Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua (original) (raw)
Related papers
Journal of Insect Physiology, 2008
During insect development, ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the ecdysteroid receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of ecdysteroid receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown. r
Archives of Insect Biochemistry and Physiology, 2008
In this report with an ecdysteroid-responsive cell line of the beet armyworm, Spodoptera exigua (Se4) selection for resistance against methoxyfenozide and the insect moulting hormone (20-hydroxyecdysone, 20E) was carried out to analyze the resulting resistant cells in order to elucidate possible mechanisms of resistance towards these compounds. From these cultures, five methoxyfenozide- and four 20E-resistant subclones were selected starting from 0.1 nM methoxyfenozide up to 100 μM and from 10 nM 20E up to 100 μM, respectively. To date, the selected cells kept their loss of susceptibility for 100 μM. Here we evaluated two processes known to be important in insecticide resistance, namely metabolism and pharmacokinetics, in the selected methoxyfenozide- and 20E-resistant subclones. Synergism experiments with piperonyl butoxide, S,S,S-tributyl phosphorotrithioate, and diethyl maleate, which are respective inhibitors of monooxygenases, esterases, and gluthation-S-transferases, did not affect the level of the resistance. To check the possible existence of active transport in the resistant cells, we used ouabain, an inhibitor of active membrane transport. In parallel, the absorption profile was studied in resistant and susceptible cells with use of 14C-methoxyfenozide. Interestingly, resistant subclones showed cross-resistance towards methoxyfenozide and 20E. The resistance was irreversible even after the compounds were removed from the medium. Arch. Insect Biochem. Physiol. 2007. © 2007 Wiley-Liss, Inc.
Archives of Insect Biochemistry and Physiology, 1997
A cell line derived from the embryos of the cotton boll weevil, Anthonomus grandis (BRL-AG-2), was used to study morphological and biochemical responses to 20-hydroxyecdysone (20E). The cells respond to 10-6 M 20E by inhibition of cell growth and enhanced production of some secreted proteins. Crude nuclear extracts containing the ecdysteroid receptor complex proteins consisting of the ecdysteroid receptor (EcR) and ultraspiracle (USP) bound ponasterone A with a K d of 6.1 nM. Bound radiolabeled ponasterone A was displaced by both 20E and the lepidopteran-specific non-steroidal ecdysteroid agonist, RH-5992, with 41-and about 1,900-fold higher K d values, respectively. We identified the ecdysteroid receptor components in this cell line, using monoclonal antibodies against the Drosophila ecdysteroid receptor (DmEcR) and ultraspiracle (DmUSP) proteins. A predominant band of about 70 kDa was detected with anti-EcR, and multiple bands ranging from 50-55 kDa were detected with anti-USP in the A. grandis extracts. Using degenerate primer RT-PCR, we isolated a 450 bp cDNA fragment of the putative AgEcR. Using this fragment as a probe, we identified a large mRNA of ca. 10 kb by Northern blot analysis. These results demonstrate the usefulness of this cell line for the study of ecdysone response and the isolation of the receptor components in A.