Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues
Identifieur interne : 004383 ( Pmc/Corpus ); précédent : 004382; suivant : 004384Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues
Auteurs : Frédéric Landmann ; Jeremy M. Foster ; Barton Slatko ; William SullivanSource :
- PLoS Neglected Tropical Diseases [ 1935-2727 ] ; 2010.
Abstract
Url:
DOI: 10.1371/journal.pntd.0000758
PubMed: 20689574
PubMed Central: 2910707
Links to Exploration step
PMC:2910707Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Asymmetric <italic>Wolbachia</italic> Segregation during Early
<italic>Brugia malayi</italic> Embryogenesis Determines Its Distribution in
Adult Host Tissues</title><author><name sortKey="Landmann, Frederic" sort="Landmann, Frederic" uniqKey="Landmann F" first="Frédéric" last="Landmann">Frédéric Landmann</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Foster, Jeremy M" sort="Foster, Jeremy M" uniqKey="Foster J" first="Jeremy M." last="Foster">Jeremy M. Foster</name><affiliation><nlm:aff id="aff2"><addr-line>New England Biolabs, Inc., Ipswich, Massachusetts, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Slatko, Barton" sort="Slatko, Barton" uniqKey="Slatko B" first="Barton" last="Slatko">Barton Slatko</name><affiliation><nlm:aff id="aff2"><addr-line>New England Biolabs, Inc., Ipswich, Massachusetts, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Sullivan, William" sort="Sullivan, William" uniqKey="Sullivan W" first="William" last="Sullivan">William Sullivan</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20689574</idno><idno type="pmc">2910707</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910707</idno><idno type="RBID">PMC:2910707</idno><idno type="doi">10.1371/journal.pntd.0000758</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">004383</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004383</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Asymmetric <italic>Wolbachia</italic> Segregation during Early
<italic>Brugia malayi</italic> Embryogenesis Determines Its Distribution in
Adult Host Tissues</title><author><name sortKey="Landmann, Frederic" sort="Landmann, Frederic" uniqKey="Landmann F" first="Frédéric" last="Landmann">Frédéric Landmann</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Foster, Jeremy M" sort="Foster, Jeremy M" uniqKey="Foster J" first="Jeremy M." last="Foster">Jeremy M. Foster</name><affiliation><nlm:aff id="aff2"><addr-line>New England Biolabs, Inc., Ipswich, Massachusetts, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Slatko, Barton" sort="Slatko, Barton" uniqKey="Slatko B" first="Barton" last="Slatko">Barton Slatko</name><affiliation><nlm:aff id="aff2"><addr-line>New England Biolabs, Inc., Ipswich, Massachusetts, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Sullivan, William" sort="Sullivan, William" uniqKey="Sullivan W" first="William" last="Sullivan">William Sullivan</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS Neglected Tropical Diseases</title><idno type="ISSN">1935-2727</idno><idno type="eISSN">1935-2735</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Wolbachia</italic> are required for filarial nematode survival and
fertility and contribute to the immune responses associated with human filarial
diseases. Here we developed whole-mount immunofluorescence techniques to
characterize <italic>Wolbachia</italic> somatic and germline transmission
patterns and tissue distribution in <italic>Brugia malayi</italic>, a nematode
responsible for lymphatic filariasis. In the initial embryonic divisions,
<italic>Wolbachia</italic> segregate asymmetrically such that they occupy
only a small subset of cells in the developing embryo, facilitating their
concentration in the adult hypodermal chords and female germline.
<italic>Wolbachia</italic> are not found in male reproductive tissues and
the absence of <italic>Wolbachia</italic> from embryonic germline precursors in
half of the embryos indicates <italic>Wolbachia</italic> loss from the male
germline may occur in early embryogenesis. <italic>Wolbachia</italic> rely on
fusion of hypodermal cells to populate adult chords. Finally, we detect
<italic>Wolbachia</italic> in the secretory canal lumen suggesting living
worms may release bacteria and/or their products into their host.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Crompton, Dw" uniqKey="Crompton D">DW Crompton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Molyneux, Dh" uniqKey="Molyneux D">DH Molyneux</name></author><author><name sortKey="Bradley, M" uniqKey="Bradley M">M Bradley</name></author><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author><author><name sortKey="Kyelem, D" uniqKey="Kyelem D">D Kyelem</name></author><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bandi, C" uniqKey="Bandi C">C Bandi</name></author><author><name sortKey="Trees, Aj" uniqKey="Trees A">AJ Trees</name></author><author><name sortKey="Brattig, Nw" uniqKey="Brattig N">NW Brattig</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author><author><name sortKey="Bandi, C" uniqKey="Bandi C">C Bandi</name></author><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author><author><name sortKey="Mand, S" uniqKey="Mand S">S Mand</name></author><author><name sortKey="Adjei, O" uniqKey="Adjei O">O Adjei</name></author><author><name sortKey="Fleischer, B" uniqKey="Fleischer B">B Fleischer</name></author><author><name sortKey="Buttner, Dw" uniqKey="Buttner D">DW Buttner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author><author><name sortKey="Specht, S" uniqKey="Specht S">S Specht</name></author><author><name sortKey="Buttner, M" uniqKey="Buttner M">M Buttner</name></author><author><name sortKey="Pfarr, K" uniqKey="Pfarr K">K Pfarr</name></author><author><name sortKey="Mand, S" uniqKey="Mand S">S Mand</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Specht, S" uniqKey="Specht S">S Specht</name></author><author><name sortKey="Mand, S" uniqKey="Mand S">S Mand</name></author><author><name sortKey="Marfo Debrekyei, Y" uniqKey="Marfo Debrekyei Y">Y Marfo-Debrekyei</name></author><author><name sortKey="Debrah, Ay" uniqKey="Debrah A">AY Debrah</name></author><author><name sortKey="Konadu, P" uniqKey="Konadu P">P Konadu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Supali, T" uniqKey="Supali T">T Supali</name></author><author><name sortKey="Djuardi, Y" uniqKey="Djuardi Y">Y Djuardi</name></author><author><name sortKey="Pfarr, Km" uniqKey="Pfarr K">KM Pfarr</name></author><author><name sortKey="Wibowo, H" uniqKey="Wibowo H">H Wibowo</name></author><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bazzocchi, C" uniqKey="Bazzocchi C">C Bazzocchi</name></author><author><name sortKey="Ceciliani, F" uniqKey="Ceciliani F">F Ceciliani</name></author><author><name sortKey="Mccall, Jw" uniqKey="Mccall J">JW McCall</name></author><author><name sortKey="Ricci, I" uniqKey="Ricci I">I Ricci</name></author><author><name sortKey="Genchi, C" uniqKey="Genchi C">C Genchi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brattig, Nw" uniqKey="Brattig N">NW Brattig</name></author><author><name sortKey="Rathjens, U" uniqKey="Rathjens U">U Rathjens</name></author><author><name sortKey="Ernst, M" uniqKey="Ernst M">M Ernst</name></author><author><name sortKey="Geisinger, F" uniqKey="Geisinger F">F Geisinger</name></author><author><name sortKey="Renz, A" uniqKey="Renz A">A Renz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author><author><name sortKey="Cross, Hf" uniqKey="Cross H">HF Cross</name></author><author><name sortKey="Bilo, K" uniqKey="Bilo K">K Bilo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gillette Ferguson, I" uniqKey="Gillette Ferguson I">I Gillette-Ferguson</name></author><author><name sortKey="Hise, Ag" uniqKey="Hise A">AG Hise</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y Sun</name></author><author><name sortKey="Diaconu, E" uniqKey="Diaconu E">E Diaconu</name></author><author><name sortKey="Mcgarry, Hf" uniqKey="Mcgarry H">HF McGarry</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Saint Andre, A" uniqKey="Saint Andre A">A Saint Andre</name></author><author><name sortKey="Blackwell, Nm" uniqKey="Blackwell N">NM Blackwell</name></author><author><name sortKey="Hall, Lr" uniqKey="Hall L">LR Hall</name></author><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author><author><name sortKey="Brattig, Nw" uniqKey="Brattig N">NW Brattig</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Turner, Jd" uniqKey="Turner J">JD Turner</name></author><author><name sortKey="Langley, Rs" uniqKey="Langley R">RS Langley</name></author><author><name sortKey="Johnston, Kl" uniqKey="Johnston K">KL Johnston</name></author><author><name sortKey="Gentil, K" uniqKey="Gentil K">K Gentil</name></author><author><name sortKey="Ford, L" uniqKey="Ford L">L Ford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Buckley, Jj" uniqKey="Buckley J">JJ Buckley</name></author><author><name sortKey="Edeson, Jf" uniqKey="Edeson J">JF Edeson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cooray, Gh" uniqKey="Cooray G">GH Cooray</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schacher, Jf" uniqKey="Schacher J">JF Schacher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Franz, M" uniqKey="Franz M">M Franz</name></author><author><name sortKey="Buttner, Dw" uniqKey="Buttner D">DW Buttner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kozek, Wj" uniqKey="Kozek W">WJ Kozek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mclaren, Dj" uniqKey="Mclaren D">DJ McLaren</name></author><author><name sortKey="Worms, Mj" uniqKey="Worms M">MJ Worms</name></author><author><name sortKey="Laurence, Br" uniqKey="Laurence B">BR Laurence</name></author><author><name sortKey="Simpson, Mg" uniqKey="Simpson M">MG Simpson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sironi, M" uniqKey="Sironi M">M Sironi</name></author><author><name sortKey="Bandi, C" uniqKey="Bandi C">C Bandi</name></author><author><name sortKey="Sacchi, L" uniqKey="Sacchi L">L Sacchi</name></author><author><name sortKey="Di Sacco, B" uniqKey="Di Sacco B">B Di Sacco</name></author><author><name sortKey="Damiani, G" uniqKey="Damiani G">G Damiani</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Williams, Sa" uniqKey="Williams S">SA Williams</name></author><author><name sortKey="Lizotte Waniewski, Mr" uniqKey="Lizotte Waniewski M">MR Lizotte-Waniewski</name></author><author><name sortKey="Foster, J" uniqKey="Foster J">J Foster</name></author><author><name sortKey="Guiliano, D" uniqKey="Guiliano D">D Guiliano</name></author><author><name sortKey="Daub, J" uniqKey="Daub J">J Daub</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author><author><name sortKey="Bilo, K" uniqKey="Bilo K">K Bilo</name></author><author><name sortKey="Cross, Hf" uniqKey="Cross H">HF Cross</name></author><author><name sortKey="Archer, Jp" uniqKey="Archer J">JP Archer</name></author><author><name sortKey="Underwood, Ap" uniqKey="Underwood A">AP Underwood</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcgarry, Hf" uniqKey="Mcgarry H">HF McGarry</name></author><author><name sortKey="Egerton, Gl" uniqKey="Egerton G">GL Egerton</name></author><author><name sortKey="Taylor, Mj" uniqKey="Taylor M">MJ Taylor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fenn, K" uniqKey="Fenn K">K Fenn</name></author><author><name sortKey="Blaxter, M" uniqKey="Blaxter M">M Blaxter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ward, S" uniqKey="Ward S">S Ward</name></author><author><name sortKey="Carrel, Js" uniqKey="Carrel J">JS Carrel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goldstein, B" uniqKey="Goldstein B">B Goldstein</name></author><author><name sortKey="Hird, Sn" uniqKey="Hird S">SN Hird</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lahl, V" uniqKey="Lahl V">V Lahl</name></author><author><name sortKey="Halama, C" uniqKey="Halama C">C Halama</name></author><author><name sortKey="Schierenberg, E" uniqKey="Schierenberg E">E Schierenberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Skiba, F" uniqKey="Skiba F">F Skiba</name></author><author><name sortKey="Schierenberg, E" uniqKey="Schierenberg E">E Schierenberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dolinski, Cjbg" uniqKey="Dolinski C">CJBG Dolinski</name></author><author><name sortKey="Thomas, Wk" uniqKey="Thomas W">WK Thomas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goldstein, B" uniqKey="Goldstein B">B Goldstein</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Malakhov, Vv" uniqKey="Malakhov V">VV Malakhov</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sulston, Je" uniqKey="Sulston J">JE Sulston</name></author><author><name sortKey="Schierenberg, E" uniqKey="Schierenberg E">E Schierenberg</name></author><author><name sortKey="White, Jg" uniqKey="White J">JG White</name></author><author><name sortKey="Thomson, Jn" uniqKey="Thomson J">JN Thomson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chisholm, Ad" uniqKey="Chisholm A">AD Chisholm</name></author><author><name sortKey="Hardin, J" uniqKey="Hardin J">J Hardin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schaner, Ce" uniqKey="Schaner C">CE Schaner</name></author><author><name sortKey="Deshpande, G" uniqKey="Deshpande G">G Deshpande</name></author><author><name sortKey="Schedl, Pd" uniqKey="Schedl P">PD Schedl</name></author><author><name sortKey="Kelly, Wg" uniqKey="Kelly W">WG Kelly</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pinskaya, M" uniqKey="Pinskaya M">M Pinskaya</name></author><author><name sortKey="Morillon, A" uniqKey="Morillon A">A Morillon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Albertson, R" uniqKey="Albertson R">R Albertson</name></author><author><name sortKey="Casper Lindley, C" uniqKey="Casper Lindley C">C Casper-Lindley</name></author><author><name sortKey="Cao, J" uniqKey="Cao J">J Cao</name></author><author><name sortKey="Tram, U" uniqKey="Tram U">U Tram</name></author><author><name sortKey="Sullivan, W" uniqKey="Sullivan W">W Sullivan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sato, H" uniqKey="Sato H">H Sato</name></author><author><name sortKey="Kusel, Jr" uniqKey="Kusel J">JR Kusel</name></author><author><name sortKey="Thornhill, J" uniqKey="Thornhill J">J Thornhill</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ferree, Pm" uniqKey="Ferree P">PM Ferree</name></author><author><name sortKey="Frydman, Hm" uniqKey="Frydman H">HM Frydman</name></author><author><name sortKey="Li, Jm" uniqKey="Li J">JM Li</name></author><author><name sortKey="Cao, J" uniqKey="Cao J">J Cao</name></author><author><name sortKey="Wieschaus, E" uniqKey="Wieschaus E">E Wieschaus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schneider, Sq" uniqKey="Schneider S">SQ Schneider</name></author><author><name sortKey="Bowerman, B" uniqKey="Bowerman B">B Bowerman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goldstein, B" uniqKey="Goldstein B">B Goldstein</name></author><author><name sortKey="Macara, Ig" uniqKey="Macara I">IG Macara</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Serbus, Lr" uniqKey="Serbus L">LR Serbus</name></author><author><name sortKey="Sullivan, W" uniqKey="Sullivan W">W Sullivan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Serbus, Lr" uniqKey="Serbus L">LR Serbus</name></author><author><name sortKey="Casper Lindley, C" uniqKey="Casper Lindley C">C Casper-Lindley</name></author><author><name sortKey="Landmann, F" uniqKey="Landmann F">F Landmann</name></author><author><name sortKey="Sullivan, W" uniqKey="Sullivan W">W Sullivan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hunter, Cp" uniqKey="Hunter C">CP Hunter</name></author><author><name sortKey="Kenyon, C" uniqKey="Kenyon C">C Kenyon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baugh, Lr" uniqKey="Baugh L">LR Baugh</name></author><author><name sortKey="Hill, Aa" uniqKey="Hill A">AA Hill</name></author><author><name sortKey="Claggett, Jm" uniqKey="Claggett J">JM Claggett</name></author><author><name sortKey="Hill Harfe, K" uniqKey="Hill Harfe K">K Hill-Harfe</name></author><author><name sortKey="Wen, Jc" uniqKey="Wen J">JC Wen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Casiraghi, M" uniqKey="Casiraghi M">M Casiraghi</name></author><author><name sortKey="Mccall, Jw" uniqKey="Mccall J">JW McCall</name></author><author><name sortKey="Simoncini, L" uniqKey="Simoncini L">L Simoncini</name></author><author><name sortKey="Kramer, Lh" uniqKey="Kramer L">LH Kramer</name></author><author><name sortKey="Sacchi, L" uniqKey="Sacchi L">L Sacchi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sakaguchi, Y" uniqKey="Sakaguchi Y">Y Sakaguchi</name></author><author><name sortKey="Tada, I" uniqKey="Tada I">I Tada</name></author><author><name sortKey="Ash, Lr" uniqKey="Ash L">LR Ash</name></author><author><name sortKey="Aoki, Y" uniqKey="Aoki Y">Y Aoki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ercan, S" uniqKey="Ercan S">S Ercan</name></author><author><name sortKey="Lieb, Jd" uniqKey="Lieb J">JD Lieb</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chuang, Pt" uniqKey="Chuang P">PT Chuang</name></author><author><name sortKey="Lieb, Jd" uniqKey="Lieb J">JD Lieb</name></author><author><name sortKey="Meyer, Bj" uniqKey="Meyer B">BJ Meyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lieb, Jd" uniqKey="Lieb J">JD Lieb</name></author><author><name sortKey="Capowski, Ee" uniqKey="Capowski E">EE Capowski</name></author><author><name sortKey="Meneely, P" uniqKey="Meneely P">P Meneely</name></author><author><name sortKey="Meyer, Bj" uniqKey="Meyer B">BJ Meyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bird, Afb" uniqKey="Bird A">AFB Bird</name></author><author><name sortKey="Bird, J" uniqKey="Bird J">J Bird</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Howells, Re" uniqKey="Howells R">RE Howells</name></author><author><name sortKey="Chen, Sn" uniqKey="Chen S">SN Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ghedin, E" uniqKey="Ghedin E">E Ghedin</name></author><author><name sortKey="Hailemariam, T" uniqKey="Hailemariam T">T Hailemariam</name></author><author><name sortKey="Depasse, Jv" uniqKey="Depasse J">JV Depasse</name></author><author><name sortKey="Zhang, X" uniqKey="Zhang X">X Zhang</name></author><author><name sortKey="Oksov, Y" uniqKey="Oksov Y">Y Oksov</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bandi, C" uniqKey="Bandi C">C Bandi</name></author><author><name sortKey="Mccall, Jw" uniqKey="Mccall J">JW McCall</name></author><author><name sortKey="Genchi, C" uniqKey="Genchi C">C Genchi</name></author><author><name sortKey="Corona, S" uniqKey="Corona S">S Corona</name></author><author><name sortKey="Venco, L" uniqKey="Venco L">L Venco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author><author><name sortKey="Volkmann, L" uniqKey="Volkmann L">L Volkmann</name></author><author><name sortKey="Nissen Paehle, K" uniqKey="Nissen Paehle K">K Nissen-Paehle</name></author><author><name sortKey="Schmetz, C" uniqKey="Schmetz C">C Schmetz</name></author><author><name sortKey="Autenrieth, I" uniqKey="Autenrieth I">I Autenrieth</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bennuru, S" uniqKey="Bennuru S">S Bennuru</name></author><author><name sortKey="Semnani, R" uniqKey="Semnani R">R Semnani</name></author><author><name sortKey="Meng, Z" uniqKey="Meng Z">Z Meng</name></author><author><name sortKey="Ribeiro, Jm" uniqKey="Ribeiro J">JM Ribeiro</name></author><author><name sortKey="Veenstra, Td" uniqKey="Veenstra T">TD Veenstra</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kozek, Wj" uniqKey="Kozek W">WJ Kozek</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brattig, Nw" uniqKey="Brattig N">NW Brattig</name></author><author><name sortKey="Buttner, Dw" uniqKey="Buttner D">DW Buttner</name></author><author><name sortKey="Hoerauf, A" uniqKey="Hoerauf A">A Hoerauf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gillette Ferguson, I" uniqKey="Gillette Ferguson I">I Gillette-Ferguson</name></author><author><name sortKey="Hise, Ag" uniqKey="Hise A">AG Hise</name></author><author><name sortKey="Mcgarry, Hf" uniqKey="Mcgarry H">HF McGarry</name></author><author><name sortKey="Turner, J" uniqKey="Turner J">J Turner</name></author><author><name sortKey="Esposito, A" uniqKey="Esposito A">A Esposito</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gillette Ferguson, I" uniqKey="Gillette Ferguson I">I Gillette-Ferguson</name></author><author><name sortKey="Daehnel, K" uniqKey="Daehnel K">K Daehnel</name></author><author><name sortKey="Hise, Ag" uniqKey="Hise A">AG Hise</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y Sun</name></author><author><name sortKey="Carlson, E" uniqKey="Carlson E">E Carlson</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS Negl Trop Dis</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosntds</journal-id><journal-title-group><journal-title>PLoS Neglected Tropical Diseases</journal-title></journal-title-group><issn pub-type="ppub">1935-2727</issn><issn pub-type="epub">1935-2735</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20689574</article-id><article-id pub-id-type="pmc">2910707</article-id><article-id pub-id-type="publisher-id">10-PNTD-RA-1005R2</article-id><article-id pub-id-type="doi">10.1371/journal.pntd.0000758</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline"><subject>Cell Biology/Microbial Growth and Development</subject><subject>Developmental Biology/Embryology</subject><subject>Developmental Biology/Microbial Growth and Development</subject><subject>Infectious Diseases/Helminth Infections</subject><subject>Infectious Diseases/Neglected Tropical Diseases</subject></subj-group></article-categories><title-group><article-title>Asymmetric <italic>Wolbachia</italic> Segregation during Early
<italic>Brugia malayi</italic> Embryogenesis Determines Its Distribution in
Adult Host Tissues</article-title><alt-title alt-title-type="running-head"><italic>Wolbachia</italic> Transmission in
<italic>Brugia malayi</italic></alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Landmann</surname><given-names>Frédéric</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Foster</surname><given-names>Jeremy M.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Slatko</surname><given-names>Barton</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sullivan</surname><given-names>William</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America</addr-line></aff><aff id="aff2"><label>2</label><addr-line>New England Biolabs, Inc., Ipswich, Massachusetts, United States of America</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>O'Neill</surname><given-names>Scott Leslie</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">The University of Queensland, Australia</aff><author-notes><corresp id="cor1">* E-mail: <email>landmann@biology.ucsc.edu</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: FL. Performed the experiments: FL.
Analyzed the data: FL WS. Contributed reagents/materials/analysis tools: FL
JMF BS. Wrote the paper: FL JMF BS WS.</p></fn></author-notes><pub-date pub-type="collection"><month>7</month><year>2010</year></pub-date><pub-date pub-type="epub"><day>27</day><month>7</month><year>2010</year></pub-date><pub-date pub-type="ecorrected"><day>29</day><month>7</month><year>2010</year></pub-date><volume>4</volume><issue>7</issue><elocation-id>e758</elocation-id><history><date date-type="received"><day>26</day><month>3</month><year>2010</year></date><date date-type="accepted"><day>7</day><month>6</month><year>2010</year></date></history><permissions><copyright-statement>Landmann 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.</copyright-statement><copyright-year>2010</copyright-year></permissions><abstract><p><italic>Wolbachia</italic> are required for filarial nematode survival and
fertility and contribute to the immune responses associated with human filarial
diseases. Here we developed whole-mount immunofluorescence techniques to
characterize <italic>Wolbachia</italic> somatic and germline transmission
patterns and tissue distribution in <italic>Brugia malayi</italic>, a nematode
responsible for lymphatic filariasis. In the initial embryonic divisions,
<italic>Wolbachia</italic> segregate asymmetrically such that they occupy
only a small subset of cells in the developing embryo, facilitating their
concentration in the adult hypodermal chords and female germline.
<italic>Wolbachia</italic> are not found in male reproductive tissues and
the absence of <italic>Wolbachia</italic> from embryonic germline precursors in
half of the embryos indicates <italic>Wolbachia</italic> loss from the male
germline may occur in early embryogenesis. <italic>Wolbachia</italic> rely on
fusion of hypodermal cells to populate adult chords. Finally, we detect
<italic>Wolbachia</italic> in the secretory canal lumen suggesting living
worms may release bacteria and/or their products into their host.</p></abstract><abstract abstract-type="summary"><title>Author Summary</title><p>Filarial diseases affect over 150 million people in tropical countries. They are
caused by parasitic nematodes like <italic>Brugia malayi</italic> that rely on
their endosymbiont <italic>Wolbachia</italic> for their survival and fertility.
These bacteria are a recognized drug target in the search for treatments killing
adult worms. To understand the transmission of <italic>Wolbachia</italic> from
the embryonic to adult stages, we developed new techniques to track these
bacteria at the cellular and tissue levels. These techniques include
immunofluorescence in whole mount adult tissues and embryos. We found that
<italic>Wolbachia</italic> segregate asymetrically in specific cells, in a
lineage-specific manner during early <italic>Brugia</italic> embryogenesis, and
rely on cell fusion to subsequently populate the adult hypodermal chords. From
the chords, the <italic>Wolbachia</italic> can be secreted in the
secretory-excretory canal, suggesting that in addition to dead worms releasing
the bacteria in the human body, living worms may also secrete
<italic>Wolbachia</italic>, whose role in stimulating the immune system in
filarial pathologies is now well established.</p></abstract><counts><page-count count="13"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Filarial nematodes are the causative agents of human filariasis, affecting over 150
million individuals. The most pathogenic diseases, lymphatic filariasis and
onchocerciasis, (river blindness) comprise a major cause of global morbidity in the
tropics, with over 1 billion people at risk of these arthropod-transmitted
infections <xref ref-type="bibr" rid="pntd.0000758-Crompton1">[1]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Molyneux1">[2]</xref>. Three filarial nematode species are responsible
for lymphatic filariasis: <italic>Wuchereria bancrofti</italic>, <italic>Brugia
malayi</italic> and <italic>Brugia timori</italic>, causing pathologies that
include hydrocoele and lymphoedema (elephantiasis). Onchocerciasis is caused by
<italic>Onchocerca volvulus</italic>, leading to skin disease,
“onchocercoma nodules” and visual impairment, including
blindness. These parasitic nematodes rely on alpha-proteobacterial
<italic>Wolbachia</italic> endosymbionts for development, viability and fertility
(for reviews see, <xref ref-type="bibr" rid="pntd.0000758-Bandi1">[3]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Taylor1">[4]</xref>). This obligate dependence was first discovered
using anti-<italic>Rickettsial</italic> tetracycline antibiotics, in <italic>in
vitro</italic> and <italic>in vivo</italic> model systems. Treatments deplete
<italic>Wolbachia</italic>, resulting in embryonic arrest and a decrease in
microfilarial (larval) production <xref ref-type="bibr" rid="pntd.0000758-Taylor1">[4]</xref>. Human trials with doxycycline or rifampicin
provide evidence for long-term sterilization and macrofilaricidal (adulticidal)
effects against both lymphatic filariasis and onchocerciasis <xref ref-type="bibr" rid="pntd.0000758-Hoerauf1">[5]</xref>–<xref ref-type="bibr" rid="pntd.0000758-Supali1">[8]</xref>.</p><p><italic>Wolbachia</italic> play a significant role in the pathogenesis of filarial
disease <xref ref-type="bibr" rid="pntd.0000758-Bazzocchi1">[9]</xref>–<xref ref-type="bibr" rid="pntd.0000758-Turner1">[14]</xref>.
<italic>Wolbachia</italic> activate inflammatory immune responses, including
antibody responses and induction of corneal keratitis in the case of <italic>O.
volvulus</italic> infection, and are implicated in the inflammation response
leading to blindness, induced by release of <italic>Wolbachia</italic> antigens from
degenerating microfilariae <xref ref-type="bibr" rid="pntd.0000758-Bandi1">[3]</xref>. In lymphatic filariasis, the major pathologies
are attributable to death and destruction of adult worms within the lymphatic
vessels and activation of innate inflammation; effects which are lost following
antibiotic depletion of bacteria and absent from soluble extracts derived from
filarial species naturally lacking <italic>Wolbachia</italic> such as
<italic>Acanthocheilonema viteae</italic><xref ref-type="bibr" rid="pntd.0000758-Taylor1">[4]</xref>.</p><p>To better understand the endosymbiont interaction with the parasitic nematode, it is
of primary importance to characterize <italic>Wolbachia</italic> localization at the
host tissue and cellular levels. The histology of the parasitic nematode <italic>B.
malayi</italic> was established more than half a century ago by differential
contrast microscopy (DIC) on whole mount adult specimens <xref ref-type="bibr" rid="pntd.0000758-Buckley1">[15]</xref>. Subsequent DIC and
electron microscopy studies carried out on cross sections revealed the presence of
intracellular bacteria <xref ref-type="bibr" rid="pntd.0000758-Cooray1">[16]</xref>–<xref ref-type="bibr" rid="pntd.0000758-McLaren1">[20]</xref> which were only
“re-discovered” and identified as <italic>Wolbachia</italic>decades later by phylogenetic analysis and genomic studies <xref ref-type="bibr" rid="pntd.0000758-Sironi1">[21]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Williams1">[22]</xref>.
<italic>Wolbachia</italic> are present primarily in the lateral hypodermal chords of
both adult males and females and in the ovaries, oocytes and embryonic stages within
the uteri of females. The absence of <italic>Wolbachia</italic> in the male
reproductive system indicates that the bacterium is vertically transmitted through
the cytoplasm of the egg and not through the sperm <xref ref-type="bibr" rid="pntd.0000758-Kozek1">[19]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Taylor3">[23]</xref>.</p><p>Although <italic>Wolbachia</italic> are observed in all stages of the host
life-cycle, there are significant variations in bacterial growth kinetics in host
development <xref ref-type="bibr" rid="pntd.0000758-McGarry1">[24]</xref>, . Bacterial numbers remain constant in microfilariae
(mf) and the mosquito-borne larval stages (L2 and L3), but the
<italic>Wolbachia</italic> multiply rapidly beginning within the first week of
infection of the mammalian host.</p><p>Features of the symbiotic relationship left unresolved include the localization and
segregation patterns of <italic>Wolbachia</italic> during embryogenesis, which are
essential to understanding the specific localization in adult somatic tissue and the
germline. To address this issue, we developed fixation, immunofluorescent staining
and imaging protocols to characterize <italic>Wolbachia</italic> in whole-mount
<italic>B. malayi</italic> embryos and adult specimens at the tissue, cellular
and sub-cellular levels. These studies demonstrate that <italic>Wolbachia</italic>localize to the posterior of the egg upon fertilization and segregate asymmetrically
during early embryogenesis, in a lineage-specific manner, resulting in only a small
fraction of the cells in the developing embryo containing the endobacteria.
Specifically, <italic>Wolbachia</italic> concentrate in the C blastomere hypodermal
descendants, and in the P blastomere germline precursors. The asymmetric and
lineage-specific segregation of <italic>Wolbachia</italic> during the initial stages
of embryogenesis resembles that of some <italic>Caenorhabditis elegans</italic>polarity and lineage-specific determinants, and suggests that
<italic>Wolbachia</italic> may interact with the counterparts of these determinants
in <italic>B. malayi</italic>.</p><p>This transmission pattern readily explains the tissue specific pattern of
<italic>Wolbachia</italic> localization in the adult hypodermal lateral chords
and female germline. The absence of the bacteria from the embryonic germ line
precursors in nearly half of the embryos suggest <italic>Wolbachia</italic> loss
from the male germline may occur during embryogenesis. We find that
<italic>Wolbachia</italic> rely on fusion of hypodermal cells to populate young
adult chords. We also detected <italic>Wolbachia</italic> in the lumen of the
secretory-excretory canals embedded in the hypodermal lateral chords, suggesting
that in addition to dead or degenerating parasites, live adult worms may also
release bacteria and/or their products through this route into the host tissues.</p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2a"><title>Specimens</title><p>Living <italic>B. malayi</italic> adult male and female worms were supplied by
TRS Laboratories (Athens Georgia). The worms were raised in jirds and the
procedures described below were performed approximately 1 to 3 days after their
removal.</p></sec><sec id="s2b"><title>Adult Worm Fixation and Immunofluorescence</title><p>To prepare worms for whole mount immuno-fluorescence analysis, they were soaked
in M9 buffer (see <underline>Buffers in Supplementary Experimental
Procedures</underline>) for 30 seconds to allow them to uncoil and immediately
placed in liquid nitrogen. M9 was then removed and replaced with (PBS+
Paraformaldehyde (PFA) 4% final- (Electron Microscopy Sciences))
1/3+2/3 Heptane on a rotator for 30 minutes at room temperature. If
required, worms were cut or open with a blade to expose the different tissues to
antibodies prior to fixation. All fixation and immunostaining steps gave better
results in eppendorf tubes in rotation compared to whole mount animals on
slides.</p><p>For propidium iodide (PI) (Molecular Probes) DNA staining, worms were incubated
overnight at room temperature in PBS + RNAse A (15 mg/mL, Sigma), in
rotating tubes followed by PI incubation (1.0 mg/mL solution) for 20 minutes in
PBS (1∶50) and a 5 minute wash. For DAPI DNA staining alone, fixed
worms were simply pulled out of the tube with a curved needle, placed on a glass
slide with thin needles in a line of PBS. The PBS was then aspirated and worms
mounted into Vectashield with DAPI (Vector Laboratories) and left at 4 degrees
overnight: DAPI penetrates all tissues and stains <italic>Wolbachia</italic>very well.</p></sec><sec id="s2c"><title>Embryo Fixation and Immunofluorescence</title><p>To prepare embryos for whole-mount immuno-fluorescent analysis, females were cut
into sections with a blade on a glass slide. The sections were collected and the
slide rinsed with PBS and collected in an 0.5mL eppendorf tube. PFA and heptane
were added as described above. The tube was vortexed for one minute at this
step. After fixation for 10 to 20 minutes, embryos were immersed in (1/4 water,
1/4 KOH 10M, 1/2 NaClO 15%) for 30 seconds to facilitate removal of
the eggshell (optional), centrifuged, and rinsed in PBST. Prior to each change
of solution or rinse, samples were centrifuged for 1 min at 4,200 rpm. This
procedure yielded hundreds of embryos per female, and allowed staining of at
least half of them. As an alternative procedure we used the freeze crack
techniques that work with <italic>C. elegans</italic> embryos but these gave
unsatisfactory results, likely due to the smaller size of the
<italic>Brugia</italic> embryos. For protocols used to determine the identity of
specific embryonic blastomeres and conditions for primary and secondary antibody
incubations see <xref ref-type="supplementary-material" rid="pntd.0000758.s008">Methods S1</xref>.</p></sec><sec id="s2d"><title>Live Analysis</title><p>For live fluorescent analysis of <italic>Wolbachia</italic> and host nuclei,
adult worms were incubated in RPMI medium with 1/10,000 Syto11 (Invitrogen) or
vital Hoechst for 30 minutes, and observed as for <italic>C. elegans</italic> on
an 2% agarose pad with Sodium Azide 25mM between slide and coverslip
(<ext-link ext-link-type="uri" xlink:href="http://www.wormbook.org/chapters/www_intromethodscellbiology/intromethodscellbiology.html">http://www.wormbook.org/chapters/www_intromethodscellbiology/intromethodscellbiology.html</ext-link>).
To observe the Secretory-Excretory canal, we added 50µL of Resorufin
(Sigma) at 10 µg/mL, 1/10,000 Syto11 for approximately 30 minutes to
an hour, and washed the worms in RPMI for 15 minutes. Worms were mounted in PBS
and anesthetized with Sodium Azide.</p></sec><sec id="s2e"><title>Microscopy</title><p>Confocal microscope images were captured on an inverted photoscope (DMIRB; Leitz)
equipped with a laser confocal imaging system (TCS SP2; Leica) using an HCX PL
APO 1.4 NA 63 oil objective (Leica) at room temperature. Images in
epifluorescence were captured on a Leica DMI 6000B microscope and a Zeiss
Axioscope 2 plus microscope.</p></sec></sec><sec id="s3"><title>Results</title><sec id="s3a"><title>Fertilization and Embryogenesis of <italic>B. malayi</italic></title><p>The fertilization and embryogenesis of <italic>B. malayi</italic> resemble that
of other secernentean nematodes. The sperm entry activates the oocyte to
complete meiosis I and II and defines the posterior pole of the egg <xref ref-type="bibr" rid="pntd.0000758-Ward1">[26]</xref>,
<xref ref-type="bibr" rid="pntd.0000758-Goldstein1">[27]</xref>. All examined species of secernentean nematodes
undergo asymmetric cleavage leading to early separation of soma and germ line,
and establishment of five somatic cell lineages <xref ref-type="bibr" rid="pntd.0000758-Lahl1">[28]</xref>–. This is
followed by a developmental phase during which organ identity is specified.
Subsequent morphogenetic events such as ventral closure and an elongation phase
due to contraction of circumferential actin bundles in the hypodermis lead to
newly hatched larvae appearing very similar among nematodes species. It has been
shown that despite this high similarity in the anatomy of the first stage larvae
(most often the species variation being acquired during larval life), variations
can exist from the first asymmetric divisions <xref ref-type="bibr" rid="pntd.0000758-Goldstein2">[31]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Malakhov1">[32]</xref>.</p><p>Although the cell lineage of <italic>B. malayi</italic> (a Rhabditia Spirurida of
clade III of the Secernentea) has not been established, parallels with the
completely defined lineage of <italic>C. elegans</italic> (a Rhabditia
Rhabditida of clade V of the Secernentea) are likely <xref ref-type="bibr" rid="pntd.0000758-Malakhov1">[32]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Sulston1">[33]</xref>.
In secernentean nematodes, the first division cleaves the zygote asymmetrically
into somatic cell AB and a smaller P1 germ line precursor cell (<xref ref-type="fig" rid="pntd-0000758-g001">Fig. 1A</xref>). Most of the
embryonic ectodermal cells (hypodermal and neuronal cells) are derived from the
anterior AB blastomere. The posterior P1 blastomere, after three rounds of
division, primarily gives rise to the somatic gonad, pharynx, ectodermal and
mesodermal derivatives (MS), gut (E), posterior hypodermal derivatives (C), body
wall muscles (D) and P4 blastomeres. During gastrulation, the posterior P4 cell
follows the gut precursors inward and divides to produce the two germline
precursors Z2 and Z3. Based on the similarity between the <italic>C.
elegans</italic> lineage and embryonic maps, putative germ line precursor cells
can also be localized (the counterparts of the <italic>C. elegans</italic> Z2
and Z3) during the process of elongation (i.e. when the embryonic tail reaches
half the length of the worm body).</p><fig id="pntd-0000758-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g001</object-id><label>Figure 1</label><caption><title>Schematic representation of the first embryonic cleavages in
secernentean nematodes, and growth of <italic>Brugia malayi</italic>embryos in utero.</title><p>(A) Cell linage adapted from <xref ref-type="bibr" rid="pntd.0000758-Goldstein2">[31]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Sulston1">[33]</xref>. Anterior daughters are to the left,
posterior ones to the right. The main derivatives are indicated by
dotted lines. (B) Uterus filled with early cleavage embryos. Note the
variety of embryo sizes (small and large arrows). (C) Three
developmental stages in one merge of confocal stacks (lower left to
upper right): embryos at approximately the 12-cell, 100-cell stage prior
to morphogenesis, and mature pretzel stage prior to hatching. (D) Length
of the <italic>B. malayi</italic> embryo eggshell according to the
embryonic stage. In each case, the longest axis of the eggshell was
measured. Each data point represents one measurement.</p></caption><graphic xlink:href="pntd.0000758.g001"></graphic></fig><p>In contrast to <italic>C. elegans</italic> embryonic development, <italic>B.
malayi</italic> embryos grow and increase their volume in the uterus (<xref ref-type="fig" rid="pntd-0000758-g001">Fig. 1B, C</xref>). The length of the
one-cell egg increases from about 16 µm to 38 µm for an egg
containing a mature worm-shaped embryo (<xref ref-type="fig" rid="pntd-0000758-g001">Fig. 1D</xref>). Thus, unlike <italic>C.
elegans</italic>, the <italic>B. malayi</italic> eggshell grows and suggests
that uptake of nutrients through the eggshell occurs while the embryo is still
in the uterus. These observations may reflect fundamental metabolic differences
during embryonic development between the parasitic <italic>B. malayi</italic>and free living <italic>C. elegans</italic>.</p></sec><sec id="s3b"><title><italic>Wolbachia</italic> Segregation during Fertilization and the Initial
Zygotic Divisions</title><p>We used propidium iodide (PI) to stain the host chromatin and the bacterial DNA,
and used an anti-WSP (<italic>wBm</italic> Surface Protein) specific to
<italic>Wolbachia</italic> to perform a fluorescent analysis of the
<italic>Wolbachia</italic> segregation in the <italic>Brugia</italic>embryo. The anti-WSP revealed that the punctate staining obtained with PI
corresponds to the <italic>Wolbachia</italic> DNA and does not stain
mitochondrial DNA (i.e. <xref ref-type="fig" rid="pntd-0000758-g002">Fig.
2</xref>). During fertilization, <italic>Wolbachia</italic> appear distributed
throughout the oocyte completing meiosis, although more concentrated in the
vicinity of the maternal chromatin in the anterior pole (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2A, B</xref>). This may reflect an interaction
with the microtubule spindle as observed at earlier stages (i.e. <xref ref-type="supplementary-material" rid="pntd.0000758.s001">Fig. S1</xref>).
As early as the pronuclei migration stage, <italic>Wolbachia</italic>dramatically relocalize towards the posterior pole of the egg (P0, <xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2 C to E</xref>, n>50).</p><fig id="pntd-0000758-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g002</object-id><label>Figure 2</label><caption><title><italic>Wolbachia</italic> concentrate to the posterior pole of the
egg and preferentially segregate to the P1 blastomere, to later enrich
the C blastomere and subsequently colonize posterior hypodermal cells.</title><p>Merges of confocal stacks of <italic>B. malayi</italic> eggs and embryos
stained for DNA (propidium iodide, red), <italic>Wolbachia</italic>(anti-WSP, blue), and cortical actin (green). (A), completion of
meiosis, metaphase I, and (B) completion of metaphase, meiosis II as
revealed by the presence of a polar body (PB, white arrow). White
arrowheads indicate the pseudocleavage. Maternal chromatin to the
anterior pole (left) and paternal chromatin to the posterior (Sp.,
sperm). (C) Pronuclei migration and (D) Pronuclei apposition while the
three polar bodies (PBs) are extruded at the anterior pole. Note that
the PBs usually lost at the 4 or 6-cell stage, can detach earlier,
probably due to the fixation technique (i.e. (E)). (F) Division of P0
into the anterior AB and the posterior P1 blastomeres. (G) 3-cell stage,
AB divides prior P1. (H) 4-cell stage, (I) 6-cell stage, and a (J)
12-cell stage embryo. Partial projections highlighting the dorsal (JI)
and ventral (JII) blastomeres. Solid lines connect sister blastomeres on
the corresponding schematic drawings. (KI to KIV) Merges of confocal
stacks of a comma-stage embryo, from the surface to half depth, anterior
to the left. White arrowheads and arrows highlight dorsal and ventral
hypodermal cells respectively, and dots indicate lateral hypodermis.
Green arrowheads highlight ventral (KII) and dorsal (KIII) muscles.
Green, white, and red ovals encompass the neuroblasts, the pharynx, and
the gut primordium respectively. The red arrowhead indicates the rectum
(KIII, KIV). Scale bar = 6
µm.</p></caption><graphic xlink:href="pntd.0000758.g002"></graphic></fig><p>We then followed <italic>Wolbachia</italic> segregation patterns in the two
rounds of division following pronuclear fusion to create a diploid P0 zygotic
nucleus. P0 division produces anterior -identified by the localization of polar
bodies at the anterior surface (<xref ref-type="fig" rid="pntd-0000758-g001">Fig. 1A</xref> and <xref ref-type="fig" rid="pntd-0000758-g002">Fig.
2F</xref>)- and posterior localized, AB and P1 blastomeres respectively.
<italic>Wolbachia</italic> always asymmetrically localize in P1 (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2E, F</xref>, n>50). P1
divides to produce EMS and P2 daughter blastomeres (<xref ref-type="fig" rid="pntd-0000758-g001">Fig. 1A</xref>). <italic>Wolbachia</italic>preferentially segregate to the posteriorly localized P2 blastomere. P2 divides
to produce a dorsal C blastomere (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2JI</xref>) and a posterior P3 blastomere (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2JII</xref>). Most of the
<italic>Wolbachia</italic> segregate to the C blastomere and a minority
segregate to the P3 blastomere.</p><p>Although during the first zygotic division, the majority of
<italic>Wolbachia</italic> preferentially localize in the P1 blastomere, a few
localize to the AB blastomere. Division of the AB blastomere produces daughter
blastomeres ABa and ABp (<xref ref-type="fig" rid="pntd-0000758-g002">Fig.
2G</xref>). <italic>Wolbachia</italic> titer in these descendants is variable
but always lower than in the direct descendants of the P1 lineage (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2H, I</xref>).</p></sec><sec id="s3c"><title><italic>Wolbachia</italic> Segregation during Embryogenesis</title><p>In the 12-cell embryo (<xref ref-type="fig" rid="pntd-0000758-g002">Fig.
2J</xref>) P2 has divided to give dorsally C (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2JI</xref>) and the posterior P3 (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2JII</xref>). Most of the
<italic>Wolbachia</italic> are in C, followed by P3. The titer in the AB
descendants, MS or E, although variable, is always lower than that in C and P3.
In the next rounds of divisions, C divides asymmetrically to give muscle cells
and hypodermal cells (<xref ref-type="fig" rid="pntd-0000758-g001">Fig.
1A</xref>) <xref ref-type="bibr" rid="pntd.0000758-Sulston1">[33]</xref>. However without specific lineage markers,
following the descendants of specific blastomeres is not possible after the
12-cell stage. Fortunately morphogenesis, as revealed by phalloidin-based actin
staining, in the early <italic>B. malayi</italic> embryo is strikingly similar
to that of <italic>C. elegans</italic> (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2KI to KIV</xref>) <xref ref-type="bibr" rid="pntd.0000758-Chisholm1">[34]</xref>. At this stage,
the cellular proliferation is over, and circumferential actin bundles in
hypodermal cells contract, transforming the round-shaped embryo into a worm. In
both <italic>B. malayi</italic> and <italic>C. elegans</italic>, the hypodermis
is composed of intercalated dorsal cells, lateral and ventral cells organized
like the <italic>C. elegans</italic> hyp7, seam and P cells (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2KI</xref>). Under the dorsal
and ventral hypodermal cells run the muscle quadrants from the anterior to the
posterior (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2KII,III</xref>).
Deeper in the embryo, the embryonic neuroblasts, pharyngeal and gut cells can
also be localized (<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2KIII,
IV</xref>). Assuming that the lineage of <italic>B. malayi</italic> is very
similar to the established lineage of <italic>C. elegans</italic>, we could
verify the vertical transmission of <italic>Wolbachia</italic> in the embryonic
blastomeres. Our observations of early stages showed that the
<italic>Wolbachia</italic> were diluted out in the AB descendants, and in the MS
and E blastomeres (<xref ref-type="fig" rid="pntd-0000758-g001">Figs. 1A</xref>,
<xref ref-type="fig" rid="pntd-0000758-g002">2G to JII</xref>). During
morphogenesis, we constantly found the <italic>Wolbachia</italic> enriched in
the dorsal posterior hypodermis, and absent from nearly all anterior cells
including neuroblast and pharyngeal cells, as well as muscle and gut cells
(<xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2KI to KIV</xref>,
n>30). This also implies that other asymmetrical segregations of
<italic>Wolbachia</italic> occur in the C lineage to exclude them from the
C-derived muscle cells and concentrate them in the hypodermis.</p><p>Because the germline precursor P4 has already divided into Z2 and Z3 during
morphogenesis and because these cells are often difficult to identify (<xref ref-type="fig" rid="pntd-0000758-g003">Fig. 3A to C</xref>) we used the
anti-histone H3K4me2. It has been demonstrated that in <italic>C.
elegans</italic> as well as in <italic>Drosophila melanogaster</italic>, a
subset of nucleosome modifications (dimethylation on lysine 4 of histone H3 and
acetylation on lysine 8 of histone H4) are absent from germline precursors but
present in all the other blastomeres. In <italic>C. elegans</italic> embryos,
H3K4me2 marks all the blastomeres, including P4, until it divides symmetrically
into Z2 and Z3 <xref ref-type="bibr" rid="pntd.0000758-Schaner1">[35]</xref>. Lysine 4 dimethylation on histone H3 is
involved in transcription regulation and its absence reveals transcriptional
quiescence <xref ref-type="bibr" rid="pntd.0000758-Pinskaya1">[36]</xref>. In <italic>B. malayi</italic> however, we
found embryos containing only one H3meK4-negative cell, suggesting that the
putative P4 blastomere reorganizes its chromatin architecture to enter
transcriptional quiescence prior to division (<xref ref-type="fig" rid="pntd-0000758-g003">Fig. 3D,E</xref>). We found half
<italic>Wolbachia</italic>-infected and half non-infected putative P4
blastomeres or putative Z2/Z3 germline cells (<xref ref-type="fig" rid="pntd-0000758-g003">Fig. 3D to L</xref>). It is likely the embryos with
uninfected blastomeres are males and those with infected blastomeres are
females.</p><fig id="pntd-0000758-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g003</object-id><label>Figure 3</label><caption><title><italic>Wolbachia</italic> are not always detected in the germline
precursors.</title><p>(A) to (C) Merges of confocal stacks of a 1,5 fold stage embryo (when the
tail reaches half of the length of the embryo) stained for DNA
(propidium iodide, red) and <italic>Wolbachia</italic> (anti-WSP, blue).
Infected putative primordial germ cells Z2/Z3 are indicated by an
arrowhead. (D) to (L) Merges of confocal stacks of <italic>B.
malayi</italic> embryos in gastrulation stained for DNA (PI, red) and
with anti-H3K4me2 (green) at the level of the putative P4 blastomere
(D,E), or putative Z2 and Z3 (F). (D) and (E) are the earliest stages
where the H3meK4-negative P4 blastomere was detected. (G to L) are
confocal stacks in cross sections focusing on Z2 or Z3. (D) Non-infected
P4; (E) Infected P4 (arrowheads in inset point to
<italic>Wolbachia</italic>); (G) Non-infected Z2/Z3; (G to I)
Non-infected Z2 or Z3 cell (arrow); (J to L) A
<italic>Wolbachia</italic>-infected Z2 or Z3 cell (the arrow points to
the cell nucleus and the arrowhead to a cluster of bacteria. Scale
bar = 10 µm.</p></caption><graphic xlink:href="pntd.0000758.g003"></graphic></fig><p>We also found the average number of <italic>Wolbachia</italic> did not differ
from early to mid embryogenesis (70+/−12
(n = 10)) and is in general agreement with the
average number detected in microfilariae using qPCR <xref ref-type="bibr" rid="pntd.0000758-McGarry1">[24]</xref>. Although this may
suggest that asymmetric relocalization prior to division is the major cause for
specific enrichment of a given blastomere, it does not rule out a possible
stimulation/repression of bacterial replication due to asymmetrically localized
cues. In fact, we noticed that in P2, prior to division,
<italic>Wolbachia</italic> appeared as doublets in the enriched antero/dorsal
pole while as individual units in the posterior/ventral pole. This was supported
by a WSP staining surrounding the doublets, suggesting active replication (<xref ref-type="supplementary-material" rid="pntd.0000758.s002">Fig.
S2A,B</xref>).</p></sec><sec id="s3d"><title>Overview of the Anatomy of Adult Female and Male Worms</title><p><italic>B. malayi</italic> adults, like any secernentean nematodes, have a simple
and conserved anatomy. Non segmented, these worms have body walls organized in
four longitudinal rows of hypodermal chords secreting the cuticle, and separated
by four muscle quadrants. Lateral chords contain the excretory-secretory canal,
while dorsal and ventral chords surround the nerves. They lack circulatory and
respiratory systems. A nerve ring located around the pharynx constitutes the
central nervous system (<xref ref-type="supplementary-material" rid="pntd.0000758.s003">Fig. S3D, E</xref>). The triradiate pharynx is
connected to the gut. Females have two gonads starting in the posterior and
ending in the anterior vulva, while the male has one gonad starting in the
anterior and ending in the posterior cloaca.</p><p>To determine <italic>Wolbachia</italic> distribution in adult tissues, we stained
whole-mount fixed adults either with DAPI or live specimens with the vital dye
Syto11 (<xref ref-type="bibr" rid="pntd.0000758-Albertson1">[37]</xref>, cf. <xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4</xref> and <xref ref-type="supplementary-material" rid="pntd.0000758.s004">Fig. S4</xref>).
Detailed measurements of body and tissues features have already been reported
<xref ref-type="bibr" rid="pntd.0000758-Buckley1">[15]</xref>.</p><fig id="pntd-0000758-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g004</object-id><label>Figure 4</label><caption><title><italic>Brugia malayi</italic> adult anatomy.</title><p>(A) Overview of the adult anatomy of fixed and DAPI-stained
<italic>Brugia malayi</italic> female and male. (B to F) Anatomical
details of the <italic>Brugia malayi</italic> female oriented anterior
top to posterior bottom. (B) Higher magnification of anterior female
reproductive apparatus. A zone of dense somatic nuclei is surrounded by
muscles, which have fewer nuclei. (C) Zone of hatching in one of the two
uteri. Fully developed pretzel-stage larvae still in their eggshell are
at the bottom. Hatched microfilariae that spontaneously align along the
antero-posterior are at the top. (D) Developing embryos in the uterus.
The perfectly aligned hypodermal nuclei of a lateral chord are visible.
(E) Distal-most part of the uterus where sperm has concentrated,
brightly stained with DAPI (i.e. in between dotted arrows, also in
bright patches in (A) between D and E). (F) Posterior part of the
female, showing the two ovaries running back and forth (“ovary
1”, arrows indicating the 180° turns -1, 2 and 3-,
idem for “ovary 2”). The amount of sperm, embryos
and microfilariae are variable among females.</p></caption><graphic xlink:href="pntd.0000758.g004"></graphic></fig><p>The two female distal gonad arms located in the posterior (several millimeters
separate the two ovaries distal ends) coil along an anterior-posterior axis
(<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4F</xref>). The ovaries
lead anteriorly to the two uteri that are also coiled around one another (i.e.
<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4B</xref>), and filled
with sperm that has migrated in their distal parts (<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4A, E</xref>). The amount of sperm is variable
between females and may reflect the time of observation after copulation.
Oogenesis begins at the distal region of the ovaries and as the oocytes mature
they are pushed proximally and are fertilized in the distal part of the uteri,
where developing embryos are present in the proximal regions (<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4A, C, D</xref>; <xref ref-type="supplementary-material" rid="pntd.0000758.s005">Fig. S5</xref>).
Thousands of microfilariae are released in the lymph of the host through the
ovejector that ends the vulva where the uteri meet, in the anterior part of the
female, specifically at the level of the posterior pharynx (<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4A, B, C</xref>; <xref ref-type="supplementary-material" rid="pntd.0000758.s003">Fig. S3A</xref> to
C). The male gonad consists of a testis posterior to the pharynx of the
nematode, connected to the sperm duct which in turn leads to a widened seminal
vesicle where mature sperm is stored (<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4A</xref>; <xref ref-type="supplementary-material" rid="pntd.0000758.s004">Fig. S4</xref>).
The gonad ends in the cloaca where two specialized spicules are used for mating
(<xref ref-type="supplementary-material" rid="pntd.0000758.s004">Fig.
S4</xref>).</p><p>The intestine is a thin empty tube connected at the anterior to the pharynx, and
at the posterior to the ventral rectum close to the posterior tip in both male
and female worms (<xref ref-type="fig" rid="pntd-0000758-g004">Fig. 4A,
F</xref>; <xref ref-type="supplementary-material" rid="pntd.0000758.s006">Fig.
S6</xref>). Gonads and intestine fill the pseudocoel contained within the
body wall.</p><p>Lateral chords are prominent in <italic>Brugia</italic>. They are formed through
fusion of hypodermal cells producing a syncytial chord surrounding the
secretory-excretory canal and in between muscle quadrants <xref ref-type="bibr" rid="pntd.0000758-Malakhov1">[32]</xref>. The lateral
chords project a thin layer of cytoplasm over the muscles to connect the dorsal
and ventral chords. These dorsal and ventral chords, containing the dorsal and
ventral nerves, are very thin and difficult to observe by differential contrast
microscopy but can be revealed by staining the surrounding muscles (<xref ref-type="supplementary-material" rid="pntd.0000758.s003">Fig. S3F to
H</xref>). Uteri appear closely apposed or embedded in the lateral chords (<xref ref-type="supplementary-material" rid="pntd.0000758.s007">Fig. S7</xref>).
The body wall possesses a slight periodicity, and hypodermal chords and muscle
quadrants turn several times around the central axis of the worm between the two
tips. The male posterior end is coiled three to four times in the region
encompassing the spicules, probably to ensure a better grip during mating (<xref ref-type="supplementary-material" rid="pntd.0000758.s004">Fig.
S4</xref>).</p></sec><sec id="s3e"><title><italic>Wolbachia</italic> in the Somatic Tissues: An Uneven Distribution</title><p>In both male and female worms (n>30), <italic>Wolbachia</italic>concentrate around the two rows of hypodermal nuclei in lateral chords. While
most worms displayed two infected lateral chords (<xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5A to D</xref>), in about 40% only
one of the two chords was infected (the left or the right chord). We also
observed worms with half of a chord infected (<xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5E, F</xref>), sometimes in a mosaic pattern
(<xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5I, J</xref>), possibly
reflecting the earlier mosaicism in <italic>Wolbachia</italic> segregation
during embryonic development. In the chords, the <italic>Wolbachia</italic> like
the nuclei are located in the basal part, while the circumferentially oriented
actin bundles are in the apical compartment (<xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5K to L</xref>′). In addition, no
adult was found with both lateral chords completely lacking
<italic>Wolbachia</italic>, suggesting that <italic>Wolbachia</italic>localization in these chords may be essential for worm survival. Conversely, the
<italic>Wolbachia</italic> are completely absent from the intestinal cells,
the somatic gonad (gonadal contractile sheath cells and epithelium, <xref ref-type="supplementary-material" rid="pntd.0000758.s006">Fig. S6</xref>)
and the muscle quadrants (<xref ref-type="fig" rid="pntd-0000758-g005">Fig.
5</xref>). It is hard to clearly draw a conclusion on the presence of
<italic>Wolbachia</italic> in the nervous system, due to the low quality of
the nerve ring chromatin staining with PI but the bacteria appear either absent
or at very low titer in this organ (Sup. <xref ref-type="fig" rid="pntd-0000758-g003">Fig. 3D, E</xref>).</p><fig id="pntd-0000758-g005" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g005</object-id><label>Figure 5</label><caption><title><italic>Wolbachia</italic> concentrate in the hypodermal lateral
chords.</title><p>DNA (propidium iodide, red), and actin (green) stainings of female (A, B)
and male (C, D) adult lateral chords. (E, F) The upper but not the lower
chord is infected. (G,H) Both chords are uninfected. (H) The phalloidin
staining reveals the gonad contractile sheath cells. (I, J) The upper
lateral chord is being invaded by <italic>Wolbachia</italic>, revealed
by propidium iodide. The uterus is embedded in the chord. (K, L) Surface
view of a chord and (K′, L′) 90° projections
showing the circumferentially oriented actin bundles in the apical part,
while the <italic>Wolbachia</italic>, surround the two rows of
hypodermal nuclei (see Material and <xref ref-type="sec" rid="s2">Methods</xref> for technical details) in the basal part, separated
by the secretory-excretory canal (arrowheads). Scale
bars = 100 µm.</p></caption><graphic xlink:href="pntd.0000758.g005"></graphic></fig><p>We stained the secretory-excretory canals with phalloidin in non-fixed worms, to
avoid the overwhelming signal coming from actin-rich tissues (i.e. muscles).
Despite variable results, we have been able to locate the secretory-excretory
pore close to the mouth (<xref ref-type="fig" rid="pntd-0000758-g006">Fig.
6A</xref>). By increasing the phalloidin signal in fixed animals, we could
reveal the lumen of the canal (<xref ref-type="fig" rid="pntd-0000758-g006">Fig.
6B to G</xref>, between arrowheads). We found propidium iodide spots present
in the lumen of the canal in variable amounts, similar to those in infected
parts of chords (<xref ref-type="fig" rid="pntd-0000758-g006">Fig. 6B to
D</xref>), but also in the lumen of the canal in non-infected parts of chords
(i.e. <xref ref-type="fig" rid="pntd-0000758-g006">Fig. 6E to G</xref>). We
confirmed these observations on worms kept alive, by visualizing the
secretory-excretory canal with the fluorescent marker resorufin. This marker is
a substrate of P-glycoproteins and multidrug resistance associated proteins,
localized in the apex of polarized cells, involved in excretory processes (cf.
<xref ref-type="bibr" rid="pntd.0000758-Sato1">[38]</xref>). Resorufin concentrates in the chords and
neighboring tissues before its excretion via the canal. We combined it with the
DNA vital dye syto11 (<xref ref-type="fig" rid="pntd-0000758-g006">Fig. 6H to
M</xref>). Altogether these data suggest that adult <italic>Brugia</italic> may
secrete/excrete low numbers of <italic>Wolbachia</italic> into their host.</p><fig id="pntd-0000758-g006" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g006</object-id><label>Figure 6</label><caption><title>Detection of <italic>Wolbachia</italic> by propidium iodide in the
lumen of the secretory-excretory canal.</title><p>(A) One canal reaching the pore near the mouth, stained with phalloidin
without fixation. (B) to (G) Confocal merges at the level of the lumen
in fixed females stained for DNA (propidium iodide, red), and cortical
actin (green). (B) to (D) At the level of an infected or (E) to (G)
non-infected chords. (E) to (G) are close to the pore. m., muscles,
arrowheads point to the lumen of the canal. (H) to (M) Live female
incubated with resorufin (red) and syto11(green). (H) to (J) one focal
plan in the largest section of the canal, (K) to (M) total projection of
the canal, (K′) to (M′) 90° projections
showing a possible excretion of <italic>Wolbachia revealed by the
propidium iodide</italic> (white arrowheads). Note that the canal
appears enlarged, as a consequence of the use of Sodium Azide to
immobilize the worm (white bars).</p></caption><graphic xlink:href="pntd.0000758.g006"></graphic></fig></sec><sec id="s3f"><title><italic>Wolbachia</italic> in the Germline: A Matter of Gender</title><p>In the female and male germinal zones, oogoniae or spermatogoniae are partially
surrounded by an actin rich membrane connected to the actin-rich central rachis
at the distal part of the ovary (Sup. <xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5A, C, D</xref>). These germ cell nuclei
initially organized in a syncitium, then cellularize and detach from the central
rachis while migrating proximally towards the uterus or the sperm duct (<xref ref-type="supplementary-material" rid="pntd.0000758.s005">Fig. S5G to
J</xref>). All female germ cells are infected and contain an average of
35+/−6.8 <italic>Wolbachia</italic>(n = 13; <xref ref-type="supplementary-material" rid="pntd.0000758.s005">Fig. S5A to
C</xref>) in the most distal part of the ovary, before complete cellularization.
This suggests a high replication rate of the bacteria in the mitotic region of
the ovary (<xref ref-type="supplementary-material" rid="pntd.0000758.s005">Fig.
S5A to C, G, H</xref>). More mature oogoniae are located more proximally in
the ovary and contain slightly more bacteria (49+/−10,
n = 8). Mature oocytes are fertilized when
encountering sperm in the distal uterus to give zygotes.</p><p>Surprisingly, analogous studies in the male germline revealed no bacteria at any
stage of spermatogenesis (n = 3 males).
Although it is difficult to distinguish bacteria from mature sperm chromatin,
cytological observations at earlier stages of spermatogenesis left no doubt on
the absence of <italic>Wolbachia</italic> in the male germline (i.e. <xref ref-type="supplementary-material" rid="pntd.0000758.s005">Fig. S5D to F, I,
J</xref>).</p></sec></sec><sec id="s4"><title>Discussion</title><sec id="s4a"><title><italic>Wolbachia</italic> Asymmetrically Segregate during Embryogenesis in a
Lineage-Specific Manner</title><p>Characterization of the transmission mechanisms, distribution pattern and titer
of <italic>Wolbachia</italic> in the germline and soma is of primary importance
for understanding the biology of the interaction between
<italic>Wolbachia</italic> and its host <italic>B. malayi</italic>. We found in
<italic>B. malayi</italic> a high <italic>Wolbachia</italic>/ host nuclei
ratio in early embryogenesis and in the adult lateral chords.
<italic>Wolbachia</italic> were also concentrated in the female germline but
absent from the male germline (summarized in <xref ref-type="fig" rid="pntd-0000758-g007">Fig. 7A</xref>). To understand the origin of this
distribution pattern, we examined <italic>Wolbachia</italic> segregation during
early embryogenesis. Despite variability in the <italic>Wolbachia</italic> titer
among embryos of the same stage, the bacteria were present in all embryonic
blastomeres until about the 6-cell stage, but greatly enriched in the posterior
pole, following fertilization. Our data indicate that <italic>Wolbachia</italic>are present in the most posterior blastomeres, P2 and EMS at the 4-cell stage,
followed by C and P3 in all embryos at the 12-cell stage, that is to say both
male and female embryos. Presence in C is the main source of transmission to the
hypodermis, while maintenance in P3 and subsequently P4 ensures transmission to
the germline (<xref ref-type="fig" rid="pntd-0000758-g007">Fig. 7B</xref>).</p><fig id="pntd-0000758-g007" position="float"><object-id pub-id-type="doi">10.1371/journal.pntd.0000758.g007</object-id><label>Figure 7</label><caption><title><italic>Wolbachia</italic> localization and segregation patterns
followed during early embryogenesis.</title><p>(A) Schematic drawing of <italic>Wolbachia</italic> localization in a
cross section of a <italic>Brugia malayi</italic> adult female. The
<italic>Wolbachia</italic> (purple dots) are present in the cell
bodies of the hypodermal lateral chords (in red), secretory-excretory
canal, in the germline and in embryos. They are absent from the somatic
gonad, the muscles and the intestine. (B) Model of likely segregation
patterns followed by <italic>Wolbachia</italic> during early
embryogenesis to colonize adult tissues. Observation of fixed embryos
and adult tissues indicate that <italic>Wolbachia</italic> must follow
the germline precursors in females and the C lineage leading to
hypodermal cells (green solid lines). In contrast, some blastomere
derivatives are not favorable to <italic>Wolbachia</italic>proliferation (i.e. blue dotted lines). In male embryos, the
<italic>Wolbachia</italic> may not be maintained in the germline
precursors.</p></caption><graphic xlink:href="pntd.0000758.g007"></graphic></fig><p>During the mitotic proliferation of oogoniae, association of
<italic>Wolbachia</italic> with the mitotic spindle is likely used to ensure an
even segregation in the female germline. Interactions of
<italic>Wolbachia</italic> with the host microtubules has been well documented
in arthropods (i.e. <xref ref-type="bibr" rid="pntd.0000758-Ferree1">[39]</xref>). During fertilization, the primary
enrichment could be due to a passive mechanism involving the deep cytoplasmic
flow oriented towards the posterior <xref ref-type="bibr" rid="pntd.0000758-Schneider1">[40]</xref>. It is also well
established that in <italic>C. elegans</italic> the sperm entry induces
partitioning of the evolutionary conserved cell polarizing factors PARs <xref ref-type="bibr" rid="pntd.0000758-Schneider1">[40]</xref>,
<xref ref-type="bibr" rid="pntd.0000758-Goldstein3">[41]</xref>. <italic>Brugia</italic> PAR orthologs and their
downstream effectors may be responsible for keeping the
<italic>Wolbachia</italic> in the posterior. Asymmetric segregation has been
described as a common feature of <italic>Wolbachia</italic> localization in
arthropods, such as in <italic>Drosophila</italic> germline and somatic cells,
in wasp species and mosquito germlines <xref ref-type="bibr" rid="pntd.0000758-Serbus1">[42]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Serbus2">[43]</xref>.
Whether the mechanisms used to enrich the posterior of the embryos, to
subsequently invade the germ cell precursors, is due to convergent evolution or
to common developmental pathways remains to be determined. The latter may
provide new targets in anti-<italic>Wolbachia</italic> based therapies in
filariasis. An evolutionarily conserved mechanism for posterior localization
maybe supported by the mode of invasion of the chords. Instead of invading the
AB lineage, the main source of hypodermal blastomeres,
<italic>Wolbachia</italic> utilize the posterior C blastomere. It is tempting to
speculate that during the evolution of the <italic>Wolbachia</italic>-nematode
interaction, the bacteria followed conserved posterior determinants to ensure
transmission to the germline, and subsequently acquired an affinity for the C
blastomere and its ectodermal derivatives.</p><p>In secernentean nematodes, fixed lineages contribute to different types of
tissues. This raises the intriguing question of the mechanisms underlying the
transmission of the bacteria to the proper differentiated blastomeres. The
segregation pattern of <italic>Wolbachia</italic> in the early embryo could
result from sensing regulatory networks patterning the embryo, to asymmetrically
segregate and proliferate. From the 2 to 12-cell stages, their transmission
pattern is very similar to the expression pattern of the <italic>C.
elegans</italic> homeodomain protein PAL-1, required for the C-lineage
expression <xref ref-type="bibr" rid="pntd.0000758-Hunter1">[44]</xref>. In a second phase, C-derived ectodermal
derivatives could trigger <italic>Wolbachia</italic> proliferation <xref ref-type="bibr" rid="pntd.0000758-Baugh1">[45]</xref>.
Likewise, germline-specific factors are likely to play a role in segregation in
the P germline lineage. It is significant however that some embryos appeared
devoid of <italic>Wolbachia</italic> in the P4 blastomere and the Z2/Z3 germline
cells, implying a possible loss of <italic>Wolbachia</italic> after
establishment of the P3 blastomere and before establishment of Z2/Z3. What
happens to these <italic>Wolbachia</italic> remains unclear, they may all
segregate into the P4 sister, the D blastomere, to be diluted out without
replicating for instance, as observed in the descendants of AB, E or MS. We
hypothesize that embryos with and without <italic>Wolbachia</italic> in the P4
blastomere are female and male embryos respectively, since we find a ratio
identical to the equal sex ratio described in larvae of the closely related
<italic>Brugia pahangi</italic><xref ref-type="bibr" rid="pntd.0000758-Casiraghi1">[46]</xref>. Our data do not allow us to rule out possible
mechanisms of transcellular invasion from neighboring tissues to non-infected
germ cells at later stages, or later loss of <italic>Wolbachia</italic> in the
male germline when <italic>Wolbachia</italic> are initially observed in the
germline precursors. In <italic>Brugia</italic>, sex determination if of XX/XY
type, and males possess a heterogametic pair of chromosomes <xref ref-type="bibr" rid="pntd.0000758-Sakaguchi1">[47]</xref>.
<italic>Wolbachia</italic> may sense the gender of the embryo prior to the
establishment of the P4 blastomere. Such an early sensing of the
embryo's gender may involve interactions with a X- chromosome dosage
compensation machinery <xref ref-type="bibr" rid="pntd.0000758-Ercan1">[48]</xref>. In <italic>C. elegans</italic> for instance,
this protein complex is active as early as the 30-cell stage, before formation
of the P4 blastomere <xref ref-type="bibr" rid="pntd.0000758-Chuang1">[49]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Lieb1">[50]</xref>.</p><p>Based on lessons from <italic>C. elegans</italic> genetics and cell biology on
embryonic cell fate establishment, immunofluorescence and RNAi techniques on
relevant <italic>Brugia</italic> orthologs should help us to understand the
molecular mechanisms of <italic>Wolbachia</italic> transmission.</p></sec><sec id="s4b"><title>Wolbachia Use Cell Fusion to Populate the Hypodermal Chords</title><p>We observed the highest bacteria titer in the adult lateral hypodermal chords.
Some worms however possess partially infected chords, or one chord lacking
<italic>Wolbachia</italic>. This observation may explain the wide range in
<italic>Wolbachia</italic> load between individual worms as measured by qPCR
<xref ref-type="bibr" rid="pntd.0000758-McGarry1">[24]</xref>. Observations at the embryonic level revealed few
infected hypodermal cells, mainly posterior-dorsal, in which the bacteria
multiplied (i.e. <xref ref-type="fig" rid="pntd-0000758-g002">Fig.2K</xref>). A
common feature of secernentean nematodes is that during development, hypodermal
cells fuse creating a syncytium in the adult <xref ref-type="bibr" rid="pntd.0000758-Bird1">[51]</xref>. Since we did not
find nuclei in the adult ventral and dorsal chords, it is likely that all the
hypodermal dorsal and ventral nuclei migrate laterally in
<italic>Brugia</italic>. Thus <italic>Wolbachia</italic> may spread through
fusion of infected with uninfected hypodermal cells. The developmental timing of
hypodermal fusion is unknown. However since we observed
<italic>Wolbachia</italic> invasion of lateral chords in young adults (<xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5I, J</xref>), hypodermal fusion
in <italic>Brugia</italic> is likely to have occurred during larval or young
adult stages. This would predict a dramatic increase in
<italic>Wolbachia</italic> titer per host nuclei during larval stages and early
adult, and is supported by quantitative PCR data <xref ref-type="bibr" rid="pntd.0000758-Fenn1">[25]</xref>. Hence, the selective
pressure for somatic invasion must be less important than in the germline, since
vertical transmission from a single hypodermal cell of a chord is theoretically
sufficient to ensure a successful colonization. We have performed our
cytological studies in young adults while <italic>B. malayi</italic> can live
for many years. This could explain the presence of these partially non-infected
chords. It would be interesting to determine whether aged adults contain fully
infected lateral chords.</p></sec><sec id="s4c"><title>A Possible Role of <italic>Wolbachia</italic> in the Chords in the Embryonic
Development</title><p>The nematode hypodermal chords have been shown to play a fundamental function in
the metabolism of stored carbohydrate and protein synthesis, as well as the
uptake of nutrients via the transcuticular route <xref ref-type="bibr" rid="pntd.0000758-Bird1">[51]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Howells1">[52]</xref>.
<italic>Wolbachia</italic> may participate in these lateral chord functions.
Moreover it has been recently demonstrated that part of the stress response
induced in <italic>Wolbachia</italic>-depleted <italic>B. malayi</italic> by
tetracycline is an upregulation of amino acids synthesis and protein
translation, suggesting an initial compensation for the lack of
<italic>Wolbachia</italic><xref ref-type="bibr" rid="pntd.0000758-Ghedin1">[53]</xref>.
Depletion of <italic>Wolbachia</italic> with antibiotics has been shown to
reduce the production of microfilariae and to affect embryogenesis <xref ref-type="bibr" rid="pntd.0000758-Ghedin1">[53]</xref>–<xref ref-type="bibr" rid="pntd.0000758-Hoerauf3">[55]</xref>. This last study
also shows that tetracycline treatments result in <italic>Wolbachia</italic>degeneration in the germline and embryos prior to <italic>Wolbachia</italic>loss in the lateral chords. Defects in embryogenesis may still be due to a
perturbed metabolism starting at the level of the hypodermal chords rather than
a direct effect on the few <italic>Wolbachia</italic> present in embryonic
hypodermal and germ line cells. Support for this idea comes from the fact that
<italic>Wolbachia</italic> are present exclusively in the female germ line
and not in the male germ line. Thus while <italic>Wolbachia</italic> are
transmitted vertically through the female germline, they may not be necessary
for germline development. Selective pressure in the germline may be greatly
reduced in endosymbionts such as <italic>Wolbachia</italic> that are involved in
metabolic mutualism. In contrast, <italic>Wolbachia</italic> are parasitic in
many arthropod species and accordingly have a profound influence on host
germline function <xref ref-type="bibr" rid="pntd.0000758-Serbus2">[43]</xref>. Second, we observed an increase in embryo
size during development suggesting nutrient uptake from the uterus. Third, both
in live specimens and in whole mount fixed adults a tight association between
lateral chords and the uterus was observed, arguing for a role of the chords in
supplying the production demands of microfilariae (i.e. <xref ref-type="fig" rid="pntd-0000758-g005">Fig. 5H</xref> and <xref ref-type="supplementary-material" rid="pntd.0000758.s007">Fig.
S7</xref>).</p></sec><sec id="s4d"><title>Adult Worms May Trigger the Host Immune Response by Directly Secreting
<italic>Wolbachia</italic></title><p>It has been established that <italic>Wolbachia</italic> release in the human
body, presumably from degenerating worms, has a crucial impact on the
development of river blindness and lymphatic filariasis, by activating the host
immune response <xref ref-type="bibr" rid="pntd.0000758-Bandi1">[3]</xref>, <xref ref-type="bibr" rid="pntd.0000758-GilletteFerguson1">[12]</xref>, <xref ref-type="bibr" rid="pntd.0000758-SaintAndre1">[13]</xref>, <xref ref-type="bibr" rid="pntd.0000758-Turner1">[14]</xref>. We detected variable amounts of
<italic>Wolbachia</italic> in the secretory-excretory canals, present in the
chords, even in non-infected regions of the chords. This suggests that in
addition to degenerating worms, live adults may release
<italic>Wolbachia</italic>, through the excretory pore. PCR analysis of short
term <italic>in vitro</italic> culture supernatant was unable to detect
<italic>Wolbachia</italic> DNA, although 90 <italic>Wolbachia</italic>proteins were detected in ES products <xref ref-type="bibr" rid="pntd.0000758-Bennuru1">[56]</xref>. Furthermore,
immunohistochemistry of <italic>O. volvulus</italic> does not detect the
abundant release of <italic>Wolbachia</italic> into the surrounding tissues
<xref ref-type="bibr" rid="pntd.0000758-Hoerauf4">[57]</xref>. Nevertheless, low numbers of
<italic>Wolbachia</italic> and/or their products may be released via the
excretory/secretory canal as previously hypothesized <xref ref-type="bibr" rid="pntd.0000758-Kozek2">[58]</xref>, and act as an
additional source of immunostimulatory components that contribute to the known
innate and adaptive immune responses typical of filarial infections <xref ref-type="bibr" rid="pntd.0000758-Brattig2">[59]</xref>–<xref ref-type="bibr" rid="pntd.0000758-GilletteFerguson3">[61]</xref>.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pntd.0000758.s001"><label>Figure S1</label><caption><p><italic>Wolbachia</italic> concentrate to the astral and spindle microtubules
in oogoniae during mitosis. Merges of confocal stacks of <italic>B.
malayi</italic> oogoniae stained for DNA (propidium iodide, red),
<italic>Wolbachia</italic> (anti-WSP, blue), and microtubules (green).
(A) Oogonia in S phase. (B) Oogonia in metaphase. (C) Oogoniae in anaphase.
Arrowheads point to <italic>Wolbachia</italic> associated with the spindle,
and arrows highlight <italic>Wolbachia</italic> around the asters. The
figures of division and the absence of polar bodies strongly suggest that
these oogoniae are in the phase of mitotic proliferation. Note than in (B)
and (C) sperm cells are in the top right–present on slide after
dissection of the gonads. Scale bars = 5
µm.</p><p>(1.99 MB PDF)</p></caption><media xlink:href="pntd.0000758.s001.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s002"><label>Figure S2</label><caption><p>Stimulation of <italic>Wolbachia</italic> replication by host factors may
participate to the asymmetrical enrichment. (A) Merge of confocal stacks of
the P2 blastomere of the 6-cell stage embryo shown in <xref ref-type="fig" rid="pntd-0000758-g002">Fig. 2H</xref>. (B) One focal plane showing
doublets of <italic>Wolbachia</italic> in the antero-dorsal pole (arrows) or
single bacteria in the postero-ventral pole (arrowheads) pole of P2.</p><p>(0.55 MB PDF)</p></caption><media xlink:href="pntd.0000758.s002.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s003"><label>Figure S3</label><caption><p>Anterior histological observations in <italic>Brugia</italic> female. (A to
C) Phalloidin (A) and PI stainings (B) of the vulva and the ovejector, in
front of the pharynx. (D, E) Phalloidin and PI stainings of the nerve ring
(arrows). (F to H) Phalloidin (F) and propidium iodide (G) stainings showing
the dorsal chord (arrow). Neither <italic>Wolbachia</italic> nor nuclei are
found in the narrow chord.</p><p>(3.15 MB PDF)</p></caption><media xlink:href="pntd.0000758.s003.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s004"><label>Figure S4</label><caption><p>Anatomical details of the <italic>Brugia malayi</italic> male. Syto11
staining of a male observed by epifluorescence. Anterior is at the bottom
right. Enlargements show key stages of spermatogenesis, and the most
posterior coiled part.</p><p>(2.46 MB PDF)</p></caption><media xlink:href="pntd.0000758.s004.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s005"><label>Figure S5</label><caption><p><italic>Wolbachia</italic> are present in high number in the female germline,
but absent from the male germline. (A to C) Distal ovary or (D to F) distal
testis stained for DNA (propidium iodide, grey and red channels), actin
(green), with the addition of anti-acetylated histone H4 staining on panel
(C) and (H) in green to discriminate <italic>Wolbachia</italic> DNA (white
arrowheads in (A)) from host chromatin. In (B) the central actin-rich rachis
is clearly visible. Higher magnifications in a more proximal localization in
a female and male gonad of a cellularized oogonium (G, H) or of cellularized
spermatocytes (I, J). In (G) and (H) somatic gonad nuclei in the background
are not surrounded by bacteria. Note in (I) and (J) the presence of mature
sperm cells brightly stained with propidium iodide (arrows). Scale
bars = 12 µm.</p><p>(2.68 MB PDF)</p></caption><media xlink:href="pntd.0000758.s005.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s006"><label>Figure S6</label><caption><p><italic>Wolbachia</italic> are absent from the somatic gonads and intestine.
Merges of confocal stacks of tissues stained for DNA (propidium iodide, (A)
and (D)), actin (green, (B) and (E)), and <italic>Brugia</italic> chromatin
(anti-acetylated Histone H4, green, (B) and (E)). (A to C) Outer view of a
gonad showing epithelial cells (arrowheads) and contractile sheath cells
(arrows). (D to F) Sagital view of intestinal cells in the widest part of
the lumen. Scale bars = 10 µm.</p><p>(1.00 MB PDF)</p></caption><media xlink:href="pntd.0000758.s006.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s007"><label>Figure S7</label><caption><p>Vital Hoechst staining (left panels) and DIC (center) imaging of a lateral
hypodermal chord above a uterus at mid length of a female (merge on the
right). The depth of focus from the cuticle is indicated on the left panels
in µm. (A) The somatic gonad (s.g.) is embedded in the lateral
chord (l.c.) and the pseudocoel is not visible between these tissues at this
level. Nuclei of the contractile sheath cells are visible (arrows). (B)
Nuclei of the lateral chord are aligned in two deep grooves where lie the
hypodermal cell bodies (B and C, arrows). <italic>Wolbachia</italic> appear
as a granulated staining in the lateral chords (i.e. (A) white circle). (D)
A thin projection of hypodermal cytoplasm covers the uterus and contains the
secretory-excretory canal (s.e.c, double white arrow) appearing as a dark
line on left panels of (D) and (E). In (D) and (E) the hypodermal
cytoplasmic projections above the uterus and the muscles do not contain any
bacteria, probably because of spatial constraints. (F) The cuticle secreted
by the hypodermis. Scale bar = 50
µm.</p><p>(0.20 MB PDF)</p></caption><media xlink:href="pntd.0000758.s007.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pntd.0000758.s008"><label>Methods S1</label><caption><p>(0.03 MB DOC)</p></caption><media xlink:href="pntd.0000758.s008.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>We would like to thank Susan Strome for critical reading and providing reagents, and
Mark Taylor for helpful ideas. We also thank the NIAID/NIH Filariasis Research
Resource Center (FR3) for supplying us with <italic>B. malayi</italic> worms.</p></ack><fn-group><fn fn-type="conflict"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p>This work was supported by the Sinsheimer Laboratories Insurance fund
(433317-09577) and New England Biolabs, Inc. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript.</p></fn></fn-group><ref-list><title>References</title><ref id="pntd.0000758-Crompton1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Crompton</surname><given-names>DW</given-names></name></person-group><year>1999</year><article-title>How much human helminthiasis is there in the world?</article-title><source>J Parasitol</source><volume>85</volume><fpage>397</fpage><lpage>403</lpage><pub-id pub-id-type="pmid">10386428</pub-id></element-citation></ref><ref id="pntd.0000758-Molyneux1"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Molyneux</surname><given-names>DH</given-names></name><name><surname>Bradley</surname><given-names>M</given-names></name><name><surname>Hoerauf</surname><given-names>A</given-names></name><name><surname>Kyelem</surname><given-names>D</given-names></name><name><surname>Taylor</surname><given-names>MJ</given-names></name></person-group><year>2003</year><article-title>Mass drug treatment for lymphatic filariasis and onchocerciasis.</article-title><source>Trends Parasitol</source><volume>19</volume><fpage>516</fpage><lpage>522</lpage><pub-id pub-id-type="pmid">14580963</pub-id></element-citation></ref><ref id="pntd.0000758-Bandi1"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bandi</surname><given-names>C</given-names></name><name><surname>Trees</surname><given-names>AJ</given-names></name><name><surname>Brattig</surname><given-names>NW</given-names></name></person-group><year>2001</year><article-title>Wolbachia in filarial nematodes: evolutionary aspects and
implications for the pathogenesis and treatment of filarial diseases.</article-title><source>Vet Parasitol</source><volume>98</volume><fpage>215</fpage><lpage>238</lpage><pub-id pub-id-type="pmid">11516587</pub-id></element-citation></ref><ref id="pntd.0000758-Taylor1"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>MJ</given-names></name><name><surname>Bandi</surname><given-names>C</given-names></name><name><surname>Hoerauf</surname><given-names>A</given-names></name></person-group><year>2005</year><article-title>Wolbachia bacterial endosymbionts of filarial nematodes.</article-title><source>Adv Parasitol</source><volume>60</volume><fpage>245</fpage><lpage>284</lpage><pub-id pub-id-type="pmid">16230105</pub-id></element-citation></ref><ref id="pntd.0000758-Hoerauf1"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoerauf</surname><given-names>A</given-names></name><name><surname>Mand</surname><given-names>S</given-names></name><name><surname>Adjei</surname><given-names>O</given-names></name><name><surname>Fleischer</surname><given-names>B</given-names></name><name><surname>Buttner</surname><given-names>DW</given-names></name></person-group><year>2001</year><article-title>Depletion of wolbachia endobacteria in Onchocerca volvulus by
doxycycline and microfilaridermia after ivermectin treatment.</article-title><source>Lancet</source><volume>357</volume><fpage>1415</fpage><lpage>1416</lpage><pub-id pub-id-type="pmid">11356444</pub-id></element-citation></ref><ref id="pntd.0000758-Hoerauf2"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoerauf</surname><given-names>A</given-names></name><name><surname>Specht</surname><given-names>S</given-names></name><name><surname>Buttner</surname><given-names>M</given-names></name><name><surname>Pfarr</surname><given-names>K</given-names></name><name><surname>Mand</surname><given-names>S</given-names></name><etal></etal></person-group><year>2008</year><article-title>Wolbachia endobacteria depletion by doxycycline as antifilarial
therapy has macrofilaricidal activity in onchocerciasis: a randomized
placebo-controlled study.</article-title><source>Med Microbiol Immunol</source><volume>197</volume><fpage>295</fpage><lpage>311</lpage><pub-id pub-id-type="pmid">17999080</pub-id></element-citation></ref><ref id="pntd.0000758-Specht1"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Specht</surname><given-names>S</given-names></name><name><surname>Mand</surname><given-names>S</given-names></name><name><surname>Marfo-Debrekyei</surname><given-names>Y</given-names></name><name><surname>Debrah</surname><given-names>AY</given-names></name><name><surname>Konadu</surname><given-names>P</given-names></name><etal></etal></person-group><year>2008</year><article-title>Efficacy of 2- and 4-week rifampicin treatment on the Wolbachia
of Onchocerca volvulus.</article-title><source>Parasitol Res</source><volume>103</volume><fpage>1303</fpage><lpage>1309</lpage><pub-id pub-id-type="pmid">18679718</pub-id></element-citation></ref><ref id="pntd.0000758-Supali1"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Supali</surname><given-names>T</given-names></name><name><surname>Djuardi</surname><given-names>Y</given-names></name><name><surname>Pfarr</surname><given-names>KM</given-names></name><name><surname>Wibowo</surname><given-names>H</given-names></name><name><surname>Taylor</surname><given-names>MJ</given-names></name><etal></etal></person-group><year>2008</year><article-title>Doxycycline treatment of Brugia malayi-infected persons reduces
microfilaremia and adverse reactions after diethylcarbamazine and
albendazole treatment.</article-title><source>Clin Infect Dis</source><volume>46</volume><fpage>1385</fpage><lpage>1393</lpage><pub-id pub-id-type="pmid">18419441</pub-id></element-citation></ref><ref id="pntd.0000758-Bazzocchi1"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bazzocchi</surname><given-names>C</given-names></name><name><surname>Ceciliani</surname><given-names>F</given-names></name><name><surname>McCall</surname><given-names>JW</given-names></name><name><surname>Ricci</surname><given-names>I</given-names></name><name><surname>Genchi</surname><given-names>C</given-names></name><etal></etal></person-group><year>2000</year><article-title>Antigenic role of the endosymbionts of filarial nematodes: IgG
response against the Wolbachia surface protein in cats infected with
Dirofilaria immitis.</article-title><source>Proc Biol Sci</source><volume>267</volume><fpage>2511</fpage><lpage>2516</lpage><pub-id pub-id-type="pmid">11197127</pub-id></element-citation></ref><ref id="pntd.0000758-Brattig1"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brattig</surname><given-names>NW</given-names></name><name><surname>Rathjens</surname><given-names>U</given-names></name><name><surname>Ernst</surname><given-names>M</given-names></name><name><surname>Geisinger</surname><given-names>F</given-names></name><name><surname>Renz</surname><given-names>A</given-names></name><etal></etal></person-group><year>2000</year><article-title>Lipopolysaccharide-like molecules derived from Wolbachia
endobacteria of the filaria Onchocerca volvulus are candidate mediators in
the sequence of inflammatory and antiinflammatory responses of human
monocytes.</article-title><source>Microbes Infect</source><volume>2</volume><fpage>1147</fpage><lpage>1157</lpage><pub-id pub-id-type="pmid">11008105</pub-id></element-citation></ref><ref id="pntd.0000758-Taylor2"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>MJ</given-names></name><name><surname>Cross</surname><given-names>HF</given-names></name><name><surname>Bilo</surname><given-names>K</given-names></name></person-group><year>2000</year><article-title>Inflammatory responses induced by the filarial nematode Brugia
malayi are mediated by lipopolysaccharide-like activity from endosymbiotic
Wolbachia bacteria.</article-title><source>J Exp Med</source><volume>191</volume><fpage>1429</fpage><lpage>1436</lpage><pub-id pub-id-type="pmid">10770808</pub-id></element-citation></ref><ref id="pntd.0000758-GilletteFerguson1"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gillette-Ferguson</surname><given-names>I</given-names></name><name><surname>Hise</surname><given-names>AG</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Diaconu</surname><given-names>E</given-names></name><name><surname>McGarry</surname><given-names>HF</given-names></name><etal></etal></person-group><year>2006</year><article-title>Wolbachia- and Onchocerca volvulus-induced keratitis (river
blindness) is dependent on myeloid differentiation factor 88.</article-title><source>Infect Immun</source><volume>74</volume><fpage>2442</fpage><lpage>2445</lpage><pub-id pub-id-type="pmid">16552075</pub-id></element-citation></ref><ref id="pntd.0000758-SaintAndre1"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Saint Andre</surname><given-names>A</given-names></name><name><surname>Blackwell</surname><given-names>NM</given-names></name><name><surname>Hall</surname><given-names>LR</given-names></name><name><surname>Hoerauf</surname><given-names>A</given-names></name><name><surname>Brattig</surname><given-names>NW</given-names></name><etal></etal></person-group><year>2002</year><article-title>The role of endosymbiotic Wolbachia bacteria in the pathogenesis
of river blindness.</article-title><source>Science</source><volume>295</volume><fpage>1892</fpage><lpage>1895</lpage><pub-id pub-id-type="pmid">11884755</pub-id></element-citation></ref><ref id="pntd.0000758-Turner1"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Turner</surname><given-names>JD</given-names></name><name><surname>Langley</surname><given-names>RS</given-names></name><name><surname>Johnston</surname><given-names>KL</given-names></name><name><surname>Gentil</surname><given-names>K</given-names></name><name><surname>Ford</surname><given-names>L</given-names></name><etal></etal></person-group><year>2009</year><article-title>Wolbachia lipoprotein stimulates innate and adaptive immunity
through Toll-like receptors 2 and 6 to induce disease manifestations of
filariasis.</article-title><source>J Biol Chem</source><volume>284</volume><fpage>22364</fpage><lpage>22378</lpage><pub-id pub-id-type="pmid">19458089</pub-id></element-citation></ref><ref id="pntd.0000758-Buckley1"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buckley</surname><given-names>JJ</given-names></name><name><surname>Edeson</surname><given-names>JF</given-names></name></person-group><year>1956</year><article-title>On the adult morphology of Wuchereria sp. (malayi?) from a monkey
(Macaca irus) and from cats in Malaya, and on Wuchereria pahangi n.sp. from
a dog and a cat.</article-title><source>J Helminthol</source><volume>30</volume><fpage>1</fpage><lpage>20</lpage><pub-id pub-id-type="pmid">13319678</pub-id></element-citation></ref><ref id="pntd.0000758-Cooray1"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cooray</surname><given-names>GH</given-names></name></person-group><year>1960</year><article-title>Some observations on filarial infection in Ceylon with special
reference to its histopathology.</article-title><source>Indian J Malariol</source><volume>14</volume><fpage>617</fpage><lpage>632</lpage><pub-id pub-id-type="pmid">13695347</pub-id></element-citation></ref><ref id="pntd.0000758-Schacher1"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schacher</surname><given-names>JF</given-names></name></person-group><year>1962</year><article-title>Developmental stages of Brugia pahangi in the final host.</article-title><source>J Parasitol</source><volume>48</volume><fpage>693</fpage><lpage>706</lpage><pub-id pub-id-type="pmid">13976564</pub-id></element-citation></ref><ref id="pntd.0000758-Franz1"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Franz</surname><given-names>M</given-names></name><name><surname>Buttner</surname><given-names>DW</given-names></name></person-group><year>1986</year><article-title>Histology of adult Brugia malayi.</article-title><source>Trop Med Parasitol</source><volume>37</volume><fpage>282</fpage><lpage>285</lpage><pub-id pub-id-type="pmid">3787124</pub-id></element-citation></ref><ref id="pntd.0000758-Kozek1"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kozek</surname><given-names>WJ</given-names></name></person-group><year>1977</year><article-title>Transovarially-transmitted intracellular microorganisms in adult
and larval stages of Brugia malayi.</article-title><source>J Parasitol</source><volume>63</volume><fpage>992</fpage><lpage>1000</lpage><pub-id pub-id-type="pmid">592054</pub-id></element-citation></ref><ref id="pntd.0000758-McLaren1"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McLaren</surname><given-names>DJ</given-names></name><name><surname>Worms</surname><given-names>MJ</given-names></name><name><surname>Laurence</surname><given-names>BR</given-names></name><name><surname>Simpson</surname><given-names>MG</given-names></name></person-group><year>1975</year><article-title>Micro-organisms in filarial larvae (Nematoda).</article-title><source>Trans R Soc Trop Med Hyg</source><volume>69</volume><fpage>509</fpage><lpage>514</lpage><pub-id pub-id-type="pmid">1228988</pub-id></element-citation></ref><ref id="pntd.0000758-Sironi1"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sironi</surname><given-names>M</given-names></name><name><surname>Bandi</surname><given-names>C</given-names></name><name><surname>Sacchi</surname><given-names>L</given-names></name><name><surname>Di Sacco</surname><given-names>B</given-names></name><name><surname>Damiani</surname><given-names>G</given-names></name><etal></etal></person-group><year>1995</year><article-title>Molecular evidence for a close relative of the arthropod
endosymbiont Wolbachia in a filarial worm.</article-title><source>Mol Biochem Parasitol</source><volume>74</volume><fpage>223</fpage><lpage>227</lpage><pub-id pub-id-type="pmid">8719164</pub-id></element-citation></ref><ref id="pntd.0000758-Williams1"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Williams</surname><given-names>SA</given-names></name><name><surname>Lizotte-Waniewski</surname><given-names>MR</given-names></name><name><surname>Foster</surname><given-names>J</given-names></name><name><surname>Guiliano</surname><given-names>D</given-names></name><name><surname>Daub</surname><given-names>J</given-names></name><etal></etal></person-group><year>2000</year><article-title>The filarial genome project: analysis of the nuclear,
mitochondrial and endosymbiont genomes of Brugia malayi.</article-title><source>Int J Parasitol</source><volume>30</volume><fpage>411</fpage><lpage>419</lpage><pub-id pub-id-type="pmid">10731564</pub-id></element-citation></ref><ref id="pntd.0000758-Taylor3"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>MJ</given-names></name><name><surname>Bilo</surname><given-names>K</given-names></name><name><surname>Cross</surname><given-names>HF</given-names></name><name><surname>Archer</surname><given-names>JP</given-names></name><name><surname>Underwood</surname><given-names>AP</given-names></name></person-group><year>1999</year><article-title>16S rDNA phylogeny and ultrastructural characterization of
Wolbachia intracellular bacteria of the filarial nematodes Brugia malayi, B.
pahangi, and Wuchereria bancrofti.</article-title><source>Exp Parasitol</source><volume>91</volume><fpage>356</fpage><lpage>361</lpage><pub-id pub-id-type="pmid">10092480</pub-id></element-citation></ref><ref id="pntd.0000758-McGarry1"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McGarry</surname><given-names>HF</given-names></name><name><surname>Egerton</surname><given-names>GL</given-names></name><name><surname>Taylor</surname><given-names>MJ</given-names></name></person-group><year>2004</year><article-title>Population dynamics of Wolbachia bacterial endosymbionts in
Brugia malayi.</article-title><source>Mol Biochem Parasitol</source><volume>135</volume><fpage>57</fpage><lpage>67</lpage><pub-id pub-id-type="pmid">15287587</pub-id></element-citation></ref><ref id="pntd.0000758-Fenn1"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fenn</surname><given-names>K</given-names></name><name><surname>Blaxter</surname><given-names>M</given-names></name></person-group><year>2004</year><article-title>Quantification of Wolbachia bacteria in Brugia malayi through the
nematode lifecycle.</article-title><source>Mol Biochem Parasitol</source><volume>137</volume><fpage>361</fpage><lpage>364</lpage><pub-id pub-id-type="pmid">15383308</pub-id></element-citation></ref><ref id="pntd.0000758-Ward1"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ward</surname><given-names>S</given-names></name><name><surname>Carrel</surname><given-names>JS</given-names></name></person-group><year>1979</year><article-title>Fertilization and sperm competition in the nematode
Caenorhabditis elegans.</article-title><source>Dev Biol</source><volume>73</volume><fpage>304</fpage><lpage>321</lpage><pub-id pub-id-type="pmid">499670</pub-id></element-citation></ref><ref id="pntd.0000758-Goldstein1"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goldstein</surname><given-names>B</given-names></name><name><surname>Hird</surname><given-names>SN</given-names></name></person-group><year>1996</year><article-title>Specification of the anteroposterior axis in Caenorhabditis
elegans.</article-title><source>Development</source><volume>122</volume><fpage>1467</fpage><lpage>1474</lpage><pub-id pub-id-type="pmid">8625834</pub-id></element-citation></ref><ref id="pntd.0000758-Lahl1"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lahl</surname><given-names>V</given-names></name><name><surname>Halama</surname><given-names>C</given-names></name><name><surname>Schierenberg</surname><given-names>E</given-names></name></person-group><year>2003</year><article-title>Comparative and experimental embryogenesis of Plectidae
(Nematoda).</article-title><source>Dev Genes Evol</source><volume>213</volume><fpage>18</fpage><lpage>27</lpage><pub-id pub-id-type="pmid">12590349</pub-id></element-citation></ref><ref id="pntd.0000758-Skiba1"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Skiba</surname><given-names>F</given-names></name><name><surname>Schierenberg</surname><given-names>E</given-names></name></person-group><year>1992</year><article-title>Cell lineages, developmental timing, and spatial pattern
formation in embryos of free-living soil nematodes.</article-title><source>Dev Biol</source><volume>151</volume><fpage>597</fpage><lpage>610</lpage><pub-id pub-id-type="pmid">1601187</pub-id></element-citation></ref><ref id="pntd.0000758-Dolinski1"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dolinski</surname><given-names>CJBG</given-names></name><name><surname>Thomas</surname><given-names>WK</given-names></name></person-group><year>2001</year><article-title>Comparative survey of early embryogenesis of Secernetea
(Nematoda), with phylogenetic implication.</article-title><source>Can J Zool</source><volume>79</volume><fpage>82</fpage><lpage>94</lpage></element-citation></ref><ref id="pntd.0000758-Goldstein2"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goldstein</surname><given-names>B</given-names></name></person-group><year>2001</year><article-title>On the evolution of early development in the Nematoda.</article-title><source>Philos Trans R Soc Lond B Biol Sci</source><volume>356</volume><fpage>1521</fpage><lpage>1531</lpage><pub-id pub-id-type="pmid">11604120</pub-id></element-citation></ref><ref id="pntd.0000758-Malakhov1"><label>32</label><element-citation publication-type="other"><person-group person-group-type="author"><name><surname>Malakhov</surname><given-names>VV</given-names></name></person-group><year>1994</year><comment>Nematodes, Structure, Development, Classification and Phylogeny:
Smithsonian Institution Press</comment></element-citation></ref><ref id="pntd.0000758-Sulston1"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sulston</surname><given-names>JE</given-names></name><name><surname>Schierenberg</surname><given-names>E</given-names></name><name><surname>White</surname><given-names>JG</given-names></name><name><surname>Thomson</surname><given-names>JN</given-names></name></person-group><year>1983</year><article-title>The embryonic cell lineage of the nematode Caenorhabditis
elegans.</article-title><source>Dev Biol</source><volume>100</volume><fpage>64</fpage><lpage>119</lpage><pub-id pub-id-type="pmid">6684600</pub-id></element-citation></ref><ref id="pntd.0000758-Chisholm1"><label>34</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Chisholm</surname><given-names>AD</given-names></name><name><surname>Hardin</surname><given-names>J</given-names></name></person-group><year>2005</year><source>Epidermal morphogenesis</source><publisher-name>WormBook</publisher-name><fpage>1</fpage><lpage>22</lpage></element-citation></ref><ref id="pntd.0000758-Schaner1"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schaner</surname><given-names>CE</given-names></name><name><surname>Deshpande</surname><given-names>G</given-names></name><name><surname>Schedl</surname><given-names>PD</given-names></name><name><surname>Kelly</surname><given-names>WG</given-names></name></person-group><year>2003</year><article-title>A conserved chromatin architecture marks and maintains the
restricted germ cell lineage in worms and flies.</article-title><source>Dev Cell</source><volume>5</volume><fpage>747</fpage><lpage>757</lpage><pub-id pub-id-type="pmid">14602075</pub-id></element-citation></ref><ref id="pntd.0000758-Pinskaya1"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pinskaya</surname><given-names>M</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>Histone H3 Lysine 4 di-methylation: a novel mark for
transcriptional fidelity?</article-title><source>Epigenetics</source><volume>4</volume><fpage>302</fpage><lpage>306</lpage><pub-id pub-id-type="pmid">19633430</pub-id></element-citation></ref><ref id="pntd.0000758-Albertson1"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Albertson</surname><given-names>R</given-names></name><name><surname>Casper-Lindley</surname><given-names>C</given-names></name><name><surname>Cao</surname><given-names>J</given-names></name><name><surname>Tram</surname><given-names>U</given-names></name><name><surname>Sullivan</surname><given-names>W</given-names></name></person-group><year>2009</year><article-title>Symmetric and asymmetric mitotic segregation patterns influence
Wolbachia distribution in host somatic tissue.</article-title><source>J Cell Sci</source><volume>122</volume><fpage>4570</fpage><lpage>4583</lpage><pub-id pub-id-type="pmid">19934219</pub-id></element-citation></ref><ref id="pntd.0000758-Sato1"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sato</surname><given-names>H</given-names></name><name><surname>Kusel</surname><given-names>JR</given-names></name><name><surname>Thornhill</surname><given-names>J</given-names></name></person-group><year>2002</year><article-title>Functional visualization of the excretory system of adult
Schistosoma mansoni by the fluorescent marker resorufin.</article-title><source>Parasitology</source><volume>125</volume><fpage>527</fpage><lpage>535</lpage><pub-id pub-id-type="pmid">12553571</pub-id></element-citation></ref><ref id="pntd.0000758-Ferree1"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ferree</surname><given-names>PM</given-names></name><name><surname>Frydman</surname><given-names>HM</given-names></name><name><surname>Li</surname><given-names>JM</given-names></name><name><surname>Cao</surname><given-names>J</given-names></name><name><surname>Wieschaus</surname><given-names>E</given-names></name><etal></etal></person-group><year>2005</year><article-title>Wolbachia utilizes host microtubules and Dynein for anterior
localization in the Drosophila oocyte.</article-title><source>PLoS Pathog</source><volume>1</volume><fpage>e14</fpage><pub-id pub-id-type="pmid">16228015</pub-id></element-citation></ref><ref id="pntd.0000758-Schneider1"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schneider</surname><given-names>SQ</given-names></name><name><surname>Bowerman</surname><given-names>B</given-names></name></person-group><year>2003</year><article-title>Cell polarity and the cytoskeleton in the Caenorhabditis elegans
zygote.</article-title><source>Annu Rev Genet</source><volume>37</volume><fpage>221</fpage><lpage>249</lpage><pub-id pub-id-type="pmid">14616061</pub-id></element-citation></ref><ref id="pntd.0000758-Goldstein3"><label>41</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goldstein</surname><given-names>B</given-names></name><name><surname>Macara</surname><given-names>IG</given-names></name></person-group><year>2007</year><article-title>The PAR proteins: fundamental players in animal cell
polarization.</article-title><source>Dev Cell</source><volume>13</volume><fpage>609</fpage><lpage>622</lpage><pub-id pub-id-type="pmid">17981131</pub-id></element-citation></ref><ref id="pntd.0000758-Serbus1"><label>42</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Serbus</surname><given-names>LR</given-names></name><name><surname>Sullivan</surname><given-names>W</given-names></name></person-group><year>2007</year><article-title>A cellular basis for Wolbachia recruitment to the host germline.</article-title><source>PLoS Pathog</source><volume>3</volume><fpage>e190</fpage><pub-id pub-id-type="pmid">18085821</pub-id></element-citation></ref><ref id="pntd.0000758-Serbus2"><label>43</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Serbus</surname><given-names>LR</given-names></name><name><surname>Casper-Lindley</surname><given-names>C</given-names></name><name><surname>Landmann</surname><given-names>F</given-names></name><name><surname>Sullivan</surname><given-names>W</given-names></name></person-group><year>2008</year><article-title>The Genetic and Cell Biology of Wolbachia-host Interactions.</article-title><source>Annu Rev Genet</source></element-citation></ref><ref id="pntd.0000758-Hunter1"><label>44</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hunter</surname><given-names>CP</given-names></name><name><surname>Kenyon</surname><given-names>C</given-names></name></person-group><year>1996</year><article-title>Spatial and temporal controls target pal-1
blastomere-specification activity to a single blastomere lineage in C.
elegans embryos.</article-title><source>Cell</source><volume>87</volume><fpage>217</fpage><lpage>226</lpage><pub-id pub-id-type="pmid">8861906</pub-id></element-citation></ref><ref id="pntd.0000758-Baugh1"><label>45</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baugh</surname><given-names>LR</given-names></name><name><surname>Hill</surname><given-names>AA</given-names></name><name><surname>Claggett</surname><given-names>JM</given-names></name><name><surname>Hill-Harfe</surname><given-names>K</given-names></name><name><surname>Wen</surname><given-names>JC</given-names></name><etal></etal></person-group><year>2005</year><article-title>The homeodomain protein PAL-1 specifies a lineage-specific
regulatory network in the C. elegans embryo.</article-title><source>Development</source><volume>132</volume><fpage>1843</fpage><lpage>1854</lpage><pub-id pub-id-type="pmid">15772128</pub-id></element-citation></ref><ref id="pntd.0000758-Casiraghi1"><label>46</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Casiraghi</surname><given-names>M</given-names></name><name><surname>McCall</surname><given-names>JW</given-names></name><name><surname>Simoncini</surname><given-names>L</given-names></name><name><surname>Kramer</surname><given-names>LH</given-names></name><name><surname>Sacchi</surname><given-names>L</given-names></name><etal></etal></person-group><year>2002</year><article-title>Tetracycline treatment and sex-ratio distortion: a role for
Wolbachia in the moulting of filarial nematodes?</article-title><source>Int J Parasitol</source><volume>32</volume><fpage>1457</fpage><lpage>1468</lpage><pub-id pub-id-type="pmid">12392911</pub-id></element-citation></ref><ref id="pntd.0000758-Sakaguchi1"><label>47</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sakaguchi</surname><given-names>Y</given-names></name><name><surname>Tada</surname><given-names>I</given-names></name><name><surname>Ash</surname><given-names>LR</given-names></name><name><surname>Aoki</surname><given-names>Y</given-names></name></person-group><year>1983</year><article-title>Karyotypes of Brugia pahangi and Brugia malayi (Nematoda:
Filarioidea).</article-title><source>J Parasitol</source><volume>69</volume><fpage>1090</fpage><lpage>1093</lpage><pub-id pub-id-type="pmid">6674460</pub-id></element-citation></ref><ref id="pntd.0000758-Ercan1"><label>48</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ercan</surname><given-names>S</given-names></name><name><surname>Lieb</surname><given-names>JD</given-names></name></person-group><year>2009</year><article-title>C. elegans dosage compensation: a window into mechanisms of
domain-scale gene regulation.</article-title><source>Chromosome Res</source><volume>17</volume><fpage>215</fpage><lpage>227</lpage><pub-id pub-id-type="pmid">19308702</pub-id></element-citation></ref><ref id="pntd.0000758-Chuang1"><label>49</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chuang</surname><given-names>PT</given-names></name><name><surname>Lieb</surname><given-names>JD</given-names></name><name><surname>Meyer</surname><given-names>BJ</given-names></name></person-group><year>1996</year><article-title>Sex-specific assembly of a dosage compensation complex on the
nematode X chromosome.</article-title><source>Science</source><volume>274</volume><fpage>1736</fpage><lpage>1739</lpage><pub-id pub-id-type="pmid">8939870</pub-id></element-citation></ref><ref id="pntd.0000758-Lieb1"><label>50</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lieb</surname><given-names>JD</given-names></name><name><surname>Capowski</surname><given-names>EE</given-names></name><name><surname>Meneely</surname><given-names>P</given-names></name><name><surname>Meyer</surname><given-names>BJ</given-names></name></person-group><year>1996</year><article-title>DPY-26, a link between dosage compensation and meiotic chromosome
segregation in the nematode.</article-title><source>Science</source><volume>274</volume><fpage>1732</fpage><lpage>1736</lpage><pub-id pub-id-type="pmid">8939869</pub-id></element-citation></ref><ref id="pntd.0000758-Bird1"><label>51</label><element-citation publication-type="other"><person-group person-group-type="author"><name><surname>Bird</surname><given-names>AFB</given-names></name><name><surname>Bird</surname><given-names>J</given-names></name></person-group><year>1991</year><comment>The structure of Nematodes: Academic Press</comment></element-citation></ref><ref id="pntd.0000758-Howells1"><label>52</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Howells</surname><given-names>RE</given-names></name><name><surname>Chen</surname><given-names>SN</given-names></name></person-group><year>1981</year><article-title>Brugia pahangi: feeding and nutrient uptake in vitro and in vivo.</article-title><source>Exp Parasitol</source><volume>51</volume><fpage>42</fpage><lpage>58</lpage><pub-id pub-id-type="pmid">7461090</pub-id></element-citation></ref><ref id="pntd.0000758-Ghedin1"><label>53</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ghedin</surname><given-names>E</given-names></name><name><surname>Hailemariam</surname><given-names>T</given-names></name><name><surname>Depasse</surname><given-names>JV</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Oksov</surname><given-names>Y</given-names></name><etal></etal></person-group><year>2009</year><article-title>Brugia malayi Gene Expression in Response to the Targeting of the
Wolbachia Endosymbiont by Tetracycline Treatment.</article-title><source>PLoS Negl Trop Dis</source><volume>3</volume><fpage>e525</fpage><pub-id pub-id-type="pmid">19806204</pub-id></element-citation></ref><ref id="pntd.0000758-Bandi2"><label>54</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bandi</surname><given-names>C</given-names></name><name><surname>McCall</surname><given-names>JW</given-names></name><name><surname>Genchi</surname><given-names>C</given-names></name><name><surname>Corona</surname><given-names>S</given-names></name><name><surname>Venco</surname><given-names>L</given-names></name><etal></etal></person-group><year>1999</year><article-title>Effects of tetracycline on the filarial worms Brugia pahangi and
Dirofilaria immitis and their bacterial endosymbionts Wolbachia.</article-title><source>Int J Parasitol</source><volume>29</volume><fpage>357</fpage><lpage>364</lpage><pub-id pub-id-type="pmid">10221636</pub-id></element-citation></ref><ref id="pntd.0000758-Hoerauf3"><label>55</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoerauf</surname><given-names>A</given-names></name><name><surname>Volkmann</surname><given-names>L</given-names></name><name><surname>Nissen-Paehle</surname><given-names>K</given-names></name><name><surname>Schmetz</surname><given-names>C</given-names></name><name><surname>Autenrieth</surname><given-names>I</given-names></name><etal></etal></person-group><year>2000</year><article-title>Targeting of Wolbachia endobacteria in Litomosoides sigmodontis:
comparison of tetracyclines with chloramphenicol, macrolides and
ciprofloxacin.</article-title><source>Trop Med Int Health</source><volume>5</volume><fpage>275</fpage><lpage>279</lpage></element-citation></ref><ref id="pntd.0000758-Bennuru1"><label>56</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bennuru</surname><given-names>S</given-names></name><name><surname>Semnani</surname><given-names>R</given-names></name><name><surname>Meng</surname><given-names>Z</given-names></name><name><surname>Ribeiro</surname><given-names>JM</given-names></name><name><surname>Veenstra</surname><given-names>TD</given-names></name><etal></etal></person-group><year>2009</year><article-title>Brugia malayi excreted/secreted proteins at the host/parasite
interface: stage- and gender-specific proteomic profiling.</article-title><source>PLoS Negl Trop Dis</source><volume>3</volume><fpage>e410</fpage><pub-id pub-id-type="pmid">19352421</pub-id></element-citation></ref><ref id="pntd.0000758-Hoerauf4"><label>57</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoerauf</surname><given-names>A</given-names></name></person-group><year>2003</year><article-title>Control of filarial infections: not the beginning of the end, but
more research is needed.</article-title><source>Curr Opin Infect Dis</source><volume>16</volume><fpage>403</fpage><lpage>410</lpage><pub-id pub-id-type="pmid">14501992</pub-id></element-citation></ref><ref id="pntd.0000758-Kozek2"><label>58</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kozek</surname><given-names>WJ</given-names></name></person-group><year>2005</year><article-title>What is new in the Wolbachia/Dirofilaria interaction?</article-title><source>Vet Parasitol</source><volume>133</volume><fpage>127</fpage><lpage>132</lpage><pub-id pub-id-type="pmid">16198819</pub-id></element-citation></ref><ref id="pntd.0000758-Brattig2"><label>59</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brattig</surname><given-names>NW</given-names></name><name><surname>Buttner</surname><given-names>DW</given-names></name><name><surname>Hoerauf</surname><given-names>A</given-names></name></person-group><year>2001</year><article-title>Neutrophil accumulation around Onchocerca worms and chemotaxis of
neutrophils are dependent on Wolbachia endobacteria.</article-title><source>Microbes Infect</source><volume>3</volume><fpage>439</fpage><lpage>446</lpage><pub-id pub-id-type="pmid">11377205</pub-id></element-citation></ref><ref id="pntd.0000758-GilletteFerguson2"><label>60</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gillette-Ferguson</surname><given-names>I</given-names></name><name><surname>Hise</surname><given-names>AG</given-names></name><name><surname>McGarry</surname><given-names>HF</given-names></name><name><surname>Turner</surname><given-names>J</given-names></name><name><surname>Esposito</surname><given-names>A</given-names></name><etal></etal></person-group><year>2004</year><article-title>Wolbachia-induced neutrophil activation in a mouse model of
ocular onchocerciasis (river blindness).</article-title><source>Infect Immun</source><volume>72</volume><fpage>5687</fpage><lpage>5692</lpage><pub-id pub-id-type="pmid">15385467</pub-id></element-citation></ref><ref id="pntd.0000758-GilletteFerguson3"><label>61</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gillette-Ferguson</surname><given-names>I</given-names></name><name><surname>Daehnel</surname><given-names>K</given-names></name><name><surname>Hise</surname><given-names>AG</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Carlson</surname><given-names>E</given-names></name><etal></etal></person-group><year>2007</year><article-title>Toll-like receptor 2 regulates CXC chemokine production and
neutrophil recruitment to the cornea in Onchocerca
volvulus/Wolbachia-induced keratitis.</article-title><source>Infect Immun</source><volume>75</volume><fpage>5908</fpage><lpage>5915</lpage><pub-id pub-id-type="pmid">17875630</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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