The authors thank Victoria Carpenter for her critical comments of this protocol.

Part I - RNAi-mediated gene knockdown in adult Aedes aegypti mosquitoes. For experiment overview see Figure 1.
1. dsRNA Synthesis
<ol>
 <li>Synthesize specific dsRNAs against genes of interest and control dsRNAs. 
 Note: We recommend developing primers for PCR fragments in the range of 300 to 500 base pairs located at the 3' end of the specific cDNA2 and with the T7 primer sequence attached at the 5' end (5'-TAA TAC GAC TCA CTA TAG GG-3'). Uniqueness of ...

<ol>
	<li>Drake, L. L., 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+Aquaporin+gene+family+of+the+yellow+fever+mosquito,+Aedes+aegypti.">The Aquaporin gene family of the yellow fever mosquito, Aedes aegypti.</a> PloS one. 5, e15578 (2010).</li><li>Shepard, A. R., Jacobson, N., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+quantitative+PCR+primer+location+for+short+interfering+RNA+efficacy+determination.">Importance of quantitative PCR primer location for short interfering RNA efficacy determination.</a> Analytical biochemistry. 344, 287-288 (2005).</li><li>Altschul, S. F., 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=Gapped+BLAST+and+PSI-BLAST:+a+new+generation+of+protein+database+search+programs.">Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.</a> Nucleic acids research. 25, 3389-3402 (1997).</li><li>Garver, L., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+RNAi+Assays+in+Adult+Mosquitoes+(A.+gambiae).">Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae).</a> J. Vis. Exp. (5), e230 (2007).</li><li>Clements, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+1+Development,+Nutrition,+and+Reproduction.">Volume 1 Development, Nutrition, and Reproduction.</a> The Biology of Mosquitoes. 2, (1992).</li><li>Hansen, I. A., Attardo, G. M., Park, J. H., Peng, Q., Raikhel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+of+rapamycin-mediated+amino+acid+signaling+in+mosquito+anautogeny.">Target of rapamycin-mediated amino acid signaling in mosquito anautogeny.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 10626-10631 (2004).</li><li>Hansen, I. A., Attardo, G. M., Roy, S. G., Raikhel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+of+rapamycin-dependent+activation+of+S6+kinase+is+a+central+step+in+the+transduction+of+nutritional+signals+during+egg+development+in+a+mosquito.">Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito.</a> The Journal of biological chemistry. 280, 20565-20572 (2005).</li><li>Pannabecker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+the+Malpighian+Tubule.">Physiology of the Malpighian Tubule.</a> Annual review of entomology. 40, 493-510 (1995).</li><li>Dow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+Malpighian+tubule+function+from+functional+genomics.">New insights into Malpighian tubule function from functional genomics.</a> Comp Biochem Phys A. 150, S135 (2008).</li><li>Dow, J. A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+Malpighian+tubule+from+functional+genomics.">Insights into the Malpighian tubule from functional genomics.</a> Journal of Experimental Biology. 212, 435-445 (2009).</li><li>Lawson, D., 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=VectorBase:+a+data+resource+for+invertebrate+vector+genomics.">VectorBase: a data resource for invertebrate vector genomics.</a> Nucleic acids research. 37, 583-587 (2009).</li><li>Beyenbach, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+mechanisms+of+diuresis+in+Malpighian+tubules+of+insects.">Transport mechanisms of diuresis in Malpighian tubules of insects.</a> J. Exp. Biol. 206 (Pt 21), 3845-3856 (2003).</li><li>Coast, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+recording+of+excretory+water+loss+from+Musca+domestica+using+a+flow-through+humidity+meter:+hormonal+control+of+diuresis.">Continuous recording of excretory water loss from Musca domestica using a flow-through humidity meter: hormonal control of diuresis.</a> Journal of insect physiology. 50, 455-468 (2004).</li><li>Kersch, C. N., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+critical+for+in+vivo+fluid+excretion+post+blood+feeding.">Mosquito Aedes aegypti (L.) leucokinin receptor is critical for in vivo fluid excretion post blood feeding.</a> FEBS letters. 585, 3507-3512 (2011).</li><li>Hays, A. R., Raikhel, A. 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We have nothing to disclose.

The RNAi protocol used has been developed in the laboratory of Alexander Raikhel at the University of California Riverside6,7 and is similar to a protocol published by Garver and Dimopoulos4. The experimental approach shown in this video protocol can be used to study genes involved in insect diuresis in an in vivo setting. The excretory organs of insects, the Malpighian tubules, have attracted the interest of generations of researchers as a 'simple' model system for diuresis. This organ is ...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of reagent or equipment</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>MEGAscript 			T7 			High Yield Kit</td>
 <td>Ambion, 			Inc.</td>
 <td>AM1334</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS 			buffer</td>
 <td>Sigma-Aldrich</td>
 <td>P4417</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 			tubing</td>
 <td>Local 			vendor</td>
 <td></td>
 <td>PVC</td>
 </tr>
 <tr>
 <td>1 			ml plastic pipette tip</td>
 <td>VWR</td>
 <td>83007-376</td>
 <td>Blue 			tip</td>
 </tr>
 <tr>
 <td>1 			ml syringe</td>
 <td>Becton, 			Dickinson and Company</td>
 <td>309602</td>
 <td></td>
 </tr>
 <tr>
 <td>Scissors </td>
 <td>Local 			vendor</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Metal 			needle</td>
 <td>Carolina 			Biologicals</td>
 <td>654307</td>
 <td>Size 			5</td>
 </tr>
 <tr>
 <td>Fly 			pad</td>
 <td>Genesee 			Scientific</td>
 <td>789060</td>
 <td></td>
 </tr>
 <tr>
 <td>Battery-powered 			aspirator w/ collection vial</td>
 <td>UPMA 			Labs</td>
 <td>IPMM 			2000</td>
 <td></td>
 </tr>
 <tr>
 <td>Fine 			tip forceps</td>
 <td>World 			Precision Instruments</td>
 <td>14095</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass 			capillary needles</td>
 <td>World 			Precision Instruments</td>
 <td>1B200-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereo 			dissection microscope </td>
 <td>Leica 			Microsystems</td>
 <td>S6D</td>
 <td></td>
 </tr>
 <tr>
 <td>Analytical 			precision balance</td>
 <td>Mettler 			Toledo</td>
 <td>AB54S</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>84097</td>
 <td></td>
 </tr>
 <tr>
 <td>One 			pint waxed lined cardboard cups</td>
 <td>Local 			vendor</td>
 <td></td>
 <td>Manufactured 			soup cups</td>
 </tr>
 <tr>
 <td>Mesh 			net</td>
 <td>Local 			vendor</td>
 <td></td>
 <td>plastic 			fly gauze</td>
 </tr>
 </tbody>
</table>

rnai-mediated gene knockdown and in vivo diuresis assay in adult female aedes aegypti mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

Watch this Scientific Journal Video about RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes at JoVE.com

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

rnai-mediated-gene-knockdown-vivo-diuresis-assay-adult-female-aedes

Research

JoVE Journal

Biology

13.4K visualizações.  New Mexico State University. Neste protocolo combinamos RNAi mediada silenciamento de genes com um In vivo Ensaio diurese para estudar a knockdown efeitos dos genes de interesse tem sobre a excreção de fluido do mosquito.