Characterisation of a functional allatotropin receptor in the bumblebee, Bombus terrestris (Hymenoptera, Apidae) Heleen Verlinden a,b,⇑ , Els Lismont c , Magdalena Bil c , Elodie Urlacher b , Alison Mercer b , Jozef Vanden Broeck a , Roger Huybrechts c a Molecular Developmental Physiology and Signal Transduction, KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium b Department of Zoology, University of Otago, 340 Great King Street, Dunedin, New Zealand c Insect Physiology and Molecular Ethology, KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium article info Article history: Received 11 May 2013 Revised 5 August 2013 Accepted 9 August 2013 Available online 19 August 2013 Keywords: AT ATR GPCR Invertebrate Neuropeptide Orexin abstract Allatotropins (ATs) are multifunctional neuropeptides initially isolated from the tobacco hornworm, Manduca sexta, where they were found to stimulate juvenile hormone synthesis and release from the cor- pora allata. ATs have been found in a wide range of insects, but appear to be absent in Drosophila. The first AT receptor (ATR) was characterised in 2008 in the lepidopteran Bombyx mori. Since then ATRs have been characterised in Coleoptera and Diptera and in 2012, an AT precursor gene was identified in hymenop- teran species. ATRs show large sequence and structural similarity to vertebrate orexin receptors (OXR). Also, AT in insects and orexin in vertebrates show some overlap in functions, including modulation of feeding behaviour and reproduction. The goal of this study was to identify a functional ATR in a hymenopteran species. We used ATRs (insect sequences) and OXRs (vertebrate sequences) to search the genome of the bumblebee, Bombus terrestris. Two receptors (XP_003402490 and XP_003394933) with resemblance to ATRs and OXRs were found. Phylogenetic analysis provided the first indication that XP_003402490 was more closely related to ATRs than XP_003394933. We investigated the transcript level distribution of both receptors and the AT pre- cursor gene by means of quantitative real-time reverse transcriptase PCR. XP_003402490 displayed a tis- sue distribution comparable with ATRs in other species, with high transcript levels in the male accessory glands. After pharmacological characterisation, it appeared that XP_003402490 is indeed a functional ATR. Activation of the receptor causes an increase in intracellular calcium and cyclic AMP levels with an EC 50 value in the low nanomolar to picomolar range. XP_003394933 remains an orphan receptor. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Allatotropins (ATs) were named after their first known biolog- ical function, namely the ability to stimulate juvenile hormone (JH) biosynthesis in the corpora allata (CA) of adult, female lepi- dopteran insects in vitro (Kataoka et al., 1989). A multitude of additional functions of ATs over a wide range of insect species and developmental stages have subsequently been identified. AT stimulates myotropic activity (Duve et al., 1999, 2000; Paemen et al., 1991), heart activity (Veenstra et al., 1994) and ventral dia- phragm oscillation (Koladich et al., 2002). It also plays a role in the photic entrainment of the circadian clock (Petri et al., 2002) and inhibits active ion transport across the larval midgut epithe- lium in vitro (Lee et al., 1998). ATs have been isolated from numerous insect species, deduced from the sequences of homol- ogous genes, or identified in silico from the nucleotide sequence databases (Elekonich and Horodyski, 2003; Veenstra et al., 2012; Weaver and Audsley, 2009). Related peptides have been isolated from mollusks (Harada et al., 1993; Li et al., 1993) and annelids (Ukena et al., 1995) based on their myotropic activity. It has been suggested that the ancestral role of this peptide family is related to its myotropic role and that the JH stimulating action evolved secondarily (Elekonich and Horodyski, 2003). The myo- tropic activity of AT on the gut has been suggested to be impor- tant for feeding behaviour, since gut contractions are necessary to allow food motility and the flow of digestive enzymes (Audsley and Weaver, 2009; Spit et al., 2012). Despite its widespread appearance in numerous insects, the AT sequence has not been identified in Drosophila melanogaster or other members of that genus (Hewes and Taghert, 2001; Vanden Broeck, 2001). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.08.006 ⇑ Corresponding author at: Molecular Developmental Physiology and Signal Transduction, KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. Fax: +32 16323902. E-mail addresses: heleen.verlinden@bio.kuleuven.be (H. Verlinden), Els.lis- mont@bio.kuleuven.be (E. Lismont), Magdalena.bil@bio.kuleuven.be (M. Bil), Elo- die.urlacher@otago.ac.nz (E. Urlacher), Alison.mercer@otago.ac.nz (A. Mercer), Jozef.vandenbroeck@bio.kuleuven.be (J. Vanden Broeck), Roger.huybrechts@bio.ku- leuven.be (R. Huybrechts). General and Comparative Endocrinology 193 (2013) 193–200 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen