Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Double-Stranded RNA Uptake through Topical Application, Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in <italic>Diaphorina citri</italic></title><author><name sortKey="Killiny, Nabil" sort="Killiny, Nabil" uniqKey="Killiny N" first="Nabil" last="Killiny">Nabil Killiny</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hajeri, Subhas" sort="Hajeri, Subhas" uniqKey="Hajeri S" first="Subhas" last="Hajeri">Subhas Hajeri</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tiwari, Siddharth" sort="Tiwari, Siddharth" uniqKey="Tiwari S" first="Siddharth" last="Tiwari">Siddharth Tiwari</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Gowda, Siddarame" sort="Gowda, Siddarame" uniqKey="Gowda S" first="Siddarame" last="Gowda">Siddarame Gowda</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Stelinski, Lukasz L" sort="Stelinski, Lukasz L" uniqKey="Stelinski L" first="Lukasz L." last="Stelinski">Lukasz L. Stelinski</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25330026</idno><idno type="pmc">4203802</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203802</idno><idno type="RBID">PMC:4203802</idno><idno type="doi">10.1371/journal.pone.0110536</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000213</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Double-Stranded RNA Uptake through Topical Application, Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in <italic>Diaphorina citri</italic></title><author><name sortKey="Killiny, Nabil" sort="Killiny, Nabil" uniqKey="Killiny N" first="Nabil" last="Killiny">Nabil Killiny</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hajeri, Subhas" sort="Hajeri, Subhas" uniqKey="Hajeri S" first="Subhas" last="Hajeri">Subhas Hajeri</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tiwari, Siddharth" sort="Tiwari, Siddharth" uniqKey="Tiwari S" first="Siddharth" last="Tiwari">Siddharth Tiwari</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Gowda, Siddarame" sort="Gowda, Siddarame" uniqKey="Gowda S" first="Siddarame" last="Gowda">Siddarame Gowda</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Stelinski, Lukasz L" sort="Stelinski, Lukasz L" uniqKey="Stelinski L" first="Lukasz L." last="Stelinski">Lukasz L. Stelinski</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P<sub>450</sub> genes family 4 (<italic>CYP4</italic>) in <italic>Diaphorina citri</italic>. We previously reported that these <italic>CYP4</italic> genes are associated with the development of insecticide resistance in <italic>D. citri</italic>. We targeted five <italic>CYP4</italic> genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five <italic>CYP4</italic> genes as a result of dsRNA topically applied to the thoracic region of <italic>D. citri</italic> when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P<sub>450</sub> proteins (45 kDa) in adult <italic>D. citri</italic> treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for <italic>D. citri</italic> treated with dsRNA that targeted specific <italic>CYP4</italic> genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of <italic>D. citri</italic> and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in <italic>D. citri</italic> populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of <italic>D. citri</italic> by targeting physiologically critical genes involved in growth and development.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Huvenne, H" uniqKey="Huvenne H">H Huvenne</name></author><author><name sortKey="Smagghe, G" uniqKey="Smagghe G">G Smagghe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Izant, Jg" uniqKey="Izant J">JG Izant</name></author><author><name sortKey="Weintraub, H" uniqKey="Weintraub H">H Weintraub</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fire, A" uniqKey="Fire A">A Fire</name></author><author><name sortKey="Xu, S" uniqKey="Xu S">S Xu</name></author><author><name sortKey="Montgomery, Mk" uniqKey="Montgomery M">MK Montgomery</name></author><author><name sortKey="Kostas, Sa" uniqKey="Kostas S">SA Kostas</name></author><author><name sortKey="Driver, Se" uniqKey="Driver S">SE Driver</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tomoyasu, Y" uniqKey="Tomoyasu Y">Y Tomoyasu</name></author><author><name sortKey="Miller, Sc" uniqKey="Miller S">SC Miller</name></author><author><name sortKey="Tomita, S" uniqKey="Tomita S">S Tomita</name></author><author><name sortKey="Schoppmeier, M" uniqKey="Schoppmeier M">M Schoppmeier</name></author><author><name sortKey="Grossmann, D" uniqKey="Grossmann D">D Grossmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wuriyanghan, H" uniqKey="Wuriyanghan H">H Wuriyanghan</name></author><author><name sortKey="Rosa, C" uniqKey="Rosa C">C Rosa</name></author><author><name sortKey="Falk, Bw" uniqKey="Falk B">BW Falk</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, N" uniqKey="Yu N">N Yu</name></author><author><name sortKey="Christiaens, O" uniqKey="Christiaens O">O Christiaens</name></author><author><name sortKey="Liu, J" uniqKey="Liu J">J Liu</name></author><author><name sortKey="Niu, J" uniqKey="Niu J">J Niu</name></author><author><name sortKey="Cappelle, K" uniqKey="Cappelle K">K Cappelle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gordon, Kh" uniqKey="Gordon K">KH Gordon</name></author><author><name sortKey="Waterhouse, Pm" uniqKey="Waterhouse P">PM Waterhouse</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Price, Drg" uniqKey="Price D">DRG Price</name></author><author><name sortKey="Gatehouse, Ja" uniqKey="Gatehouse J">JA Gatehouse</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhou, X" uniqKey="Zhou X">X Zhou</name></author><author><name sortKey="Wheeler, Mm" uniqKey="Wheeler M">MM Wheeler</name></author><author><name sortKey="Oi, Fm" uniqKey="Oi F">FM Oi</name></author><author><name sortKey="Scharf, Me" uniqKey="Scharf M">ME Scharf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Y" uniqKey="Wang Y">Y Wang</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author><author><name sortKey="Li, H" uniqKey="Li H">H Li</name></author><author><name sortKey="Miao, X" uniqKey="Miao X">X Miao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, F" uniqKey="Zhu F">F Zhu</name></author><author><name sortKey="Xu, J" uniqKey="Xu J">J Xu</name></author><author><name sortKey="Palli, R" uniqKey="Palli R">R Palli</name></author><author><name sortKey="Ferguson, J" uniqKey="Ferguson J">J Ferguson</name></author><author><name sortKey="Palli, Sr" uniqKey="Palli S">SR Palli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rangasamy, M" uniqKey="Rangasamy M">M Rangasamy</name></author><author><name sortKey="Siegfried, Bd" uniqKey="Siegfried B">BD Siegfried</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Feyereisen, R" uniqKey="Feyereisen R">R Feyereisen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Scott, Jg" uniqKey="Scott J">JG Scott</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author><author><name sortKey="Rogers, Me" uniqKey="Rogers M">ME Rogers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Clayson, Pj" uniqKey="Clayson P">PJ Clayson</name></author><author><name sortKey="Kuhns, Eh" uniqKey="Kuhns E">EH Kuhns</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Feyereisen, R" uniqKey="Feyereisen R">R Feyereisen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, X" uniqKey="Li X">X Li</name></author><author><name sortKey="Schuler, Ma" uniqKey="Schuler M">MA Schuler</name></author><author><name sortKey="Berenbaum, Mr" uniqKey="Berenbaum M">MR Berenbaum</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Gondhalekar, A" uniqKey="Gondhalekar A">A Gondhalekar</name></author><author><name sortKey="Mann, Rs" uniqKey="Mann R">RS Mann</name></author><author><name sortKey="Scharf, Me" uniqKey="Scharf M">ME Scharf</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Halbert, Se" uniqKey="Halbert S">SE Halbert</name></author><author><name sortKey="Manjunath, Kl" uniqKey="Manjunath K">KL Manjunath</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Manjunath, Kl" uniqKey="Manjunath K">KL Manjunath</name></author><author><name sortKey="Halbert, Se" uniqKey="Halbert S">SE Halbert</name></author><author><name sortKey="Ramadugu, C" uniqKey="Ramadugu C">C Ramadugu</name></author><author><name sortKey="Webb, S" uniqKey="Webb S">S Webb</name></author><author><name sortKey="Lee, Rf" uniqKey="Lee R">RF Lee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Setamou, M" uniqKey="Setamou M">M Sétamou</name></author><author><name sortKey="Rodriguez, D" uniqKey="Rodriguez D">D Rodriguez</name></author><author><name sortKey="Saldana, R" uniqKey="Saldana R">R Saldana</name></author><author><name sortKey="Schwarzlose, G" uniqKey="Schwarzlose G">G Schwarzlose</name></author><author><name sortKey="Parlang, D" uniqKey="Parlang D">D Parlang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Mann, Rs" uniqKey="Mann R">RS Mann</name></author><author><name sortKey="Rogers, Me" uniqKey="Rogers M">ME Rogers</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Grafton Cardwell, E" uniqKey="Grafton Cardwell E">E Grafton-Cardwell</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author><author><name sortKey="Stansly, Pa" uniqKey="Stansly P">PA Stansly</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qureshi, Ja" uniqKey="Qureshi J">JA Qureshi</name></author><author><name sortKey="Stansly, Pa" uniqKey="Stansly P">PA Stansly</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Serikawa, Rh" uniqKey="Serikawa R">RH Serikawa</name></author><author><name sortKey="Backus, Ea" uniqKey="Backus E">EA Backus</name></author><author><name sortKey="Rogers, Me" uniqKey="Rogers M">ME Rogers</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Pelz Stelinski, K" uniqKey="Pelz Stelinski K">K Pelz-Stelinski</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tiwari, S" uniqKey="Tiwari S">S Tiwari</name></author><author><name sortKey="Pelz Stelinski, K" uniqKey="Pelz Stelinski K">K Pelz-Stelinski</name></author><author><name sortKey="Mann, Rs" uniqKey="Mann R">RS Mann</name></author><author><name sortKey="Stelinski, Ll" uniqKey="Stelinski L">LL Stelinski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="El Shesheny, I" uniqKey="El Shesheny I">I El-Shesheny</name></author><author><name sortKey="Hajeri, S" uniqKey="Hajeri S">S Hajeri</name></author><author><name sortKey="El Hawary, I" uniqKey="El Hawary I">I El-Hawary</name></author><author><name sortKey="Gowda, S" uniqKey="Gowda S">S Gowda</name></author><author><name sortKey="Killiny, N" uniqKey="Killiny N">N Killiny</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brogdon, Wg" uniqKey="Brogdon W">WG Brogdon</name></author><author><name sortKey="Mcallister, Jc" uniqKey="Mcallister J">JC McAllister</name></author><author><name sortKey="Vulule, J" uniqKey="Vulule J">J Vulule</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Livak, Kj" uniqKey="Livak K">KJ Livak</name></author><author><name sortKey="Schmittgen, Td" uniqKey="Schmittgen T">TD Schmittgen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wheeler, Mm" uniqKey="Wheeler M">MM Wheeler</name></author><author><name sortKey="Tarver, Mr" uniqKey="Tarver M">MR Tarver</name></author><author><name sortKey="Coy, Mr" uniqKey="Coy M">MR Coy</name></author><author><name sortKey="Scharf, Me" uniqKey="Scharf M">ME Scharf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bradford, Mm" uniqKey="Bradford M">MM Bradford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hunter, Cp" uniqKey="Hunter C">CP Hunter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rajagopal, R" uniqKey="Rajagopal R">R Rajagopal</name></author><author><name sortKey="Sivakumar, S" uniqKey="Sivakumar S">S Sivakumar</name></author><author><name sortKey="Agrawal, N" uniqKey="Agrawal N">N Agrawal</name></author><author><name sortKey="Malhotra, P" uniqKey="Malhotra P">P Malhotra</name></author><author><name sortKey="Bhatnagar, Rk" uniqKey="Bhatnagar R">RK Bhatnagar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Surakasi, Vp" uniqKey="Surakasi V">VP Surakasi</name></author><author><name sortKey="Mohamed, Aam" uniqKey="Mohamed A">AAM Mohamed</name></author><author><name sortKey="Kim, Y" uniqKey="Kim Y">Y Kim</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Whyard, S" uniqKey="Whyard S">S Whyard</name></author><author><name sortKey="Singh, Ad" uniqKey="Singh A">AD Singh</name></author><author><name sortKey="Wong, S" uniqKey="Wong S">S Wong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mao, Yb" uniqKey="Mao Y">YB Mao</name></author><author><name sortKey="Cai, Wj" uniqKey="Cai W">WJ Cai</name></author><author><name sortKey="Wang, Jw" uniqKey="Wang J">JW Wang</name></author><author><name sortKey="Hong, Gj" uniqKey="Hong G">GJ Hong</name></author><author><name sortKey="Tao, Xy" uniqKey="Tao X">XY Tao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baum, Ja" uniqKey="Baum J">JA Baum</name></author><author><name sortKey="Bogaert, T" uniqKey="Bogaert T">T Bogaert</name></author><author><name sortKey="Clinton, W" uniqKey="Clinton W">W Clinton</name></author><author><name sortKey="Heck, Gr" uniqKey="Heck G">GR Heck</name></author><author><name sortKey="Feldmann, P" uniqKey="Feldmann P">P Feldmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Clemens, Mj" uniqKey="Clemens M">MJ Clemens</name></author><author><name sortKey="Elia, A" uniqKey="Elia A">A Elia</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 One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</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">25330026</article-id><article-id pub-id-type="pmc">4203802</article-id><article-id pub-id-type="publisher-id">PONE-D-14-33137</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0110536</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Agriculture</subject><subj-group><subject>Pest Control</subject></subj-group></subj-group><subj-group><subject>Biochemistry</subject></subj-group><subj-group><subject>Biotechnology</subject></subj-group><subj-group><subject>Genetics</subject></subj-group></subj-group></article-categories><title-group><article-title>Double-Stranded RNA Uptake through Topical Application, Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in <italic>Diaphorina citri</italic></article-title><alt-title alt-title-type="running-head">Silencing of Cytochrome P<sub>450</sub> in <italic>Diaphorina citri</italic></alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Killiny</surname><given-names>Nabil</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>Hajeri</surname><given-names>Subhas</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Tiwari</surname><given-names>Siddharth</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Gowda</surname><given-names>Siddarame</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Stelinski</surname><given-names>Lukasz L.</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 Entomology and Nematology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, Florida, United States of America</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Hansen</surname><given-names>Immo A.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>New Mexico State University, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>nabikilliny@ufl.edu</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: NK SH ST SG LLS. Performed the experiments: NK SH SG. Analyzed the data: NK SH ST SG LLS. Contributed reagents/materials/analysis tools: NK SH ST SG LLS. Contributed to the writing of the manuscript: NK SH ST SG LLS.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>20</day><month>10</month><year>2014</year></pub-date><volume>9</volume><issue>10</issue><elocation-id>e110536</elocation-id><history><date date-type="received"><day>1</day><month>8</month><year>2014</year></date><date date-type="accepted"><day>16</day><month>9</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Killiny et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P<sub>450</sub> genes family 4 (<italic>CYP4</italic>) in <italic>Diaphorina citri</italic>. We previously reported that these <italic>CYP4</italic> genes are associated with the development of insecticide resistance in <italic>D. citri</italic>. We targeted five <italic>CYP4</italic> genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five <italic>CYP4</italic> genes as a result of dsRNA topically applied to the thoracic region of <italic>D. citri</italic> when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P<sub>450</sub> proteins (45 kDa) in adult <italic>D. citri</italic> treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for <italic>D. citri</italic> treated with dsRNA that targeted specific <italic>CYP4</italic> genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of <italic>D. citri</italic> and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in <italic>D. citri</italic> populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of <italic>D. citri</italic> by targeting physiologically critical genes involved in growth and development.</p></abstract><funding-group><funding-statement>This project was supported by a grant to LS from the Citrus Research and Development Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="8"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.</p></notes></front><body><sec id="s1"><title>Introduction</title><p>RNA interference (RNAi) is a promising tool for studying functional genomics in eukaryotes and insects in particular <xref rid="pone.0110536-Scharf1" ref-type="bibr">[1]</xref>, <xref rid="pone.0110536-Huvenne1" ref-type="bibr">[2]</xref>. Anti-sense (nonsense) RNA strand transcription has been used for over three decades to inhibit gene activity <xref rid="pone.0110536-Izant1" ref-type="bibr">[3]</xref>. The efficacy of anti-sense silencing depends on hybridization between the injected RNA and an endogenous messenger. Since the discovery of double-stranded RNA (dsRNA) mediated gene-specific silencing in the nematode, <italic>Caenorhabditis elegans</italic> (Maupas) <xref rid="pone.0110536-Fire1" ref-type="bibr">[4]</xref>, dsRNA-mediated RNAi has been employed with various insects to silence specific genes <xref rid="pone.0110536-Huvenne1" ref-type="bibr">[2]</xref>. RNAi has been widely used in various insect orders, including Coleoptera, Dictyoptera, Diptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Neuroptera, and Orthoptera <xref rid="pone.0110536-Tomoyasu1" ref-type="bibr">[5]</xref>, <xref rid="pone.0110536-Wuriyanghan1" ref-type="bibr">[6]</xref>, <xref rid="pone.0110536-Yu1" ref-type="bibr">[7]</xref>. The systemic nature of dsRNA-mediated RNAi has allowed this tool to be used in the management of various insect pests <xref rid="pone.0110536-Gordon1" ref-type="bibr">[8]</xref>–<xref rid="pone.0110536-Rangasamy1" ref-type="bibr">[13]</xref>.</p><p>The cytochrome P<sub>450</sub> monooxygenases are an important group of enzymes that are involved in the metabolism of xenobiotic compounds in insects. This group of enzymes is associated with insecticide resistance and metabolism of a wide range of endogenous and exogenous compounds that includes hormones, pheromones, insecticides, and plant secondary compounds in insects <xref rid="pone.0110536-Hodgson1" ref-type="bibr">[14]</xref>–<xref rid="pone.0110536-Tiwari2" ref-type="bibr">[18]</xref>. Overtranscription of families 4, 6, 9, and 12 has been frequently linked to insecticide metabolism and resistance <xref rid="pone.0110536-Feyereisen2" ref-type="bibr">[19]</xref>–<xref rid="pone.0110536-Tiwari4" ref-type="bibr">[22]</xref>.</p><p>The Asian citrus psyllid, <italic>Diaphorina citri</italic> Kuwayama (Hemiptera: Psyllidae), is perhaps the most destructive pest of citrus, mainly because it is a vector for the putative causal agent of huanglongbing (HLB), <italic>Candidatus</italic> Liberibacter asiaticus (<italic>C</italic>Las) <xref rid="pone.0110536-Halbert1" ref-type="bibr">[23]</xref>. HLB is a deadly citrus disease with no known cure <xref rid="pone.0110536-Halbert1" ref-type="bibr">[23]</xref>, <xref rid="pone.0110536-Manjunath1" ref-type="bibr">[24]</xref>. Currently, the main tools that limit the spread of the disease are insecticides to manage the vector <xref rid="pone.0110536-Tiwari2" ref-type="bibr">[18]</xref>, <xref rid="pone.0110536-Stamou1" ref-type="bibr">[25]</xref>, <xref rid="pone.0110536-Tiwari5" ref-type="bibr">[26]</xref>. <italic>D. citri</italic> are susceptible to several insecticide classes, which includes the pyrethroids, organophosphates, carbamates, neonicotinoids, insect growth regulators, horticultural oils, and lipid synthesis inhibitors <xref rid="pone.0110536-GraftonCardwell1" ref-type="bibr">[27]</xref>. Foliar treatments may suppress populations for 3 weeks following application <xref rid="pone.0110536-GraftonCardwell1" ref-type="bibr">[27]</xref>. Broad-spectrum insecticides (pyrethroids, organophosphates, and neonicotinoids) are more effective against <italic>D. citri</italic> than IGRs or oils, and insecticide use against <italic>D. citri</italic> is most effective when populations are not actively reproducing <xref rid="pone.0110536-Qureshi1" ref-type="bibr">[28]</xref>. Systemic soil-applied insecticides provide a much longer duration of population control (months) than foliar insecticides (weeks) <xref rid="pone.0110536-GraftonCardwell1" ref-type="bibr">[27]</xref>. The neonicitonoids have been the main class of effective systemic insecticides for <italic>D. citri</italic> control during the past decade <xref rid="pone.0110536-GraftonCardwell1" ref-type="bibr">[27]</xref>. Systemic neonicotinoids are particularly effective in protecting young trees as they mature into production <xref rid="pone.0110536-Serikawa1" ref-type="bibr">[29]</xref>.</p><p>Intense insecticide use has led to the development of varying levels of insecticide resistance in populations of <italic>D. citri</italic> in Florida, USA <xref rid="pone.0110536-Tiwari5" ref-type="bibr">[26]</xref>. This is particularly concerning for the neonicotinoid class, since these are the main current tools for protecting young trees from <italic>C</italic>Las infection <xref rid="pone.0110536-Serikawa1" ref-type="bibr">[29]</xref>. A metabolic mechanism for the evolution of insecticide resistance in populations of <italic>D. citri</italic>, particularly for neonicotinoids, is supported by increased activities of detoxifying enzymes and overexpression of Cytochrome P<sub>450</sub> genes family 4 (<italic>CYP4</italic>) <xref rid="pone.0110536-Tiwari1" ref-type="bibr">[17]</xref>, <xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref>, <xref rid="pone.0110536-Tiwari5" ref-type="bibr">[26]</xref>, <xref rid="pone.0110536-Tiwari6" ref-type="bibr">[30]</xref>, <xref rid="pone.0110536-Tiwari7" ref-type="bibr">[31]</xref>. In the present study, we targeted the abovementioned <italic>CYP4</italic> genes for silencing by topical application of specific dsRNA to the thorax of newly emerged <italic>D. citri</italic> adults. Additionally, we tested the effect of dsRNA treatment on insecticide resistance by comparing mortality of known susceptible and resistant populations of <italic>D. citri</italic>.</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title><sec id="s2a"><title>Insect populations</title><p>A laboratory susceptible population (LS) of <italic>D. citri</italic> was maintained in a greenhouse at the Citrus Research and Education Center, Lake Alfred, Florida. The culture was established in 2000 using field populations from Polk County, Florida and maintained on sweet orange (<italic>Citrus sinensis</italic> (L.) Osbeck) without exposure to insecticides in a greenhouse at 27–28°C, with 60–65% relative humidity and a 14∶10 (light:dark) photocycle hours. Three field populations of <italic>D. citri</italic> were collected from commercial citrus groves in Florida during 2013. The populations were collected <underline>with permissions</underline> from private groves. Name of groves, counties, and GPS coordinates are as the following: <italic>i</italic>) GapWay Groves (Private managed grove), Polk County (PL) (28° 05′ 40.14″ N; 81° 43′ 19.03″ W); <italic>ii</italic>) Winter Garden, Conserve II (Private managed grove), Lake County (LA) (28° 27′ 52.17″ N; 81° 39′ 31.69″ W); and <italic>iii)</italic> Uncle Matt’s Organic (Organically managed grove), Lake County (OG) (28° 31′ 00.88″ N; 81° 40′ 01.90″ W). Adults were collected using sweep nets and aspirators, transferred to the laboratory, released onto citrus plants within Plexiglas cages (40×40×40 cm), and used in bioassays shortly thereafter.</p></sec><sec id="s2b"><title>Constructing dsRNA</title><p>A consensus sequence, derived from five previously published <italic>CYP4</italic> sequences <xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref>, was used to design <italic>CYP4</italic>-specific primers (<xref ref-type="table" rid="pone-0110536-t001">Table 1</xref>). The <italic>CYP4</italic>-specific primers were tailed with a T7 promoter sequence to generate sense and antisense transcripts separately.</p><table-wrap id="pone-0110536-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.t001</object-id><label>Table 1</label><caption><title>Primers used in this investigation.</title></caption><alternatives><graphic id="pone-0110536-t001-1" xlink:href="pone.0110536.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Purpose</td><td align="left" rowspan="1" colspan="1">Gene</td><td align="left" rowspan="1" colspan="1">Sequence</td><td align="left" rowspan="1" colspan="1">Reference</td></tr></thead><tbody><tr><td colspan="4" align="left" rowspan="1"><italic>ds-RNA synthesis</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>CYP4</bold></td><td align="left" rowspan="1" colspan="1">Forward CACG<italic>ttaattaa</italic><underline>ACGTTCATGTTCGAGGGGCACGATACAACAAC</underline></td><td align="left" rowspan="1" colspan="1">This</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>Sense</bold></td><td align="left" rowspan="1" colspan="1">Reverse GAGC<italic>aggcct</italic>GAAGGGTACATAGGAGTAAGGATGACGTTTCTG</td><td align="left" rowspan="1" colspan="1">investigation</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>CYP4 Antisense</bold></td><td align="left" rowspan="1" colspan="1">Forward GCAGCA<italic>TAATACGACTCACTATAGGGAGA</italic>GAAGGGTACATAGGAGTAAGGATGACGTTTCTG Reverse <named-content content-type="gene">ACGTTCATGTTCGAGGGGCACGATACAACAAC</named-content></td><td align="left" rowspan="1" colspan="1">This investigation</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>GFP</bold></td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">GCGAACTAGTATGGCTAGCAAAGGAGAAGAACTTTTCACTG</named-content></td><td align="left" rowspan="1" colspan="1">El-Shesheny</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">GAGACCGCGGCTACCCCTCGAGTTATTTGTAGAGCTCATC</named-content></td><td align="left" rowspan="1" colspan="1">et al. <xref rid="pone.0110536-ElShesheny1" ref-type="bibr">[32]</xref></td></tr><tr><td colspan="4" align="left" rowspan="1"><italic>Gene expression</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td colspan="3" align="left" rowspan="1"><italic>CYP4 genes</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CYP4C67</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">TGGAACGTGTCATCAAGGAG</named-content></td><td align="left" rowspan="1" colspan="1">Tiwari et al.</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">CCGGATTGAAACTGTTAGGC</named-content></td><td align="left" rowspan="1" colspan="1"><xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CYP4DA1</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">AGTGGTGTCGGAAATTGAGG</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">GTTCGAGCCACCTGGAGATA</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CYPC68</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">CTAGCCTGGACCCTCTTCCT</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">ACCCTCCCTATGAACGGAAC</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CYPG70</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">GCCGGAAGTTCTTTCTTCCT</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">TAACGGGTACTGGTGGGAAC</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">CYPDB1</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">CTGTACGCTCTGGGACATCA</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">TTGAGCGGTGCATAGAGTTG</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td colspan="3" align="left" rowspan="1"><italic>Reference genes</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">α-tubulin</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">CAGGTCTTGTGTGGGACGTA</named-content></td><td align="left" rowspan="1" colspan="1">Tiwari et al. <xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">GGCCACAGTTTGTTTCTTGC</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Actin</td><td align="left" rowspan="1" colspan="1">Forward <named-content content-type="gene">CCCTGGACTTTGAACAGGAA</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Reverse <named-content content-type="gene">CTCGTGGATACCGCAAGATT</named-content></td><td align="left" rowspan="1" colspan="1"></td></tr></tbody></table></alternatives></table-wrap><p>Total RNA isolation was performed on groups of 40–50 psyllids using the SV total RNA isolation kit (Promega, Madison, WI, USA). One microgram of RNA was used to synthesize cDNA using the <italic>CYP4</italic>-specific reverse primers and iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Sense and antisense PCR products were generated in separate PCR reaction using specific combination of primers (<xref ref-type="table" rid="pone-0110536-t001">Table 1</xref>). To generate plus-sense transcripts, sense primer with T7 promoter sequence and regular antisense primer were used; while to generate antisense transcripts, regular sense primer and antisense primer with T7 promoter sequence were used. Sense and antisense transcripts were annealed by denaturing at 70°C for 10 min, followed by slowly cooling to room temperature for 20 min. To eliminate DNA template and single-stranded RNA, dsRNA was treated with DNase I and RNase A. The dsRNA was then purified of proteins and free nucleotides using the phenol-chloroform purification method. The amount of purified dsRNA was measured with a NanoDrop Spectrophotometer. We used dsRNA-<italic>gfp</italic> as an irrelevant dsRNA (control). dsRNA-<italic>gfp</italic> was produced as described above. Green fluorescent protein (GFP) mRNA is 732 bp in length. Specific primers (<xref ref-type="table" rid="pone-0110536-t001">Table 1</xref>) were used to amplify the full-length of GFP gene by using TMV-30BGFP according to El-Shesheny et al. <xref rid="pone.0110536-ElShesheny1" ref-type="bibr">[32]</xref>.</p></sec><sec id="s2c"><title><italic>D. citri</italic> treatment with dsRNA</title><p>Purified dsRNA was serially diluted using RNase-free water to obtain desired concentrations of dsRNA. Three concentrations of dsRNA (50, 75, and 100 ng/µl) and a control (0 ng/µl) were used to treat <italic>D. citri</italic> adults. <italic>D. citri</italic> adults were anaesthetized under CO<sub>2</sub> within a few hours of eclosion. A 0.2 µl droplet containing10, 15, or 20 ng of dsRNA was topically applied to the ventral side of the thorax using a 10 µl Hamilton syringe. To investigate the effect of dsRNA- <italic>P<sub>450</sub></italic> on gene and protein expression and enzymatic activity, treated adults were placed into 60 mm plastic disposable Petri dishes that were lined with citrus leaf disks, as a food source, over agar beds as described in Tiwari et al. <xref rid="pone.0110536-Tiwari1" ref-type="bibr">[17]</xref>. Petri dishes with treated adults were kept at 25±1°C and 50±5% RH, with a 14∶10 h light:dark photoperiod, in a growth chamber for 72 h. Insects were collected and stored in −20°C until use. dsRNA-<italic>gfp</italic> was used as a non-relevant dsRNA control.</p></sec><sec id="s2d"><title>Cytochrome <italic>P<sub>450</sub></italic> (general oxidase) assay</title><p>The activity of cytochrome <italic>P<sub>450</sub></italic> was quantified and expressed in terms of general oxidase level, which is an indirect measure of cytochrome <italic>P<sub>450</sub></italic> by using heme peroxidation as described in Tiwari et al. <xref rid="pone.0110536-Tiwari7" ref-type="bibr">[31]</xref>. This method has been considered a reliable tool for comparing differences in general oxidase levels based on hemoprotein levels. Because heme constitutes the majority of cytochrome <italic>P<sub>450</sub></italic> in non-blood-fed insects, quantification of heme activity has been used to compare the levels of cytochrome <italic>P<sub>450</sub></italic> on the basis of general oxidase levels <xref rid="pone.0110536-Brogdon1" ref-type="bibr">[33]</xref>. In brief, heme peroxidase activity was measured using 3,3′5,5′-tetra-methylbenzidine (TMBZ) (Sigma Aldrich) as the substrate. Five replicates, each consisting of three insects, were performed for each treatment.</p></sec><sec id="s2e"><title>Gene expression analysis</title><p>Live adult <italic>D. citri</italic> from each treatment were subjected to RNA isolation and cDNA synthesis. RNA isolations were performed in three biological replicates using the SV total RNA isolation kit (Promega, Madison, WI, USA). The quantity and quality of RNA from each sample was measured on a NanoDrop 1000 Spectrophotometer using the absorbance at 260 nm and the A260/A280 ratio, respectively. Subsequently, cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) for each replicate within each treatment. Quantitative real-time PCR (qPCR) was performed using iQ SYBR Green Supermix with an iCycler iQ real-time PCR detection system (Bio-Rad). Primers for five <italic>CYP4</italic> genes, <italic>Alpha-tubulin</italic> and the endogenous gene, <italic>Actin</italic>, were used to measure the gene expression of cytochrome <italic>P<sub>450</sub></italic>, as described in Tiwari et al. <xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref> (<xref ref-type="table" rid="pone-0110536-t001">Table 1</xref>). Six biological replicates were performed for each treatment. The production of gene-specific products and absence of ‘primer dimers’ was verified by 1% agarose electrophoresis in TAE buffer with ethidium bromide staining.</p><p>The 2<sup>−ΔΔ<italic>C</italic>T</sup> method was used to compare the relative expression of the consensus sequence among PCR products derived from the three dsRNA concentrations and control treatments <xref rid="pone.0110536-Livak1" ref-type="bibr">[34]</xref>. This was done by first normalizing the expression level of dsRNA treated samples to <italic>Actin</italic><xref rid="pone.0110536-Tiwari3" ref-type="bibr">[21]</xref> gene expression, followed by normalization to the treatment giving the lowest gene expression. <italic>Alpha-tubulin</italic> was used as a non-targeted gene (control). Five biological replicates, each consisting of three insects and three technical replicates, were performed for each treatment.</p></sec><sec id="s2f"><title>Western blot assay</title><p>Subcellular protein fractions were extracted using the methods described by Wheeler et al. <xref rid="pone.0110536-Wheeler1" ref-type="bibr">[35]</xref> from adults in each treatment. The protein concentration was determined by the Bradford method <xref rid="pone.0110536-Bradford1" ref-type="bibr">[36]</xref> using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with ovalbumin as the standard. Since cytochrome P<sub>450</sub> proteins were clearly detected in the microsomal protein <xref rid="pone.0110536-Tiwari4" ref-type="bibr">[22]</xref>, we used the microsomal fractions to perform the Western blot analysis, as described by Tiwari et al. <xref rid="pone.0110536-Tiwari4" ref-type="bibr">[22]</xref>.</p></sec><sec id="s2g"><title>Survival assay</title><p>The survival assay was carried out on <italic>D. citri</italic> treated with dsRNA-<italic>gfp</italic>, dsRNA- <italic>P<sub>450</sub></italic>, or RNase-free water as a control. Insects were placed on an autoclaved clear plant tissue culture container (75×75×100 mm) lined with 0.5 mm filter paper saturated with 20% sucrose. Fifty insects were placed per container and five replicates were performed for each treatment. Live insects were counted daily.</p></sec><sec id="s2h"><title>Residual activity of dsRNA- <italic>P<sub>450</sub></italic></title><p>In order to assess the duration of the RNAi effect, 70 insects treated with RNase-free water or dsRNA-<italic>P450</italic> (20 ng/insect) were placed into plant tissue culture containers with filter paper saturated with 20% sucrose. Samples consisting of three insects were taken daily and kept at −20°C. Cytochrome <italic>P<sub>450</sub></italic> (general oxidase) activity was measured in all samples as described above. Five replicates were performed for both treatments.</p></sec><sec id="s2i"><title>Pesticide application</title><p>To investigate the effect of treatment with dsRNA- <italic>P<sub>450</sub></italic> on insecticide resistance, <italic>D. citri</italic> adults were treated with the dsRNAs (20 ng/insect) as described above. <italic>D. citri</italic> were initially maintained on Petri dishes with untreated citrus leaf disks for 72 h and thereafter transferred to new Petri dishes that contained leaf discs treated with insecticide solution. Briefly, the leaf disks (60 mm diameter) were excised, dipped in the insecticide solution made in acetone for 30 s, and allowed to air dry in a fume hood for 1 h prior to placement into the Petri dishes as described by Tiwari et al. <xref rid="pone.0110536-Tiwari5" ref-type="bibr">[26]</xref>. We used analytical-grade imidacloprid at the LD<sub>50</sub> dosage (0.02 ng Al/µl acetone) previously determined by Tiwari et al. <xref rid="pone.0110536-Tiwari5" ref-type="bibr">[26]</xref>. The mortality of <italic>D. citri</italic> adults was assessed after 24 h. dsRNA-<italic>gfp</italic> was used as a negative control for dsRNA- <italic>P<sub>450</sub></italic>. Fives replicates (Petri dishes), with five insects each, were performed for each of the four <italic>D. citri</italic> populations tested. Each population was subjected to four different treatments: <italic>D. citri</italic> treated with RNase-free water on 1) RNase-free water- or 2) imidacloprid-treated disks, as well as <italic>D. citri</italic> treated with dsRNA- <italic>P<sub>450</sub></italic> on 3) RNase-free water- or 4) imidacloprid-treated leaf disks.</p></sec><sec id="s2j"><title>Statistical analysis</title><p>All analyses were performed using SPSS version 19.0. Survival was calculated during the interval from initial treatment to when all insects died. Overall survival (OS) curves were obtained using the Kaplan-Meier method and comparisons were made using log rank and Wilcoxon tests. Analysis of variance (ANOVA) was used to compare: <italic>i</italic>) Calculated <italic>D. citri</italic> lifespans between various treatments, <italic>ii</italic>) The effect of dsRNA treatments on <italic>CYP4</italic> activity and insecticide resistance, and <italic>iii</italic>) The duration of the RNAi effect. Post hoc pairwise comparisons between treatments were performed with the Tukey honestly significant difference test. Statistical significance was established as <italic>P</italic><0.05.</p></sec></sec><sec id="s3"><title>Results</title><sec id="s3a"><title>Treatment with dsRNA-<italic>P<sub>450</sub></italic> causes down regulation of five <italic>CYP4</italic> genes</title><p>Relative expression levels for the five <italic>CYP4</italic> genes were compared between dsRNA- <italic>P</italic><bold><italic><sub>450</sub></italic></bold>, dsRNA-<italic>gfp</italic>-treated and control psyllids (<xref ref-type="fig" rid="pone-0110536-g001">Figure 1</xref>). Treatment with dsRNA- <italic>P</italic><bold><italic><sub>450</sub></italic></bold> caused reduced expression of the five <italic>CYP4</italic> genes. The effect of dsRNA- <italic>P</italic><bold><italic><sub>450</sub></italic></bold> was positively correlated with the quantity applied per treatment. The expression level of α-tubulin (non-target gene) remained constant among all treatments indicating the specificity of dsRNA- <italic>P</italic><bold><italic><sub>450</sub></italic></bold> to <italic>CYP4</italic> genes. In contrast, there was no effect of the dsRNA-<italic>gfp</italic> control treatments targeting irrelevant psyllid genes on expression levels of <italic>CYP4</italic> genes. The greatest reduction in expression level was found with <italic>CYP4G70</italic>, while the lowest effect was with <italic>CYP4C68</italic>. This observation may help in evaluating gene candidates for RNAi technology for <italic>D. citri</italic>.</p><fig id="pone-0110536-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.g001</object-id><label>Figure 1</label><caption><title>Relative expression levels of the five <italic>CYP4</italic> genes targeted by RNAi in <italic>Diaphorina citri</italic> adults 72 h after treatment with dsRNA.</title><p>Ct values were first normalized to the endogenous control gene <italic>Actin</italic> followed by normalization to the treatment giving the lowest gene expression using the 2<sup>−ΔΔCT</sup> method. Standard deviations were calculated based on three independent experiments, each with three technical replicates. Alpha-tubulin was used as a non-target gene control. dsRNA-<italic>gfp</italic> treatment was used as a control targeting an irrelevant gene.</p></caption><graphic xlink:href="pone.0110536.g001"></graphic></fig></sec><sec id="s3b"><title>Treatment with dsRNA-<italic>P<sub>450</sub></italic> reduces the protein expression and the enzymatic activity of <italic>CYP4</italic></title><p>We investigated the effect of treating <italic>D. citri</italic> with dsRNA on general oxidase activity. The activities were similar for all doses of dsRNA-<italic>gfp</italic> treatment, while reduced when <italic>D. citri</italic> were treated with dsRNA-<italic>P<sub>450</sub></italic>. The activity decreased as the concentration of applied dsRNA- <italic>P<sub>450</sub></italic> was increased. Additionally, Western blots performed using the microsomal fractions revealed the presence of a band corresponding to a 45 kDa protein that cross-reacted with the primary antibody of cytochrome P<sub>450</sub> protein. Twenty-five micrograms of microsomal proteins for each treatment were used to perform the Western blot which indicated the highest amount of cytochrome P<sub>450</sub> proteins (detoxifying enzymes) in <italic>D. citri</italic> treated with 0 ng/µl of dsRNA, followed by <italic>D. citri</italic> treated with 10, 15, and 20 ng/insect of dsRNA (<xref ref-type="fig" rid="pone-0110536-g002">Figure 2</xref>). There was no signal detected in <italic>D. citri</italic> treated with 20 ng/insect of dsRNA. Expression levels of target <italic>CYP4</italic> and oxidase activity, as a result of dsRNA treatment, directly correlated with the protein expression in adults treated with dsRNA.</p><fig id="pone-0110536-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.g002</object-id><label>Figure 2</label><caption><title>Cytochrome P<sub>450</sub> (general oxidase) activity and protein expression in dsRNA-treated <italic>D.citri</italic>.</title><p>A) Box blot representing general oxidase activity. Boxes indicate the interquartile range, including 50% of results, and the mid-horizontal lines represent the median; different letters above deviation error bars represent significant differences between treatments (<italic>P</italic><0.05). B) Protein analysis of dsRNA-treated and control <italic>D. citri</italic> using Western blot. Western blot was performed using the microsomal proteins prepared from adults treated with three quantities of dsRNA and a control. dsRNA-<italic>gfp</italic> treatment was used as a control targeting an irrelevant gene.</p></caption><graphic xlink:href="pone.0110536.g002"></graphic></fig></sec><sec id="s3c"><title>Silencing of <italic>CYP4</italic> reduced the lifespan of <italic>D. citri</italic></title><p>Survival of <italic>D. citri</italic> was quantified following treatment with dsRNA-<italic>gfp</italic>, dsRNA- <italic>P<sub>450</sub></italic>, and the control (water). The experiment was conducted under the conditions described earlier (<xref ref-type="fig" rid="pone-0110536-g003">Figure 3A</xref>). In this experiment, a 20 ng/insect concentration was used for both dsRNA-<italic>gfp</italic> and dsRNA- <italic>P<sub>450</sub></italic>. A Kaplan-Meier survival plot indicated significant differences among all treatments (log rank = 154.63, <italic>P</italic><0.001). No significant differences in survival were found between the control and dsRNA-<italic>gfp-</italic>treated <italic>D. citri</italic> (log rank = 1.54, <italic>P</italic> = 0.64). The lifespan in dsRNA- <italic>P<sub>450</sub></italic>-treated <italic>D. citri</italic> was significantly shorter than that observed for other treatments. Mean lifespans for the treatments are presented in <xref ref-type="fig" rid="pone-0110536-g003">Figure 3B</xref>. This suggests that reduced expression of <italic>CYP4</italic> genes shortens the lifespan of <italic>D. citri</italic>.</p><fig id="pone-0110536-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.g003</object-id><label>Figure 3</label><caption><title>Effect of dsRNA- <italic>P<sub>450</sub></italic> on <italic>D. citri</italic> survival.</title><p>A) Kaplan-Meier survival curve showing the effect of dsRNA- <italic>P<sub>450</sub></italic> treatment on lifespan of <italic>D. citri.</italic> B) Average life span of <italic>D. citri.</italic> Bars represent the standard deviations; different letters above error bars represent significant differences between treatments (<italic>P</italic><0.05). dsRNA-<italic>gfp</italic> treatment was used as a control targeting an irrelevant gene.</p></caption><graphic xlink:href="pone.0110536.g003"></graphic></fig></sec><sec id="s3d"><title>Residual activity of dsRNA- <italic>P<sub>450</sub></italic> treatment</title><p>The residual activity of dsRNA- <italic>P<sub>450</sub></italic> was measured at 20 ng/insect concentration. We used general oxidase activity as an indicator for the residual of dsRNA- <italic>P<sub>450</sub></italic>. We compared oxidase activity between the control and dsRNA- <italic>P<sub>450</sub></italic>-treated <italic>D. citri</italic> daily after the treatment application. Oxidase activity was significantly reduced following application of dsRNA- <italic>P<sub>450</sub></italic> for up to 8 days (<xref ref-type="fig" rid="pone-0110536-g004">Figure 4</xref>).</p><fig id="pone-0110536-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.g004</object-id><label>Figure 4</label><caption><title><italic>P<sub>450</sub></italic> as measured by general oxidase activity.</title><p>Asterisks indicate significant differences between dsRNA- <italic>P<sub>450</sub></italic> treated and non-treated <italic>D. citri,</italic> while ns indicates no significant differences(<italic>P</italic><0.05).</p></caption><graphic xlink:href="pone.0110536.g004"></graphic></fig></sec><sec id="s3e"><title>Silencing of <italic>CYP4</italic> increased insecticide susceptibility</title><p>In order to determine the effect of dsRNA- <italic>P<sub>450</sub></italic> on insecticide susceptibility of <italic>D. citri</italic>, we used imidacloprid at the LD<sub>50</sub> dosage. Two susceptible and two resistant populations were used in this experiment (<xref ref-type="fig" rid="pone-0110536-g005">Figure 5</xref>). <italic>D. citri</italic> that were treated with RNase-free water and then exposed to leaf discs treated with imidacloprid exhibited differing susceptibilities, depending on the population tested (<xref ref-type="fig" rid="pone-0110536-g005">Figure 5</xref>). Specifically, the two populations from commercially managed citrus groves that had received imidacloprid treatment over the previous several years (LA (Lake County) and PL (Polk County)) were less susceptible to imidacloprid at the LD<sub>50</sub> dosage than <italic>D. citri</italic> collected from our Laboratory Susceptible culture (LS) and from the Organic Grove (OG) where imidacloprid had not been used previously (<xref ref-type="fig" rid="pone-0110536-g005">Figure 5</xref>). Mortality of <italic>D. citri</italic> exposed to imidacloprid after treatment with dsRNA- <italic>P<sub>450</sub></italic> was increased for each of the four populations as compared with the water control (<xref ref-type="fig" rid="pone-0110536-g005">Figure 5</xref>). Mortality of <italic>D. citri</italic> from the two resistant populations (LA, PL), at the LD<sub>50</sub> dosage of imidacloprid, was significantly higher after treatment with dsRNA- <italic>P<sub>450</sub></italic> as compared with the water control (<xref ref-type="fig" rid="pone-0110536-g005">Figure 5</xref>). Given that dsRNA- <italic>P<sub>450</sub></italic> also increased mortality of <italic>D. citri</italic> from the two susceptible populations (LS and OG) further suggests that cytochrome P<sub>450</sub> is implicated in imidacloprid resistance in <italic>D. citri</italic>.</p><fig id="pone-0110536-g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0110536.g005</object-id><label>Figure 5</label><caption><title>Effect of dsRNA- <italic>P<sub>450</sub></italic> treatment on <italic>D. citri</italic> susceptibility to imidacloprid using the LD<sub>50</sub> dosage.</title><p>A) Illustration of the protocol used to test the effect. B) Percent mortality of <italic>D. citri</italic> after exposure to imidacloprid for 24 h. Insects were maintained on non-treated leaf discs for 48 h after the dsRNA- <italic>P<sub>450</sub></italic> treatment prior to exposure to imidacloprid. LS: Laboratory susceptible population. OG: Susceptible population collected from an organic grove. PL: Resistant population collected from commercially managed Polk County, Florida. LA: Resistant population collected from commercially managed Lake County, Florida. Asterisks indicate significant differences, while ns indicates no significant differences (<italic>P</italic><0.05).</p></caption><graphic xlink:href="pone.0110536.g005"></graphic></fig></sec></sec><sec id="s4"><title>Discussion</title><p>Most insect RNAi studies have relied on the delivery of specific dsRNA through either microinjections <xref rid="pone.0110536-Fire1" ref-type="bibr">[4]</xref> or ingestion through feeding <xref rid="pone.0110536-Zhou1" ref-type="bibr">[10]</xref>, <xref rid="pone.0110536-Rangasamy1" ref-type="bibr">[13]</xref>. Each of these methods has advantages and disadvantages. The microinjection method requires intense training and is a notably time-consuming technique. In addition, optimization is required for volume selection, place of injection, and needle size for successful dsRNA injection into the insect body <xref rid="pone.0110536-Yu1" ref-type="bibr">[7]</xref>. Delivery of dsRNA through ingestion also has limitations, such as reduced effectiveness for inducing RNAi <xref rid="pone.0110536-Hunter1" ref-type="bibr">[37]</xref>, reduced efficacy of dsRNA due to the unfavorable gut environment <xref rid="pone.0110536-Rajagopal1" ref-type="bibr">[38]</xref>, and difficulties in quantifying the amount of dsRNA ingested <xref rid="pone.0110536-Surakasi1" ref-type="bibr">[39]</xref>. The current work highlights a novel method of dsRNA delivery through topical microapplication to the abdomen of adult <italic>D. citri</italic>. The ventral microapplication allows dsRNA uptake through the exoskeleton of insect. The uptake occurs via the intersegmental membranes. This investigation describes a relatively easy and efficient method for delivering and allowing the dsRNA to enter the insect’s body to induce RNAi. This method has also been described for <italic>D. citri</italic> nymphs with high efficiency of activity <xref rid="pone.0110536-ElShesheny1" ref-type="bibr">[32]</xref>. Also, a similar delivery method was reported to induce RNAi in <italic>Ostrinia nubilalis</italic> larvae <xref rid="pone.0110536-Wang1" ref-type="bibr">[11]</xref>.</p><p>A dosage as low as 50 ng/µl of dsRNA down-regulated the expression of the consensus sequence derived from five <italic>CYP4</italic> genes from <italic>D. citri,</italic> as verified by qPCR and Western blot. The lifespan of <italic>D. citri</italic> following dsRNA treatment was statistically shorter as compared with untreated controls in the current investigation. In another example, mortality in <italic>O. nubilalis</italic> larvae ranged between approximately 40–50% following topical treatment with dsRNA <xref rid="pone.0110536-Wang1" ref-type="bibr">[11]</xref>. The mortality, coupled with the lack of any other abnormality observed in the dsRNA-treated adult <italic>D. citri</italic>, suggests that the <italic>CYP4</italic> specific dsRNA are highly target specific. Target specificity of dsRNA is also useful considering the potential for dsRNA exposure to non-target organisms under field conditions. Designing target specific dsRNA is not uncommon; species-specific dsRNA has been shown to work like an insecticide by killing specifically targeted insect pests <xref rid="pone.0110536-Whyard1" ref-type="bibr">[40]</xref>. The low concentrations needed for induction of RNAi and the highly specific nature of dsRNA suggest it might be a tool for managing insecticide resistance in <italic>D. citri</italic>. Our results indicate that dsRNA- <italic>P<sub>450</sub></italic> reduced oxidase activity, which presumably increased insecticide susceptibility in both resistant and susceptible populations of <italic>D. citri</italic>. In comparison, dsRNA-<italic>gfp</italic> (our negative control treatment) did not affect <italic>CYP4</italic> gene expression or oxidase activity. These findings indicate specificity of RNAi for <italic>D. citri</italic> with the genes targeted in the present investigation.</p><p>An important challenge for the application of dsRNA for practical pest control is developing a delivery method for commercial field deployment. Another practical limitation of RNAi that needs to be addressed is that large quantities of dsRNA are expensive to produce. Currently, we are working on inserting the previously described dsRNA into citrus plants for direct ingestion by <italic>D</italic>. <italic>citri</italic> during feeding. Delivery of dsRNA through transgenic plants (Plant mediated RNAi) has been achieved in <italic>Helicoverpa armigera</italic> and <italic>Diabrotica vergifera vergifera</italic><xref rid="pone.0110536-Mao1" ref-type="bibr">[41]</xref>, <xref rid="pone.0110536-Baum1" ref-type="bibr">[42]</xref>. The absence of interferon-regulated innate immunity pathways in insects allows the possibility of employing longer dsRNA for maximal RNAi <xref rid="pone.0110536-Clemens1" ref-type="bibr">[43]</xref>. Another potentially feasible way of delivering dsRNA would be to incorporate target-specific dsRNA into bacteria with an appropriate transfection reagent and then spraying the transformed bacteria onto citrus trees. However, future work is needed to evaluate the most efficient transfection reagents and bacterial formulations to prevent the breakdown of dsRNA under field conditions. Once the entire genome of <italic>D. citri</italic> is sequenced, this delivery method could be a convenient way to conduct high-throughput loss-of-function research for determining gene functions. In addition, the current results suggest that further work is needed to understand the mechanism of dsRNA entry into cells following topical application of dsRNA onto <italic>D. citri</italic> to induce RNAi.</p></sec></body><back><ack><p>We acknowledge Ian Jackson for <italic>D. citri</italic> collection and technical assistance.</p></ack><ref-list><title>References</title><ref id="pone.0110536-Scharf1"><label>1</label><mixed-citation publication-type="book">Scharf ME, Zhou X, Schwinghammer MA (2008) Application of RNA interference in functional genomics studies of a social insect. In: Barik S, editor. Methods in molecular biology, siRNA, shRNA and miRNA protocols. Vol. 442. Totowa, New Jersey: Humana Press. p. 205–229.</mixed-citation></ref><ref id="pone.0110536-Huvenne1"><label>2</label><mixed-citation publication-type="journal"><name><surname>Huvenne</surname><given-names>H</given-names></name>, <name><surname>Smagghe</surname><given-names>G</given-names></name> (<year>2010</year>) <article-title>Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review</article-title>. <source>J Insect Physiol</source><volume>56</volume>: <fpage>227</fpage>–<lpage>235</lpage>.<pub-id pub-id-type="pmid">19837076</pub-id></mixed-citation></ref><ref id="pone.0110536-Izant1"><label>3</label><mixed-citation publication-type="journal"><name><surname>Izant</surname><given-names>JG</given-names></name>, <name><surname>Weintraub</surname><given-names>H</given-names></name> (<year>1984</year>) <article-title>Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysis</article-title>. <source>Cell</source><volume>36</volume>: <fpage>1007</fpage>–<lpage>1015</lpage>.<pub-id pub-id-type="pmid">6323013</pub-id></mixed-citation></ref><ref id="pone.0110536-Fire1"><label>4</label><mixed-citation publication-type="journal"><name><surname>Fire</surname><given-names>A</given-names></name>, <name><surname>Xu</surname><given-names>S</given-names></name>, <name><surname>Montgomery</surname><given-names>MK</given-names></name>, <name><surname>Kostas</surname><given-names>SA</given-names></name>, <name><surname>Driver</surname><given-names>SE</given-names></name>, <etal>et al</etal> (<year>1998</year>) <article-title>Potent and specific genetic interference by double-stranded RNA in <italic>Caenorhabditis elegans</italic></article-title>. <source>Nature</source><volume>391</volume>: <fpage>806</fpage>–<lpage>811</lpage>.<pub-id pub-id-type="pmid">9486653</pub-id></mixed-citation></ref><ref id="pone.0110536-Tomoyasu1"><label>5</label><mixed-citation publication-type="journal"><name><surname>Tomoyasu</surname><given-names>Y</given-names></name>, <name><surname>Miller</surname><given-names>SC</given-names></name>, <name><surname>Tomita</surname><given-names>S</given-names></name>, <name><surname>Schoppmeier</surname><given-names>M</given-names></name>, <name><surname>Grossmann</surname><given-names>D</given-names></name>, <etal>et al</etal> (<year>2008</year>) <article-title>Exploring systemic RNA interference in insects: A genome wide survey for RNAi genes in Tribolium</article-title>. <source>Genome Biol</source><volume>9</volume>: <fpage>R10</fpage>.<pub-id pub-id-type="pmid">18201385</pub-id></mixed-citation></ref><ref id="pone.0110536-Wuriyanghan1"><label>6</label><mixed-citation publication-type="journal"><name><surname>Wuriyanghan</surname><given-names>H</given-names></name>, <name><surname>Rosa</surname><given-names>C</given-names></name>, <name><surname>Falk</surname><given-names>BW</given-names></name> (<year>2011</year>) <article-title>Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, <italic>Bactericerca cockerelli</italic></article-title>. <source>PLoS ONE</source><volume>6</volume>: <fpage>e27736</fpage>.<pub-id pub-id-type="pmid">22110747</pub-id></mixed-citation></ref><ref id="pone.0110536-Yu1"><label>7</label><mixed-citation publication-type="journal"><name><surname>Yu</surname><given-names>N</given-names></name>, <name><surname>Christiaens</surname><given-names>O</given-names></name>, <name><surname>Liu</surname><given-names>J</given-names></name>, <name><surname>Niu</surname><given-names>J</given-names></name>, <name><surname>Cappelle</surname><given-names>K</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>Delivery of dsRNA for RNAi in insect: An overview and future directions</article-title>. <source>Insect Sci</source><volume>20</volume>: <fpage>4</fpage>–<lpage>14</lpage>.<pub-id pub-id-type="pmid">23955821</pub-id></mixed-citation></ref><ref id="pone.0110536-Gordon1"><label>8</label><mixed-citation publication-type="journal"><name><surname>Gordon</surname><given-names>KH</given-names></name>, <name><surname>Waterhouse</surname><given-names>PM</given-names></name> (<year>2007</year>) <article-title>RNAi for insect-proof plants</article-title>. <source>Nat Biotechnol</source><volume>25</volume>: <fpage>1231</fpage>–<lpage>1232</lpage>.<pub-id pub-id-type="pmid">17989682</pub-id></mixed-citation></ref><ref id="pone.0110536-Price1"><label>9</label><mixed-citation publication-type="journal"><name><surname>Price</surname><given-names>DRG</given-names></name>, <name><surname>Gatehouse</surname><given-names>JA</given-names></name> (<year>2008</year>) <article-title>RNAi-mediated crop protection against insects</article-title>. <source>Trends Biotechnol</source><volume>26</volume>: <fpage>393</fpage>–<lpage>400</lpage>.<pub-id pub-id-type="pmid">18501983</pub-id></mixed-citation></ref><ref id="pone.0110536-Zhou1"><label>10</label><mixed-citation publication-type="journal"><name><surname>Zhou</surname><given-names>X</given-names></name>, <name><surname>Wheeler</surname><given-names>MM</given-names></name>, <name><surname>Oi</surname><given-names>FM</given-names></name>, <name><surname>Scharf</surname><given-names>ME</given-names></name> (<year>2008</year>) <article-title>RNA interference in the termite <italic>Reticulitermes flavipes</italic> through ingestion of double-stranded RNA</article-title>. <source>Insect Biochem Molec Biol</source><volume>38</volume>: <fpage>805</fpage>–<lpage>815</lpage>.<pub-id pub-id-type="pmid">18625404</pub-id></mixed-citation></ref><ref id="pone.0110536-Wang1"><label>11</label><mixed-citation publication-type="journal"><name><surname>Wang</surname><given-names>Y</given-names></name>, <name><surname>Zhang</surname><given-names>H</given-names></name>, <name><surname>Li</surname><given-names>H</given-names></name>, <name><surname>Miao</surname><given-names>X</given-names></name> (<year>2011</year>) <article-title>Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control</article-title>. <source>PLoS ONE</source><volume>6(4)</volume>: <fpage>e18644</fpage>.<pub-id pub-id-type="pmid">21494551</pub-id></mixed-citation></ref><ref id="pone.0110536-Zhu1"><label>12</label><mixed-citation publication-type="journal"><name><surname>Zhu</surname><given-names>F</given-names></name>, <name><surname>Xu</surname><given-names>J</given-names></name>, <name><surname>Palli</surname><given-names>R</given-names></name>, <name><surname>Ferguson</surname><given-names>J</given-names></name>, <name><surname>Palli</surname><given-names>SR</given-names></name> (<year>2011</year>) <article-title>Ingested RNA interference for managing the populations of the Colorado potato beetle, <italic>Leptinotarsa decemlineata</italic></article-title>. <source>Pest Manag Sci</source><volume>67</volume>: <fpage>175</fpage>–<lpage>182</lpage>.<pub-id pub-id-type="pmid">21061270</pub-id></mixed-citation></ref><ref id="pone.0110536-Rangasamy1"><label>13</label><mixed-citation publication-type="journal"><name><surname>Rangasamy</surname><given-names>M</given-names></name>, <name><surname>Siegfried</surname><given-names>BD</given-names></name> (<year>2012</year>) <article-title>Validation of RNA interference in western corn rootworm <italic>Diabrotica virgifera virgifera</italic> LeConte (Coleoptera: Chrysomelidae) adults</article-title>. <source>Pest Manag Sci</source><volume>68</volume>: <fpage>587</fpage>–<lpage>591</lpage>.<pub-id pub-id-type="pmid">22500293</pub-id></mixed-citation></ref><ref id="pone.0110536-Hodgson1"><label>14</label><mixed-citation publication-type="book">Hodgson E (1985) Microsomal monooxygenases. In: Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology, biochemistry, and pharmacology. Vol. 11 Pharmacology. Elmsford, New York: Pergamon. p. 225–322.</mixed-citation></ref><ref id="pone.0110536-Feyereisen1"><label>15</label><mixed-citation publication-type="journal"><name><surname>Feyereisen</surname><given-names>R</given-names></name> (<year>1999</year>) <article-title>Insect P<sub>450</sub> enzymes</article-title>. <source>Annu Rev Entomol</source><volume>44</volume>: <fpage>507</fpage>–<lpage>533</lpage>.<pub-id pub-id-type="pmid">9990722</pub-id></mixed-citation></ref><ref id="pone.0110536-Scott1"><label>16</label><mixed-citation publication-type="journal"><name><surname>Scott</surname><given-names>JG</given-names></name> (<year>1999</year>) <article-title>Cytochromes P<sub>450</sub> and insecticide resistance</article-title>. <source>Insect Biochem Mol Biol</source><volume>29</volume>: <fpage>757</fpage>–<lpage>777</lpage>.<pub-id pub-id-type="pmid">10510498</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari1"><label>17</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name>, <name><surname>Rogers</surname><given-names>ME</given-names></name> (<year>2012</year>) <article-title>Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid</article-title>. <source>J Econ Entomol</source><volume>105</volume>: <fpage>540</fpage>–<lpage>548</lpage>.<pub-id pub-id-type="pmid">22606825</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari2"><label>18</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Clayson</surname><given-names>PJ</given-names></name>, <name><surname>Kuhns</surname><given-names>EH</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name> (<year>2012</year>) <article-title>Effects of buprofezin and diflubenzuron on various developmental stages of Asian citrus psyllid, <italic>Diaphorina citri</italic></article-title>. <source>Pest Manag Sci</source><volume>68</volume>: <fpage>1405</fpage>–<lpage>1412</lpage>.<pub-id pub-id-type="pmid">22653617</pub-id></mixed-citation></ref><ref id="pone.0110536-Feyereisen2"><label>19</label><mixed-citation publication-type="journal"><name><surname>Feyereisen</surname><given-names>R</given-names></name> (<year>2006</year>) <article-title>Evolution of insect P<sub>450</sub></article-title>. <source>Biochem Soc Trans</source><volume>34</volume>: <fpage>1252</fpage>–<lpage>1255</lpage>.<pub-id pub-id-type="pmid">17073796</pub-id></mixed-citation></ref><ref id="pone.0110536-Li1"><label>20</label><mixed-citation publication-type="journal"><name><surname>Li</surname><given-names>X</given-names></name>, <name><surname>Schuler</surname><given-names>MA</given-names></name>, <name><surname>Berenbaum</surname><given-names>MR</given-names></name> (<year>2007</year>) <article-title>Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics</article-title>. <source>Annu Rev Entomol</source><volume>52</volume>: <fpage>231</fpage>–<lpage>253</lpage>.<pub-id pub-id-type="pmid">16925478</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari3"><label>21</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Gondhalekar</surname><given-names>A</given-names></name>, <name><surname>Mann</surname><given-names>RS</given-names></name>, <name><surname>Scharf</surname><given-names>ME</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name> (<year>2011</year>) <article-title>Characterization of five <italic>CYP4</italic> genes of Asian citrus psyllid and their expression levels in <italic>Candidatus</italic> Liberibacter asiaticus infected and uninfected adults</article-title>. <source>Insect Mol Biol</source><volume>20</volume>: <fpage>733</fpage>–<lpage>744</lpage>.<pub-id pub-id-type="pmid">21919983</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari4"><label>22</label><mixed-citation publication-type="other">Tiwari S, Killiny N, Mann RS, Wenninger EJ, Stelinski LL (2012) Abdominal color of the Asian citrus psyllid, <italic>Diaphorina citri</italic>, is associated with susceptibility to various insecticides. Pest Manag Sci. 7 p. (wileyonlinelibrary.com) DOI 10.1002/ps.3407.</mixed-citation></ref><ref id="pone.0110536-Halbert1"><label>23</label><mixed-citation publication-type="journal"><name><surname>Halbert</surname><given-names>SE</given-names></name>, <name><surname>Manjunath</surname><given-names>KL</given-names></name> (<year>2004</year>) <article-title>Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida</article-title>. <source>Fla Entomol</source><volume>87</volume>: <fpage>330</fpage>–<lpage>353</lpage>.</mixed-citation></ref><ref id="pone.0110536-Manjunath1"><label>24</label><mixed-citation publication-type="journal"><name><surname>Manjunath</surname><given-names>KL</given-names></name>, <name><surname>Halbert</surname><given-names>SE</given-names></name>, <name><surname>Ramadugu</surname><given-names>C</given-names></name>, <name><surname>Webb</surname><given-names>S</given-names></name>, <name><surname>Lee</surname><given-names>RF</given-names></name> (<year>2008</year>) <article-title>Detection of ‘<italic>Candidatus</italic> Liberibacter asiaticus’ in <italic>Diaphorina citri</italic> and its importance in the management of citrus Huanglongbing in Florida</article-title>. <source>Phytopathology</source><volume>98</volume>: <fpage>387</fpage>–<lpage>396</lpage>.<pub-id pub-id-type="pmid">18944186</pub-id></mixed-citation></ref><ref id="pone.0110536-Stamou1"><label>25</label><mixed-citation publication-type="journal"><name><surname>Sétamou</surname><given-names>M</given-names></name>, <name><surname>Rodriguez</surname><given-names>D</given-names></name>, <name><surname>Saldana</surname><given-names>R</given-names></name>, <name><surname>Schwarzlose</surname><given-names>G</given-names></name>, <name><surname>Parlang</surname><given-names>D</given-names></name>, <etal>et al</etal> (<year>2010</year>) <article-title>Efficacy and uptake of soil-applied imidacloprid in the control of Asian citrus psyllid and a citrus leafminer, two foliar feeding citrus pests</article-title>. <source>J Econ Entomol</source><volume>103</volume>: <fpage>1711</fpage>–<lpage>1719</lpage>.<pub-id pub-id-type="pmid">21061971</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari5"><label>26</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Mann</surname><given-names>RS</given-names></name>, <name><surname>Rogers</surname><given-names>ME</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name> (<year>2011</year>) <article-title>Insecticide resistance in field populations of Asian citrus psyllid in Florida</article-title>. <source>Pest Manag Sci</source><volume>67</volume>: <fpage>1258</fpage>–<lpage>1268</lpage>.<pub-id pub-id-type="pmid">21538798</pub-id></mixed-citation></ref><ref id="pone.0110536-GraftonCardwell1"><label>27</label><mixed-citation publication-type="journal"><name><surname>Grafton-Cardwell</surname><given-names>E</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name>, <name><surname>Stansly</surname><given-names>PA</given-names></name> (<year>2013</year>) <article-title>Biology and management of Asian citrus psyllid, vector of huanglongbing pathogens</article-title>. <source>Annu Rev Entomol</source><volume>58</volume>: <fpage>413</fpage>–<lpage>432</lpage>.<pub-id pub-id-type="pmid">23317046</pub-id></mixed-citation></ref><ref id="pone.0110536-Qureshi1"><label>28</label><mixed-citation publication-type="journal"><name><surname>Qureshi</surname><given-names>JA</given-names></name>, <name><surname>Stansly</surname><given-names>PA</given-names></name> (<year>2010</year>) <article-title>Dormant season foliar sprays of broad spectrum insecticides: An effective component of integrated management for <italic>Diaphorina citri</italic> (Hemiptera: Psyllidae) in citrus orchards</article-title>. <source>Crop Prot</source><volume>29</volume>: <fpage>860</fpage>–<lpage>866</lpage>.</mixed-citation></ref><ref id="pone.0110536-Serikawa1"><label>29</label><mixed-citation publication-type="journal"><name><surname>Serikawa</surname><given-names>RH</given-names></name>, <name><surname>Backus</surname><given-names>EA</given-names></name>, <name><surname>Rogers</surname><given-names>ME</given-names></name> (<year>2012</year>) <article-title>Effects of soil-applied imidacloprid on Asian citrus psyllid (Hemiptera: Psyllidae) feeding behavior</article-title>. <source>J Econ Entomol</source><volume>105</volume>: <fpage>1492</fpage>–<lpage>1502</lpage>.<pub-id pub-id-type="pmid">23156142</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari6"><label>30</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Pelz-Stelinski</surname><given-names>K</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name> (<year>2011</year>) <article-title>Effect of <italic>Candidatus</italic> Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, <italic>Diaphorina citri</italic>, to selected insecticides</article-title>. <source>Pest Manag Sci</source><volume>67</volume>: <fpage>94</fpage>–<lpage>99</lpage>.<pub-id pub-id-type="pmid">20960471</pub-id></mixed-citation></ref><ref id="pone.0110536-Tiwari7"><label>31</label><mixed-citation publication-type="journal"><name><surname>Tiwari</surname><given-names>S</given-names></name>, <name><surname>Pelz-Stelinski</surname><given-names>K</given-names></name>, <name><surname>Mann</surname><given-names>RS</given-names></name>, <name><surname>Stelinski</surname><given-names>LL</given-names></name> (<year>2011</year>) <article-title>Glutathione S-transferase and cytochrome P<sub>450</sub> (general oxidase) activity levels in <italic>Candidatus</italic> Liberibacter asiaticus-infected and uninfected Asian citrus psyllid (Hemiptera: Psyllidae)</article-title>. <source>Ann Entomol Soc Am</source><volume>104</volume>: <fpage>297</fpage>–<lpage>305</lpage>.</mixed-citation></ref><ref id="pone.0110536-ElShesheny1"><label>32</label><mixed-citation publication-type="journal"><name><surname>El-Shesheny</surname><given-names>I</given-names></name>, <name><surname>Hajeri</surname><given-names>S</given-names></name>, <name><surname>El-Hawary</surname><given-names>I</given-names></name>, <name><surname>Gowda</surname><given-names>S</given-names></name>, <name><surname>Killiny</surname><given-names>N</given-names></name> (<year>2013</year>) <article-title>Silencing abnormal wing disc gene of the Asian Citrus Psyllid, <italic>Diaphorina citri</italic> disrupts adult wing development and increases nymph mortality</article-title>. <source>PLoS ONE</source><volume>8(5)</volume>: <fpage>e65392</fpage>.<pub-id pub-id-type="pmid">23734251</pub-id></mixed-citation></ref><ref id="pone.0110536-Brogdon1"><label>33</label><mixed-citation publication-type="journal"><name><surname>Brogdon</surname><given-names>WG</given-names></name>, <name><surname>McAllister</surname><given-names>JC</given-names></name>, <name><surname>Vulule</surname><given-names>J</given-names></name> (<year>1997</year>) <article-title>Heme peroxidase activity measured in single mosquitoes identifies individuals expressing an elevated oxidase for insecticide resistance</article-title>. <source>J Am Mosquito Contr</source><volume>13</volume>: <fpage>233</fpage>–<lpage>237</lpage>.</mixed-citation></ref><ref id="pone.0110536-Livak1"><label>34</label><mixed-citation publication-type="journal"><name><surname>Livak</surname><given-names>KJ</given-names></name>, <name><surname>Schmittgen</surname><given-names>TD</given-names></name> (<year>2001</year>) <article-title>Analysis of relative gene expression data using realtime quantitative PCR and the 2<sup>−ΔΔC</sup><sub>T</sub> method</article-title>. <source>Methods</source><volume>25</volume>: <fpage>402</fpage>–<lpage>408</lpage>.<pub-id pub-id-type="pmid">11846609</pub-id></mixed-citation></ref><ref id="pone.0110536-Wheeler1"><label>35</label><mixed-citation publication-type="journal"><name><surname>Wheeler</surname><given-names>MM</given-names></name>, <name><surname>Tarver</surname><given-names>MR</given-names></name>, <name><surname>Coy</surname><given-names>MR</given-names></name>, <name><surname>Scharf</surname><given-names>ME</given-names></name> (<year>2010</year>) <article-title>Characterization of four esterase genes and esterase activity from the gut of the termite <italic>Reticulitermes flavipes</italic></article-title>. <source>Arch Insect Biochem Physiol</source><volume>73</volume>: <fpage>30</fpage>–<lpage>48</lpage>.<pub-id pub-id-type="pmid">19802899</pub-id></mixed-citation></ref><ref id="pone.0110536-Bradford1"><label>36</label><mixed-citation publication-type="journal"><name><surname>Bradford</surname><given-names>MM</given-names></name> (<year>1976</year>) <article-title>A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding</article-title>. <source>Anal Biochem</source><volume>72</volume>: <fpage>248</fpage>–<lpage>254</lpage>.<pub-id pub-id-type="pmid">942051</pub-id></mixed-citation></ref><ref id="pone.0110536-Hunter1"><label>37</label><mixed-citation publication-type="journal"><name><surname>Hunter</surname><given-names>CP</given-names></name> (<year>1999</year>) <article-title>Genetics: A touch of elegance with RNAi</article-title>. <source>Curr Biol</source><volume>9</volume>: <fpage>R440</fpage>–<lpage>R442</lpage>.<pub-id pub-id-type="pmid">10375522</pub-id></mixed-citation></ref><ref id="pone.0110536-Rajagopal1"><label>38</label><mixed-citation publication-type="journal"><name><surname>Rajagopal</surname><given-names>R</given-names></name>, <name><surname>Sivakumar</surname><given-names>S</given-names></name>, <name><surname>Agrawal</surname><given-names>N</given-names></name>, <name><surname>Malhotra</surname><given-names>P</given-names></name>, <name><surname>Bhatnagar</surname><given-names>RK</given-names></name> (<year>2002</year>) <article-title>Silencing of midgut aminopeptidase N of <italic>Spodoptera litura</italic> by double-stranded RNA establishes its role as <italic>Bacillus thuringiensis</italic> toxin receptor</article-title>. <source>J Biol Chem</source><volume>277</volume>: <fpage>46849</fpage>–<lpage>46851</lpage>.<pub-id pub-id-type="pmid">12377776</pub-id></mixed-citation></ref><ref id="pone.0110536-Surakasi1"><label>39</label><mixed-citation publication-type="journal"><name><surname>Surakasi</surname><given-names>VP</given-names></name>, <name><surname>Mohamed</surname><given-names>AAM</given-names></name>, <name><surname>Kim</surname><given-names>Y</given-names></name> (<year>2011</year>) <article-title>RNA interference of beta 1 integrin subunit impairs development and immune responses of the beet armyworm, <italic>Spodoptera exigua</italic></article-title>. <source>J Insect Physiol</source><volume>57</volume>: <fpage>1537</fpage>–<lpage>1544</lpage>.<pub-id pub-id-type="pmid">21856307</pub-id></mixed-citation></ref><ref id="pone.0110536-Whyard1"><label>40</label><mixed-citation publication-type="journal"><name><surname>Whyard</surname><given-names>S</given-names></name>, <name><surname>Singh</surname><given-names>AD</given-names></name>, <name><surname>Wong</surname><given-names>S</given-names></name> (<year>2009</year>) <article-title>Ingested double-stranded RNAs can act as species-specific insecticides</article-title>. <source>Insect Biochem Molec Biol</source><volume>39</volume>: <fpage>824</fpage>–<lpage>832</lpage>.<pub-id pub-id-type="pmid">19815067</pub-id></mixed-citation></ref><ref id="pone.0110536-Mao1"><label>41</label><mixed-citation publication-type="journal"><name><surname>Mao</surname><given-names>YB</given-names></name>, <name><surname>Cai</surname><given-names>WJ</given-names></name>, <name><surname>Wang</surname><given-names>JW</given-names></name>, <name><surname>Hong</surname><given-names>GJ</given-names></name>, <name><surname>Tao</surname><given-names>XY</given-names></name>, <etal>et al</etal> (<year>2007</year>) <article-title>Silencing a cotton bollworm <italic>P<sub>450</sub></italic> monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol</article-title>. <source>Nature Biotechnol</source><volume>25</volume>: <fpage>1307</fpage>–<lpage>1313</lpage>.<pub-id pub-id-type="pmid">17982444</pub-id></mixed-citation></ref><ref id="pone.0110536-Baum1"><label>42</label><mixed-citation publication-type="journal"><name><surname>Baum</surname><given-names>JA</given-names></name>, <name><surname>Bogaert</surname><given-names>T</given-names></name>, <name><surname>Clinton</surname><given-names>W</given-names></name>, <name><surname>Heck</surname><given-names>GR</given-names></name>, <name><surname>Feldmann</surname><given-names>P</given-names></name>, <etal>et al</etal> (<year>2007</year>) <article-title>Control of coleopteran insect pests through RNA interference</article-title>. <source>Nature Biotechnol</source><volume>25</volume>: <fpage>1322</fpage>–<lpage>1326</lpage>.<pub-id pub-id-type="pmid">17982443</pub-id></mixed-citation></ref><ref id="pone.0110536-Clemens1"><label>43</label><mixed-citation publication-type="journal"><name><surname>Clemens</surname><given-names>MJ</given-names></name>, <name><surname>Elia</surname><given-names>A</given-names></name> (<year>1997</year>) <article-title>The double-stranded RNA-dependent protein kinase PKR: Structure and function</article-title>. <source>J Interferon Cytokine Res</source><volume>17</volume>: <fpage>503</fpage>–<lpage>524</lpage>.<pub-id pub-id-type="pmid">9335428</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Bois/explor/OrangerV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000213 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000213 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Bois
|area= OrangerV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.25. |  |