Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes
Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques o...
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| creator | Ramakrishnan, Chandra Rademacher, Annika Soichot, Julien Costa, Giulia Waters, Andrew P Janse, Chris J Ramesar, Jai Franke-Fayard, Blandine M Levashina, Elena A |
| description | Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines. |
| doi_str_mv | 10.1371/journal.pone.0036376 |
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Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0036376</identifier><identifier>PMID: 22574152</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Animals, Genetically Modified ; Anopheles ; Anopheles gambiae ; Aquatic insects ; Biology ; Cloning ; Culicidae ; Culicidae - parasitology ; Dehydrogenases ; Dihydrofolate reductase ; Disease transmission ; Down-Regulation ; Evaluation ; Fluorescence ; Fluorescence microscopy ; Gene expression ; Gene silencing ; Genes ; Genes, Reporter - genetics ; Genetic engineering ; Genomics ; Green fluorescent protein ; Humans ; Immunology ; Infections ; Infectious diseases ; Infectivity ; Insect Vectors - parasitology ; Laboratories ; Life Sciences ; Loads (forces) ; Luciferase ; Malaria ; Medicine ; Mosquitoes ; Organ Specificity ; Parasite Load - methods ; Parasites ; Parasitology ; Plasmodium ; Plasmodium berghei ; Plasmodium berghei - genetics ; Plasmodium berghei - physiology ; Proteins ; Reporter gene ; Salivary gland ; Salivary Glands - parasitology ; Salivary Proteins and Peptides - deficiency ; Sporozoites ; Sporozoites - physiology ; Time Factors ; Transgenic ; Vaccines ; Vector-borne diseases</subject><ispartof>PloS one, 2012-05, Vol.7 (5), p.e36376-e36376</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Ramakrishnan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>Ramakrishnan et al. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c759t-97b907ce5257cc4b1dfcc1ba04ae9d2d2f806fa4b9424b59aa745dfe8165f4db3</citedby><cites>FETCH-LOGICAL-c759t-97b907ce5257cc4b1dfcc1ba04ae9d2d2f806fa4b9424b59aa745dfe8165f4db3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3344870/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3344870/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22574152$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-02346278$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramakrishnan, Chandra</creatorcontrib><creatorcontrib>Rademacher, Annika</creatorcontrib><creatorcontrib>Soichot, Julien</creatorcontrib><creatorcontrib>Costa, Giulia</creatorcontrib><creatorcontrib>Waters, Andrew P</creatorcontrib><creatorcontrib>Janse, Chris J</creatorcontrib><creatorcontrib>Ramesar, Jai</creatorcontrib><creatorcontrib>Franke-Fayard, Blandine M</creatorcontrib><creatorcontrib>Levashina, Elena A</creatorcontrib><title>Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines.</description><subject>Animals</subject><subject>Animals, Genetically Modified</subject><subject>Anopheles</subject><subject>Anopheles gambiae</subject><subject>Aquatic insects</subject><subject>Biology</subject><subject>Cloning</subject><subject>Culicidae</subject><subject>Culicidae - parasitology</subject><subject>Dehydrogenases</subject><subject>Dihydrofolate reductase</subject><subject>Disease transmission</subject><subject>Down-Regulation</subject><subject>Evaluation</subject><subject>Fluorescence</subject><subject>Fluorescence microscopy</subject><subject>Gene expression</subject><subject>Gene silencing</subject><subject>Genes</subject><subject>Genes, Reporter - genetics</subject><subject>Genetic engineering</subject><subject>Genomics</subject><subject>Green fluorescent protein</subject><subject>Humans</subject><subject>Immunology</subject><subject>Infections</subject><subject>Infectious diseases</subject><subject>Infectivity</subject><subject>Insect Vectors - parasitology</subject><subject>Laboratories</subject><subject>Life Sciences</subject><subject>Loads (forces)</subject><subject>Luciferase</subject><subject>Malaria</subject><subject>Medicine</subject><subject>Mosquitoes</subject><subject>Organ Specificity</subject><subject>Parasite Load - methods</subject><subject>Parasites</subject><subject>Parasitology</subject><subject>Plasmodium</subject><subject>Plasmodium berghei</subject><subject>Plasmodium berghei - genetics</subject><subject>Plasmodium berghei - physiology</subject><subject>Proteins</subject><subject>Reporter gene</subject><subject>Salivary gland</subject><subject>Salivary Glands - parasitology</subject><subject>Salivary Proteins and Peptides - deficiency</subject><subject>Sporozoites</subject><subject>Sporozoites - physiology</subject><subject>Time Factors</subject><subject>Transgenic</subject><subject>Vaccines</subject><subject>Vector-borne diseases</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk01vEzEQhlcIREvhHyCwhIToIcH2ete7F6SoAhopUhEFrpY_xokrZ53auxEffx6HpCWpeqh8WGv2ed-xZzxF8ZLgMSk5eX8VhthJP16FDsYYl3XJ60fFMWlLOqopLh_v7Y-KZyldYVyVTV0_LY4orTgjFT0u_lxK79Yy_kJzLzszSivQzjqNvoyRgjhfgEMRViH2EJF3HSQEnVQeUJQrZxCspR9k70KHgkW9S2mA_yYpC8Pv4HpAPkiTkOvQMqTrwfUB0vPiiZU-wYvd96T4_unjt7Pz0ezi8_RsMhtpXrX9qOWqxVxDlQ-tNVPEWK2JkphJaA011Da4tpKpllGmqlZKzipjoSF1ZZlR5Unxeuu78iGJXd2SICVlNW7bps3EdEuYIK_EKrplrogI0ol_gRDnQsbeaQ9CQd1QZqRhsmYVV0pyhQlYq8E2TNvs9WGXbVBLMBq6Pkp_YHr4p3MLMQ9rUZaMNRxng9OtweKO7HwyE5sYpiWrKW_WJLPvdsliuB4g9WLpkgafewlhyHfEhLecE9w8BKWcNdk6o2_uoPcXbUfNZa6L62zI19EbUzFhOSflVbXxGt9D5WVg6XR-u9bl-IHg9ECQmR5-9nM5pCSml18fzl78OGTf7rELkL5fpOCHzdtNhyDbgjqGlCLY2yYQLDajd1MNsRk9sRu9LHu13_db0c2slX8Brs8rSQ</recordid><startdate>20120504</startdate><enddate>20120504</enddate><creator>Ramakrishnan, Chandra</creator><creator>Rademacher, Annika</creator><creator>Soichot, Julien</creator><creator>Costa, Giulia</creator><creator>Waters, Andrew P</creator><creator>Janse, Chris J</creator><creator>Ramesar, Jai</creator><creator>Franke-Fayard, Blandine M</creator><creator>Levashina, Elena A</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>F1W</scope><scope>H95</scope><scope>H97</scope><scope>L.G</scope><scope>1XC</scope><scope>VOOES</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120504</creationdate><title>Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes</title><author>Ramakrishnan, Chandra ; Rademacher, Annika ; Soichot, Julien ; Costa, Giulia ; Waters, Andrew P ; Janse, Chris J ; Ramesar, Jai ; Franke-Fayard, Blandine M ; Levashina, Elena A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c759t-97b907ce5257cc4b1dfcc1ba04ae9d2d2f806fa4b9424b59aa745dfe8165f4db3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>Animals, Genetically Modified</topic><topic>Anopheles</topic><topic>Anopheles gambiae</topic><topic>Aquatic insects</topic><topic>Biology</topic><topic>Cloning</topic><topic>Culicidae</topic><topic>Culicidae - parasitology</topic><topic>Dehydrogenases</topic><topic>Dihydrofolate reductase</topic><topic>Disease transmission</topic><topic>Down-Regulation</topic><topic>Evaluation</topic><topic>Fluorescence</topic><topic>Fluorescence microscopy</topic><topic>Gene expression</topic><topic>Gene silencing</topic><topic>Genes</topic><topic>Genes, Reporter - genetics</topic><topic>Genetic engineering</topic><topic>Genomics</topic><topic>Green fluorescent protein</topic><topic>Humans</topic><topic>Immunology</topic><topic>Infections</topic><topic>Infectious diseases</topic><topic>Infectivity</topic><topic>Insect Vectors - parasitology</topic><topic>Laboratories</topic><topic>Life Sciences</topic><topic>Loads (forces)</topic><topic>Luciferase</topic><topic>Malaria</topic><topic>Medicine</topic><topic>Mosquitoes</topic><topic>Organ Specificity</topic><topic>Parasite Load - methods</topic><topic>Parasites</topic><topic>Parasitology</topic><topic>Plasmodium</topic><topic>Plasmodium berghei</topic><topic>Plasmodium berghei - 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Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22574152</pmid><doi>10.1371/journal.pone.0036376</doi><tpages>e36376</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | PloS one, 2012-05, Vol.7 (5), p.e36376-e36376 |
| issn | 1932-6203 1932-6203 |
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| subjects | Animals Animals, Genetically Modified Anopheles Anopheles gambiae Aquatic insects Biology Cloning Culicidae Culicidae - parasitology Dehydrogenases Dihydrofolate reductase Disease transmission Down-Regulation Evaluation Fluorescence Fluorescence microscopy Gene expression Gene silencing Genes Genes, Reporter - genetics Genetic engineering Genomics Green fluorescent protein Humans Immunology Infections Infectious diseases Infectivity Insect Vectors - parasitology Laboratories Life Sciences Loads (forces) Luciferase Malaria Medicine Mosquitoes Organ Specificity Parasite Load - methods Parasites Parasitology Plasmodium Plasmodium berghei Plasmodium berghei - genetics Plasmodium berghei - physiology Proteins Reporter gene Salivary gland Salivary Glands - parasitology Salivary Proteins and Peptides - deficiency Sporozoites Sporozoites - physiology Time Factors Transgenic Vaccines Vector-borne diseases |
| title | Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-03T20%3A59%3A57IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Salivary%20gland-specific%20P.%20berghei%20reporter%20lines%20enable%20rapid%20evaluation%20of%20tissue-specific%20sporozoite%20loads%20in%20mosquitoes&rft.jtitle=PloS%20one&rft.au=Ramakrishnan,%20Chandra&rft.date=2012-05-04&rft.volume=7&rft.issue=5&rft.spage=e36376&rft.epage=e36376&rft.pages=e36376-e36376&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0036376&rft_dat=%3Cgale_plos_%3EA477127556%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1324609989&rft_id=info:pmid/22574152&rft_galeid=A477127556&rft_doaj_id=oai_doaj_org_article_be6824dad4a6457bba7b01effcef84cf&rfr_iscdi=true |