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

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

New Mexico State University

The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti.

The Aquaporin gene family of the yellow fever mosquito, Aedes aegypti.

Insect yolk protein precursor gene expression is regulated by nutritional and endocrine signals. A surge of amino acids in the hemolymph of blood-fed female mosquitoes activates a nutrient signaling system in the fat bodies, which subsequently derepresses yolk protein precursor genes and makes them responsive to activation by steroid hormones. Orphan transporters of the SLC7 family were identified as essential upstream components of the nutrient signaling system in the fat body of fruit flies and the yellow fever mosquito, Aedes aegypti. However, the transport function of these proteins was unknown. We report expression and functional characterization of AaCAT1, cloned from the fat body of A. aegypti. Expression of AaCAT1 transcript and protein undergoes dynamic changes during postembryonic development of the mosquito. Transcript expression was especially high in the third and fourth larval stages; however, the AaCAT1 protein was detected only in pupa and adult stages. Functional expression and analysis of AaCAT1 in Xenopus oocytes revealed that it acts as a sodium-independent cationic amino acid transporter, with unique selectivity to L-histidine at neutral pH (K(0.5)(L-His) = 0.34 ± 0.07 mM, pH 7.2). Acidification to pH 6.2 dramatically increases AaCAT1-specific His(+)-induced current. RNAi-mediated silencing of AaCAT1 reduces egg yield of subsequent ovipositions. Our data show that AaCAT1 has notable differences in its transport mechanism when compared with related mammalian cationic amino acid transporters. It may execute histidine-specific transport and signaling in mosquito tissues.

AaCAT1 of the yellow fever mosquito, Aedes aegypti: a novel histidine-specific amino acid transporter from the SLC7 family.

El cuerpo de grasa es el principal &oacute;rgano del metabolismo intermediario en insectos y la principal fuente de prote&iacute;nas de la hemolinfa. Como parte de nuestros continuos esfuerzos para entender la fisiolog&iacute;a de grasa corporal de mosquito y para identificar nuevas dianas para control de insectos, hemos realizado un an&aacute;lisis de transcriptoma de la grasa corporal de Aedes aegypti antes y en respuesta a la alimentaci&oacute;n de la sangre.

Los transcriptomas de grasa corporal del fiebre amarilla mosquito Aedes aegypti, pre- y post-harina de sangre.

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The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding.

The fat body transcriptomes of the yellow fever mosquito Aedes aegypti, pre- and post- blood meal.

Le corps gras est l&#39;organe principal du m&eacute;tabolisme interm&eacute;diaire chez les insectes et la principale source de prot&eacute;ines de l&#39;h&eacute;molymphe. Dans le cadre de nos efforts pour comprendre la physiologie du tissu adipeux moustique et identifier de nouvelles cibles pour la lutte contre les insectes, nous avons effectu&eacute; une analyse du transcriptome du tissu adipeux de l&#39;Aedes aegypti avant et en r&eacute;ponse &agrave; nourrir de sang.

Les transcriptions de corps gras de la fi&egrave;vre jaune moustique Aedes aegypti, pr&eacute;- et post-repas de sang.

Der Fett-K&ouml;rper ist das wichtigste Organ der intermedi&auml;re Metabolismus bei Insekten und die Hauptquelle der H&auml;molymphe Proteine. Im Rahmen unserer fortlaufenden Bem&uuml;hungen, Moskito Fett K&ouml;rper Physiologie zu verstehen und neue Ziele f&uuml;r die Insektenbek&auml;mpfung zu identifizieren haben wir eine Transkriptom-Analyse des K&ouml;rpers Fett von Aedes Aegypti vor und als Reaktion auf die F&uuml;tterung Blut durchgef&uuml;hrt.

Der K&ouml;rper Fett Transcriptomes von Gelbfieber Moskito Aedes Aegypti, vor- und Nachbedingungen Blutmahlzeit.

Il corpo di grasso &egrave; l&#39;organo principale del metabolismo intermedio in insetti e la principale fonte di proteine di emolinfa. Come parte dei nostri sforzi in corso per capire la fisiologia del corpo grasso zanzara e per identificare nuovi bersagli per il controllo degli insetti, abbiamo condotto un&#39;analisi del transcriptome del corpo grasso di Aedes aegypti prima e in risposta alla somministrazione di sangue.

Il corpo grasso transcriptomes della febbre gialla zanzara Aedes aegypti, pre- e post-pasto di sangue.

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An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.

Lisa L. Drake

Biology Department

New Mexico State University

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

Lisa L. Drake

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Biology Department

New Mexico State University

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

Biology Department