title: Determination of juvenile hormone titers by means of LC-MS/MS/MS and a juvenile hormone-responsive Gal4/UAS system in Aedes aegypti mosquitoes.
abstract: In anautogenous mosquitoes, juvenile hormone III (JH) plays an essential role in female post-eclosion (PE) development, preparing them for subsequent blood feeding and egg growth. We re-examined the JH titer during the reproductive cycle of female Aedes aegypti mosquitoes. Using liquid chromatography coupled with triple tandem mass spectrometry (LC-MS/MS/MS), we have shown that it reaches its peak at 48-54 h PE in the female hemolymph and at 72 h PE in whole body extracts. This method represents an effective assay for determination of JH titers. The 2.1-kb 5' promoter region of the Early Trypsin (ET) gene, which is specifically expressed in the female midgut under the control of JH during the PE phase, was utilized to genetically engineer the Ae. aegypti mosquito line with the ET-Gal4 activator. We then established the ET-GAL4>UAS-enhanced green fluorescent protein (EGFP) system in Ae. aegypti. In ET-Gal4>UAS-EGFP female mosquitoes, the intensity of the midgut-specific EGFP signal was observed to correspond to the ET gene transcript level and follow the JH titer during the PE phase. The EGFP signal and the EGFP transcript level were significantly diminished in midguts of transgenic female mosquitoes after RNA interference depletion of the JH receptor Methoprene-tolerant (Met), providing evidence of the control of ET gene expression by Met. Topical JH application caused premature enhancement of the EGFP signal and the EGFP transcript level in midguts of newly eclosed ET-Gal4>UAS-EGFP female mosquitoes, in which endogenous JH titer is still low. Hence, this novel ET-Gal4>UAS system permits JH-dependent gene overexpression in the midgut of Ae. aegypti female mosquitoes prior to a blood meal.
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA; College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Kokoza
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA.
authors.forename: Tusar T
authors.initials: TT
authors.lastname: Saha
authors.affiliation: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Xue-Li
authors.initials: XL
authors.lastname: Wang
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address: zouzhen@ioz.ac.cn.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zou
authors.affiliation: Department of Entomology, University of California, Riverside, CA, 92521, USA; The Institute for Integrative Genome Biology, University of California, Riverside, CA, 92521, USA. Electronic address: alexander.raikhel@ucr.edu.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Raikhel
mesh_terms: D000330:Aedes / Q000235:genetics / Q000502:physiology*; D000818:Animals; D030801:Animals, Genetically Modified; D002853:Chromatography, Liquid; D005260:Female; D015870:Gene Expression; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007605:Juvenile Hormones / Q000235:genetics* / Q000378:metabolism; D008726:Methoprene / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D007605:Juvenile Hormones; D014157:Transcription Factors; D008726:Methoprene
keywords: Early trypsin; Hormone action; Juvenile hormone; Methoprene-tolerant; Mosquito; Transgenesis