REVIEW ARTICLE published: 30 November 2012 doi: 10.3389/fendo.2012.00151 More than two decades of research on insect neuropeptide GPCRs: an overview

Jelle Caers , Heleen Verlinden , Sven Zels , Hans Peter Vandersmissen , Kristel Vuerinckx and Liliane Schoofs*

Animal Physiology and Neurobiology, Department of Biology, Zoological Institute, KU Leuven, Leuven, Belgium

Edited by: This review focuses on the state of the art on neuropeptide receptors in insects. Most of Hubert Vaudry, University of Rouen, these receptors are G protein-coupled receptors (GPCRs) and are involved in the regulation France of virtually all physiological processes during an insect’s life. More than 20 years ago a Reviewed by: milestone in invertebrate endocrinology was achieved with the characterization of the Dick R. Nässel, Stockholm University, Sweden first insect neuropeptide receptor, i.e., the Drosophila tachykinin-like receptor. However, Hoffmann Klaus H., University of it took until the release of the Drosophila genome in 2000 that research on neuropeptide Bayreuth, Germany receptors boosted. In the last decade a plethora of genomic information of other insect *Correspondence: species also became available, leading to a better insight in the functions and evolution of Liliane Schoofs, Department the neuropeptide signaling systems and their intracellular pathways. It became clear that of Biology, Research Group of Functional Genomics and some of these systems are conserved among all insect species, indicating that they fulfill Proteomics, Naamsestraat 59, crucial roles in their physiological processes. Meanwhile, other signaling systems seem to KU Leuven, 3000 Leuven, Belgium. be lost in several insect orders or species, suggesting that their actions were superfluous e-mail: liliane.schoofs@ in those insects, or that other neuropeptides have taken over their functions. It is striking bio.kuleuven.be that the deorphanization of neuropeptide GPCRs gets much attention, but the subsequent unraveling of the intracellular pathways they elicit, or their physiological functions are often hardly examined. Especially in insects besides Drosophila this information is scarce if not absent. And although great progress made in characterizing neuropeptide signaling systems, even in Drosophila several predicted neuropeptide receptors remain orphan, awaiting for their endogenous ligand to be determined. The present review gives a précis of the insect neuropeptide receptor research of the last two decades. But it has to be emphasized that the work done so far is only the tip of the iceberg and our comprehensive understanding of these important signaling systems will still increase substantially in the coming years.

Keywords: insects, neuropeptides, G protein-coupled receptors, signal transduction, neurobiology

INTRODUCTION et al., 2009; Verlinden et al., 2009; Weaver and Audsley, 2009; The class of Insecta, which consists of more than 30 orders, forms Altstein and Nässel, 2010; Bendena, 2010; Nässel and Winther, the most diverse animal group on earth. With about one million 2010; Van Hiel et al., 2010; Van Loy et al., 2010; Nässel and documented species and presumably 10–30 million awaiting to be Wegener, 2011; Herrero, 2012; Spit et al., 2012; Taghert and described, insects probably account for 50–70% of all existing ani- Nitabach, 2012). mals (Scherkenbeck and Zdobinsky, 2009; Bellés, 2010; Van Hiel Neuropeptides exert their physiological functions by interact- et al., 2010). Basically all the physiological processes during an ing with specific signal-transducing membrane receptors, result- insect’s life cycle are regulated by neuropeptides, including devel- ing in intracellular responses (Zupanc, 1996). Most of these opmental processes, behavioral functions, metabolic events and neuropeptide receptors belong to the G protein-coupled recep- reproduction. As such, neuropeptides are the largest (very ver- tors (GPCRs), the largest family of cell surface proteins. However, satile) class of extracellular signaling molecules that are involved there are some exceptions like the prothoracicotropic hormone in communication between insect cells (Gäde and Goldsworthy, (PTTH), which executes its role in metamorphosis through the 2003; Meeusen et al., 2003; Claeys et al., 2005a). The insect neu- activation of a receptor tyrosine kinase (RTK) (Rewitz et al., ropeptides and their actions have extensively been reviewed in 2009). Most of the insulin-like peptides (ILPs) also interact with the past (Nässel, 2002; Gäde and Auerswald, 2003; Gäde and RTKs (Fernandez et al., 1995; Graf et al., 1997; Brogiolo et al., Goldsworthy, 2003; Meeusen et al., 2003; Altstein, 2004; Gäde, 2001; Wheeler et al., 2006; Wen et al., 2010; Iga and Smagghe, 2004; Isaac et al., 2004; Simonet et al., 2004; Claeys et al., 2005a; 2011). The eclosion hormone (EH), involved in ecdysis, interacts Coast and Garside, 2005; Ewer, 2005; Predel and Wegener, 2006; with a membrane-bound guanylate cyclase receptor (Chang et al., Mertens et al., 2007; Stay and Tobe, 2007; De Loof, 2008; Audsley 2009) as does the neuropeptide-like precursor peptide 1 (NPLP1) and Weaver, 2009; Scherkenbeck and Zdobinsky, 2009; Verleyen (Overend et al., 2012).

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The functional characterization of the first insect neuropeptide the insect neuropeptide GPCRs will be reviewed. To conclude, a receptor, the Drosophila melanogaster tachykinin-like receptor short discussion about the importance of neuropeptide research (DTKR) took place in 1991 (Li et al., 1991). Subsequently, another in insects will be given. Drosophila tachykinin-like receptor (NKD) (Monnier et al., 1992) and a neuropeptide Y (NPY)-like receptor (Li et al., 1992)were G PROTEIN-COUPLED RECEPTORS identified. The latter has recently been deorphanized as the Several GPCR-(sub)families originated prior to the divergence of Drosophila RYamide receptor (Collin et al., 2011; Ida et al., 2011a). protostomian and deuterostomian animals. This led to a great In the following years only a few more insect GPCRs were cloned, diversification in chemical specificity to external stimuli like neu- e.g., the diuretic hormone receptors of Manduca sexta and Acheta ropeptides, glycoproteins, nucleotides, biogenic amines, odor- domesticus (Reagan, 1994, 1996), the Drosophila gonadotropin- ants, taste ligands, and photons. Although GPCRs do not share releasing hormone receptor (Hauser et al., 1998), which later any overall sequence homology, they do expose a similar topo- on was deorphanized as an adipokinetic hormone (AKH) recep- graphical structure which is remarkably well conserved during tor (Staubli et al., 2002)andtheDrosophila allatostatin (AST) evolution. They are typically composed of seven transmembrane receptor (DAR-1) (Birgül et al., 1999). (7TM) α-helices, each consisting of 20–30 hydrophobic amino The real breakthrough in the field of insect neuropeptide acids, and three extracellular and intracellular loops connecting receptor research came with the publication of the Drosophila thedifferenthelices.TheN-terminusislocatedattheextra- genome in 2000 (Adams et al., 2000). This opened the oppor- cellular site and often possesses several glycosylation sites; the tunity to predict receptors based on genomic data (Hewes and C-terminus, on the other hand, is orientated into the cytoplasm Taghert, 2001), which clearly boosted the receptor deorphaniza- and offers some potential phosphorylation sites. The extracellular tion rate. At present, 35 GPCRs are functionally characterized in parts are involved in ligand-specific binding, while the intracellu- Drosophila. One receptor (Dmel\SPR) is activated by seemingly lar areas interact with a member of the family of heterotrimeric different neuropeptides, the myoinhibitory peptide (MIP) and GTP-binding proteins (G proteins), consisting of an α-, β-, and the sex peptide SP. The others mainly respond to one neuropep- γ-subunit (Bockaert and Pin, 1999).Basedonsharedsequence tide type, which underlines the specificity of the receptor/ligand motifs, the GPCRs are categorized into at least six subfamilies. couples. Another 14 GPCRs are predicted to be involved in neu- The evolutionary relationship between the different families is ropeptide signaling pathways, but their ligands are still unknown still unclear because of the lack of significant sequence homol- and therefore they are classified as “orphan” receptors (Table 1) ogy. They probably evolved independently of each other or have (Meeusen et al., 2003; Hauser et al., 2006, 2008; Clynen et al., adopted the G protein signal transduction pathways through 2010a). In section “Methuselah (CG6936) and Methuselah-like convergent evolution (Brody and Cravchik, 2000; Gether, 2000; Receptors” the methuselah receptor is also briefly discussed. In Horn et al., 2000). All the neuropeptide GPCRs belong to spite the fact that several studies have been performed on this the rhodopsin-like (family A) or the secretin-like (family B) receptor, it still is not clear if it is really a neuropeptide receptor subfamily. and if the stunted gene really encodes for its endogenous ligands. When a GPCR becomes activated by its ligand, the extra- Despite the diversity in their endogenous ligands, GPCRs have cellular signal will be transduced into intracellular physiological been rather well conserved during evolution. This has facilitated responses. An activated receptor will undergo a conformational the search for neuropeptide receptors in newly released genomes change, which in turn leads to the activation of the associ- like those of Apis mellifera (Hauser et al., 2006), Tribolium casta- ated G protein. This promotes the release of GDP from the neum (Hauser et al., 2008), and Bombyx mori (Yamanaka et al., α-subunit, followed by binding of GTP. Next, the GTP-bound 2008; Fan et al., 2010). Research in other insects also revealed a α-subunit dissociates from the βγ-dimer and both will be released set of new neuropeptide signaling systems that are not present in the cytoplasm. Subsequently, they can interact with their spe- in Drosophila, e.g., AKH/corazonin-related peptide (ACP) dis- cific effector proteins to elicit cellular signaling pathways. The covered in Anopheles gambiae (Hansen et al., 2010), allatotropin effector proteins involved depend on the type of the α-subunit. (AT) discovered in B. mori (Yamanaka et al., 2008), and inotocin The most common α-subunits are Gq,Gs,andGi/o.TheGq discoverd in T. castaneum (Stafflinger et al., 2008)(Table 1). subunits interact with phospholipase Cβ (PLCβ)inorderto Hitherto, 149 insect genome projects are either com- initiate the hydrolysis of the membrane-bound phosphoinositol- pleted or in progress (http://www.ncbi.nlm.nih.gov/sites/entrez? biphospholipid-bisphosphates resulting in diacylglycerol (DAG) db=bioproject) and in 2011, the i5K project was initiated, and inositol triphosphate (IP3). DAG activates protein kinase 2+ which aims to sequence 5000 insect genomes in the next 5 years C(PKC)andIP3 mobilizes Ca from intracellular stores like (Robinson et al., 2011). With this overload of genomic informa- the endoplasmic reticulum. The Gs and Gi/o subunits, respec- tion coming up, we intend to give the reader a clear overview of tively, activate or inhibit adenylyl cyclase provoking a subsequent what is currently known on insect neuropeptide receptors. First, increase or decrease of the cyclic adenosine monophosphate we will discuss some general characteristics of GPCRs and the (cAMP) concentration within the cell. The Gs proteins are also 2+ deorphanizing strategies. Next, we will highlight the area of pep- capable of activating Ca channels, while the Gi/o proteins are tidomics, which facilitated the prediction and detection of ligands able to interact with K+-channels. The intrinsic GTPase activity enormously, followed by a genetics part to discuss some com- of Gα induces the hydrolysis of GTP to GDP, resulting in the reas- monly used tools to unravel the physiological functions of the sociation of the subunits (Hepler and Gilman, 1992; Lustig et al., neuropeptide-receptor systems. Thereafter, the current status of 1993; Vanden Broeck, 1996, 2001; Brody and Cravchik, 2000).

Frontiers in Endocrinology | Neuroendocrine Science November 2012 | Volume 3 | Article 151 | 2 Caers et al. Insect neuropeptide GPCRs: an overview (Continued) Cazzamali et al., 2003 Iversen et al., 2002b Cazzamali and Sudo et al., 2005 Radford et al., 2002 Egerod et al., 2003a Egerod et al., 2003a Garczynski et al., 2002 Hyun et al., 2005; Johnson et al., 2003a Cazzamali et al., 2005 Rosenkilde et al., 2003 Rosenkilde et al., 2003 Luo et al., 2005 Ida et al., 2011a Yapici et al., 2008; Kim Mertens et al., 2002 Jørgensen et al., 2006 Kubiak et al., 2002 Poels et al., 2009 Birse et al., 2006 Ida et al., 2011b Staubli et al., 2002 Larsen et al., 2001 Larsen et al., 2001 Kreienkamp et al., 2002 Kreienkamp et al., 2002 Johnson et al., 2005 Iversen et al., 2002a Hansen et al., 2011 Hansen et al., 2011 Chen et al., 2012 Cazzamali et al., 2002 Johnson et al., 2004 Hector et al., 2009 Grimmelikhuijzen, 2002 Mertens et al., 2005 et al., 2010 CG17673/CG33495 and CG6456 neuropeptide receptors. Drosophila —Orphan myoinhibiting peptide precursor Drosophila G33344/CG6111/CG14547 Cardioacceleratory peptide CG4910 Symbol Receptor gene Endogenous ligand Ligand gene Reference* neuropeptide receptors—Neuropeptide receptors not present in Drosophila Drosophila melanogaster receptor Ecdysis triggering hormone receptorFMRFamide receptorGlycoprotein A2/Glycoprotein B5 receptorKinin Dmel\ETHR receptorMyosuppressin receptor 1 Dmel\Lgr1Myosuppressin receptor 2Neuropeptide F receptorPigment CG5911 dispersing factor receptor Dmel\FR CG7665Proctolin receptorPyrokinin 1 receptor Dmel\DmsR-1Pyrokinin 2 receptor Dmel\Pdfr Dmel\DmsR-2Pyrokinin 2 receptorRickets CG2114 Dmel\Lkr Dmel\NPFR1RYamide receptor CG8985 EcdysisSex CG43745/CG13803 triggering peptide hormone receptor CG13758 GPA2/GPB5 CG1147Short CG10626 CG18105 Dmel\Proc-R neuropeptide F receptor Dmel\Pk1rSIFamide receptor Dmel\CG8784 DromyosuppressinSulfakinin receptor Dmel\CG8795Tachykinin receptor FMRFamide CG6986Tachykinin receptor DromyosuppressinTrissin CG8784 CG9918 Dmel\NepYr receptor Dmel\SPR Pigment-dispersing Dmel\sNPF-R factor CG17878/CG40041 CG8795 CG6440 Neuropeptide Dmel\rk F Leucokinin CG5811 CG6496 CG6440 CG7395/CG18639 Dmel\SIFR CG16752/CG12731 CG2346 Dmel\CCKLR-17D3 Dmel\Takr86C Proctolin CG8930 Dmel\Takr99D CG32540/CG6894/CG6881 Drm-PK-2 Drm-PK-1 CG10342 Drm-PK-2 CG10823 Short neuropeptide Sex F peptides** Dmel\TrissinR CG6515 and Drosulfakinin CG13480 CG7887 RYamide CG34381/CG14003 CG13968 CG7105 Bursicon/Partner of burs CG6371 CG15520 SIFamide CG6371 CG18090 Trissin Tachykinin Tachykinin CG13419/CG15284 CG40733 CG33527 CG14734 CG14734 CG14871 Adipokinetic hormone receptorAllatostatin A receptor 1Allatostatin A receptor 2Allatostatin C receptor 1Allatostatin C receptor 2Calcitonin-like diuretic hormone Dmel\GRHR receptorCAPA receptorCCHamide-1 receptor Dmel\Dh31-R1CCHamide-2 receptor Dmel\AlstRCholecystokinin (CCK)-like CG11325 receptor Dmel\AR-2Corazonin receptor Dmel\star1 CG32843/CG17415/CG17043CRF-like diuretic hormone receptor Dmel\AlCR2 1CRF-like diuretic hormone receptor Diuretic 2 CG2872 hormone 31Crustacean Dmel\CCKLR-17D1 cardioactive peptide receptor CG10001 CG7285 Dmel\CCHa1r CG42301/CG6857 Dmel\Dh44-R1 CG13702 Dmel\CcapR Dmel\capaR Dmel\CCHa2r Dmel\Dh44-R2 Adipokinetic hormone CG13094 CG8422 CG30106/CG14484 Dmel\GRHRII CG12370 CG14593 C CG14575 Drosulfakinin Allatostatin A CG1171 Allatostatin A CG10698 Allatostatin C Allatostatin CCHamide-1 C Diuretic hormone 44 Diuretic CG18090 CCHamide-2 hormone 44 Capa-1/Capa-2 CG13633 CG13633 CG14919 Corazonin CG14358 CG14919 CG8348 CG8348 CG15520 CG14375 CG3302 Table 1 | Characterized

www.frontiersin.org November 2012 | Volume 3 | Article 151 | 3 Caers et al. Insect neuropeptide GPCRs: an overview . e u l a v Hansen et al., 2010 Yamanaka et al., 2008 Stafflinger et al., 2008 50 C E t n a v e l e r l a c i g o l o i s y h p a n i g n i t l u s e r h c a o r p p a y g o l o c a m r a h p e s r e v e r a g n i s u d e z i n a h p r o e d d n a d e t c e f s n a r t s a w r o t p e c e r e h t f o A Symbol Receptor gene Endogenous ligand Ligand gene Reference* N D c g n i d o c l l u f e h t h c i h Drosophila w n i r e receptors p a p d e h s i l b u p Drosophila t s r fi e Accessory gland peptide 70A (Acp70A)/Ductus ejaculatorius peptide 99B (Dup99B). h T Receptors not present in Orphan HectorLgr3Lgr4Moody receptorTrapped in endoderm 1Orphan receptorOrphan receptorOrphan receptorOrphan receptorOrphan receptorOrphan receptorOrphan receptorOrphan Dmel\Tre1 receptor Dmel\hec Dmel\moodyOrphan receptor Dmel\Lgr3 Dmel\CG4313 Dmel\CG34411 Dmel\CG12290 CG3171 CG4322 Dmel\CG32547 CG4395 Dmel\CG13229 CG34411/CG4187 CG4313 CG31096/CG5042 CG12290 Dmel\CG13995 CG32547/CG12610 Dmel\CG33696 CG13229 Dmel\CG33639 CG13995 Dmel\CG30340 CG33696/CG16726 Dmel\CG13575 CG33639/CG5936 CG30340 CG13575 AKH/corazonin-related peptide receptorAllatotropin receptorInotocin receptor GPRGNR3 XP_321591 NGR-A16 ITR ACP NP_001127714 NP_001078830 Allatotropin Inotocin 3290616 692738 100038343 Table 1 | Continued ** *

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DEORPHANIZING STRATEGIES libraries containing potential neuropeptides. The possibility to There is a clear distinction between the techniques used to create such libraries coincided with the availability of the first deorphanize receptors before and after the genomic era. In the whole genome databases. This also launched the era of pep- past, one started with a bioactive ligand, purified from tissue tidomics, which encloses the purpose to simultaneously identify extracts, in order to identify its corresponding receptor (the clas- and/or visualize all peptides present in a cell, tissue, body liq- sic approach). Nowadays, an orphan receptor is used to explore its uid, or organism. Peptidomics studies are based on two major activating ligand from a library of synthetic compounds consist- elements, the in silico prediction of neuropeptides and the discov- ing of predicted neuropeptides (reverse pharmacology) (Meeusen ery and identification of neuropeptides using mass spectrometric et al., 2003). This strategy makes use of appropriate cellular devices (Baggerman et al., 2002; Predel et al., 2004; Wegener et al., expression systems used to express orphan receptors of interest. 2006). Thesesystemsholdtheopportunitytomeasureoneofthemany Endogenous neuropeptides are enclosed in larger preprohor- second messenger reporter molecules released after receptor acti- mones, mostly between 50 and 500 amino acids long (Baggerman vation. The most commonly used expression systems are mam- et al., 2005a). They can code for multiple structurally related or malian cell lines (Chinese Hamster Ovary [CHO] cells or Human unrelated neuropeptides, as well as for just one neuropeptide. The Embryonic Kidney [HEK] 293 cells) and Xenopus oocytes. These only common feature of preprohormones is the presence of an are used in the bioluminescence-based assay (CHO cells), the amino-terminal signal peptide, with exception of a predicted AST fluorescence-based assay (HEK293 cells), the luciferase-based CC neuropeptide in Drosophila which has an amino-terminal assay (HEK293 cells) and the electrophysiological assay (Xenopus peptide anchor (Veenstra, 2009a). This peptide is immediately oocytes). cleaved off after arrival in the endoplasmic reticulum. The resid- BecauseitisnearlyimpossibletopredictwhichkindofG ual prohormone undergoes enzymatic cleavage at mono- or diba- protein interacts with an orphan receptor, a universal tool was sic amino acid residues to release the neuropeptides (Hook et al., required to predict the signaling cascade. This problem was cir- 2008; Rholam and Fahy, 2009). Most neuropeptides require post- cumvented with the discovery of the promiscuous G protein translational modifications to become bioactive or to improve α subunits Gα16 (human) and Gα15 (murine). These Gα pro- stability. teins regulate PLCβ, and possess the ability to interact with Because of the poor sequence conservation between prepro- most GPCRs and, as such, their signaling pathways are redirected hormones and the short length of the neuropeptides, the majority toward the release of Ca2+ (Offermanns and Simon, 1995). Both, consists only of 4–20 amino acids, their prediction from genome the bioluminescence and the fluorescence assay are based on the databases is not straightforward. Nevertheless, classical BLAST measurement of the release of intracellular Ca2+ upon receptor analyses have revealed 36 neuropeptide genes in D. melanogaster activation. The bioluminescence assay makes use of biolumi- (Hewes and Taghert, 2001; Vanden Broeck, 2001), and 35 in nescent proteins such as aequorine, purified from the jellyfish, A. gambiae (Riehle et al., 2002). Later on, the combined use Aequoria victoria,thatinteractwithCa2+ (Prasher et al., 1987; of different bioinformatic tools, to overcome the low sensitiv- Stables et al., 1997). In the fluorescence assay usually HEK293 ity of a BLAST analysis alone, revealed a total of 119 potential cells are charged with a Ca2+ sensitive fluorophore that serves neuropeptide-coding genes in Drosophila (Liu et al., 2006; Clynen as readout (Bender et al., 2002). The luciferase assay makes use et al., 2010a). All neuropeptides predicted by these methods can of a reporter gene plasmid consisting of a cAMP response ele- be synthesized to construct synthetic peptide libraries applied in ment (CRE) as readout for measuring intracellular cAMP levels the reverse pharmacology assays. (Janssen et al., 2008; Horodyski et al., 2011; Vuerinckx et al., The bioinformatic predictions, though, do not reveal which 2011). For the electrophysiological assay, Xenopus oocytes are neuropeptides are ultimately produced, and endogenous bioac- injected with a mix of the orphan receptor and the G protein gated tive neuropeptides may be overlooked in the genomic data. The inwardly rectifying K+ (GIRK) channels that are activated upon processing of a precursor can also differ during developmental ligand binding. This leads to subsequent inward K+ currents that stages or between tissues, and post-translational modifications can be measured (Kofuji et al., 1995; Ho and Murrell-Lagnado, are hard to predict based on sequence information. Therefore, a 1999; Ulens et al., 1999). biochemical characterization of neuropeptides is necessary. There Besides the use of these heterologous expression systems, one are several possible peptidomics methods available to provide can also make use of a homologous expression system in which in these needs, all based on mass spectrometry. The most com- the orphan receptor is expressed in Drosophila Schneider-2 (S2) mon tool is a combination of liquid chromatography, tandem cells (Vanden Broeck et al., 1998). The use of heterologous mass spectrometry and database mining, which allows the detec- expression systems, however, prevents that compounds present tion and sequencing of low concentrations of neuropeptides from in an insect extract, or predicted insect ligands would activate complex mixtures (Clynen et al., 2010b). Mass spectrometry endogenous mammalian or amphibian receptors (for reviews, applications led to the discovery of hundreds of neuropeptides. As see: Meeusen et al., 2003; Mertens et al., 2004; Beets et al., 2011; is often the case, Drosophila peptidomics (Baggerman et al., 2002; Bendena et al., 2012). Baggermanet al., 2005b; Schoofs and Baggerman, 2003)paved the way for peptidomic studies in other insects, e.g., A. mellifera NEUROPEPTIDES AND PEPTIDOMICS (Hummon et al., 2006; Boerjan et al., 2010a), Nasonia vitripen- An important feature of the currently used deorphanizing strate- nis (Hauser et al., 2010), T. castaneum (Li et al., 2008), and Aedes gies is the ability to screen orphan receptors with compound aegypti (Predel et al., 2010). Also in insects with no completely

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sequenced genome, peptidomics may prove useful, e.g., Locusta characterized for a selection of model insects. In the next sec- migratoria (Clynen et al., 2006; for reviews, see: Hummon et al., tion we aim to give a brief summary of what is known so far 2006; Boonen et al., 2008; Menschaert et al., 2010). relating to these insect neuropeptide receptors. For convenience all intertitles are accompanied with the corresponding computed FUNCTIONAL GENOMICS gene (CG) numbers of the Drosophila receptors. These numbers Upon the characterization of a neuropeptide receptor and its were used for genes identified during the annotation of the whole ligand, the question remains which function they possess in a spe- Drosophila genome sequence. For those receptor genes not anno- cific organism. These functions can be determined with genetic tated in Drosophila, the accession number of the receptor gene for tools. In the classic approach the phenotype of interest is chosen the insect in which it was first deorphanized is added. first and then attempts are made to identify the genes responsi- ble for this phenotype (forward genetics). With the rise of the DEORPHANIZED NEUROPEPTIDE RECEPTORS whole genome era, a tremendous number of genes with unknown ADIPOKINETIC HORMONE RECEPTORS (CG11325 ORTHOLOGS) functions were identified. This made it possible to start with The first structural characterization of an AKH neuropeptide was a gene of interest and to study its function (reverse genetics). achieved in 1976 (Stone et al., 1976). Currently, around 55 iso- Currently, the most used techniques to perform reverse genet- forms, derived from various insect species, have been described ics are silencing of genes of interest by RNA interference (RNAi), (Gäde, 2009; Caers et al., 2012; Gäde and Marco, 2012; Jedlickaˇ generating knockouts, and overexpressing specific genes using the et al., 2012; Malik et al., 2012; Weaver et al., 2012). They con- GAL4/UAS system. sist of 8–10 amino acids, and are characterized by a blocked The generation of loss-of-function phenotypes through the N-terminus (pyroglutamate) and C-terminus (amidation) (Gäde application of RNAi is a fairly new technique as it was described and Auerswald, 2003).ThemainfunctionofAKHistheregula- for the first time in 1998 in Caenorhabditis elegans (Fire et al., tion of the energy metabolism. During energy requiring processes 1998),immediatelyfollowedbyareportofRNAiusageinD. like flight, the AKH neuropeptides are released from the corpora melanogaster (Kennerdell and Carthew, 1998). RNAi studies are cardiac (CC) and will interact with their receptors, present in the widely used in the field of insect research and have proven to membrane of the fat body adipocytes. This will induce the release be appropriate to unravel functions of neuropeptides and their of energy rich substrates (lipids, trehalose, or proline) (Lorenz receptors in various species (Bellés, 2010; An et al., 2012). There is and Gäde, 2009). The kind of substrates released, depends on the a genome-wide transgenic RNAi library available for Drosophila, coupled G protein. When AKH binds to a Gq protein-coupled consisting of short gene fragments cloned as inverted repeats and receptor, glycogen phosphorylase will be activated and trehalose expressed using the binary GAL4/UAS system (Dietzl et al., 2007). will be set free. If the signaling pathway acts by a Gs protein- TheusageoftheGAL4/UASsystemtoperformRNAiexperiments coupled receptor, triacylglycerol lipase will be activated, resulting becomes also more established in other insects like B. mori (Dai in the production of DAG or free fatty acids (Gäde and Auerswald, et al., 2008)andT. castaneum (Schinko et al., 2010). 2003). The last years it became clear that the function of AKH is RNAi can not entirely impede the expression of a gene of not restricted to locomotory activity alone, but that it acts as a interest. To generate a complete knockout of a gene, muta- general regulator of homeostasis in insects, influencing all energy genic or homologous recombination tools are frequently used. requiring processes (e.g., egg production, feeding behavior, lar- Mutagenesis relies on the incorporation of mutations, which can val growth, molting, and immune response) (Goldsworthy et al., be obtained by the application of chemical mutagenesis or by 2002, 2003; Lorenz, 2003; Lee and Park, 2004; Isabel et al., 2005; transposable element mutagenesis, followed by a thorough screen Grönke et al., 2007; Bharucha et al., 2008; Lorenz and Gäde, 2009; to detect the samples containing mutations in the gene of inter- Arrese and Soulages, 2010; Attardo et al., 2012; Konuma et al., est. Homologous recombination is based on the host DNA repair 2012).AKHalsoservesasananti-stresshormoneinoxidative system for the alteration of a target sequence in the genome by a stress situations (Kodrík et al., 2007; Veceˇ raˇ et al., 2007; Kodrík, donor sequence. This donor sequence exhibits homology to the 2008; Huang et al., 2011a). target sequence, but contains the desired genetic modifications. The AKH receptors (AKHR) are closely related to the ACP The alteration is preceded by the generation of a double strand receptors and constitute the invertebrate AKH/ACP receptor break in the target or donor sequence, inducing the homolo- family. Together with the invertebrate corazonin/gonadotropin gous recombination repair system (for reviews, see: Reumer et al., releasing hormone (GnRH) receptor family and the verte- 2008; Wesolowska and Rong, 2010; An et al., 2012). brate/protochordate GnRH receptor family they compose the Besides studying the effects of a knockdown or a complete GnRH receptor superfamily (Lindemans et al., 2011; Roch et al., knockout of a certain gene, overexpressing a gene can also yield 2011). The first AKHR was determined in M. sexta by using fat important information about its function. To obtain overexpres- body fractions to ascertain the optimal binding conditions for sion, the gene of interest can be coupled to a binary GAL4/UAS tritium-labeled Manse-AKH (Ziegler et al., 1995). The Drosophila system as well. AKHR was the first to be cloned and was deorphanized by The previous described techniques to identify, deorphanize making use of the electrophysiological assay (Park et al., 2002), and determine the functions of neuropeptide signaling systems and its characterization was confirmed by Staubli et al. (2002) are widely applied in insect research, yielding an enormous using a bioluminescence assay. Later, AKHRs were also identified amount of information. Table 2 summarizes the neuropep- and characterized in other insect species: Periplaneta americana tide receptors that have been predicted and/or functionally (Hansen et al., 2006; Wicher et al., 2006a), A. gambiae (Belmont

Frontiers in Endocrinology | Neuroendocrine Science November 2012 | Volume 3 | Article 151 | 6 Caers et al. Insect neuropeptide GPCRs: an overview (Continued) NP_001127725 NP_001037007 NP_001036929 NP_001127732 NP_001127724 NP_001127746 NP_001127714 NP_001127740 NP_001127726 NP_001127745 NP_001037035 NP_001127736 NP_001037033 na NP_001127721 NP_001127710 NP_001037049 NP_001127741 NP_001165739 Bombyx mori XP_003245221 XP_001943863 NP_001127711 XP_001949220 na XP_001945436 XP_003245941 XP_001952361 XP_001948574 NP_001127712 XP_001947807 na XP_001950448 XP_001950333 NP_001127723 XP_003244979 na XP_001945379 XP_001944708 XP_001943752 XP_001945278 XP_001944842 na XP_003240503 XP_003245097 XP_001944756 XP_001944816 Acyrthosiphon pisum XP_973738 XP_974772 XP_974786 XP_969392 XP_969504 NP_001076809 ABX52400 XP_975416 NP_001076796 NP_001076795 NP_001078830 na XP_971178 XP_969030 XP_973937 NP_001167548 NP_001076792 NP_001076793 XP_968907 Tribolium castaneum XP_001606711 XP_003427176 XP_001607985 na XP_001601649 XP_003424370 XP_001604352 XP_001604277 XP_001602277 XP_001605409 na XP_001601584 NP_001161243 XP_001605342 XP_001600654 XP_001600587 XP_001606566 na NP_001165745 vitripennis na NP_001091702 NP_001073563 XP_395101 XP_396992 NP_001137393 naXP_397268 naACI90286 naACI90287 NP_001127719 NP_001035354 XP_397024 XP_396335 XP_396046 XP_001122652 na na XP_396025 XP_001120367 Apis mellifera Nasonia XP_318029 XP_001120335 XP_001604582 AAX84796 XP_317851 XP_313395 AAQ67361 XP_315468 AAQ73620 XP_314133 XP_001687839 XP_321591 XP_320274 XP_312952 XP_312953 XP_321555 AAS77205 XP_312031 XP_003436278 XP_309589 na EAU77489 gambiae na na XP_001655248 XP_001655249 ABX57919 XP_001652590 XP_003435928 AEN03789 XP_001660594 XP_318856 XP_001656825 XP_001662526 XP_001655399 ABX57920 XP_001659389 ABI93273 ABI93274 XP_001649032 na AAT95982 XP_001651852 XP_001653920 XP_001662510 XP_001663106 Aedes aegypti Anopheles CG11325 CG14575 CG30106 CG14593 CG10698 CG8422 CG12370 CG5911 CG2114 na CG8985 CG13803 CG2872 CG10001 CG7285 CG13702 CG32843 CG33344 CG7665 CG10626 na Drosophila melanogaster Diptera Diptera Diptera Hymenoptera Hymenoptera Coleoptera Hemiptera Lepidoptera Table 2 | Characterized and predicted neuropeptide receptors in insect species of varying insect orders. Adipokinetic hormone receptor Glycoprotein A2/Glycoprotein B5 receptor Myosuppressin receptor 2 Allatostatin C receptor 1 Allatostatin C receptor 2 Crustacean cardioactive peptide receptor CAPA receptor Ecdysis triggering hormone receptor Calcitonin-like diuretic hormone receptor CRF-like diuretic hormone receptor 2 Allatostatin A receptor 1 Allatostatin A receptor 2 Allatotropin receptor na CCHamide-1 receptor CRF-like diuretic hormone receptor 1 Inotocin receptor Kinin receptor Myosuppressin receptor 1 AKH/corazonin-related peptide receptor FMRFamide receptor Corazonin receptor CCHamide-2 receptor

www.frontiersin.org November 2012 | Volume 3 | Article 151 | 7 Caers et al. Insect neuropeptide GPCRs: an overview NP_001127707 NP_001127708 NP_001127742 NP_001127733 NP_001036913 NP_001037033 NP_001127739 na NP_001108346 DAA35192 NP_001127748 NP_001127749 NP_001127722 NP_001036977 na NP_001127717 NP_001127720 NP_001127744 Bombyx mori na XP_003241084 XP_001948776 XP_001952283 na XP_001944453 XP_001947491 XP_003247524 XP_001946954 XP_001946949 na XP_001950091 XP_001946360 XP_001946314 XP_003241610 na Acyrthosiphon pisum XP_967689 XP_971738 EEZ97715 XP_975514 XP_966794 XP_970225 EFA01332 EEZ97729 EEZ97728 NP_001106940 XP_971383 ADZ17181 XP_972750 XP_975226 XP_970102 na Tribolium castaneum na XP_003427082 XP_001606201 XP_001605837 XP_001601972 XP_001600098 na NP_001153387 XP_003427563 XP_003427654 na na na XP_001607956 na vitripennis na XP_395896 na NP_001157480 NP_001153387 XP_395206 na XP_001123033 NP_001106756 XP_395081 na XP_395084 XP_003250082 na Apis mellifera Nasonia AAT81602 XP_313426 na AAX84797 XP_317111 ABW86945 ABD96049 XP_314231 CAD27763 XP_003435887 XP_321512 AAX84798 XP_310762 XP_311031 XP_001237302 XP_001237203 XP_309215 XP_312088 na gambiae na XP_001660966 na XP_001662936 XP_001649742 AAY46405 XP_001653188 XP_001647749 ABW86944 XP_001663694 EAT38404 XP_001654357 na XP_001652376 na XP_001657210 Aedes aegypti Anopheles CG13758 CG1147 CG6986 CG9918 CG8795 CG8784 CG8930 CG5811 CG16752 CG7395 CG10823 CG6881 CG42301 CG6515 CG7887 CG34381 Drosophila melanogaster Diptera Diptera Diptera Hymenoptera Hymenoptera Coleoptera Hemiptera Lepidoptera SIFamide receptor Tachykinin receptor Proctolin receptor Pyrokinin 2 receptor RYamide receptor Table 2 | Continued Neuropeptide F receptor Black box: functionally characterized neuropeptide receptors. Gray box: previously predicted neuropeptide receptors. White box: predicted neuropeptide receptors based on ourna: own not blast annotated. analysis. Pigment dispersing factor receptor Pyrokinin 1 receptor Short neuropeptide F receptor Sulfakinin receptor CCK-like receptor Tachykinin receptor Trissin receptor Rickets Sex peptide receptor

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et al., 2006), B. mori (Staubli et al., 2002; Zhu et al., 2009; the SP receptor [see section “Sex Peptide/Myoinhibiting Peptide Huang et al., 2010), and T. castaneum (Li, unpublished data). Receptor (CG16752/CG12731 Orthologs)”]. Two putative AKHR variants have been predicted in A. aegypti The A-type AST-As, or FGLamides were first isolated of brain (Kaufmann et al., 2009) and one AKHR is identified in the Apis extracts of cockroaches (Woodhead et al., 1989; Pratt et al., genome (Hauser et al., 2006). However, it remains doubtful if 1991), and have since been found in every investigated insect this receptor is really functional in the honeybee, because mass species, except for T. castaneum (Li et al., 2008). They are char- spectrometric techniques have failed to detect the predicted Apis acterized by a conserved (Y/F)XFG(L/I)-NH2 sequence (Hayes AKH neuropeptide (Veenstra et al., 2012). Besides expression in et al., 1994; Audsley et al., 1998). AST-As regulate JH biosyn- the fat body (Kaufmann and Brown, 2006; Ziegler et al., 2011), thesis in cockroaches, crickets, and termites (Pratt et al., 1989, the AKHR is also expressed in various neurons of P. americana, 1991; Woodhead et al., 1989, 1994; Bellés et al., 1994; Weaver including the abdominal dorsal unpaired medial (DUM) neu- et al., 1994; Lorenz et al., 1995, 1999; Yagi et al., 2005;fora rons, which are responsible for the release of octopamine. As such, review, see: Stay and Tobe, 2007). A property attributed to all octopamine may be the link between elevated AKH-titers and AST-As is myoinhibition of visceral muscles (Hoffmann et al., the increase in locomotion (Wicher et al., 2006b, 2007; Verlinden 1999; Stay, 2000; Aguilar et al., 2003; Weaver and Audsley, 2009; et al., 2010). Zandawala et al., 2012). Recently, Drosophila AST-A was linked to food intake and foraging behavior (Hergarden et al., 2012; ADIPOKINETIC HORMONE/CORAZONIN-RELATED PEPTIDE RECEPTOR Wang et al., 2012). In Drosophila, two A-type AST receptors (XP_321591 ORTHOLOGS) are identified: DAR-1 and DAR-2 (Birgül et al., 1999; Larsen In 2006, an A. gambiae receptor was annotated and cloned that et al., 2001). DAR-1, when expressed in Xenopus oocytes was was closely related to the AKH and corazonin receptors, but could showntocoupletoaG-proteinoftheGi/o family. When not be activated by these neuropeptides (Belmont et al., 2006). expressed in CHO cells, DAR-1 and -2 are activated by AST-A and + Hansen et al. (2010) detected a neuropeptide closely related to mobilize intracellular Ca2 (Larsen et al., 2001). AST-A recep- both AKH and corazonin and named it ACP. This neuropeptide tors were also characterized in P. americana (Auerswald et al., was able to activate the receptor expressed in CHO/Gα16 cells in 2001), B. mori (Secher et al., 2001), and Diploptera punctata a dose-responsive manner (Hansen et al., 2010). Subsequently, (Lungchukiet et al., 2008). Northern blot experiments showed the ACP receptor was also characterized in T. castaneum (Hansen that the B. mori receptor is expressed in the midgut of fifth lar- et al., 2010). Recently, two predicted ACP receptors of B. mori val instars and to a much lesser extend in the brain (Secher et al., (Yamanaka et al., 2008; Hansen et al., 2010) were also character- 2001). ized, but were indicated as AKHR (Shi et al., 2011). The ACP neu- ropeptides were in fact already described in L. migratoria (Siegert, ALLATOSTATIN C RECEPTORS (CG7285 AND CG13702 ORTHOLOGS) 1999)andinA. gambiae (Kaufmann and Brown, 2006), but ThefirstC-orPISCF-typeASTwascharacterizedinthelate were classified as AKH neuropeptides with unknown functions. pupae of M. sexta. AST-Cs contain a typical C-terminal PISCF- ACP and its receptor are structurally intermediate between the OH sequence, a blocked N-terminus and a disulfide bridge link- AKH and corazonin neuropeptides and their receptors, which is ing Cys-7 and Cys-14 (Kramer et al., 1991). Orthologs are found a prominent example of receptor/ligand co-evolution. An ances- in other lepidopteran, dipteran and coleopteran species (Li et al., tral receptor and ligand gene have probably duplicated several 2006). In several insects, C-type or C-type-like ASTs can have times followed by mutations and evolutionary selection, leading both allatostatic and allatotropic properties, depending on the age to three signaling systems. However, the ACP signaling system of the animal (Abdel-Latief et al., 2004; Clark et al., 2008; Griebler is absent in all investigated Drosophila species as well as in A. et al., 2008; Abdel-Latief and Hoffmann, 2010). In Diptera two mellifera, Acyrthosiphon pisum, Pediculus humanus,andinthe AST-C receptors have been characterized for Drosophila using crustacean Daphnia pulex,suggestingthatitmayhavebeenlost Xenopus oocytes (Kreienkamp et al., 2002)andforAedes using several times during arthropod evolution (Hansen et al., 2010). HEK cells (Mayoral et al., 2010). Only one AST-C receptor was So far no functions are assigned to the ACP signaling system, but found to be present in Bombyx (Yamanaka et al., 2008)and the high expression shortly before and after hatching of T. casta- in Tribolium (Audsley et al., 2012). Activation of the Bombyx neum suggests a role in early larval development (Hansen et al., AST-C receptor elicits an increase in intracellular cAMP levels 2010). (Yamanaka et al., 2008), while the Tribolium receptor was deor- phanized in HEK cells, inducing a Ca2+ response (Audsley et al., ALLATOSTATIN A RECEPTORS (CG2872 AND CG10001 ORTHOLOGS) 2012). In adult fruit flies, both drostar genes are expressed in the The endogenous ligands of the AST A receptor are the A-type optic lobes and the pars intercerebralis, where the AST-C neu- AST neuropeptides which belong to the group of allatoregula- ropeptide was also found to be present. This suggests a function tory neuropeptides together with the B-, and C-type allatostins in the modulation of visual information processing. In the last lar- and the ATs (for a review, see: Weaver and Audsley, 2009) val stage, receptor expression was found in the brain and corpora and the recently discovered AST CC neuropeptides (Veenstra, allata (CA) (Kreienkamp et al., 2002). In Aedes significant differ- 2009a). Allatoregulatory peptides are named after their ability ences were observed in tissue distribution and expression levels to either inhibit (ASTs) or stimulate (ATs) juvenile hormone for the two receptor paralogs (Mayoral et al., 2010). In Tribolium (JH) synthesis (Audsley et al., 2008). The B-type ASTs are also the highest transcript levels were noticed in the head and the gut, known as myoinhibiting peptides and were found to activate with variable amounts in the fat body and reproductive organs.

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These transcript levels were also shown to be sex-dependent membrane V-ATPases via cAMP as second messenger (Coast (Audsley et al., 2012). et al., 2001)andinAnopheles the fluid excretion in Malpighian The recently discovered AST CC neuropeptide (AST CC) tubulesisalsocAMPdriven(Coast et al., 2005). In Rhodnius, (Veenstra, 2009a) was also identified in Tribolium and showed to diuresis by CT/DH seems to be independent of cAMP (Te Brugge be capable of activating the AST-C receptor in a dose-dependent et al., 2011). CT/DH is also involved in contractions of the gut manner (Audsley et al., 2012). A knock out of the Drosophila Ast- and associated glands (Te Brugge et al., 2009) and may play a role CC gene is embryonic lethal, suggestingthatitisanessentialgene in ecdysis (Kim et al., 2006a,b). The Drosophila CT/DH recep- (Veenstra, 2009a). tor (DH31-R1) is activated by Drome-DH31 and is expressed in the Malpighian tubules. The signaling in HEK293 cells was ALLATOTROPIN RECEPTORS (NP_001127714 ORTHOLOGS) dependent upon co-expression of the receptor component pro- AT was named after its ability to stimulate JH biosynthesis in tein (RCP), which is critical for downstream signaling from the the CA but is also linked to other functions like myostimula- mammalian calcitonin-like receptor (Johnson et al., 2005). One tion, cardio-acceleration, regulation of photic entrainment, ion CT/DH receptor has been predicted in A. aegypti, A. gambiae, A. exchange regulation, and the up-regulation of the secretion of mellifera, N. vitripennis, and T. castaneum and two CT/DH recep- digestive enzymes (Veenstra et al., 1994; Würden and Homberg, tors were found in A. pisum,althoughitisnotyetclearwhether 1995; Lee et al., 1998; Koladich et al., 2002; Petri et al., 2002; both paralogues encode a functional CT/DH receptor. Homberg et al., 2003; Hofer and Homberg, 2006; Lwalaba et al., 2010; Sterkel et al., 2010), of which the myotropic role of AT is CAPA RECEPTORS (CG14575 ORTHOLOGS) probably the most ancestral (Elekonich and Horodyski, 2003). The insect capa neuropeptides, or periviscerokinin peptides, usu- ATs are found in several invertebrate EST and genomic databases ally possess the C-terminal sequence FPRVamide. The insect (for reviews, see: Clynen and Schoofs, 2009; Weaver and Audsley, capability gene encodes a preprohormone containing two capa 2009) and they all have a TARGF/Y motif at the C-terminus. neuropeptides (capa-1 and capa-2) and one or more pyrokinin-1 In Manduca and Bombyx, also AT-like (ATL) neuropeptides (Kean et al., 2002), but they do not activate each other’s recep- were found, which arise by alternative splicing of the AT gene tors (Iversen et al., 2002a; Rosenkilde et al., 2003; Cazzamali (Horodyski et al., 2001; Nagata et al., 2012a). In 2008, the AT et al., 2005). Capa neuropeptides have a diuretic effect on the receptor (ATR) was characterized in B. mori (Yamanaka et al., Malpighian tubules of Drosophila (Pollock et al., 2004), but in 2008). Remarkable, this receptor was mainly localized in the Short R. prolixus and other insects they act antidiuretic (Coast and neuropeptide F (sNPF)-producing cells in the CC, but not in the Garside, 2005; Paluzzi and Orchard, 2006). Recently, it was shown JH producing CA. It was suggested that AT regulates the produc- that the Aedes capa neuropeptide can induce either diuretic or tion and/or release of sNPFs from the CC and that these sNPFs antidiuretic effects depending on the dose (Ionescu and Donini, are responsible for some of the allatotropic functions assigned 2012). In addition, capa neuropeptides have myotropic effects in to the ATs (Yamanaka et al., 2008). In 2011, the ATRs of M. a variety of insects (Wegener et al., 2002; Predel and Wegener, sexta, T. castaneum, and A. aegypti were characterized (Horodyski 2006). Capa receptors have been characterized in Drosophila and et al., 2011; Vuerinckx et al., 2011; Nouzova et al., 2012)and in Anopheles (Iversen et al., 2002a; Park et al., 2002; Olsen et al., show, unlike the ligand, remarkable similarity with the verte- 2007; Terhzaz et al., 2012). Both capa-1 and capa-2 elicited a brate orexin receptors (Yamanaka et al., 2008; Vuerinckx et al., dose-dependent response. The gene encoding the Drosophila capa 2011). Upon activation by AT or ATLs, Manse-ATR, and Trica- receptor is highly expressed in larval and adult tubules (Terhzaz ATR elevate both intracellular Ca2+ and cAMP concentrations et al., 2012). in cellular expression systems (Horodyski et al., 2011; Vuerinckx Capa receptors are found in different mosquito species, et al., 2011). Expression of ATs and ATRs in the different insect although not in A. aegypti. In other holometabolous insects, species is likely to be strongly regulated, since large differences orthologs are found in representatives of the major orders, were measured between developmental stages, sexes, feeding con- including Hymenoptera, Coleoptera, and Lepidoptera. The honey ditions, etc.(Elekonich and Horodyski, 2003; Horodyski et al., bee genome contains two paralogues, as does the B. mori 2011; Vuerinckx et al., 2011; Nouzova et al., 2012). Possibly addi- and M. sexta genome. Also N. vitripennis contains a par- tional ATRs may be present in some insect species, since very alogue (XP_001600587.2), formerly suggested lacking this recep- similar additional receptors have been predicted from Bombyx tor (Hauser et al., 2006, 2010; Yamanaka et al., 2008). Also in and Aedes genomes (Yamanaka et al., 2008; Nouzova et al., 2012). Coleoptera, a capa receptor is found in Tribolium (Hauser et al., 2008). CALCITONIN-LIKE DIURETIC HORMONE RECEPTORS (CG32843/CG17415/CG17043 ORTHOLOGS) CCHAMIDE-1 AND -2 RECEPTORS (CG30106/CG14484 AND CG14593 The first calcitonin-like diuretic hormone (CT/DH), called ORTHOLOGS) Dippu-DH31 was identified in D. punctata (Furuya et al., The first CCHamide neuropeptide has only recently been iden- 2000). More orthologs were discovered by phylogenetic anal- tified in B. mori and it was found to be expressed in the central ysis (Zandawala, 2012). CT/DH stimulates fluid secretion by nervous system and the midgut (Roller et al., 2008). Subsequently, Malpighian tubules and seems to work via a Ca2+-dependent two CCHamide neuropeptides were detected in all insects with mechanism in D. punctata (Furuya et al., 2000). In Drosophila, a sequenced genome (Hansen et al., 2011). In D. melanogaster, CT/DH stimulates fluid secretion by activating the apical cognate receptors have been identified for both CCHamide

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neuropeptides. CG30106 expressed in CHO/Gα16 cells was acti- consisting of 41 amino acids that shows sequence similarity to vated by CCHamide-1 at nanomolar concentrations but also corticotropin releasing factor, urotensin I and sauvagine (Kataoka responded to high concentrations of CCHamide-2. CG14593 was et al., 1989). A second CRF/DH was also discovered in M. activated by nanomolar concentrations of CCHamide-2 as well sexta (Blackburn et al., 1991). CRF/DHs are also referred to as by micromolar concentrations of CCHamide-1 (Hansen et al., as DH44, after the number of amino acids in the CRF/DH of 2011). Previously, CG30106 had been described as a receptor for D. melanogaster (Cabrero et al., 2002). CRF/DH increases fluid myoinhibiting neuropeptides (Johnson et al., 2003b), but as sev- excretion in vivo (Kataoka et al., 1989)andin vitro (Kay et al., eral independent attempts to repeat this result were fruitless, this 1991, 1992; Lehmberg et al., 1991; Clottens et al., 1994)and was likely an erroneous characterization. increases cAMP levels in Malpighian tubules (Lehmberg et al., 1991; Kay et al., 1992; Clottens et al., 1994; Furuya et al., 1995). CORAZONIN RECEPTORS (CG10698 ORTHOLOGS) Besides its diuretic function, CRF/DH negatively influences feed- The first corazonin was isolated and identified from the CC of ing and reproduction (Keeley et al., 1992; Van Wielendaele et al., P. americana and was presented as a new cardioaccelerating neu- 2012) and stimulates gut contractions (Te Brugge et al., 2009). ropeptide (Veenstra, 1989). Corazonin is present in most insects The M. sexta CRF/DH receptor was the first to be cloned and was (excluding beetles and aphids) (for reviews, see: Gäde et al., 2008; activated by Manse-DH, making use of cAMP as second messen- Li et al., 2008; Weaver and Audsley, 2008; Huybrechts et al., 2010) ger (Reagan, 1995). Also the CRF/DH receptor in A. domesticus and the most common corazonin sequence among insects is pQT- uses cAMP as secondary messenger (Reagan, 1996). The first D. FQYSRGWTNamide (Predel et al., 2007). The role of corazonin, melanogaster CRF/DH receptor (DH44-R1), encoded by CG8422, however, is not restricted to cardio-excitatory actions. In locusts, may couple to multiple second messengers as both cAMP and + corazonin is involved in cuticular melanization in the gregarious Ca2 were stimulated upon binding of Drome-DH to the recep- phase (Tawfik et al., 1999; Tanaka et al., 2002), in M. sexta arole tor (Johnson et al., 2004). The second D. melanogaster CRF/DH in the initiation of ecdysis behavior is noticed (Kim et al., 2004; receptor (DH44-R2), encoded by CG12370, is also activated by Žitnanˇ et al., 2007) and it has been suggested that corazonin is Drome-DH resulting in an increase of intracellular cAMP and involved in sex-dependent stress responses (Zhao et al., 2010) causes specific β-arrestin translocation to the plasma membrane. and in the regulation of insulin producing cells in Drosophila DH44-R2 is probably the receptor that modulates DH sensitivity ((Kapan et al., 2012); for reviews, see: Veenstra, 2009b; Boerjan at the level of the microtubules (Hector et al., 2009). A CRF/DH et al., 2010b). receptor was also cloned in B. mori and in A. aegypti (Ha et al., The corazonin receptor was first characterized in Drosophila 2000). The Aedes DH-I receptor is by far the most abundant by making use of a bioluminescence assay (Cazzamali et al., receptor in Malpighian tubules and its transcript levels increase 2002), which was confirmed using Xenopus oocytes (Park et al., after a blood meal (Jagge and Pietrantonio, 2008). More CRF/DH 2002). Subsequently, the corazonin receptors for M. sexta (Kim receptor orthologs were found in T. castaneum and A. pisum,but et al., 2004), A. gambiae (Belmont et al., 2006)andB. mori (Shi only one orthologue is found in A. gambiae, A. mellifera, and N. et al., 2011) were characterized, and a putative corazonin recep- vitripennis up to date. Although the number of receptors seems tor for Musca domestica,wascloned(Sha et al., 2012). Neither to differ, CRF/DH signaling is likely to be conserved in all major the corazonin neuropeptide nor its receptor could be identified insect orders. in Tribolium (Hauser et al., 2008)orAcyrthosiphon.InN. vit- ripennis, despite the presence of a corazonin neuropeptide, so far CRUSTACEAN CARDIOACTIVE PEPTIDE RECEPTORS no corazonin receptor could be predicted (Hauser et al., 2010). (CG33344/CG6111/CG14547 ORTHOLOGS) The invertebrate corazonin receptors are part of the GnRH recep- Crustacean cardioactive peptide (CCAP) was originally identified tor superfamily [see section “Adipokinetic Hormone Receptors in the shore crab Carcinus maenas and exhibited an accelera- (CG11325 Orthologs)”] (Lindemans et al., 2011; Roch et al., tory effect on semi-isolated heart tissue (Stangier et al., 1987). An 2011). The Drosophila receptor is expressed in all developmental identical neuropeptide was subsequently isolated from L. migra- stages (Cazzamali et al., 2002). The Manduca corazonin receptor toria (Stangier et al., 1989). The structure of CCAP is identical is present in endocrine Inka cells, the source of preecdysis- and in all examined insects and consists of the cyclic nonapep- ecdysis-triggering hormones, suggesting a role upstream of ecd- tide PFCNAFTGCamide. CCAP stimulates heart contractions ysis triggering hormone (ETH) (Kim et al., 2004). In Anopheles, (Cheung et al., 1992; Furuya et al., 1993; Li et al., 2011a)and there are pronounced spikes of corazonin receptor expression in contractions of visceral muscles (Stangier et al., 1989; Donini 2nd instar larvae and around the transition from pupa to adult et al., 2001, 2002; Donini and Lange, 2002), and promotes the (Hillyer et al., 2012). In Musca, a high level of corazonin receptor release of AKH (Veelaert et al., 1997) and digestive enzymes (Sakai expression was noticed in the larval salivary glands and a moder- et al., 2006). CCAP also plays a role in ecdysis in several insects ate level in the central nervous system. In adults, the receptor was (Gammie and Truman, 1997; Ewer et al., 1998; Kim et al., 2006a,b; expressed both in the head and body (Sha et al., 2012). Arakane et al., 2008). Drosophila and Anopheles CCAP recep- tors have been expressed in CHO/Gα16 cells and are activated CRF-LIKE DIURETIC HORMONE RECEPTORS (CG8422 AND CG12370 by CCAP (Cazzamali et al., 2003; Belmont et al., 2006). In T. ORTHOLOGS) castaneum, two genes encode for CCAP receptors (Hauser et al., The first corticotropin-releasing factor like diuretic hormone 2008) and both showed a dose-dependent response to CCAP (Li (CRF/DH) was identified in M. sexta as a diuretic peptide (DP) et al., 2011a). Functional analysis using RNAi revealed that only

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TcCCAPR-2 is essential for cardioacceleratory activity (Li et al., modulate heart and gut contractions in insects (Banner and 2011a). CCAP receptor orthologs have been found in A. mellif- Osborne, 1989; Robb and Evans, 1990; Duttlinger et al., 2002). era (Hauser et al., 2006), A. aegypti, A. pisum, B. mori, and N. The FMRFamide neurons become active at the early stages of vitripennis and thus the CCAP receptor seems to be conserved in pre-ecdysis in D. melanogaster, suggesting a role in the ecd- many insect orders. ysis process (Kim et al., 2006b). The Drosophila FMRFamide receptor is the only deorphanized insect FMRFamide recep- ECDYSIS TRIGGERING HORMONE RECEPTORS (CG5911 ORTHOLOGS) tor so far and was found to be activated by six of the seven To be able to grow and undergo metamorphosis, insects need to endogenous D. melanogaster extended FMRFamides (Cazzamali shed their exoskeleton, the process known as ecdysis (Truman, and Grimmelikhuijzen, 2002; Meeusen et al., 2002). Orthologous 1996). This process is initiated and regulated by the ETH (for a FMRFamide receptors are found in A. gambiae (Duttlinger et al., review, see: Žitnanˇ et al., 2007). The eth gene encodes for two 2003), A. mellifera, N. vitripennis, T. castaneum, A. pisum, and B. active neuropeptides named pre-ETH and ETH in moths and mori but have not been characterized up to date. FMRFamide ETH1 and ETH2 in other insects. The ETHs have a common receptors are conserved throughout insects, but our knowledge PRX1-amide (X1 is I, V, L, or M) sequence at the C-terminus about these receptors is very limited. (Park et al., 2002). In Drosophila, Manduca, and Bombyx,the two ETHs differ in length. In Drosophila and Manduca the short INOTOCIN RECEPTOR (NP_001078830 ORTHOLOGS) form only can elicit a part of the ecdysis behaviors, whereas the This neuropeptide was first discovered in the 1980s in L. migra- long one can elicit whole ecdysis (Žitnanˇ et al., 1999; Park et al., toria and showed similarity to the oxytocin/vasopressin peptide 2002). In Bombyx and Aedes, both neuropeptides seemed to be family in Mammalia. The antiparallel dimer of the neuropep- equally potent (Žitnanˇ et al., 2002; Dai and Adams, 2009). In tide was described to have diuretic properties (Proux et al., Apis, Nasonia, and Acyrthosiphon only one form is found, that 1987). Although the neuropeptide could not be identified in most in Apis is shown to be sufficient to elicit ecdysis (Žitnanˇ et al., insect species with sequenced genomes, it was recently found in 1999; Park et al., 2002). These neuropeptides are released in the T. castaneum and N. vitripennis. The mature neuropeptide shows bloodstream and activate the ETH receptors (ETHRs) situated in C-terminal amidation. The T. castaneum inotocin receptor was the central nervous system. The ethr gene encodes for two splice characterized in CHO/Gα16 cells displaying strong activation in variants of the receptor, ETRH-A and ETRH-B (Iversen et al., the nanomolar range. For both the neuropeptide precursor and its 2002b; Park et al., 2002; Dai and Adams, 2009; Roller et al., 2010), receptor transcript levels have been reported throughout develop- and the first ETHRs were identified in Drosophila (Iversen et al., ment of T. castaneum, but in larvae and the head of adult beetles 2002b; Park et al., 2002). The two forms are expressed in differ- high levels were detected (Aikins et al., 2008; Stafflinger et al., ent central neurons (Kim et al., 2006a,b). ETHR-A is expressed 2008). Inotocin was shown to act indirectly as a diuretic factor in inhibitory and/or excitatory neuropeptide producing neurons, on Tenebrio molitor Malpighian tubules in the presence of central releasing the neuropeptides in response to ETH to regulate ecdysis nervous system and CC-CA (Aikins et al., 2008). (Kim et al., 2006a,b). In B. mori ETHR-B is highly expressed in the CA, pointing to a possible allatoregulatory function (Yamanaka KININ (MYOKININ) RECEPTORS (CG10626 ORTHOLOGS) et al., 2008). In Drosophila, Manduca, and Aedes activation of Insect kinins are small neuropeptides that function as myotropic, both receptors expressed in CHO cells could increase intracel- neuromodulatory, and diuretic hormones in the insect + lular Ca2 levels (Iversen et al., 2002b; Park et al., 2002; Kim Malphigian tubules (Hayes et al., 1989; Terhzaz et al., 1999; et al., 2006a,b; Dai and Adams, 2009). In Bombyx,ETHR-Bwas Coast and Garside, 2005). These neuropeptides, which are expressed in HEK293 cells and was shown to be able to increase characterized by the C-terminal sequence FX1X2WGamide intracellular cAMP levels (Yamanaka et al., 2008). In Tribolium, (where X1 is F, H, N, S or Y and X2 is A, P, or S), were first the function of the ETRHs was confirmed through RNAi experi- isolated from Leucophea maderae (Holman et al., 1987; Hayes ments (Arakane et al., 2008). ETRHs were also found in several et al., 1989). The Drosophila kinin receptor was deorphanized holo- and hemimetabolous insects (Riehle et al., 2002; Žitnanˇ in S2 cells using a bioluminescence assay (Radford et al., 2002). et al., 2003; Clynen et al., 2006; Roller et al., 2010). Antibodies raised against the receptor identified sites of myokinin action like stellate cells of the Malphigian tubules, two triplets FMRFAMIDE RECEPTORS (CG2114 ORTHOLOGS) of cells in the pars intercerebralis of the adult central nervous The family of (N-terminally extended) FMRFamides is named sytem and additional cells in the larval nervous system. Western after the tetrapeptide FMRFamide that was identified in the blots and reverse transcription-PCR confirmed these locations, sunray venus clam Macrocallista nimbosa (Price and Greenberg, but also identified expression in male and female gonads. These 1977), but not all extended FMRFamides retain the exact C- tissues also displayed elevated Ca2+ in response to myokinin, terminal motif. The first extended FMRFamide in insects was demonstrating novel roles for these neuropeptides (Radford cloned and characterized in D. melanogaster (Nambu et al., 1988; et al., 2002). In A. aegypti the myokinin receptor was shown Schneider and Taghert, 1988). More extended FMRFamides were to be critical for in vivo fluid excretion post blood feeding detected by mass spectrometric analysis in various major insect (Kersch and Pietrantonio, 2011). In Drosophila the receptor was orders (Verleyen et al., 2004a; Neupert and Predel, 2005; Li et al., shown to be involved in appetite, chemosensory responses, and 2008; Ons et al., 2009; Rahman et al., 2009; Huybrechts et al., metabolism (Al-Anzi et al., 2010; de Haro et al., 2010; Cognigni 2010; Audsley et al., 2011; Zoephel et al., 2012). FMRFamides et al., 2011; López-Arias et al., 2011). Receptor orthologs are also

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present in A. mellifera (Hauser et al., 2006), A. gambiae, Culex bursicon-regulated in contrast to previous results (Kimura et al., quinquefasciatus, A. pisum, P. humanus, and B. mori, but seem to 2004). With regard to wing expansion, it has been proposed be absent in N. vitripennis and T. castaneum. that the bursicon secreting neurons in the abdominal ganglion are responsible for neurohemal release, whereas the bursicon- LEUCINE-RICH REPEATS CONTAINING GPCRs (LGRs) positive neurons in the subesophageal ganglion would orchestrate These receptors, which belong to the rhodopsin-like GPCRs, can wing expansion behavior (Peabody et al., 2008). Also, there are be considered “the odd ones out” within this receptor family as indications that bursicon is released preceding the initiation of they display ectodomains that are much larger than is generally larval ecdysis and that it is responsible for tanning the pupal the case for rhodopsin-like GPCRs. Based on the structure of the case (Loveall and Deitcher, 2010). Additionally, recent data indi- ectodomain and the hinge region which links the ectodomain to cate that homodimers of the bursicon α-andβ-subunits induced the serpentine domain, three major types can be identified within innate immunity genes in the fruit fly (An et al., 2012). the LGR family (Hsu et al., 2000; Van Hiel et al., 2012). In addition to D. melanogaster, LGR2 homologues have been identified in representatives of most insect orders including in A. Type A LGRs (CG7665 orthologs) mellifera, T. castaneum, and A. pisum (Hauser et al., 2006, 2008, Type A LGRs typically have 7–9 leucine-rich repeats (LRRs) in 2010; Van Hiel et al., 2012). Interestingly, in A. mellifera asin- their ectodomain. Although little data are available on these glegenewasfoundtoencodebursicon.Thisproteinfeaturestwo receptors in insects, they are thought to be of significant impor- cystine knot domains similar tothedimeroftwocystine-knot tance as they are homologous to the three vertebrate receptors proteinsasisthecaseinthefruitflyandthesilkmoth(Mendive for the glycoprotein hormones (follicle stimulating hormone, et al., 2005). thyroid stimulating hormone, luteinizing hormone, and choriog- onadotropin). In contrast to the situation in vertebrates, inver- Type C LGRs (CG31096/CG6857 and CG34411/CG4187 orthologs) tebrate genomes encode only one type A LGR and the receptor In contrast to the vertebrate type C LGRs which are activated is conserved in most sequenced insect genomes, but seems to be by members of the insulin-relaxin peptide family, in insects lost in Hymenoptera (Hauser et al., 2006, 2010; Fan et al., 2010). these receptors are largely uncharacterized. In D. melanogaster, Another exception is the T. castaneum genome which encodes two two members of the type C LGRs can be identified, dLGR3 type A LGRs (Hauser et al., 2008; Van Hiel et al., 2012). and dLGR4. In contrast, in A. mellifera and T. castaneum,only LGR1 from D. melanogaster is activated by a heterodimer one receptor has been found which is, respectively, most closely formed by GPA2 and GPB5 (Sudo et al., 2005)whicharepro- related to dLGR3 and dLGR4 (Hauser et al., 2008). The ligands of duced in neuroendocrine cells of the ventral nervous system these receptors are still unknown. (Sellami et al., 2011).Asisthecaseforthevertebrateglycoprotein hormones, both of these subunits are cystine knot proteins with MYOSUPPRESSIN RECEPTORS (CG8985 AND CG43745/CG13803 complex three dimensional structures (Vitt et al., 2001). Based ORTHOLOGS) on transcript studies, dLgr1 gene expression has been detected Myosuppressins have a conserved C-terminal FLRFamide. The throughout all developmental stages of the fruit fly (Hauser et al., first myosuppressin was isolated from L. maderae (Holman et al., 1997; Graveley et al., 2011). In wandering larvae and adults, high 1986). Myosuppressins inhibit gut contractions and regulate transcript abundance has been reported for the hindgut and the heart contractions (Holman et al., 1986; Lange and Orchard, salivary glands (Chintapalli et al., 2007). 1998; Wasielewski and Skonieczna, 2008; Maestro et al., 2011). They also contribute to the regulation of digestive processes by Type B LGRs (CG8930 orthologs) controlling the release of several digestive enzymes in the ali- LGRs from type B feature 16–18 LRRs, about twice the number mentary canal (Harshini et al., 2002; Hill and Orchard, 2005). found in the other two types (Van Hiel et al., 2012). In vertebrates, Furthermore, myosuppressins inhibit food uptake and thus seem three type B LGRs can be identified, whereas in insect genomes to classify as anorexic factors (Matthews et al., 2008; Vilaplana only one type B has been found. The D. melanogaster member of et al., 2008; Down et al., 2011; Nagata et al., 2011). The first the type B LGRs, LGR2 (rk) was cloned in 2000 (Eriksen et al., putative myosuppressin receptor was characterized in L. migra- 2000)andwasactivatedbybursicon(Luo et al., 2005; Mendive toria. Cold competition binding studies and kinetic binding et al., 2005). Analogous to the known ligands of the LGR type A assays with a radiolabeled ligand were used to calculate the dis- receptors, this hormone is a heterodimer of cystine knot glyco- sociation constant of the receptor (Kwok and Orchard, 2002). proteins. The bursicon hormone itself had already been described D. melanogaster possesses two myosuppressin receptors, DMSR-1 in the 1960s (Fraenkel et al., 1966), but it took until 2004 before (CG8985) and DMSR-2 (CG43745/CG13803), and were activated its sequence was unraveled (Dewey et al., 2004; Honegger et al., by D. melanogaster myosuppressin in a dose-dependent manner. 2004). Bursicon was found to induce the hardening and darken- Another myosuppressin receptor was characterized in A. gam- ing of the cuticle of newly eclosed adult flies as well as the expan- biae (Schöller et al., 2005). Additional myosuppressin receptors sion of the wings (Luo et al., 2005; Mendive et al., 2005). More have been annotated in A. aegypti, A. mellifera, N. vitripennis, recently, bursicon has been shown to be responsible for the matu- T. castaneum, A. pisum,andB. mori. DMSR-2 is expressed in the ration of the wing, driving the epithelial-mesenchymal transition head and the body and possibly regulates the actions of myosup- of the wing epithelial cells (Natzle et al., 2008), but the authors pressin on visceral muscles. DMSR-1 is only expressed in the reported that apoptosis associated with wing maturation was not head (Egerod et al., 2003a). Myosuppressin receptors are not

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evolutionary related to FMRFamide receptors and both represent preference behavior screen in Drosophila, Hyun et al. (2005) two separately evolved signaling systems, despite the resemblance identified a mutant that preferred colder temperatures during of their ligands (Schöller et al., 2005). the night and named it han (Korean for cold). Han seems to be a mutant of a P element controlling the CG13758 gene. But NEUROPEPTIDE F RECEPTORS (CG1147 ORTHOLOGS) mutations in the latter gene did not cause temperature preference Invertebrate neuropeptide F (NPF) peptides are structural homo- difference. Instead it shows arrhythmic circadian behavior in logues of the vertebrate NPY family. The Drosophila NPF neu- constant darkness as seen in pdf null mutants. PDF specifically ropeptide was the first full length member of the NPY/NPF binds to S2 cells expressing HAN and thereby elevates the cAMP family identified in insects (Brown et al., 1999). The insect level. The third research group also identified a mutant with the NPF neuropeptides are characterized by the consensus sequence same disrupted circadian behavior as pfd mutants and named xnPxRxnYLx2Lx2YYx4RPRFamide (Nässel and Wegener, 2011). it groom-of-PDF (gop)(Lear et al., 2005). Later studies showed, NPF is involved in various processes in Drosophila like forag- however, that only the advanced evening activity is common with ing, feeding, alcohol sensitivity, stress, aggression, reproduction, the pdf mutants. pdfr mutants, in contrast to pdf mutants, did learning, and locomotion (Shen and Cai, 2001; Wu et al., 2003, have a morning peak (Im and Taghert, 2010). There are several 2005a,b; Wen et al., 2005; Lee et al., 2006; Dierick and Greenspan, indications that pdfr is regulated at steady-state level by the clock 2007; Lingo et al., 2007; Chen et al., 2008; Krashes et al., 2009; Xu gene period (Lear et al., 2005; Mertens et al., 2005). Localization et al., 2010; Hermann et al., 2012; Shohat-Ophir et al., 2012,for studies showed PDFR expression in the brain and visual system a review, see: Nässel and Wegener, 2011). In several other insects in close correspondence to PDF expression. PDFR expression NPF is also (predicted to be) involved in feeding behavior (Zhu shows also similarities to the clock pacemaker network of neu- et al., 1998; Stanek et al., 2002; Garczynski et al., 2005; Gonzalez rons. Furthermore expression is found dispersed in the anterior and Orchard, 2008, 2009; Nuss et al., 2008, 2010; Ament et al., and posterior surfaces of the central brain and subesophageal 2011; Huang et al., 2011b). NPF has also an effect on cardiac activ- ganglion (Shafer et al., 2008; Im and Taghert, 2010). In embryos ity in the blowfly Protophormia terraenovae (Setzu et al., 2012). no expression was noticed (Hyun et al., 2005). Drosophila is The Drosophila NPF receptor was characterized by means of a the only insect where the PDFR has been deorphanized so far. radioreceptor approach. The signaling pathway probably acts via However, homologous sequences are found in many insects like Gi and adenylate cyclase as determined by NPF-induced inhibi- several Drosophila species, A. gambiae, A. mellifera, N. vitripennis, tion of forskolin-stimulated cAMP production (Garczynski et al., B. mori, and T. castaneum. 2002). The NPF receptor was also characterized in Anopheles (Garczynski et al., 2005) and has been predicted in several other PROCTOLIN RECEPTORS (CG6986 ORTHOLOGS) insects like Bombyx and Tribolium (Hauser et al., 2008; Yamanaka Proctolin or RYLPT is a myo- and neurostimulatory neuropep- et al., 2008; Fan et al., 2010). The proposed Nasonia NPF receptor tide of which the appearance seems to be restricted to arthropods (Hauser et al., 2010) is more likely to be a short NPF recep- (Starratt and Brown, 1975; Nässel, 2002). It stimulates or poten- tor; consequently there is probably no NPF receptor present in tiates muscle contraction, is cardio-acceleratory and acts as a Nasonia. Expression of the Drome-NPF receptor was observed in neurohormone (Orchard et al., 1989; Lange, 2002; Clark et al., cells of the midgut and numerous neurons in the brain and ven- 2006; Lange and Orchard, 2006; Nässel and Winther, 2010). The tral nerve cord of the third instar larva (Garczynski et al., 2002). Drosophila gene for the proctolin receptor was identified and The NPF receptor was also located in the adult brain (Wen et al., cloned (Egerod et al., 2003b; Johnson et al., 2003a,b; Taylor 2005; Krashes et al., 2009). The Anoga-NPF receptor was detected et al., 2004; Orchard et al., 2011). When the receptor was sta- in all life stages except for eggs (Garczynski et al., 2005). bly expressed in CHO/Gα16 cells, a dose-dependent response was measured for proctolin (Egerod et al., 2003b). In competition- PIGMENT DISPERSING FACTOR RECEPTORS (CG13758 ORTHOLOGS) based studies, the proctolin receptor binds proctolin with high The first pigment dispersing factor (PDF) neuropeptide in insects affinity (Johnson et al., 2003a). The proctolin and/or proctolin was characterized in Romalea microptera (Rao et al., 1987). The receptor gene was found in the genomes of only a few insect best know function of PDF is its role in the circadian clock as a species, including T. castaneum, T. molitor, P. humanus, and A. network coordinator, output factor and regulator of its plasticity pisum (Hauser et al., 2008; Li et al., 2008; Weaver and Audsley, similar to the vertebrate vasoactive intestinal peptide (VIP). 2008; Huybrechts et al., 2010). No proctolin gene has been iden- Further processes that where associated with PDF are activity, tified in genomes of A. aegypti, A. gambiae, A. mellifera, N. reproduction, arousal, and geotaxis (for a review, see: Meelkop vitripennis, B. mori, or Acromyrmex echinatior and three other ant et al., 2011). Recently, also a role for PDF in the control of visceral species (Hauser et al., 2006, 2010; Roller et al., 2008; Predel et al., physiology in Drosophila was described, thereby extending the 2010; Nygaard et al., 2011), where proctolin and its receptor are similarities between fly PDF and VIP in mammals (Talsma et al., now considered absent. 2012). In 2005, three research groups simultaneously identified the PDF receptor in Drosophila. Mertens et al. (2005)foundthe PYROKININ RECEPTORS (CG8784, CG8795 AND CG9918 ORTHOLOGS) receptor to be specifically responsive to PDF and to couple with Pyrokinins are characterized by the C-terminal sequence Gs, leading to an elevated cAMP concentration upon receptor FXPRLamide (X = S, T, K, A, or G) (Holman et al., 1986; Predel activation. Mutants showed an aberrant behavioral rhythmicity et al., 2001). They are involved in the stimulation of gut motility, and a severe negative geotaxis. In a large-scale temperature the production and release of sex pheromones, diapause, and

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pupariation (Holman et al., 1986; Predel et al., 2001; Nässel, glands and transferred with the seminal fluid during copula- 2002; Altstein, 2004; Verleyen et al., 2004a; Homma et al., 2006). tion. It induces egg laying and loss of receptivity for additional The pyrokinins can be subdivided into two groups, pyrokinin- mating (Chen et al., 1988), alters the female’s sleep pattern 1 (C-terminus WFGPRLamide) and pyrokinin-2 (C-terminus (Isaac et al., 2010) and provokes antimicrobial peptide expression PFKPRLamide) (Cazzamali et al., 2005). The first identified (Peng et al., 2005; Domanitskaya et al., 2007). Additionally, the insect pyrokinin receptors were those of D. melanogaster,where food uptake and preference of females is altered after copulation three pyrokinin receptors occur. CG9918 seems to be specific (Carvalho et al., 2006; Barnes et al., 2007; Kubli, 2010; Ribeiro for pyrokinin-1 and CG8784 and CG8795 for pyrokinin-2 (Park and Dickson, 2010; Vargas et al., 2010). The SP receptor (SPR) et al., 2002; Rosenkilde et al., 2003; Cazzamali et al., 2005). Two from D. melanogaster has been characterized and homologues of pyrokinin receptors were cloned and pharmacologically charac- this receptor were identified in various insects with the exception terized in A. gambiae, one being more specific for pyrokinin-1, of Hymenoptera (Yapici et al., 2008; Kim et al., 2010). Expression the other for pyrokinin-2 (Olsen et al., 2007). The pyrokinin-2 of this receptor is found in the female reproductive organs, espe- receptor orthologue of Helicoverpa zea expressed in Spodoptera cially the spermatheca, and the central nervous system of both frugiperda (Sf) 9 cells also responded to pheromone biosynthesis- males and females in very similar patterns (Yapici et al., 2008; activating neuropeptide (PBAN) in the low nanomolar range Häsemeyer et al., 2009; Poels et al., 2010). (Choi et al., 2003). In addition to SP, the related ductus ejaculatorius peptide A. mellifera has two pyrokinin receptor orthologs, but since (DUP) 99B (Saudan et al., 2002) can activate SPR (Yapici et al., they both have the same sequence identities (55–56%) to the 2008). Although both SP and DUP99B have only been identified Drosophila genes, it is difficult to classify them as pyrokinin-1 in Drosophila species, they can also elicit physiological responses or -2 receptors (Hauser et al., 2006). The T. castaneum genome in the lepidopteran Helicoverpa armigera (Fan et al., 1999). As contains probably three pyrokinin receptors, which are currently SP and DUB99B so far have only been found in most—not classified according to their highest amino acid residue identi- all—Drosophila species, the receptor’s evolutionary conservation ties (Hauser et al., 2008). Pyrokinin receptors have been found in was a puzzle that was only recently solved. SPR can be acti- allinsectssofar,butitisdifficulttoclassifythemaspyrokinin- vated not only by SP, but also by myoinhibiting peptides (MIPs, 1 or -2 receptors (Jurenka and Nusawardani, 2011). This will also known as B-type ASTs) (Kim et al., 2010; Poels et al., remain problematic until in vivo studies using genetics will have 2010). These neuropeptides show the same evolutionary conser- solved this issue (Melcher et al., 2006). vation as SPR and therefore likely correspond to the ancestral ligands of SPR. MIPs display a characteristic WX6Wamide C- RYAMIDE RECEPTORS (CG5811 ORTHOLOGS) terminal motif and were first purified from L. migratoria (Schoofs In 2010, a new class of neuropeptides was discovered from et al., 1991), but members of the neuropeptide family were the genome of N. vitripennis. These RYamides are character- also identified in other species such as Gryllus bimaculatus, D. ized by the C-terminal motif FFxxxRYamide (Hauser et al., melanogaster,andR. prolixus (Lorenz et al., 1995; Williamson 2010). Thereupon, RYamides were identified for all insects with et al., 2001; Lange et al., 2012). MIPs display myoinhibiting activ- a sequenced genome, except for some ant species (Hauser et al., ity in visceral muscle preparations in vitro (Schoofs et al., 1991; 2010; Nygaard et al., 2011). Recently, the RYamide receptors Blackburn et al., 1995, 2001; Predel et al., 2001). In G. bimacu- for D. melanogaster and T. castaneum were characterized using latus, they inhibit JH biosynthesis (Lorenz et al., 1995), and in CHO/Gα16 cells. Both Drosophila RYamides were capable of acti- D. melanogaster and M. sexta,MIPmaysilenceneuronsthatare vating the receptor in the nanomolar range. For T. castaneum not required during the ecdysis program (Kim et al., 2006a,b). it was observed that Trica-RYamide-2 is somewhat more potent Evidence from B. mori indicates that expression of the MIP recep- than Trica-RYamide-1 to activate the receptor (Collin et al., 2011; tor is strongly upregulated following a sudden decline of the Ida et al., 2011a). Although the Drosophila receptor was also acti- 20-hydroxyecdysone titer. Therefore, MIP receptor signaling may vated by high concentrations of mammalian NPY and NPYY (Li be involved in the fine-tuning of ecdysteroid titers (Yamanaka et al., 1992), a phylogenetic analysis seems to indicate that there is et al., 2010). no significant structural relationship between NPY and RYamide receptors (Collin et al., 2011). A first study to unravel the function SHORT NEUROPEPTIDE F RECEPTORS (CG7395/CG18639 ORTHOLOGS) of the RYamides was performed in Phormia regina.Injectionsof sNPF neuropeptides were first identified in A. aegypti and Drosophila RYamide-1 attenuate the feeding motivation of these indicated as “Aedes head peptides” (Matsumoto et al., 1989). flies (Ida et al., 2011a). The receptor is mainly expressed in the Nowadays, sNPFs are predicted in all insect with a sequenced hindgut, while it is not, or hardly present in other investigated genome and they are characterized by the C-terminal consensus tissues in Drosophila males and females. This strengthens the sequence xPxLRLRFamide (Nässel and Wegener, 2011). The main hypothesis that the signaling system has a role in digestion, or functions of sNPF seem to be linked to the regulation of feeding maybe water reabsorption (Collin et al., 2011; Ida et al., 2011a). behavior (Lee et al., 2004, 2008, 2009; Chen and Pietrantonio, 2006; Kahsai et al., 2010; Ament et al., 2011; Lu and Pietrantonio, SEX PEPTIDE/MYOINHIBITING PEPTIDE RECEPTOR (CG16752/CG12731 2011; Nagata et al., 2011, 2012b; Root et al., 2011; Hong et al., 2012; ORTHOLOGS) Mikani et al., 2012). Other processes in which sNPF is probably SP induces the post-mating effects that occur in female fruit flies involved in are diapause, learning behavior, ovarian growth stim- (Kubli and Bopp, 2012). It is produced in the male accessory ulation, metabolic stress, cardiac activity, the circadian rhythm,

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and the regulation of hormone production and hormonal release 1986a,b; Marciniak et al., 2011), but inhibits contractions (Schoofs et al., 2001; Huybrechts et al., 2004; Johard et al., 2008; of the heart, oviduct and ejaculatory duct (Marciniak et al., Nässel et al., 2008; Kahsai et al., 2010; Lu and Pietrantonio, 2011). Only one insect SK receptor, the D. melanogaster SK 2011; Kapan et al., 2012; Setzu et al., 2012; for a review, see: receptor 1 (Drome-SKR1) has been deorphanized so far. It is Nässel and Wegener, 2011). As previously discussed in section activated by [Leu7]-Drome-SK-1 at nanomolar concentrations. “Allatotropin Receptors (NP_001127714 Orthologs),” sNPF pep- [Leu7]-Drome-SK-1 was tested instead of the endogenous tides may also possess allatotropic activity (Yamanaka et al., [Met7]-Drome-SK-1 for stability reasons. The sulphate residue 2008). The first sNPF receptor was identified in Drosophila and is essential for high-affinity receptor binding in all tested cellular all four predicted Drosophila sNPF peptides activate the receptor assays (Kubiak et al., 2002). SK receptors are widespread in in physiological concentrations (Mertens et al., 2002; Feng et al., insects: T. castaneum and A. gambiae have two SK receptors, 2003). Also in Solenopsis invicta (Chen and Pietrantonio, 2006), while A. aegypti, A. mellifera, and B. mori contain at least one. A. gambiae (Garczynski et al., 2007), and B. mori (Yamanaka Drosophila contains a second, recently characterized, SK recep- et al., 2008) the sNPF receptor has been deorphaned. When tor, the CCK-like receptor (Chen et al., 2012). As both SK recep- co-expressed in Xenopus oocytes, the Drosophila sNPF receptor tors probably arose through a gene duplication and because of activates exogenously expressed inwardly rectifying K+ chan- the high homology between the two, it is likely that they also dis- nels (Reale et al., 2004). The sNPF receptor is present in a play similar ligand specificity (Hewes and Taghert, 2001; Kubiak limited number of neurons in the nervous system of all devel- et al., 2002). Both, CCKLR and DSK are strong positive growth opmental stages. Throughout development, the receptor is also regulators of the D. melanogaster larval neuromuscular junction expressed in peripheral tissues including the gut, Malpighian (Chen and Ganetzky, 2012), by signaling via the cAMP-protein tubules, fat body, and ovaries as has been shown in various insects kinase A (PKA)-CRE binding protein (CREB) pathway, known (Mertens et al., 2002; Feng et al., 2003; Chen and Pietrantonio, for its role in structural synaptic plasticity in learning and mem- 2006; Garczynski et al., 2007; Yamanaka et al., 2008; Lu and ory (Chen and Ganetzky, 2012). A β-arrestin translocation assay Pietrantonio, 2011; Kahsai et al., 2012; Nagata et al., 2012b). in HEK cells was used to show that sulfated drosulfakinins are the endogenous ligands for CCKLR-17D1. Binding of DSK-1S or SIFAMIDE RECEPTORS (CG10823 ORTHOLOGS) DSK-2S to the receptor promotes larval locomotion and evokes SIFamides are highly conserved during evolution and have been stress-induced larval escape behavior (Chen et al., 2012). isolated from various insects (Verleyen et al., 2004b; Audsley and Weaver, 2006). SIFamide is present in four neurons in the TACHYKININ RECEPTORS (CG6515 AND CG7887 ORTHOLOGS) insect pars intercerebralis and this specific pattern suggests a Insect tachykinins differ from mammalian tachykinins by their neuromodulatory role in combining visual, tactile and olfactory C-terminal consensus sequence, which is FX1GX2Ramide, rather input. Targeted cell ablation and RNAi has revealed that SIFamide than FXGLMamide as in mammals. There are many different modulates sexual behavior in fruit flies (Terhzaz et al., 2007). tachykinin isoforms in each insect, which are all encoded by a The Drosophila SIFamide receptor is activated by the SIFamide single gene (Siviter et al., 2000). They play various roles in neu- (Jørgensen et al., 2006). The identification of well-conserved ronal signaling and gut activity (Vanden Broeck, 2001; Nässel, SIFamide receptor orthologs in all insects with a sequenced 2002; Coast and Garside, 2005; Predel et al., 2005; Van Loy genome, suggests that SIFamide signaling regulates an essential et al., 2010). The first insect GPCR capable of sensing tachykinin- function in arthropods (Hauser et al., 2006, 2008; Jørgensen et al., related neuropeptides was cloned from Drosophila and is termed 2006; Verleyen et al., 2009). Drosophila tachykinin receptor (DTKR and CG7887) (Li et al., 1991). Drosophila tachykinin-related neuropeptides (Drome-TKs) SULFAKININ AND CHOLECYSTOKININ (CCK)-LIKE RECEPTORS are the endogenous ligands of DTKR and dose-dependently (CG32540/CG6894/CG6881 AND CG42301/CG6857 ORTHOLOGS) increased intracellular Ca2+ concentrations, as well as cyclic AMP Sulfakinins (SKs) are the insect homologues of the vertebrate levels, when applied on DTKR-expressing HEK293 or S2 cells cholecystokinin (CCK) and gastrin neuropeptides (Nachman (Birse et al., 2006; Poels et al., 2007). DTKR is involved in the reg- et al., 1986a,b; Staljanssens et al., 2011). They are named ulation of insulin signaling and the olfactory sensory processing after the sulfated tyrosyl residue in their active core sequence in the antennal lobe (Ignell et al., 2009; Birse et al., 2011). YGHMRFamide that is usually required for biological activity. A second tachykinin receptor in Drosophila is the neurokinin ThefirstinsectSKswereisolatedfromL. maderae and stimulated receptor (NKD and CG6515) (Monnier et al., 1992). Drome-TK-6 hindgut contractions (Nachman et al., 1986a,b). Peptidomic (with an Ala instead of Gly) is the only known fly neuropep- techniques elucidated SK peptides in all major insect orders tide with clear agonist activity on NKD-expressing cells (Poels (Verleyen et al., 2004a; Li et al., 2008; Ons et al., 2009; Hauser et al., 2009), which suggests that NKD is able to discriminate et al., 2010; Huybrechts et al., 2010; Audsley et al., 2011; Zoephel between Ala- and Gly-containing isoforms of tachykinin ligands, et al., 2012). SK regulates food uptake and works as a satiety a feature that does not apply to DTKR (Van Loy et al., 2010). factor that inhibits feeding in several insect species (Wei et al., A similar tachykinin receptor has been cloned from Stomoxys 2000; Maestro et al., 2001; Downer et al., 2007; Meyering-Vos calcitrans (STKR) (Guerrero, 1997). Its endogenous ligand, Stoca- and Müller, 2007). Drosulfakinins are coreleased with ILPs TK, which contains an Ala-residue instead of the highly conserved and influence food choice in D. Melanogaster (Söderberg et al., Gly-residue, behaves as a partial agonist (Poels et al., 2009; Van 2012). It stimulates hindgut contractions (Nachman et al., Loy et al., 2010).

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A putative tachykinin receptor has been cloned from brain tis- embryonic Drosophila neural stem cells (neuroblasts) (Yoshiur a sue of L. maderae (Johard et al., 2001). One or two tachykinin et al., 2012). receptor orthologs have been identified in all insects with a sequenced genome (Hauser et al., 2006, 2008), pointing at an METHUSELAH (CG6936) AND METHUSELAH-LIKE indispensable role oftheseproteins. RECEPTORS The methuselah (mth) gene encodes a family B GPCR and is TRISSIN RECEPTOR (CG34381/CG14003 ORTHOLOGS) involved in stress response and biological ageing in Drosophila Trissin is a recently identified neuropeptide that contains six (Lin et al., 1998). Also 15 methuselah-like (mthl) relatives were Cys residues which form three intramolecular disulfide bridges. identified in Drosophila, most of them characterized by a unique Trissin has been shown to activate the D. melanogaster GPCR motif in the extracellular domain consisting of up to ten cysteine CG34381 stably expressed in CHO/Gα16 cells at picomolar con- residues and several glycosylation sites (West et al., 2001; Patel centrations. Given the toxic and antimicrobial properties of many et al., 2012). Two peptides were identified capable of activating Cys containing neuropeptides, one hypothesis is that trissin may the Mth receptor in a dose-dependent manner. These peptides have an antimicrobial function. Transcript profiling data for correspond with the splice variants A and B of the Drosophila trissin and its receptor on the other hand indicated that tran- gene stunted (sun), which codes for the ε-subunit of mitochondrial scripts for both were present in the central nervous system of third ATP synthase (Cvejic et al., 2004; Kidd et al., 2005). The EC50 instar larvae and adults, suggesting that trissin might be a neu- values for both polypeptides, however, were quite high with 4 μM ropeptide (Ida et al., 2011b). Neuropeptides with high sequence for Sun A and 3.8 μM for Sun B. A second splice variant of the similarity to trissin have been found in several Drosophila species receptor was activated in somewhat lower doses with an EC50 and in three mosquitoes, A. aegypti, A. gambiae, and C. quinque- value of 0.6 μM for Sun A and Sun B. The stunted gene was also fasciatus as well as in B. mori (Ida et al., 2011b). In A. mellifera, shown to be involved in ageing and oxidative stress (Cvejic et al., raalin displayed sequence similarity with trissin but this neu- 2004). Later on, the Drosophila SP and a non-physiological peptide ropeptide only features 5 Cys residues (Kaplan et al., 2007). Since with a randomly generated sequence, the serendipitous peptide currently no signal sequence or C-terminal processing sites have activator of Mth (SPAM), were also identified as agonists of the been identified for this neuropeptide, its sequence may still be Mth receptor. The peptides share almost no sequence homology incomplete. with Sun A and B, indicating the promiscuity of Mth for activation (Ja et al., 2009). However, mth mutants do not affect behaviors REMAINING ORPHAN DROSOPHILA RECEPTORS controlledby SP,soitisdoubtfulthatactivationofthe Mth receptor At least 14 GPCRs predicted to have a neuropeptide as cog- by these ligands is of any biological significance (Ja et al., 2009). nate ligand are still orphan and include CG4313, CG12290, An extensive developmental expression and sequence divergence CG32547/CG12610, CG13229, CG13995, CG33696/CG16726, study was performed by Patel et al. (2012). More studies are needed CG33639/CG5936, CG30340, and CG13575. to affirm which are the cognate ligands of the Mth receptor and to The orphan receptor hector (CG4395) is involved in the reg- unravel the physiological roles of the methuselah-like receptors. ulation of Drosophila male courtship behavior. It is expressed in numerous brain cells, mainly in the mushroom bodies, the cen- REMAINING ORPHAN NEUROPEPTIDES tral complex and, at lower levels, in a subset of glomeruli in the For several neuropeptides, including the ion transport pep- antennal lobes. However, only those cells that co-express fruitless tides (ITPs), neuroparsins, orcokinins, and amnesiac, the cognate (fru—one of the two main regulators of male courtship behav- receptor is unknown at the moment. ITPs function as antidiuretic ior besides doublesex)andhector are critical for male courtship hormones in locusts (Audsley et al., 1992; Phillips et al., 2001). (Li et al., 2011b). ITPs are found in the genomes of many insect orders including The moody gene (CG4322) encodes two splice variants, dipterans, lepidopterans, and coleopterans (Dircksen et al., 2008; Moody-α and Moody-β that differ in their long carboxy-terminal Begum et al., 2009; Dircksen, 2009). domains. Both receptors are coexpressed in glial cells that sur- Neuroparsins are pleiotropic neuropeptides and are inter alia round and insulate the nervous system, which is required for the involved in reproduction and serve as molecular markers of the formation and maintenance of the Drosophila blood-brain barrier process of phase transition in locusts (Brown et al., 1998; Girardie (Bainton et al., 2005; Schwabe et al., 2005; Hatan et al., 2011). The et al., 1998; Claeys et al., 2005b, 2006; Badisco et al., 2007). It moody receptors are also involved in drug sensitivity (Bainton is noteworthy that in the genus Drosophila, the gene coding for et al., 2005; for a review, see: Daneman and Barres, 2005). the neuroparsins is absent from the melanogaster subgroup of the The receptor encoded by trapped in endoderm 1 (tre1, CG3171) subgenus Sophophora, although present in other species of the is a functional analog of the CXCR4 receptor of vertebrates, which genus (Veenstra, 2010). is involved in tumor metastasis (Kamps et al., 2010). Tre1 is Insect orcokinins were first identified in B. germanica and S. essential for the transepithelial migration of Drosophila germ cells gregaria (Pascual et al., 2004; Hofer et al., 2005)andweresub- from the posterior midgut toward the gonads (Kunwar et al., sequently detected in various other insects, excluding Drosophila 2003). The receptor probably plays a role in three phases of and Tribolium (Roller et al., 2008). A study in L. maderae indicates early migration: polarization of germ cells, dispersal into indi- that orcokinins are involved in circadian behavior (Hofer and vidual cells, and transepithelial migration (Kunwar et al., 2008). Homberg, 2006). In B. mori it was demonstrated that orcokinins Tre1 also regulates the relative orientation of cortical polarity in act as prothoracicotropic factors and as such are involved in

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ecdysteroidogenesis (Yamanaka et al., 2011). Recently, a new The usefulness of research on insect neuropeptide signaling family of neuropeptides was discovered in R. prolixus,named systems goes beyond the world of insects as several mammalian Orcokinin B, because it arises due to alternative splicing of the neuropeptides and/or their receptors have orthologs in insects. orcokinin gene. Orcokinin B expression is observed in several Well studied examples of such conserved signaling systems are insects, except Drosophila spp. and A. pisum (Sterkel et al., 2012). the GnRH (Lindemans et al., 2011; Roch et al., 2011; De Loof The amnesiac (amn) gene, which encodes a putative neuropep- et al., 2012), the tachykinin (Pennefather et al., 2004; Van Loy tide precursor (Feany and Quinn, 1995; Moore et al., 1998), is et al., 2010), the NPF/NPY (Nässel and Wegener, 2011), the capa- important for stabilizing olfactory memory, and is involved in pyrokinin/neuromedin (Melcher et al., 2006; Terhzaz et al., 2012), various aspects of other associative and non-associative learn- the AST C/somatostatin (Birgül et al., 1999; Veenstra, 2009a), ing (Quinn et al., 1979; Gong et al., 1998; DeZazzo et al., the myoinhibiting peptide/galanin (Blackburn et al., 1995), the 1999; Keene et al., 2004, 2006; Yu et al., 2006; Motosaka et al., PDF/VIP (Vosko et al., 2007; Talsma et al., 2012), the diuretic hor- 2007). Additional studies have indicated that amn is also involved mone/corticotropin releasing hormone (Lovejoy et al., 2009; De in ethanol sensitivity, sleep, temperature preference behavior Loof et al., 2012), the diuretic hormone/calcitonin (Zandawala, and nociception (Moore et al., 1998; Hong et al., 2008; Liu 2012), and the SK/CCK (Staljanssens et al., 2011)orthologs, et al., 2008; Aldrich et al., 2010). The amn gene was found to meaning that these signaling systems arose before the divergence code for neuropeptides closely related to the vertebrate pituitary of the Proto- and Deuterostomia (more than 700 million years adenylate cyclase-activating polypeptide (PACAP) and submam- ago). This strengthens the reasons to study insect endocrinology malian glucagon/growth hormone-releasing hormone (GHRH) as these studies can help and learn vertebrate endocrinologists and were shown to possess phylogenetically conserved functions more about the current vertebrate receptors (Grimmelikhuijzen (Hashimoto et al., 2002). and Hauser, 2012a). It should be clear that the importance of neuropeptides and DISCUSSION AND FUTURE PROSPECTS their receptors in insect physiology can hardly be overestimated. This review clearly shows that during the last two decades a The great variety among the neuropeptides and their receptors tremendous progress has been made on the field of insect neu- between different insect species makes them also potential tar- ropeptide signaling systems. This progress is mostly attributable gets for the development of a new generation of insecticides with to the increased availability of insect genomes and the advancing high species specificity (Grimmelikhuijzen et al., 2007; Bendena, fields of genomics and peptidomics. It became clear that several of 2010; Grimmelikhuijzen and Hauser, 2012b). Such insecticides these systems are well conserved in all insect species, suggesting would ideally only be harmful for pest insects like insects act- that they are indispensable in general insect physiological func- ing as vectors for diseases or herbivorous insects detrimental tions. Other neuropeptides and their receptors were apparantly for agriculture, while beneficial insects would be unharmed. lost during evolution in several insect species or orders, suggest- The development of such new controlling agents is necessary ing that they were otiose, or that their functions were taken over because of the detrimental effects of the currently used insecti- by other ligands. To gain more insight into the evolution of neu- cides on the environment and their toxicity to non-target organ- ropeptide GPCRs across the Insecta, more insect genomes need isms. The increasing resistance of pest insects against the used to be sequenced, which may soon be accomplished due to the i5K insecticides is also an expanding problem (Casida and Quistad, project (Robinson et al., 2011). But despite the great progression 1998; Van Hiel et al., 2010). As such, more and more research made in insect endocrinology, the knowledge about the functions is performed on neuropeptides in order to develop synthetic of many of the neuropeptides and their receptors involved is still ligands that can disturb the proper functioning of neuropep- scarce. Furthermore, even in Drosophila, the preeminent insect tide signaling systems, provoking detrimental effects on the model organism, various receptors are still orphan and the phys- insect’s fitness. Once again we want to emphasize the impor- iological roles they play are still a mystery. It may be obvious tance of elucidating the biochemical pathways and the functions that a lot of work has to be performed before the functions of of the neuropeptides in order to be able to design these so the different signaling systems will be clearly understood and to called peptidomimetics. Hitherto, no neuropeptide-based insec- unravel how these systems are intertwined with each other. This ticidesareinuse,butalotofprogressismadeonanum- information is also necessary to get a better view on the evolution- ber of interesting neuropeptides (Teal et al., 1999; Gäde and ary origin of the peptide-receptor couples and how they changed Goldsworthy, 2003; Altstein, 2004; Scherkenbeck and Zdobinsky, during evolution among species. It should be emphasized that 2009; Altstein and Nässel, 2010; Nachman and Pietrantonio, sequence similarity between different insects does not necessarily 2010). implies functional similarity or vice versa. So, it remains a prereq- uisite to functionally characterize neuropeptide GPCRs in several ACKNOWLEDGMENTS insect model species. Reverse genetic tools including RNAi, or the The authors acknowledge the Research Foundation Flanders application of the fairly new technique of genome editing using (FWO-Vlaanderen, Belgium, G.0417.08) and the KU Leuven engineered zinc finger nucleases (Urnov et al., 2010)areonly Research Foundation GOA/11/002. Heleen Verlinden is a post- some of the methods being developed in several insects, which doctoral research fellow of the FWO-Vlaanderen. Sven Zels is will likely boost GPCR functional research. Nevertheless, cross supported by the Flemish government agency for Innovation by genome clustering of receptors based on sequence homology may Science and Technology (IWT-Vlaanderen, Belgium). Hans Peter be a good starting point to acquire a better view on their putative Vandersmissen and Kristel Vuerinckx are supported by the KU functions (Metpally and Sowdhamini, 2005). Leuven.

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