We thank Sergei Demin and Tatyana Karamysheva for their help with chromosome preparation and FISH on Anopheles. We also thank David Severson for providing us Aedes and Culex genomic DNA BAC clones and Melissa Wade for editing the text. This work was supported by two grants from the National Institutes of Health: 1R21 AI88035-01 to Maria V. Sharakhova and 1R21 AI094289-01 to Igor V. Sharakhov.

<ol>
	<li>Sharakhov, I. V., Sharakhova, M. V., Verrity, J. F., Abbington, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromosome Mapping Research Developments. , (2008).</li><li>Holt, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+the+malaria+mosquito+Anopheles+gambiae.">The genome sequence of the malaria mosquito Anopheles gambiae.</a> Science. 298, 129-149 (2002).</li><li>Sharakhova, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+the+Anopheles+gambiae+PEST+genome+assembly.">Update of the Anopheles gambiae PEST genome assembly.</a> Genome biology. 8, R5 (2007).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+preparing+polytene+chromosomes+from+Aedes+aegypti+(Diptera,+Culicinae).">A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 387-390 (2003).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malpighian+tubule+polytene+chromosomes+of+Culex+quinquefasciatus+(Diptera,+Culicinae).">Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 383-386 (2003).</li><li>McAbee, R. D., Christiansen, J. A., Cornel, A. 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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs--a+new+source+of+chromosomes+for+genome+mapping+of+the+yellow+fever+mosquito+Aedes+aegypti.">Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti.</a> PLoS neglected tropical diseases. 5, e1335 (2011).</li><li>Brown, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+physical+map+of+Aedes+aegypti.">Toward a physical map of Aedes aegypti.</a> Insect Mol. Biol. 4, 161-167 (1995).</li><li>Steiniger, G. E., Mukherjee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+chromosome+banding:+technique+for+G-+and+Q-banding+pattern+in+the+mosquito+Aedes+albopictus.">Insect chromosome banding: technique for G- and Q-banding pattern in the mosquito Aedes albopictus.</a> Can. J. Genet. Cytol. 17, 241-244 (1975).</li><li>Garimberti, E., Tosi, S., Bridger, J. M., Volpi, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence in situ hybridization (FISH). , (2010).</li><li>Scott, J. A., Brogdon, W. G., Collins, F. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ribosomal+RNA+gene+probe+differentiates+member+species+of+the+Anopheles+gambiae+complex.">A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.</a> The American journal of tropical medicine and hygiene. 37, 37-41 (1987).</li><li>Kumar, A., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+localization+and+copy+number+of+18S+28S+ribosomal+RNA+genes+in+evolutionary+diverse+mosquitoes+(Diptera,+Culicidae).">Chromosomal localization and copy number of 18S+28S ribosomal RNA genes in evolutionary diverse mosquitoes (Diptera, Culicidae).</a> Hereditas. 113, 277-289 (1990).</li><li>Brown, S. E., Knudson, D. 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No conflicts of interest declared.

Nonfluorescent in situ hybridization on mitotic chromosomes of mosquitoes was performed for the first time in 1990 by A. Kumar and K. Rai 17. In that study, 18S and 28S ribosomal DNA genes, cloned together in one plasmid, were placed to the chromosomes of 20 species of mosquitoes. The DNA probe was radioactively labeled and hybridized to the chromosomes from brain ganglia. Among three mosquito genera, a FISH technique has been developed only for mitotic chromosomes from the cell line of Ae. aegypti 10,18,19 and has never been performed on mitotic chromosomes from live mosquitoes. Recently, we developed a simple, robust technique for obtaining high-quality chromosome preparations from IDs of 4th instar larvae 9. This method allows a high number of chromosomes to be obtained in one slide and can be universally used for all species of mosquitoes. The necessity of using only larval, not pupal or adult stages of mosquitoes, for slide preparation is probably the only limitation of the method. The standard FISH method 12 was optimized for using genomic BAC clone and IGS rDNA as probes for the mitotic chromosomes of Aedes, Culex, and Anopheles.
In addition to these specific applications, the FISH protocols described here can also be used for other purposes. The advanced FISH protocol, which utilizes C0t DNA fractions for blocking unspecific hybridization, can also be applied for the hybridization of BAC clones or any other large DNA fragments in heterochromatic regions of Anopheles. Heterochromatic regions are enriched with transposable elements and other repeats, and probes from these regions normally produce strong background on the chromosomes3. Using unlabeled C0t DNA fractions will help to reduce unspecific hybridization of the probe to the chromosomes. The simple version of the FISH protocol can be used for any rDNA or repetitive DNA probes on mitotic chromosomes of mosquitoes and other insects. In addition, it also can be applied for the hybridization of BAC clone DNA in species with low repetitive DNA content in euchromatic regions such as Anopheles or Drosophila. The protocol proposed here will help to obtain highly-finished chromosome-based genome assemblies for mosquitoes and can be broadly used for various cytogenetic applications in other groups of insects.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>MZ6 Leica stereomicroscope</td>
 <td>Leica</td>
 <td>VA-OM-E194-354</td>
 <td>A different stereomicroscope can be used </td>
 </tr>
 <tr>
 <td>Olympus CX41 phase microscope</td>
 <td>Olympus</td>
 <td>CX41</td>
 <td>A different phase microscope can be used </td>
 </tr>
 <tr>
 <td>Olympus BX61 fluorescent microscope</td>
 <td>Olympus</td>
 <td>BX61</td>
 <td>A different fluorescent microscope can be used </td>
 </tr>
 <tr>
 <td>ThermoBrite Slide Denaturation/Hybridization System</td>
 <td>Abbott Molecular</td>
 <td>30-144110</td>
 <td>Serves as a heating block and a humid chamber</td>
 </tr>
 <tr>
 <td>Dissecting needles </td>
 <td>Fine ScienceTools </td>
 <td>10130-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holders</td>
 <td>Fine Science Tools</td>
 <td>26018-17</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td>Fine Science Tools </td>
 <td>15000-03</td>
 <td></td>
 </tr>
 <tr>
 <td>75x25 double frosted micro slides </td>
 <td>Corning</td>
 <td>2949-75x25</td>
 <td></td>
 </tr>
 <tr>
 <td>22x22 mm microscope coverslips</td>
 <td>Fisher Scientific</td>
 <td>12-544-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Fisher Scientific</td>
 <td>13-374-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Rubber Cement</td>
 <td>Fisher Scientific</td>
 <td>50-949-105 </td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Fisher Scientific</td>
 <td>A491-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Alcohol 200 Proof</td>
 <td>Decon Laboratories</td>
 <td>2701</td>
 <td></td>
 </tr>
 <tr>
 <td>Propionic acid </td>
 <td>Sigma-Aldrich</td>
 <td>402907</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydrochloric acid</td>
 <td>Fisher Scientific</td>
 <td>A144-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate dihydrate</td>
 <td>Fisher Scientific</td>
 <td>S279-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium acetate trihydrate</td>
 <td>Fisher Scientific</td>
 <td>BP334-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Fisher Scientific</td>
 <td>BP366-500</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher Scientific</td>
 <td>S311-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris base </td>
 <td>Fisher Scientific</td>
 <td>BP152-1</td>
 <td></td>
 </tr>
 <tr>
 <td>10x PBS</td>
 <td>Invitrogen </td>
 <td>P5493</td>
 <td></td>
 </tr>
 <tr>
 <td>10% NBF (neutral buffered formalin)</td>
 <td>Sigma-Aldrich</td>
 <td>HT501128</td>
 <td></td>
 </tr>
 <tr>
 <td>99% formamide</td>
 <td>Fisher Scientific</td>
 <td>BP227500 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran sulfate sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>D8906</td>
 <td></td>
 </tr>
 <tr>
 <td>20x SSC buffer</td>
 <td>Invitrogen </td>
 <td>AM9765</td>
 <td></td>
 </tr>
 <tr>
 <td>1 mM YOYO-1 iodide (491/509) solution</td>
 <td>Invitrogen </td>
 <td>Y3601</td>
 <td></td>
 </tr>
 <tr>
 <td>Antifade Prolong Gold reagent</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a></td>
 <td></td>
 </tr>
 <tr>
 <td>dATP, dCTP, dGTP, dTTP</td>
 <td>Fermentas</td>
 <td>R0141, R0151, R0161, R0171 </td>
 <td></td>
 </tr>
 <tr>
 <td>Cy3-dUTP, Cy5-dUTP</td>
 <td>GE Healthcare</td>
 <td>PA53022, PA55022</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma-Aldrich</td>
 <td>A3294</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA Polymerase I</td>
 <td>Fermentas</td>
 <td>EP0041</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase I </td>
 <td>Fermentas</td>
 <td>EN0521</td>
 <td></td>
 </tr>
 <tr>
 <td>S1 Nuclease</td>
 <td>Fermentas</td>
 <td>EN0321</td>
 <td></td>
 </tr>
 <tr>
 <td>Taq DNA Polymerase</td>
 <td>Invitrogen</td>
 <td>18038-042</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase</td>
 <td>Sigma-Aldrich</td>
 <td>9001-99-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Pepsin</td>
 <td>USB</td>
 <td>9001-75-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Salmon sperm DNA</td>
 <td>Sigma-Aldrich</td>
 <td>D7656</td>
 <td></td>
 </tr>
 <tr>
 <td>Nonidet-P40 (NP40)</td>
 <td>US Biological</td>
 <td>NC9375914 </td>
 <td></td>
 </tr>
 <tr>
 <td>Qiagen Blood and Cell Culture Maxikit</td>
 <td>Qiagen</td>
 <td>13362</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiagen Large Construct Kit</td>
 <td>Qiagen</td>
 <td>12462</td>
 <td></td>
 </tr>
 </tbody>
</table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Watch this Scientific Journal Video about Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes at JoVE.com

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

fluorescent-in-situ-hybridization-on-mitotic-chromosomes-of-mosquitoes

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Immunology and Infection

25.5K visualizações.  Virginia Tech. Dentre os três gêneros de mosquito, ou seja, Anopheles, Aedes, E Culex, Técnicas de mapeamento do genoma físicas foram estabelecidos apenas para Anopheles, Cujos membros possuem cromossomos politênicos legíveis. Para os gêneros de Aedes E Culex No entanto, o mapeamento de citogenética permanece desafiador por causa da má qualidade dos cromossomos politênicos. Aqui apresenta...