The adipokinetic hormone system in Culicinae (Diptera: Culicidae): Molecular identification and characterization of two adipokinetic hormone (AKH) precursors from Aedes aegypti and Culex pipiens and two putative AKH receptor variants from A. aegypti Christian Kaufmann a, b, d, * , Hans Merzendorfer a , Gerd Ga ¨de a, c a Department of Biology/Chemistry, University of Osnabru ¨ck, 49069 Osnabru ¨ck, Germany b Institute of Zoology, University of Neucha ˆtel, Neucha ˆtel CH-2007, Switzerland c Zoology Department, University of Cape Town, Private Bag, ZA-7700 Rondebosch, South Africa d University of Zu ¨rich, Institute of Parasitology, Winterthurerstrasse 266a, CH-8057 Zu ¨rich, Switzerland article info Article history: Received 10 June 2009 Received in revised form 2 September 2009 Accepted 4 September 2009 Keywords: Insect Mosquito Neuropeptide Adipokinetic hormone Metabolism abstract Insect neuropeptides of the adipokinetic hormone (AKH) family induce the mobilization of energy stores to fuel flight, but also affect the nutritional balance during diapause and oogenesis. They are therefore important regulators for flight, hibernation, and reproduction in mosquitoes including those that transmit human pathogens. In this study, we identified and analyzed the genes encoding two AKH preprohormones in the Yellow fever mosquito, Aedes aegypti: Aedae-AKH-I encodes the octapeptide pELFTPSWa and Aedae- AKH-II the decapeptide pEVTFSRDWNAa. Identical AKHs were identified in the West Nile virus vector, Culex pipiens, whose genes were characterized in this study as Culpi-AKH-I and Culpi-AKH-II. Using Northern blot, transcript expression was shown in A. aegypti, for Aedae-AKH-I in the head/thorax tissues of pupae and females, as well as in the abdomen of adult males; Aedae-AKH-II was only expressed in adults. In an immunocytological study using an AKH-antibody, the corpus cardiacum (CC), the intrinsic CC-cells (X-cells), the nervi corporis cardiaci, cells in the brain and thoracic ganglia were stained. In addition, two splice variants of the AKH-receptor gene were characterized in A. aegypti,(Aedae-AKHR-I and -II). RT-PCR revealed that both variants of these typical G-protein-coupled receptors were expressed in all life stages. Aedae-AKHR-I expression was also detected in the ovaries, indicating once more the influence of the AKH/ AKHR system during the insect’s oogenesis. Based on phylogenetic data, we postulate two closely related types of AKH-receptors that could bind selectively the two AKH peptides found in A. aegypti. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction In insects, neuropeptides fulfil a large variety of regulatory functions. One of the most extensively investigated neuropeptide families is the adipokinetic hormone (AKH)/red pigment-concen- trating hormone (RPCH) family members of which occur in both of the large arthropod sister groups, the insects and the crustaceans (Ga ¨de, 2004; Ga ¨de and Marco, 2009). The structure of the classical AKH/RPCH members is quite conserved: an amino terminus blocked by pyroglutamate, a C-terminus blocked by amidation, aromatic amino acids at positions 4 (Phe or Tyr) and 8 (Trp), a Gly residue at position 9 (which is used for the amidation in the shorter octapeptides), a chain length of at least eight but maximally ten amino acids, and a charged amino acid (Asp) only occurring in very few cases (Ga ¨de, 2004; Ga ¨de and Marco, 2006). Whereas only one homologues neuropeptide (designated Panbo-RPCH) has been identified in decapod crustaceans, almost 50 different forms have been reported in various insect species. Only one member of the AKH/RPCH family occurs in phylogenetically more basal insect orders, but gene duplication occurred in a number of higher orders, and one species may contain up to 4 AKH/RPCH family homologues (Ga ¨de, 2004; Ga ¨de and Marco, 2009). As most neuropeptides from vertebrates and invertebrates alike, members of the AKH/RPCH family have a wide variety of functions and work as neurohormones, -transmitters and -modulators. The major pleiotropic actions noted for RPCH in crustaceans are a chromatophorotropic effect (concen- tration of the pigment granules in the integumental cells and pigment movement in the ommatidial cells of the eye), modulation * Corresponding author. Current address: University of Zu ¨ rich, Institute of Para- sitology, CH-8057 Zu ¨ rich, Switzerland. Tel.: þ41 44 658 8521; fax: þ41 44 635 8907. E-mail address: ch.kaufmann@access.uzh.ch (C. Kaufmann). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2009.09.002 Insect Biochemistry and Molecular Biology 39 (2009) 770–781