Isolation, endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver, Ips pini (Coleoptera: Scolytidae)

Abstract

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named “Ipi10G08”, encoded a 410 amino acid translation product that shared 26–37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 μg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45 min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of ∼22.5-fold control levels at 4 h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions.

Introduction

The juvenile hormones (JHs) are a class of acyclic sesquiterpenoids produced de novo in the corpora allata (CA) of insects (Feyereisen and Farnsworth, 1987). Although they control a variety of biological processes (Nijhout, 1994), the molecular mechanisms fundamental to JH action are still not clearly understood (Riddiford, 1994; Jones, 1995; Wyatt and Davey, 1996; Davey, 2000; Gilbert et al., 2000; Wheeler and Nijhout, 2003). Models based on nuclear or cytosolic receptors are supported by evidence that Ultraspiracle (USP) and the methoprene-tolerant (Met) gene products function as JH-dependent transcription factors (Xu et al., 2002; Fang et al., 2005; Miura et al., 2005). In addition, there are experiments supporting interaction with a membrane-bound receptor that triggers a signal transduction cascade involving protein kinase C (PKC) (Ilenchuk and Davey, 1987; Yamamoto et al., 1988; Sevala and Davey, 1989, Sevala and Davey, 1993; Sevala et al., 1995). Given the assortment of roles that JH must govern, there may be multiple signal transduction mechanisms for JH action.

Regardless of the receptor–ligand interaction, the signal transduction pathway ends with the response of primary responder genes, of which very few have been reliably identified for JH. The JH esterase gene from Choristoneura fumiferana (Cfjhe) has been identified as a primary JH response gene as it is directly induced by JH I within 1 h after treatment, even in the presence of the protein synthesis inhibitor, cycloheximide (Feng et al., 1999). Cfjhe mRNA expression is suppressed by 20-hydroxyecdysone (20-HE) (Feng et al., 1999), acting at the same 30-bp promoter region as JH (Kethidi et al., 2004). Modulation by 20-HE is common in many JH-responsive genes involved in insect development. The interplay between JH and 20-HE is apparently regulated by PKC-mediated phosphorylation of the nuclear proteins that bind to the response element (Kethidi et al. 2006). JH rapidly induces transcription of many genes, including juvenile hormone esterase (jhe) (Wroblewski et al., 1990; Feng et al., 1999), E75 nuclear receptors (Dubrovskaya et al., 2004; Dubrovsky et al., 2004), and mevalonate pathway genes (rev. Seybold and Tittiger, 2003). Other JH-responsive genes, including vitellogenin (Comas et al., 1999), calmodulin (Iyengar and Kunkel, 1995), jhp21 (Zhang et al., 1996), and others (Glinka and Wyatt, 1996; Graham et al., 1996; Hirai et al., 1998; Orth et al., 1999; Cho et al., 2000; Dubrovsky et al., 2000, Dubrovsky et al., 2002; Barchuk et al., 2004) are induced slowly with JH application, suggesting that they may not be primary response genes.

Finding true primary responders to JH that act without the involvement of ecdysone would help further elucidate the mechanism of JH action. To this end, we have been investigating JH-responsive genes in Ips pini. Feeding stimulates elevated JH III biosynthesis in male CA, resulting in aggregation pheromone biosynthesis (Tillman et al., 1999). The implication that pheromone-biosynthetic genes respond to elevated JH III titers has been subsequently confirmed (Hall et al., 2002; Tillman et al., 2004; Gilg et al., 2005; Bearfield et al., 2006; Keeling et al., 2006). Microarray expression analysis of I. pini midgut tissue revealed over 100 genes that respond to topical JH III treatment (Keeling et al., 2006). In this study, the gene displaying the earliest and most pronounced increase in mRNA following JH treatment was Ipi10G08. Here, we show the dose- and time-dependent induction of Ipi10G08 mRNA following JH III treatment, as well as its tissue distribution in male and female I. pini. The effect of JH III treatment on protein levels was also determined by immunoblot analysis. Sequence comparisons with other organisms suggest that Ipi10G08 is a member of an insect-specific gene family containing conserved motifs, thereby implying a conserved function for these genes within insects. The data suggest Ipi10G08 is likely a primary responder gene to JH III.