Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: Chemical Biology of Insect and Plant Signaling Systems

Location: Chemistry Research Unit

Title: Methyl farnesoate action, and morphogenetic signaling through the ligand binding pocket of the ortholog of the retinoid X receptor, in higher dipter

Authors
item Jones, Grace -
item Bocanegra, Jose -
item Smolka, John -
item Jones, Davy -
item Teal, Peter
item Henrich, Vince -
item Niewiadomska-Cimicka, Anna -
item Kochman, Marian -
item Krzywonos, Anna -
item Sapa, Agnes -
item Wozniak, Mietek -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 26, 2013
Publication Date: N/A

Technical Abstract: Most attention on metamorphic signaling by small terpenoids has focused action by juvenile hormone (JH) through bHLH-PAS proteins (e.g., MET and GCE), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a long-standing series of endocrine and pharmacological studies on pupariation in Diptera have remained persistently refractory to explanation with the above two-axis model. Larval Corpora Allata Secretion Necessary for Puparium Formation. Endocrine transplantation studies of Vogt [1] and Possompes [2] demonstrated that secretion from the corpora allata of the ring gland during the mid-late 3rd instar feeding stage is necessary for morphogenesis of the shape, sclerotization, and color of the puparium. Implantation of ecdysone-secreting ring glands at the early 3rd instar resulted in apolysis of the epidermis but the 3rd instar cuticle manifested a highly larviform puparium [1, 2]. Normal and Altered Titers of Circulating Methyl Farnesoids. Using a highly sensitive GC-MS on methyl farnesoid extracts of D.melanogaster blood, we determined that during the mid-late 3rd instar feeding stage, a large increase to near half micromolar levels* occurs for methyl farnesoate (MF) and bisepoxy-JH III, but not JH III (Jones et al. [3]). We then genetically reduced methyl farnesoate to <1% of normal during late 3rd instar feeding; bisepoxy JH III was 4-26% of normal, and JH III 26-125% of normal. These 3rd instar larvae exhibited the typical behaviors of ceasing feeding and wandering, but then only feebly attempted puparium formation, instead remaining essentially larviform. These above studies on endogenous secretions suggest that at the midlate 3rd instar, larval methyl farnesoids interact with a small peak in 20E to enable subsequent pupariumformation. However, the above data do not chemically identify the necessary corpora allata secretion(s). JH III Reception Not Required for Puparium Formation. Recently Abdou et al. [4] showed that D. melanogaster null for its two acknowledged JH III receptors (MET and GCE) formed a puparium essentially normal in shape, sclerotization and color. Dietary inclusion of JH III or methoprene during the 3rd instar feeding period (when JH is l owest vs. MF and bisJHIII) failed to prevent essentially normal puparium formation (Riddiford and Ashburner [5]). Ligand Pocket of RXR Ortholog Ultraspiracle: Competent to Bind and Respond to Sesquiterpene Ligand. Clayton et al. [6] estimated even under crystal formation conditions, ca. 25% of the Drosophila USP molecules were physically in apo conformation, available to receive ligand. In functional performance, Drosophila USP prepared under less severe conditions is bound by the known RXR synthetic ligand tributyltin (TBT), and by various sesquiterpenes at the respective saturating concentration of each [6]. In fact, TBT displaces sesquiterpene binding to USP in an equilibrium manner [6]. Henrich et al. [8] showed that in cultured mammalian cells Drosophila USP is competent to transduce binding of sesquiterpene via a potentiation of 20E signaling to the USP heterodimer partner, EcR. Fang et al. [8] obtained similar results in insect Sf9 cells. In a plant cell system ligand transduced USP homodimerization and transcriptional activation [9]. Of the three methyl farnesoids confirmed to be in larval circulation, MF binds USP by far the most strongly (Kd ca. 50 nM [6], similar to 9-cis RA for vertebrate RXR) and is the only sesquiterpene circulating at a titer corresponding favorably to its Kd for USP [5]. In vivo, TBT was recently shown to modulate 20E signaling in larvae of the dipteran Chironomus (Morales et al. [7]). Ligand Pocket of Ultraspiracle: Required for Puparium Formation. When USP point-mutated for reduced affinity for MF was challenged to function in larvae genetically null for usp, the larvae developed through the ecdysone-driven first two larval molts. Although the 3rd instar larvae ceased feeding, wandered, and apolysed to the pupal body, the 3rd instar cuticle remained larviform, unsclerotized and untanned (Jones et al. [5]). Genome expression analysis (by microarray, confirmed by qPCR) showed that genes for enzymes (e.g. DDC) and structural proteins associated with formation of the late 3rd instar integument were being misexpressed. The above data evoke a model for puparium formation in which MF and USP necessarily intersect with 20E/EcR signaling during late 3rd instar feeding, the JH/MET-GCE axis is not required, and the role of bisJHIII is unresolved.

Last Modified: 9/22/2014
Footer Content Back to Top of Page