italic>et al.</italic> <xref ref-type="bibr" rid="ref015">2006<italic>a</italic></xref>) seems to indicate that its presence is somehow compatible with the survival and reproduction of the host tick (see discussion in Lo <italic>et al</italic>. <xref ref-type="bibr" rid="ref015">2006<italic>a</italic></xref> and <xref ref-type="bibr" rid="ref016"><italic>b</italic></xref>).</p><p>While data have been acquired for <italic>M. mitochondrii</italic> in <italic>I. ricinus</italic>, which have permitted its description as a new genus and species, there is only circumstantial evidence for the presence of DNA sequences from related bacteria in other tick species (see <xref ref-type="sec" rid="sec004">Discussion section</xref>). The aim of this work was thus to obtain a more detailed picture of the distribution of bacteria related to <italic>Midichloria</italic> in hard ticks (family Ixodidae). The discovery of other tick species infected by bacteria related to <italic>M. mitochondrii</italic> could enhance investigations aimed at uncovering its biological role. For instance, a tick species could be found in which the prevalence of <italic>Midichloria</italic>-related symbionts is far from 100%, or in which different populations show different infection levels. This might permit both a comparison of the biology, fitness parameters and vector competence of infected versus uninfected ticks, and experiments on possible reproductive parasitism phenomena (Bandi <italic>et al.</italic> <xref ref-type="bibr" rid="ref001">2001</xref>). Furthermore, <italic>I. ricinus</italic> is the sole animal thus far shown to harbour an intra-mitochondrial bacterium and <italic>M. mitochondrii</italic> is the unique intra-mitochondrial bacterium thus far described. Determining whether intra-mitochondrial bacterial presence is an exception or a more widespread phenomenon was the main goal of this work.</p><p>The screening for <italic>Midichloria</italic>-related bacteria reported here focused on hard ticks. Most of the ~900 species of ticks thus far described (suborder Ixodida in the order Acarina) are assigned to 2 main families: the Ixodidae (hard ticks) and the Argasidae (soft ticks). The Ixodidae are subdivided into 2 main lineages, the Prostriata and Metastriata (Hoogstraal and Aeschlimann, <xref ref-type="bibr" rid="ref010">1982</xref>). The Prostriata encompass the subfamily Ixodinae (over 200 species in the single genus <italic>Ixodes</italic>). The Metastriata group comprises 370 species in 4 subfamilies: the Amblyomminae, Haemaphysalinae, Hyalomminae and Rhipicephalinae (Barker and Murrel, <xref ref-type="bibr" rid="ref002">2004</xref>). Our screening included representatives of all of the subfamilies of the Ixodidae and led to the detection of bacteria in tick specimens belonging to 7 different species (in addition to the previously investigated <italic>I. ricinus</italic>). 16S rRNA sequence data generated for these bacteria revealed a very close phylogenetic relationship with <italic>M. mitochondrii</italic>. Nucleotide identities between the 16S rRNA from <italic>M. mitochondrii</italic> of <italic>I. ricinus</italic> and the sequences from the other tick species were above 99%. We have thus referred to all of these tick bacteria using the species name <italic>M. mitochondrii</italic> (Sassera <italic>et al.</italic> <xref ref-type="bibr" rid="ref020">2006</xref>).</p></sec><sec id="sec002" sec-type="materialsandmethods"><title>MATERIALS AND METHODS</title><sec id="sec002-001"><title>Tick species screened for bacteria related to <italic>Midichloria mitochondrii</italic></title><p>Tick samples representing a total of 21 species of the Ixodidae were examined for the presence of bacteria related to <italic>M. mitochondrii</italic>. The Metastriata were represented by the genera <italic>Rhipicephalus</italic> (4 species), <italic>Dermacentor</italic> (4 species), <italic>Hyalomma</italic> (2 species), <italic>Amblyomma</italic> (3 species) and <italic>Haemaphysalis</italic> (3 species). The Prostriata were represented by 5 species of the genus <italic>Ixodes</italic>.</p><p>A total of 197 tick specimens were collected in Italy (Lazio, Sicily and Lombardia), Iceland (Grimsey) and USA (New Jersey, Florida, Missouri, South Carolina, Oregon, California and Georgia). Of these specimens, 125 were ethanol-preserved before DNA extraction, while the other 72 were processed immediately after collection. Ticks were collected at different developmental stages (larvae, nymphs, adults, and engorged females). Forty-two of the samples were pooled prior to DNA extraction. For the remaining 155 samples DNA was extracted from individual specimens. See <xref ref-type="table" rid="tab001">Table 1</xref> for a complete list of the specimens. All ticks were identified using standard taxonomic keys; in addition, for confirmation of tick identification, a fragment of the small subunit mitochondrial ribosomal RNA gene (12S rRNA) was sequenced for a subset of the examined specimens/pools of specimens.</p><p><table-wrap id="tab001" position="float"><label>Table 1.</label><caption><p>List of taxa, geographical origin, Accession numbers for bacterial 16S rRNA and tick 12S rRNA sequences, and screening results</p></caption><graphic alt-version="no" mime-subtype="gif" mimetype="image" position="float" xlink:href="S0031182007004052_tab1"></graphic><table-wrap-foot><fn id="tfn001_001" symbol="1"><label><sup>1</sup></label><p>DNA extracted from living specimens.</p></fn><fn id="tfn001_002" symbol="2"><label><sup>2</sup></label><p>DNA extracted from samples preserved in ethanol.</p></fn><fn id="tfn001_003" symbol="3"><label><sup>3</sup></label><p>Nymphs or larvae.</p></fn><fn id="tfn001_004"><p>MSNPV, Museo di Storia Naturale, Università di Pavia; RML, U.S. National Tick Collection.</p></fn><fn id="tfn001_005"><p>N.A., Sample Accession no. not available, or sequencing not performed because the samples were negative for midichlorians.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="sec002-002"><title>DNA extraction and polymerase chain reaction (PCR) amplification of tick 12S rRNA</title><p>DNA was extracted using Qiagen tissue kit (Qiagen, Hilden, Germany). Ethanol-preserved ticks were washed and dried for 45 min before DNA extraction. Afterwards, ticks were triturated into a solution containing 180 μl of ATL lysis buffer (Qiagen) and 200 ng/μl proteinase K (Sigma Aldrich, St Louis, USA). The lysis was carried out overnight with shaking at 56°C. The elution step was performed by 2 additions of 25 μl of hot (72°C) deionized water in the columns; the remaining extraction steps were performed according to the manufacturer's instructions. DNA was stored at −20°C.</p><p>PCR for amplification of 12S rRNA was performed as described by Beati and Keirans (<xref ref-type="bibr" rid="ref003">2001</xref>). PCR products were gel-purified using the QIAquick™ Gel Extraction Kit (Qiagen) according to the manufacturer's protocols, resuspended in 30 μl of deionized water and sequenced with PCR primers using the ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit, version 3.1 (Applera Europe, Warrington, UK), and run on an automated sequencer (ABI Prism 310 DNA sequencer, Applied Biosystems, Foster City, CA).</p></sec><sec id="sec002-003"><title>PCR screening for bacteria related to <italic>Midichloria mitochondrii</italic></title><p>PCR screening for bacteria related to <italic>M. mitochondrii</italic> was performed using 2 sets of primers: Midi-F (5′-GTACATGGGAATCTACCTTGC-3′) and Midi-R (5′-CAGGTCGCCCTATTGCTTCTTT-3′); Midi-F2 (5′-CAACGAGCGCAACCCT TAT-3′) and MidiR2 (5′-CAGTCGTCAACCTT ACCGT-3′). All samples were tested with both primer copies, in order to double check the results obtained. These primers, targeted the 16S rRNA gene, amplify fragments of ~1100 (Midi-F –R) and ~350 bp (Midi-F2 –R2) and were designed to be conserved between the sequence of <italic>M. mitochondrii</italic> from <italic>I. ricinus</italic> (AJ566640) and the closely related sequences available in the data bases. In particular, an alignment was generated with the aim of representing all of the main lineages of the order Rickettsiales and the sequences already available that cluster as a monophyletic group with <italic>M. mitochondrii</italic> (see phylogenetic trees in Beninati <italic>et al.</italic> <xref ref-type="bibr" rid="ref004">2004</xref>; Lo <italic>et al.</italic> <xref ref-type="bibr" rid="ref015">2006<italic>a</italic></xref>; Sassera <italic>et al.</italic> <xref ref-type="bibr" rid="ref020">2006</xref>). Primers were then designed to be conserved and specific for bacteria in the <italic>M. mitochondrii</italic> cluster. Primer specificity was then checked by BLAST searches and by PCR on biological samples positive for bacteria of the genera <italic>Rickettsia</italic>, <italic>Ehrlichia</italic>, <italic>Wolbachia</italic> and <italic>Anaplasma</italic>.</p><p>Amplifications were performed, with both primer sets, in 20 μl of buffer (10 m<sc>m</sc> Tris-HCl (pH 8·3), 50 m<sc>m</sc> KCl, 1·5 m<sc>m</sc> MgCl<sub>2</sub>, 0·001% gelatine) with 0·2 m<sc>m</sc> each deoxynucleoside triphosphate, 10 pmol of each primer, 0·5 U of <italic>Taq</italic> Polymerase (Eppendorf) and 2 μl of DNA sample. The thermal profile was: 2 min at 95°C; 40 cycles of 95°C for 30 sec, 56°C for 30 sec and 68°C for 45 sec; the elongation was completed at 10 min at 68°C. PCR products obtained with primer combination MidiF and MidiR were purified and sequenced as previously described using primers Midi-F and Midi-R and the internal primers P3b (5′-CTGTTTGCTCCCCACGCTTTC-3′) and P2b (5′-GATATTAGGAGGAACACCGC-3′).</p></sec><sec id="sec002-004"><title>Phylogenetic analyses</title><p>The bacterial 16S rRNA sequences obtained from ticks were subjected to BLAST analysis. These sequences were then aligned using as a mask an alignment downloaded from the Ribosomal Database Project (Cole <italic>et al.</italic> <xref ref-type="bibr" rid="ref007">2003</xref>). The alignment was generated considering 16S rRNA secondary structures and included the sequence of <italic>M. mitochondrii</italic> from <italic>I. ricinus</italic> and the homologous <italic>Midichloria</italic>-related sequences available in the data bases (excluding sequences below 1000 bp). Representatives of the main lineages of the order Rickettsiales were also included. Phylogenetic analyses were performed using the programmes Mega 3.1 (Kumar <italic>et al.</italic> <xref ref-type="bibr" rid="ref013">2004</xref>) and MrBayes (Huelsenbeck and Ronquist, <xref ref-type="bibr" rid="ref011">2001</xref>). Trees were generated using Neighbour Joining, Maximum Parsimony and Bayesian Inference methods. For Bayesian Inference, parameters for the model of substitution (General Time Reversible with Gamma correction for among-site heterogeneity) were estimated from the data. A total of 10 000 trees were obtained (ngen=1 000 000, samplefreq=100), and the first 3000 of these were considered as the ‘burn in’ and discarded. A 50% majority-rule consensus tree of the remaining trees, including branch lengths (sumt) was produced. For macro-taxonomic purposes, analyses were performed on alignments including representatives of the Rickettsiales (see tree in <xref ref-type="fig" rid="fig001">Fig. 1</xref>), while for investigating the relationships at a lower taxonomic level, sequences from Rickettsiales which are not closely related with <italic>M. mitochondrii</italic> were excluded (see tree in <xref ref-type="fig" rid="fig002">Fig. 2</xref>). The sequences obtained have been deposited in the EMBL data library (see Accession numbers in <xref ref-type="table" rid="tab001">Table 1</xref>).</p><p><fig fig-type="fig" id="fig001" position="float"><label>Fig. 1.</label><caption><p>Example of tree based on 16S rRNA gene sequences showing the position of <italic>Midichloria mitochondrii</italic> from different ticks relative to representatives of the order Rickettsiales. The tree was generated using MEGA (Neighbour Joining; Kimura correction). The Accession number for each sequence is indicated. Numbers adjacent to each node represent the bootstrap percentages (5000 repetitions). <italic>Acetobacter aceti</italic> (Rhodospirillales) was included as an outgroup. Additional analyses in which other Alphaproteobacteria were included as outgroups, or using other tree-building approaches, generated trees showing the same overall topology. Bar: 0·02 inferred substitutions per site.</p></caption><graphic alt-version="no" mime-subtype="gif" mimetype="image" position="float" xlink:href="S0031182007004052_fig1"></graphic></fig></p><p><fig fig-type="fig" id="fig002" position="float"><label>Fig. 2.</label><caption><p>Comparison of the phylogenies of <italic>Midichloria mitochondrii</italic> and ixodid ticks. Nucleotide sequence Accession numbers for ixodid ticks and for the different ‘strains’ of <italic>M. mitochondrii</italic> are reported in <xref ref-type="table" rid="tab001">Table 1</xref>. Accession numbers for outgroup sequences are indicated. (A) Example of tree based on 16S rRNA gene sequences showing the inferred phylogeny of <italic>M. mitochondrii</italic>. Names at the terminal nodes are those of the host ticks (for full-length genus names, see (B). The tree was generated using MEGA (Neighbour Joining; Kimura correction). Numbers adjacent to each node represent the bootstrap values (5000 repetitions). An endosymbiont of <italic>Acanthamoeba</italic> sp. was used as outgroup. Additional analyses in which other Alphaproteobacteria were included as outgroups, or using other tree-building approaches, generated trees showing the same overall topology. Bar: 0·001 inferred substitutions per site. (B) Example of phylogenetic tree based on partial 12S rRNA gene sequences showing the relationships of the examined ticks. The + symbol indicates tick species that were found to be positive for <italic>M. mitochondrii</italic>. The tree was generated using MEGA (Neighbour Joining; Kimura correction). Numbers adjacent to each node represent the bootstrap percentages (5000 repetitions). Nodes with bootstrap values below 50% are shown as unresolved. Analyses using other tree-building approaches did not lead to a better resolution of these nodes. The argasid tick <italic>Argas persicus</italic> was included as outgroup. Bar: 0·02 inferred substitutions per site.</p></caption><graphic alt-version="no" mime-subtype="gif" mimetype="image" position="float" xlink:href="S0031182007004052_fig2"></graphic></fig></p><p>The 12S mitochondrial rRNA sequences obtained were subjected to BLAST analysis (<uri xlink:href="http://www.ncbi.nlm.nih.gov/blast">http://www.ncbi.nlm.nih.gov/blast</uri>) and compared to the sequences available in the data bases in order to confirm morphology-based species identification. The sequences were aligned with ClustalX (Thompson <italic>et al.</italic> <xref ref-type="bibr" rid="ref023">1997</xref>) and manually adjusted taking into account secondary structures (Hickson <italic>et al.</italic> <xref ref-type="bibr" rid="ref009">1996</xref>). Phylogenetic analyses were then performed as described for 16S rRNA sequences. These sequences have been deposited in the EMBL data library (see Accession numbers in <xref ref-type="table" rid="tab001">Table 1</xref>).</p></sec><sec id="sec002-005"><title>Electron microscopy</title><p>Specimens of the tick species <italic>Rhipicephalus bursa</italic> were found positive for <italic>M. mitochondrii</italic> during the PCR screening; this tick species was thus chosen for transmission electron microscopy (TEM) examination. Eleven adult engorged females were collected from the field (Lazio, Italy), brought to the lab within 48 h and immediately dissected. Half of the ovary from each female specimen was fixed for TEM examination, while the remaining half was used for DNA extraction and PCR. The 4 ovarian specimens that were found to be PCR positive for <italic>M. mitochondrii</italic> as well as a negative ovarian specimen were then subjected to TEM examination. For TEM examination, ovaries were fixed in 0·1 <sc>m</sc> cacodylate buffer (pH 7·2) containing 2·5% glutaraldehyde for 3 h at 4°C. The samples were then washed in the same buffer and post-fixed in 1% OsO<sub>4</sub> in the same buffer for 1·5 h at 4°C. Successively all samples were dehydrated in ethanol and embedded in Epon 812. The semithin sections (1 μm) for light microscopy were stained with 0·5% toluidine blue; thin sections (80 nm) were stained with uranyl acetate and lead citrate and examined under an EM900 transmission electron microscope (Zeiss).</p></sec></sec><sec id="sec003" sec-type="results"><title>RESULTS</title><p>All of the ticks examined have been identified using morphological keys. In addition, all specimens were examined by PCR amplification of mitochondrial 12S rRNA to check for the quality of the DNA. A fragment of about 350 bp of the 12S rRNA was sequenced for a subset of samples, in order to confirm morphological identification. The fragment was also sequenced for all samples positive for <italic>M. mitochondrii</italic>. In total, forty 12S rRNA sequences were generated. BLAST search of 12S rRNA sequences confirmed morphological identifications in all cases. The 12S rRNA sequences obtained have been deposited in the EMBL data library (see Accession numbers in <xref ref-type="table" rid="tab001">Table 1</xref>).</p><p>The 2 PCR protocols designed to be specific for the 16S rRNA of <italic>M. mitochondrii</italic> and related bacteria gave amplifications of the expected size from tick specimens belonging to 8 species out of the 21 species included in this study. In total, 15 tick specimens were found positive out of the 155 individual ticks examined. In addition, out of 42 pooled tick samples, 7 were found to be positive for <italic>M</italic>. <italic>mitochondrii</italic>. <xref ref-type="table" rid="tab001">Table 1</xref> summarizes the results obtained.</p><p>The ~1100 bp PCR-amplified 16S rRNA gene fragments from positive specimens were then sequenced, and the sequences were, in all cases, unambiguous. The sequences obtained were run against the databases and gave the highest similarity scores with the 16S rRNA sequences of <italic>M. mitochondrii</italic> from <italic>I</italic>. <italic>ricinus</italic> and with those sequences which have already been shown to form a monophyletic group with this bacterium (see <xref ref-type="fig" rid="fig001">Fig. 1</xref> and Sassera <italic>et al.</italic> <xref ref-type="bibr" rid="ref020">2006</xref>).</p><p>Phylogenetic analyses were performed on 3 different data sets. The first included 16S rRNA sequences from bacteria of the main branches of the Rickettsiales and from <italic>M. mitochondrii</italic> and related bacteria (<xref ref-type="fig" rid="fig001">Fig. 1</xref>). The second alignment involved bacterial 16S rRNA from <italic>M. mitochondrii</italic> of <italic>I. ricinus</italic> and all closely related tick endosymbionts (<xref ref-type="fig" rid="fig002">Fig. 2</xref>A). The last alignment focused on mitochondrial 12S rRNA from ixodid ticks (<xref ref-type="fig" rid="fig002">Fig. 2</xref>B). The results of the analyses performed for each data set were similar irrespective of the phylogenetic method employed.</p><p><xref ref-type="fig" rid="fig001">Figure 1</xref> shows an example of 16S rRNA-based tree (Neighbour Joining) including representatives of the genera and families of the Rickettsiales. All of the sequences generated in this study form a monophyletic branch, which also includes the sequence of <italic>M. mitochondrii</italic> from <italic>I. ricinus</italic>, and the bacterial sequences that were already available in the data bases from the ticks <italic>Ixodes persulcatus</italic> (Mediannikov <italic>et al.</italic> <xref ref-type="bibr" rid="ref017">2004</xref>) and <italic>Haemaphysalis wellingtoni</italic> (Parola <italic>et al.</italic> <xref ref-type="bibr" rid="ref018">2003</xref>), from the marine sponge <italic>Cymbastela concentrica</italic> (Taylor <italic>et al.</italic> <xref ref-type="bibr" rid="ref022">2005</xref>) and from the amoeba <italic>Achantamoeba</italic> sp. (Fritsche <italic>et al.</italic> <xref ref-type="bibr" rid="ref008">1999</xref>). Support for this branch was very high in all of the analyses performed (Neighbour Joining; Maximum Parsimony; Bayesian Inference), with bootstrap or posterior probability values of 97–100%. Within this branch, molecular diversity at the nucleotide level is limited: with the exclusion of the sequences from <italic>Acanthamoeba</italic> sp., <italic>C. concentrica</italic> and from <italic>I. persulcatus</italic>, pairwise nucleotide differences ranged from 3 to 22 nt out of 1000.</p><p><xref ref-type="fig" rid="fig002">Figure 2</xref>A is a 16S rRNA-based tree (Neighbour Joining) including the <italic>M. mitochondrii</italic> sequences generated in this study and the sequence from the endosymbiont of <italic>Acanthamoeba</italic> sp. (included as an outgroup). Sequences from <italic>M. mitochondrii</italic> are assigned to 3 main branches, supported by high bootstrap values. <xref ref-type="fig" rid="fig002">Figure 2</xref>B shows a 12S rRNA-based tree (Neighbour Joining) including the tick species examined in this study. The tree is not fully resolved, but the main branches correspond to the traditional systematic arrangement of hard tick genera into subfamilies and of the subfamilies into the groups Prostriata and Metastriata (Hoogstraal and Aeschlimann, <xref ref-type="bibr" rid="ref010">1982</xref>), with the exception of <italic>Ixodes uriae</italic> whose positioning relative to the two groups is not resolved. The uncertain positioning of <italic>I. uriae</italic> is not surprising if we consider the results of total evidence phylogenetics of hard ticks, placing this and other species of the genus <italic>Ixodes</italic> in different positions depending on the methods of analysis (Klompen <italic>et al.</italic> <xref ref-type="bibr" rid="ref012">2000</xref>). Indeed, phylogenetic analyses of our tick data set performed using other algorithms (Maximum Parsimony and Bayesian Inference; see <xref ref-type="sec" rid="sec002">Materials and Methods section</xref>) did not lead to a better resolution. A detailed comparison of the host and symbiont phylogenies was therefore outside the scope of this work, considering that (i) tick phylogeny is still not fully resolved and (ii) the level of divergence here reported in the 16S rRNA of <italic>M. mitochondrii</italic> is not sufficient for the reconstruction of a robust phylogeny. Examining the symbiont (<xref ref-type="fig" rid="fig002">Fig. 2</xref>A) and host trees (e.g. the tree in <xref ref-type="fig" rid="fig002">Fig. 2</xref>B or the trees in Fig. 4 in Klompen <italic>et al.</italic> <xref ref-type="bibr" rid="ref012">2000</xref>) it is clear that the positioning of <italic>M. mitochondrii</italic> endosymbionts is not always congruent with the taxonomic/phylogenetic placements of the hosts. For example, endosymbionts from ticks of the <italic>Ixodes</italic> genus (the Prostriata of traditional taxonomic arrangements) cluster with endosymbionts from ticks of the Metastriata group. In some cases, tick species harbouring closely related <italic>M. mitochondrii</italic> are highly divergent at the level of their 12S rRNA gene. For example, <italic>M. mitochondrii</italic> from distantly related ticks of the genus <italic>Ixodes</italic> (<italic>I. ricinus</italic> and <italic>I. uriae</italic>) are identical to each other in their 16S rRNA, and are also identical to <italic>M. mitochondrii</italic> of the Metastriata ticks <italic>R. turanicus</italic> and <italic>Ha. punctata</italic>. There are also single-tick species in which different individuals harbour <italic>M. mitochondrii</italic> with different 16S rRNA sequences (<italic>Hy. truncatum</italic>, <italic>Hy. marginatum</italic> and <italic>R. bursa</italic>).</p><p>Out of the 7 new tick species that were found positive for <italic>M. mitochondrii</italic>, we had the possibility to collect samples on 2 different occasions only for the species <italic>R. bursa</italic>. The number of specimens collected allowed us to obtain a first picture of the prevalence of <italic>M. mitochondrii</italic> in this tick species, with 7 positive females out of the 21 examined and 2 males out of 16. We emphasize that all positive specimens were from a single population, while the specimens from the other population examined were all negative. In general, for all of the species that were found positive for <italic>M. mitochondrii</italic> in this screening, not all of the specimens examined were positive (see <xref ref-type="table" rid="tab001">Table 1</xref>), though only for <italic>R. bursa</italic> were we able to collect a number of specimens sufficient for concluding that prevalence is far from 100%.</p><p>Female specimens of <italic>R. bursa</italic> were prepared for both PCR and TEM examinations. TEM was performed on ovaries from 4 female specimens that were found positive for <italic>M. mitochondrii</italic>, and from 1 specimen that was found negative. TEM examinations of the ovaries from positive females revealed the presence of bacteria in the mitochondria of the oocytes (<xref ref-type="fig" rid="fig003">Fig. 3</xref>A). The few bacteria that were observed outside the mitochondria were free in the cytoplasm (i.e. without surrounding vacuoles). The size of these bacteria is ~1 μm in length and ~0·25 μm in diameter. The number of bacteria in the mitochondria ranged from a single bacterium to over 20 observed in a single section. Infected mitochondria in <italic>R. bursa</italic> do not present clear signs of reduction/degeneration of the matrix, even in cases where numerous bacteria infect the mitochondria (<xref ref-type="fig" rid="fig003">Fig. 3</xref>B).</p><p><fig fig-type="fig" id="fig003" position="float"><label>Fig. 3.</label><caption><p>Electron micrographs of ovarian cells of <italic>Rhipicephalus bursa</italic> showing mitochondria harbouring bacteria (A). Mitochondrion containing several bacteria does not present clear signs of reduction or degeneration of the matrix (B).</p></caption><graphic alt-version="no" mime-subtype="gif" mimetype="image" position="float" xlink:href="S0031182007004052_fig3"></graphic></fig></p></sec><sec id="sec004" sec-type="discussion"><title>DISCUSSION</title><p>The screening reported here shows that <italic>M. mitochondrii</italic> is commonly found in ticks. Eight species out of 21 were positive, and only in 1 of the 7 genera examined (<italic>Dermacentor</italic>) were positive individuals not detected. The prevalence of <italic>M. mitochondrii</italic> in the various tick species examined here differs markedly from that observed in <italic>I. ricinus</italic>, where 100% of females and 44% of males have been found positive (Lo <italic>et al.</italic> <xref ref-type="bibr" rid="ref015">2006<italic>a</italic></xref>). In general, the results here reported on the distribution of <italic>M. mitochondrii</italic> in ticks likely represent an underestimation. Firstly, for most of the species a limited number of specimens was examined, which implies that future screenings could lead to the detection of positive tick individuals from further tick species. Secondly, our screening included ethanol-preserved specimens. Partial degradation of the DNA cannot thus be excluded, even though control PCR for mitochondrial tick genes provided evidence for a good DNA quality.</p><p><xref ref-type="fig" rid="fig002">Figure 2</xref>A shows a phylogenetic tree of <italic>M. mitochondrii</italic>; a tree representing a phylogeny of the ticks included in this study is also shown for comparison (<xref ref-type="fig" rid="fig002">Fig. 2</xref>B). The overall phylogeny of ticks is still unresolved (Barker and Murrel, <xref ref-type="bibr" rid="ref002">2004</xref>), and it was not the goal of this work to address this issue. However, an incomplete congruence in the phylogenies of ticks and their endosymbionts can be inferred, both comparing the <italic>M. mitochondrii</italic> tree with the host tree in <xref ref-type="fig" rid="fig002">Fig. 2</xref>B (or with trees in other studies; e.g. Black <italic>et al.</italic> <xref ref-type="bibr" rid="ref005">1997</xref>; Klompen <italic>et al.</italic> <xref ref-type="bibr" rid="ref012">2000</xref>), as well as considering that ticks belonging to different genera (<italic>Ixodes</italic> spp. and <italic>R. turanicus</italic>) harbour <italic>M. mitochondrii</italic> with identical 16S rRNA sequences. Considering the <italic>Midichloria</italic>-like 16S rRNA sequences already available in the data bases, we note that the endosymbiont of <italic>I. persulcatus</italic> belongs to the sister group of the main cluster that includes the sequences generated in this study plus those from <italic>Ha. wellingtoni</italic> and <italic>Acanthamoeba</italic> sp. In the tree in <xref ref-type="fig" rid="fig001">Fig. 1</xref>, the endosymbiont of <italic>I. persulcatus</italic> clusters with a bacterial sequence obtained from a sponge. The distance between this <italic>Ixodes</italic> bacterium and those found in other representatives of the genus <italic>Ixodes</italic> is relatively large.</p><p>Previous studies have provided microscopical and molecular evidence for vertical transmission of <italic>M. mitochondrii</italic> in <italic>I. ricinus</italic> (Sacchi <italic>et al.</italic> <xref ref-type="bibr" rid="ref019">2004</xref>; Lo <italic>et al.</italic> <xref ref-type="bibr" rid="ref015">2006<italic>a</italic></xref>). The results reported here reveal that in some tick species different individuals harbour <italic>M. mitochondrii</italic> with identical gene sequences (i.e. in <italic>I. ricinus</italic>, <italic>A. tuberculatum</italic>, <italic>Ha. punctata</italic>). There is thus an overall consistency of information which indicates an effective role of vertical transmission in the diffusion of these bacteria. On the other hand, the results reported above also indicate a partial lack of congruence in the phylogenies of <italic>M. mitochondrii</italic> endosymbionts and their hosts, and suggest that horizontal transmission of the symbionts might have occurred. How horizontal transmission could take place is unknown. However, based on the presence of DNA from <italic>M. mitochondrii</italic> in the blood of roe deer (Skarphédinsson <italic>et al.</italic> <xref ref-type="bibr" rid="ref021">2005</xref>), and the possible detection of a related bacterium in samples from human patients (Mediannikov <italic>et al.</italic> <xref ref-type="bibr" rid="ref017">2004</xref>), we might suggest that transmission to tick hosts could occur, and that blood could represent a potential source of infection for ticks. In any case, based on the above studies, the possibility that <italic>M. mitochondrii</italic> could infect humans and animals is worthy of further investigation.</p><p>In <italic>R. bursa</italic>, the species for which we could obtain the most samples, the prevalence of <italic>M. mitochondrii</italic> was 33% in females and 13% in males. The discovery of a tick species in which the prevalence of <italic>M. mitochondrii</italic> is far from fixation in both males and females will facilitate experiments aimed at uncovering the biological role of these endosymbionts. For example, fitness parameters could be compared in positive and negative individuals, as could the relative prevalence of tick-borne pathogens in positive and negative individuals. Experiments examining the possible role of <italic>M. mitochondrii</italic> as a reproductive parasite (e.g. cytoplasmic incompatibility, as in the case of <italic>Wolbachia</italic>; see Werren, <xref ref-type="bibr" rid="ref024">1997</xref>) could also be envisioned.</p><p>TEM observations on the ovaries from <italic>R. bursa</italic> gave results comparable to those obtained in <italic>I. ricinus</italic>, in terms of the size, shape and overall appearance of the bacterial symbionts (Sacchi <italic>et al.</italic> <xref ref-type="bibr" rid="ref019">2004</xref>). Evidence was also obtained that the bacteria reside in mitochondria. <italic>I. ricinus</italic> mitochondria harbouring numerous bacteria (i.e. over 10/20 per section) always present clear signs of degeneration of the matrix, in some cases resembling empty sacs; this has been interpreted as a consumption of the matrix by the bacteria (Sacchi <italic>et al.</italic> <xref ref-type="bibr" rid="ref019">2004</xref>). Such degeneration was less evident in <italic>R. bursa</italic>, even in mitochondria containing up to 20 bacteria per section. These TEM observations, associated with the lower prevalence of <italic>M. mitochondrii</italic> in <italic>R. bursa</italic>, may indicate that the kind of interaction between the host tick and symbionts is different in <italic>R. bursa</italic> and in <italic>I. ricinus</italic>. We note that within the monophyletic branch of <italic>M. mitochondrii</italic>, the bacteria of these 2 tick species are assigned to 2 different branches.</p><p>The capacity to invade the host mitochondria is thus present in different lineages of <italic>M. mitochondrii</italic>, infecting different tick species belonging to different genera and subfamilies. We could therefore hypothesize that the tropism for the mitochondria could represent an ancestral trait of <italic>M. mitochondrii</italic>, and it is reasonable to expect that other tick species in addition to the two that we investigated at the microscopic level harbour bacteria with the capacity to invade the mitochondria. In summary, intra-mitochondrial symbiosis is probably not just a curiosity limited to <italic>I. ricinus</italic>, but could represent a more widespread biological phenomenon, at least in ticks of the family Ixodidae, a group of arthropods encompassing over 600 species, many of significant medical and veterinary importance. The fact that 16S rRNA gene fragments almost identical to those from <italic>M. mitochondrii</italic> have been found in samples from humans and deer (Mediannikov <italic>et al.</italic> <xref ref-type="bibr" rid="ref017">2004</xref>; Skarphédinsson <italic>et al.</italic> <xref ref-type="bibr" rid="ref021">2005</xref>) opens the intriguing question of whether the invasion of mitochondria by these bacteria also occurs in mammals.</p></sec></body><back><ack id="sec005"><p>We thank the French Polar Institute (IPEV programme n°333) and A. Petersen for enabling collection of <italic>I. uriae</italic> in Iceland. N. L. is supported by an Australian Research Council Postdoctoral Fellowship. The authors are most grateful to George Lucas for his permission to derive the genus name <italic>Midichloria</italic> after the fictional creatures midichlorians (see Sassera <italic>et al.</italic> <xref ref-type="bibr" rid="ref020">2006</xref>). 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Epis and others</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species</title></titleInfo><name type="personal"><namePart type="given">S.</namePart><namePart type="family">EPIS</namePart><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation><affiliation>These authors contributed equally to this work.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">D.</namePart><namePart type="family">SASSERA</namePart><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation><affiliation>These authors contributed equally to this work.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">T.</namePart><namePart type="family">BENINATI</namePart><affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</affiliation><affiliation>§Present address: Faculty of Veterinary Science, The University of Sydney, New South Wales, Australia.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">N.</namePart><namePart type="family">LO</namePart><affiliation>School of Biological Sciences, The University of Sydney, New South Wales, Australia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">L.</namePart><namePart type="family">BEATI</namePart><affiliation>Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">J.</namePart><namePart type="family">PIESMAN</namePart><affiliation>Division of Vector Borne Infectious Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">L.</namePart><namePart type="family">RINALDI</namePart><affiliation>Dipartimento di Patologia e Sanità Animale, Universita degli Studi di Napoli Federico II, Napoli, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">K. D.</namePart><namePart type="family">McCOY</namePart><affiliation>Génétique et Evolution des Maladies Infectieuses, UMR CNRS-IRD 2724, IRD, Montpellier, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">A.</namePart><namePart type="family">TORINA</namePart><affiliation>Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">L.</namePart><namePart type="family">SACCHI</namePart><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">E.</namePart><namePart type="family">CLEMENTI</namePart><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">M.</namePart><namePart type="family">GENCHI</namePart><affiliation>Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 9, Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">S.</namePart><namePart type="family">MAGNINO</namePart><affiliation>Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Pavia, Italy</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C.</namePart><namePart type="family">BANDI</namePart><affiliation>Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy</affiliation><affiliation>E-mail: claudio.bandi@unimi.it</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>Cambridge University Press</publisher><place><placeTerm type="text">Cambridge, UK</placeTerm></place><dateIssued encoding="w3cdtf">2008</dateIssued><copyrightDate encoding="w3cdtf">2008</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract type="normal" lang="en">The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.</abstract><note type="footnotes">These authors contributed equally to this work.</note><subject><genre></genre><topic>Midichloria mitochondrii</topic><topic>Ixodes ricinus</topic><topic>hard ticks</topic><topic>intra-mitochondrial</topic><topic>PCR screening</topic><topic>phylogeny</topic></subject><relatedItem type="host"><titleInfo><title>Parasitology</title></titleInfo><titleInfo type="abbreviated"><title>Parasitology</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0031-1820</identifier><identifier type="eISSN">1469-8161</identifier><identifier type="PublisherID">PAR</identifier><part><date>2008</date><detail type="volume"><caption>vol.</caption><number>135</number></detail><detail type="issue"><caption>no.</caption><number>4</number></detail><extent unit="pages"><start>485</start><end>494</end><total>10</total></extent></part></relatedItem><identifier type="istex">00EF801AD23CA06DA52754AFF1D5F6433D9AFB1A</identifier><identifier type="ark">ark:/67375/6GQ-BFLRDC9S-T</identifier><identifier type="DOI">10.1017/S0031182007004052</identifier><identifier type="PII">S0031182007004052</identifier><identifier type="ArticleID">00405</identifier><accessCondition type="use and reproduction" contentType="copyright">Copyright © Cambridge University Press 2008</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-G3RCRD03-V">cambridge</recordContentSource><recordOrigin>Copyright © Cambridge University Press 2008</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/document/00EF801AD23CA06DA52754AFF1D5F6433D9AFB1A/metadata/json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/document/00EF801AD23CA06DA52754AFF1D5F6433D9AFB1A/annexes/gif</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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