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

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

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.

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.

Sarah E. Aguirre

Biology Department

New Mexico State University

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

Sarah E. Aguirre

.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