Evolutionary conservation of bursicon in the animal kingdom

Abstract

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.

Introduction

The importance of the neurohormone bursicon was established more than 40 years ago. By using a bursicon bioassay it was shown that nervous system extracts of multiple insect species induce tanning, i.e. melanization and sclerotization, of the exoskeleton in neck-ligated, freshly eclosed flies (Fraenkel and Hsiao, 1962, Fraenkel et al., 1966, Kostron et al., 1995). The active compound in these extracts was referred to as bursicon. Its molecular nature remained enigmatic for a very long period, indicating the complex nature of this hormone. Recently we identified Drosophila melanogaster bursicon as a heterodimer composed of two ±15 kDa cystine knot proteins (Mendive et al., 2005). When co-expressed, these two cystine knot proteins, referred to as bursicon α (CG13419) and bursicon β (CG15284), respectively, constitute bursicon bioactivity and, in addition, activate the fruit fly leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2) (Luo et al., 2005, Mendive et al., 2005).

Bursicon functions downstream of a cascade of peptides that initiate and regulate the ecdysis behavioral motor program, including ecdysis triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP) (recently reviewed by Truman, 2005). Postecdysially, bursicon is released into the hemolymph where it initiates the tanning of the insect’s cuticle. Genetic studies in the fruit fly also clearly demonstrated the regulatory role of bursicon in wing spreading behavior after adults emerge from their pupal case (Dewey et al., 2004).

So far, bursicon activity has only been described in insects. To obtain an idea about the evolutionary conservation of this important neurohormone, we report here on an in silico screen of protostomian and deuterostomian genome and complementary DNA (cDNA) databases in the search for potential bursicon homologues. Genome sequencing and/or expressed sequence tag (EST) projects of several animal models including nematodes, insects, crustaceans and echinoderms have been completed or are currently in progress. In the particular case of the honeybee, Apis mellifera, reverse transcription PCR analysis was carried out to evaluate a former in silico prediction that in this species bursicon α and bursicon β subunits might be fused into a single open reading frame (ORF). This is the first report describing bursicon related sequences that occur in non-insect animal species.