Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2008
Publication Date: 6/15/2010
Citation: Willis, D.K., Wang, J., Stanford, J.R., Orth, A., Goodman, W.G. 2010. Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells. Journal of Insect Science. 10(6). Available: http://www.insectscience.org/10.66/i1536-2442-10-66.pdf. Interpretive Summary: Insect juvenile hormones play important roles in the development of animal and plant insect pests. Several commercial insecticides such as methoprene are chemically synthesized juvenile hormone derivatives that aid in the control of insect pests in both animals and plants. We used microarrays containing probes for all 14,010 genes of the fruit fly Drosophila melanogaster to identify the array of genes that are affected by addition of juvenile hormone. We detected 32 genes whose expression was either increased or decreased by exposure to juvenile growth hormone. A subset of these genes were further analyzed using real-time RT-PCR and this technique confirmed that 3 out of 3 genes that were thought to increase actually did increase. One of these genes, Epac is significantly induced by juvenile hormone. However, only 1 out of 6 genes that were decreased based on microarray analysis could be confirmed to be decreased using real-time RT-PCR. These results emphasize the need to confirm the initial results of microarray analysis. The identification of the juvenile hormone regulatory cascade may provide novel targets for future insecticide action of use to both plant and animal agriculture.
Technical Abstract: A microchip array encompassing probes for 14,010 genes of Drosophila melanogaster was used to analyze the effect of juvenile hormone (JH) on genome-wide gene expression. JH is a member of a key group of insect hormones involved in regulating larval development and adult reproductive processes. Although well-studied from the physiological standpoint, the molecular actions of JH remain unclear. Total RNA was isolated from Drosophila S2 cells after 4 hours treatment with 250 ng/ml JHIII or 250 ng/ml methyl linoleate (MLA). JHIII was isolated from racemic mixtures of the homologs by chiral HPLC chromatography. MLA was has a similar structure to JHIII but lacks hormonal activity and was used as a control for the potential effects of JHIII metabolism by the S2 cells. A collection of 32 known or putative genes demonstrated a significant change with JHIII treatment (r > 2.0, P = 0.005). Of these, 13 were significantly up regulated and 19 were reduced in expression. The expression of a subset of these loci were analyzed by real-time reverse transcriptase PCR (RT-PCR). Three loci that exhibited constant expression in the presence and absence of JHIII (RP49 [FBgn0002626], FBgn0023529, and FBgn0034354) were evaluated and found to be reliable invariant reference transcripts using BestKeeper software. All three of the up-regulated genes analyzed were confirmed to increase expression in the presence of JHII by real-time RT-PCR analysis, however only one of six loci that exhibited reduced expression on microarrays could be confirmed as significantly reduced (P = 0.05). Among the confirmed JHIII up-regulated genes were two loci of unknown function (FBgn0040887 and FBgn0037057) and Epac (exchange protein directly activated by cyclic AMP), a guanine nucleotide exchange factor for Rap1 small GTPase.