Genomics and Peptidomics of Neuropeptides and Protein Hormones

Genomics and Peptidomics of Neuropeptides and Protein Hormones Present in the Parasitic Wasp Nasonia vitripennis| Frank Hauser,*,† Susanne Neupert,‡ Michael Williamson,† Reinhard Predel,‡ Yoshiaki Tanaka,§ and Cornelis J. P. Grimmelikhuijzen† Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, DK-2100 Copenhagen, Denmark, Institute of Zoology, University of Jena, D-07743 Jena, Germany, and National Institute of Agrobiological Science, Division of Insect Science, Tsukuba, Ibaraki 305-86 34, Japan Received June 8, 2010

Neuropeptides and protein hormones constitute a very important group of signaling molecules, regulating central physiological processes such as reproduction, development, and behavior. Using a bioinformatics approach, we screened the recently sequenced genome of the parasitic wasp, Nasonia vitripennis, for the presence of these signaling molecules and annotated 30 precursor genes encoding 51 different mature neuropeptides or protein hormones. Twenty-four of the predicted mature Nasonia neuropeptides could be experimentally confirmed by mass spectrometry. We also discovered a completely novel neuropeptide gene in Nasonia, coding for peptides containing the C-terminal sequence RYamide. This gene has orthologs in nearly all arthropods with a sequenced genome, and its expression in mosquitoes was confirmed by mass spectrometry. No precursor could be identified for N-terminally extended FMRFamides, even though their putative G protein coupled receptor (GPCR) is present in the Nasonia genome. Neither the precursor nor the putative receptor could be identified for allatostatin-B, capa, the glycoprotein hormones GPA2/GPB5, kinin, proctolin, sex peptide, and sulfakinin, arguing that these signaling systems are truly absent in the wasp. Also, antidiuretic factors, allatotropin, and NPLPlike precursors are missing in Nasonia, but here the receptors have not been identified in any insect, so far. Nasonia (Hymenoptera) has the lowest number of neuropeptide precursor genes compared to Drosophila melanogaster, Aedes aegypti (both Diptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Acyrthosiphon pisum (Hemiptera). This lower number of neuropeptide genes might be related to Nasonia’s parasitic life. Keywords: neuropeptide • protein hormone • PTTH • GPCR • genomics • peptidomics • MALDI-TOF • CID • mass spectrometry • insect

Introduction The tiny parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae) has been used for genetic, ecological, evolutionary and developmental research for many decades.1,2 Nasonia lays eggs into the pupae of various fly species, primarily blowflies and fleshflies, and this interesting life style has stimulated research on host finding, host selectivity, and many other aspects of parasitic behavior.3 The usefulness of parasitic wasps for the biological control of pest insects together with its potentials as a model insect for biological research have recently led to the sequencing of the genome of N. vitripennis and two other parasitic wasps belonging to the genus Nasonia.4 The cloned cDNA sequences reported in this paper have GenBank accession numbers GU937435, HM461994 and HM461995. * To whom correspondence should be addressed. F. Hauser, Center for Functional and Comparative Insect Genomics, Department of Biology, Universitetsparken 15, DK-2100 Copenhagen, Denmark; Tel. +45 3532 1206; Fax +45 3532 1200; e-mail: [email protected]. † University of Copenhagen. ‡ University of Jena. § National Institute of Agrobiological Science. |

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In addition to the three Nasonia species, the genomes of at least 25 other insect species have been sequenced or are in the pipeline to be sequenced5 (http://www.ncbi.nlm.nih.gov/ sutils/genom_table.cgi?organism)insects). These insects belong mainly to the Holometabola (insects with a complete metamorphosis), of which they represent all major orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera). With the recently sequenced genome from Nasonia we now have an excellent tool to do comparative genomics studies, especially because another hymenopteran, the honey bee Apis mellifera, which in contrast to Nasonia has a social life style, also has been sequenced recently.6 One important group of proteins to be analyzed using the comparative genomics approach are neuropeptides and protein hormones, which are signaling molecules that control central physiological processes such as development, reproduction, behavior and homeostasis. The receptors for these neuropeptides are usually G protein-coupled receptors (GPCRs), which are transmembrane proteins located in the cell membrane of the target cells.7,8 10.1021/pr100570j

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Global Analysis of Neuropeptides in Nasonia The availability of the sequenced Nasonia genome now allows us to identify the complete set of neuropeptides and protein hormones in this organism and to compare this set with those recently found in the genomes from the honeybee A. mellifera,9,10 the fruit fly Drosophila melanogaster,11,12 the mosquito Aedes aegypti,13 the silkworm Bombyx mori,14 the red flour beetle Tribolium castaneum,15,16 and the pea aphid Acyrthosiphon pisum.17 This comparative genomics approach should give us valuable information to understand the evolution of neuropeptides, and if possible to correlate insect life style with neuropeptide gene content. Here, we searched the Nasonia genome with known insect neuropeptide and protein hormone precursors and identified 30 precursor genes encoding 51 different putative mature neuropeptides or protein hormones. The presence and structure of 24 of these neuropeptides could be confirmed by direct peptide profiling of brain tissue by MALDI-TOF mass spectrometry (MS). Because neuropeptides and protein hormones require specific receptors to function in the organism, we have, in parallel, also identified all Nasonia neuropeptide and protein hormone receptors belonging to the GPCR superfamily (Hauser et al., unpublished). The presence or absence of a neuropeptide receptor in the analyzed Nasonia genome further supports our current findings of the presence or absence of the neuropeptide and protein hormone genes.

Experimental Section Genomics and Software. The whole genome sequence of Nasonia vitripennis, version 1.0, was used for homology searches with known invertebrate neuropeptide sequences at the Web site of the Human Genome Sequencing Centre, Baylors College of Medicine (http://blast.hgsc.bcm.tmc.edu/blast.hgsc), using TBLASTN. The assembled version 1.0 covers 98% of BAC and 97% of EST sequences. In the remaining unassembled trace sequences, we could not detect any neuropeptide genes. For TBLASTN homology searches in other species, the nucleotide collection, nonhuman nonmouse ESTs or whole-genome shotgun reads databases at NCBI (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) were chosen. Prediction of the gene structure and open reading frame was done in GENBOREE (http://www. genboree.org/java-bin/login.jsp) that contains the GLEAN2 predictions incorporating the results from multiple gene prediction programs and by manual correction. Signal peptides were predicted by the SIGNALP server (http://www.cbs.dtu.dk/ services/SignalP/). Multiple sequence alignments were performed in ClustalW (http://www.ebi.ac.uk/Tools/clustalw/), or using the Lasergene software package (DNASTAR inc.) with manual adjustments. Phylogenetic analyses were done, using Lasergene software. Peptide processing was predicted using the ProP program (http://www.cbs.dtu.dk/services/ProP/). Dissection and Sample Preparation for Mass Spectrometry. N. vitripennis (adults and larvae) were fixed with microneedles and submerged in insect saline (pH 7.25) of the following composition: NaCl (7.50 g/L), KCl (0.20 g/L), CaCl2 (0.20 g/L) and NaHCO3 (0.10 g/L). The body cavity or the head capsule was opened with fine forceps and ultrafine scissors. Different ganglia of the ventral nerve cord, brain/subesophageal ganglion (see Supporting Information, Figure S1), and corpora cardiaca were dissected (at least five preparations of each tissue sample) and cut in smaller pieces, which were transferred with a glass capillary into a drop of distilled water on the sample plate for MALDI-TOF MS. The water was subsequently removed using a glass capillary. Subsequently, the tissue was air-dried and

covered with approximately 50 nL of matrix solution (saturated R-cyano-4-hydroxycinnamic acid dissolved in methanol/water 1:1, v/v) over a period of about 5 s using a Nanoliter injector (World Precision Instruments, Berlin, Germany). Each preparation was air-dried again and finally covered with destilled water for a few seconds, which was removed by cellulose paper. MALDI-TOF MS. MALDI-TOF analyses were performed on an ABI 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA). To determine the parent masses, the instrument was initially operated in reflectron mode. For the tandem MS experiments (performed in gas off mode), a CID acceleration of 1 kV was used in all cases. The number of laser shots used to obtain a spectrum varied from 800-4000, depending on signal quality. The fragmentation patterns were used to manually determine the sequence of the peptides by using the Data ExplorerT software package. cDNA Cloning. For PCR, total RNA was isolated from whole adult animals (mixed males and females), using the NucleoSpin RNA II kit (Macherey-Nagel). cDNA was synthesized and amplified using the FirstChoice RLM-RACE kit (Ambion). Primers are given in the Supporting Information section Table S1.

Results Table 1 gives an alphabetical list of the 51 different neuropeptides that are encoded by the 30 neurohormone precursor genes we identified in the Nasonia genome (for FASTAsequences of the precursors see Supporting Information, Figure S2). Only 12 of these precursor genes have been automatically identified as genes by the genome project software, whereas the other 18 have been manually identified by us. Table 2 divides these 30 precursor genes into two different groups, A or B. For all members of group A, a likely receptor gene could be identified in Nasonia, giving additional support for the presence of these neuropeptides in Nasonia. For members of group B: a receptor gene can not be given, because these receptors have not been identified (deorphanized) yet in any insect.7,8 The neuropeptides that we could not identify, although we found a putative receptor in Nasonia, are given in group C. In group D, absent neuropeptides are shown for which we also could not find their receptors. These results, therefore, confirm that these peptides are truly absent in Nasonia. Finally, group E lists absent peptides for which a receptor is unknown in any insect. Below, we discuss these groups in more details. A. Neuropeptides or Protein Hormones That Can Be Matched with Putative Receptors. We found 22 wasp neuropeptide and protein hormone precursor genes that could be matched with putative receptors (Table 2, Group A). Products of seven of these neuropeptide genes (adipokinetic hormone, allatostatin-A, allatostatin-C, corazonin, myosuppressin, orcokinin, and short neuropeptide F) were detected in the corpora cardiaca, using mass spectrometry (both MALDI-TOF and CID), which suggests that they are neurohormones (Figure 1). Additional neuropeptides were identified by mass spectrometry in specific brain regions such as pars intercerebralis, accessory medulla, and antennal lobe (Figure 2, Figure 3), or in the thoracic ganglia (Figure 4). ACP. ACP (adipokinetic hormone/corazonin-related peptide) and its receptor represent a recently discovered novel insect signaling system closely related to, but functionally independent from the insect adipokinetic hormone (AKH) and corazonin systems.18 The Nasonia ACP precursor encodes a single putative neuropeptide, the decapeptide pQVTFSKGWGPamide Journal of Proteome Research • Vol. 9, No. 10, 2010 5297

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Table 1. Alphabetical List of Mature Neuropeptides and Protein Hormones Present in Nasonia

a For longer peptide sequences, the accession no. assigned by the Nasonia Genome Project is given. confirmed by mass match; ion intensity was not sufficient for fragment analysis.

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b

Allatostatin-A-4 and RYamide-1 are only

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Global Analysis of Neuropeptides in Nasonia Table 2. Neurohormone Precursors Present (A and B) or Absent (C-E) in Nasonia

a nd, not detected in Nasonia; na, not applicable (no receptor known in any insect). Supporting Information).

(Table 1 and Supporting Information, Figure S2). In Figure 5, the sequence of the Nasonia ACP precursor is aligned with ACP precursors found in other insects. This alignment clearly shows the similar overall structure of all insect ACP precursors, where the ACP sequence is directly located after the signal peptide

b

The cDNAs of these genes have been cloned (Figures S3-S5,

sequence. Also the C-terminal parts of the precursors contain several highly conserved residues, including a potential cystine bridge. This organization resembles that of the AKH and corazonin precursors.18 Interestingly, ACP and its receptor are absent in Drosophila, honey bee, pea aphid and the body louse.18 Journal of Proteome Research • Vol. 9, No. 10, 2010 5299

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Figure 1. MALDI-TOF mass spectrum typical of a preparation of a single pair of corpora cardiaca from Nasonia. The assigned neuropeptides are labeled and the identity was subsequently confirmed by fragment analysis (not shown). The presence of these neuropeptides in the corpora cardiaca suggests a role as neurohormones. Not all ion signals can be assigned to products of annotated neuropeptide genes. Detection of only a single orcokinin is in accordance with the copy number of ORC-2 (6×) in the orcokinin preprohormone. The other orcokinins were detectable in spectra with higher orcokinin abundance (see Figure 2). We do not regard NVP as a neuropeptide, because, although originally identified in the honeybee,9 this peptide sequence is not conserved in insects (see Supporting Information, Figure S6). The NVP signal shown in this and other mass spectra comes from a peptide without the NVP sequence located in a region of the NVP protein different from the NVP region in the honeybee protein (see regions highlighted in red and green in Supporting Information, Figure S6). AKH, adipokinetic hormone; AST, allatostatin; AST-PP, allatostatin precursor peptide (AYTYRSEY-OH); CC, corpora cardiaca; COR, corazonin; MS, myosuppressin; ORC, orcokinin; sNPF, short neuropeptide F.

AKH. Adipokinetic hormones (AKHs) are 8 to 10 amino acid residues long insect neuropeptides expressed in endocrine cells from the corpora cardiaca.19 They control lipid and sugar mobilization from the fat body during energy-consuming activities such as flight and intense locomotion. In the Nasonia genome, a single putative AKH precursor gene was found encoding the peptide pQLNFSTGWamide (Table 1 and Supporting Information, Figure S2). We confirmed the existence of this hormone in Nasonia by cDNA cloning of the entire AKH prepropeptide (Supporting Information, Figure S3) and by MS (Figure 1, Table 1). Allatostatin-A. In various insects, type A allatostatins inhibit the corpora allata to produce juvenile hormone or block muscle contraction in the gut.11 The Nasonia allatostatin-A precursor encodes 5 putative amidated peptides with the common C-terminal motif Y/FXFGLamide (Table 1 and Supporting Information, Figure S2). Four of these peptides and an additional precursor peptide (AYTYRSEY-OH) could be confirmed by MS (Figure 1, Table 1). Due to the lack of amino acids with charged side chains, AST-A-1, -2, and -4 show a very low signal intensity in mass spectra and only the [M + Na/K]+ adduct ions of these peptides were detectable. The nondetection of the predicted AST-A-5 suggests that the predicted monobasic cleavage site (Lys) preceding the N-terminus is not being used. Allatostatin-C and Allatostatin-CC. Another insect neuropeptide inhibiting the juvenile hormone production of the 5300


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Figure 2. MALDI-TOF mass spectra typical of preparations from specific brain regions. (A) Pars intercerebralis of the protocerebrum. In addition to peptides observed in the corpora cardiaca, ion signals of tachykinins (TK), SIFamide, and diuretic hormone (DH-31) can be identified. (Inset) Different signal intensities of nearly identical orcokinins which originate from different copy numbers in the preprohormone. (B) Accessory medulla of the optic lobes with a number of neuropeptides, including weak signals of pigment dispersing factor (PDF). We do not regard ITG as a neuropeptide, because, although originally identified in the honeybee,9 this peptide sequence is not conserved in most other insects, although the protein itself is conserved (Figure S7, Supporting Information). The ITG peptide sequence shown in this and other mass spectra is highlighted in green in Figure S7 (Supporting Information).

corpora allata (for example in the moth Manduca sexta) is allatostatin-C, a cyclic neuropeptide that is structurally unrelated to allatostatin A.11,20-22 In Nasonia, there is an allatostatin C gene coding for the peptide NYWRQCAFNAVSCFamide (Table 1 and Supporting Information, Figure S2). By mass spectrometry, this peptide was found throughout the CNS and also in the corpora cardiaca (Figures 1-3). Nasonia allatostatin-C is very similar to allatostatin-C found in the honey bee A. mellifera (Hymenoptera, Holometabola) and several hemimetabolous insects (all amidated at the C-terminus), but quite different from the nonamidated allatostatins-C occurring in holometabolous insects other than the hymenopterans.22 In addition to this allatostatin-C precursor, a partial precursor gene coding for an allatostatin-C paralog, allatostatin-CC, has recently been annotated in Nasonia and other insects22 (Table 1). The function of allatostatin-CC is still unknown.22 To confirm the existence of allatostatin-C and allatostatin-CC in Nasonia, we cloned the cDNAs encoding the entire prepropeptides of both paralogs (Supporting Information, Figure S4 and S5). Bursicon. The molting glycoprotein hormone bursicon is, like all mammalian glycoprotein hormones, a heterodimer composed of two larger N-glycosylated cystine-knot polypeptides, alpha- and beta-bursicon.23,24 Bursicon causes tanning and hardening of the soft cuticle after adult ecdysis and induces wing expansion and apoptosis of the wing epithelial cells after

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Global Analysis of Neuropeptides in Nasonia 25-27

Figure 3. Mass spectrum analysis of neuropeptides in the antennal lobe. (A) MALDI-TOF mass spectrum typical of a preparation of a single antennal lobe of Nasonia (approximately 50 µm in length). The assigned neuropeptides are likely involved in the processing of the olfactory information: sNPF, short neuropeptide F; AST, allatostatin; TK, tachykinin. Particularly impressive in this context is the abundance of TK 1-5. (B) CID mass spectrum of TK-2 at [M + H]+: 993.4, taken from the same sample. Fragment ions are labeled and confirm the amino acid sequence of TK-2.

Figure 4. MALDI-TOF mass spectrum of a preparation of a thoracic ganglion from Nasonia. The arrow marks a substance that is mass-identical with a putative product of the RYamide preprohormone (pQDNFYASGRFamide). Due to low signal intensity, it was not possible to perform fragment analysis of this peptide.

completed expansion. In the Nasonia genome, we found one precursor for alpha- and one for beta-bursicon. Both glycoprotein subunits contain the typical pattern of 11 cystein residues that can form 5 intramolecular and one intermolecular disulfide bridges (Supporting Information, Figure S2). CCAP. The crustacean cardioactive peptide CCAP (PFCNAFTGCamide) is a highly conserved cyclic neuropeptide originally isolated from the shore crab Carcinus maenas, but present in identical form also in insects.11 It is coexpressed with bursicon in several neurons of the insect nervous system, especially in the abdominal ganglia, where it might support the action of bursicon.25,28 CCAP is a multifunctional peptide that has cardioexcitatory and AKH-releasing activity, but also controls the motor behavior program associated with ecdysis.29 The Nasonia CCAP precursor gene encodes, like in other insects, a single copy of this neuropeptide (Table 1 and Supporting Information, Figure S2). Corazonin. Corazonin was originally isolated from cockroaches based on its cardio-excitatory actions on the isolated cockroach heart.30 In locusts, corazonin induces a darkening of the exoskeleton in association with swarm formation and migration.31 Furthermore, corazonin is proposed to be associated with behavioral and physiological responses to stress.32-34 Corazonin is found in most insects, but not in Coleoptera and the pea aphid.16,17,35,36 Within the Hymenoptera, at least four different isoforms of corazonin occur which is the greatest diversity found for this peptide in any insect order.35 In Nasonia, we found one precursor gene that encodes the peptide [Arg7]-corazonin (pQTFQYSRGWTNamide, Supporting Information, Figure S2), which is identical to the ancient corazonin found in most insects and also in crustaceans.35 The expression and structure of this peptide could be confirmed by MS (Figure 1, Table 1). Thus, the remarkable sequence variation of corazonin in the Hymenoptera seems to be restricted to the aculeatan lineage (“stinging wasps”) of Hymenoptera. DH31 and DH44. In Drosophila, two structurally unrelated diuretic hormones, DH31 (31 amino acid residues long) and DH44 (44 amino acid residues long), act on the Malpighian tubules to control water homeostasis.37 DH31 is structurally related to mammalian calcitonin, whereas DH44 resembles corticotropin-releasing factor (CRF).38 In Nasonia, we annotated one orthologue to each of these hormones (Table 1 and Supporting Information, Figure S2). The Nasonia DH31 could be confirmed by MS (Figure 2, Table 1). EH. Eclosion hormone (EH) is produced by a single pair of brain neurons during each insect molt.39 In the central nervous system, EH induces the release of CCAP, whereas it acts peripherally on Inka cells in the epitracheal glands and induces the release of ecdysis triggering hormone.40 Recently, a membrane-bound guanylyl cyclase was identified as an EH receptor in Inka cells.41 In Nasonia, we identified a precursor gene coding for an EH ortholog (Table 1 and Supporting Information, Figure S2). Interestingly, an alignment of EH sequences from various insects shows that the Nasonia EH contains only two of the three disulfide bridges usually present in EHs (Figure 6).42,43 ETH. In insects, ecdysis triggering hormone (ETH) is expressed in Inka cells and initiates molting.44 We found one precursor gene coding for a single ETH orthologue with the sequence DEPPAFFLKIAKNIPRIamide (Figure 7 and Supporting Information, Figure S2). The overall structure of the Nasonia ETH preprohormone resembles the ETH preprohormone found in honey bee,9 but is different from the ETH precursors found Journal of Proteome Research • Vol. 9, No. 10, 2010 5301

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Figure 5. Alignment of the ACP preprohormones from the parasitic wasp N. vitripennis (Nv-ACP, GenBank accession no. NM_001167727), the mosquitoes A. gambiae (Ag-ACP, NM_001166025), A. aegypti (Aa-ACP, XM_001661147, manually corrected), C. pipiens (Cp-ACP, FN391989), the silkmoth B. mori (Bm-ACP, NM_001134241), the European corn borer O. nubilalis (On-ACP, annotated from the expressed sequence tags [EST] database accession no. GH995131), the red flour beetle T. castaneum (Tc-ACP, NM_001166025), the bloodsucking bug R. prolixus (Rp-ACP, annotated from the whole genome shotgun sequences [WGS] database accession no. ACPB01036401) and the black-legged tick I. scapularis (Is-ACP, annotated from WGS ABJB010491828). Amino acid residues that are common in at least five sequences are highlighted. The immature ACP sequences are boxed. The arrows indicate the sites where signal peptidase (SP) removes the signal peptide, or where preprohormone convertase (PC) liberates immature ACPs from the prohormone. Intron positions are indicated by small boxes.

Figure 6. Alignment of the eclosion hormone (EH) preprohormones from N. vitripennis (Nv-EH, XM_001603287), A. mellifera (Am-EH, XM_001122120, manually corrected), D. melanogaster (Dm-EH, NM_079662), A. aegypti (Aa-EH, XM_001652153), A. gambiae (Ag-EH, XM_001230804), C. pipiens (Cp-EH, XM_001864393, manually corrected), B. mori (Bm-EH, NM_001043842), T. castaneum (Tc-EH, XM_964071), A. pisum (Ap-EH, XM_001943582) and I. scapularis (Is-EH, XM_002399230). Amino acid residues that are common in at least five sequences are highlighted. The arrow indicates the position where signal peptidase (SP) removes the signal peptide. The conserved cysteine residues forming disulfide bonds are shown in bold. Lines below the sequences represent the position of the three disulfide bonds usually present in EHs, lines above the sequences indicate the two putative disulfide bonds present in the Nasonia EH.

in other insects that, with the exception of pea aphid, always contain two ETH peptides (Figure 7).45 Inotocin. Inotocin is a recently discovered insect neuropeptide structurally related to mammalian oxytocin and vasopressin.46 Inotocin activates the identified inotocin receptor, but otherwise the physiological role of inotocin is unknown.46 Also in Nasonia there is an inotocin peptide with the sequence CLITNCPRLamide (Table 1). Not only the peptide sequence, but the entire overall structure of the Nasonia inotocin prepropeptide resembles the mammalian preprohormones, as it also contains a neurophysin-like sequence with 14 highly conserved cystein residues at the C-terminal end of the prepropeptide (Supporting Information, Figure S2).46 5302


Myosuppressin. Myosuppressin is a decapeptide that inhibits contraction of a variety of visceral muscles.11 We annotated one precursor gene encoding a single Nasonia myosuppressin peptide with the sequence pQDVDHVFLRFamide (Supporting Information, Figure S2). MS analysis confirmed the presence and identity of this peptide in Nasonia (Figure 1, Figure 2, Table 1). Amazingly, in the brain and corpora cardiaca the N-terminally blocked form (pQ) and the nonblocked form (Q) were found with about equimolar amounts (Figure 1, Figure 2). NPF. In Drosophila, neuropeptide F is a 36 amino acid residue long peptide with the C-terminal sequence RVRFamide.11 It is structurally related to the vertebrate neuropeptide

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Figure 7. Alignment of the ecdysis triggering hormone (ETH) preprohormones from N. vitripennis (Nv-ETH, NM_001142635), A. mellifera (Am-ETH, NM_001142607), D. melanogaster (Dm-ETH, NM_079960), A. gambiae (Ag-ETH, XM_308702), T. castaneum (Tc-ETH, NM_001172273), B. mori (Bm-ETH, NM_001172272) and A. pisum (Ap-ETH, NM_001163212). Amino acid residues that are common in at least three sequences are highlighted. The arrows indicate the positions where signal peptidase (SP) removes the signal peptide, or where preprohormone convertase (PC) liberates immature peptides from the prohormone. The immature ETH sequences are boxed. Note that Nasonia and the honey bee have only one ETH peptide, whereas higher holometabolous insects have two ETHs.

Figure 8. Alignment of the prothoracicotropic hormone (PTTH) precursors from N. vitripennis (Nv-PTTH, annotated from WGS AC185337), B. mori (Bm-PTTH, NM_001043884), A. gambiae (Ag-PTTH, XM_555854), T. castaneum (Tc-PTTH, EEZ99381) and D. melanogaster (DmPTTH, NM_134693, manually corrected). The signal peptides are highlighted in blue. Amino acid residues that are common in at least three sequences are highlighted in gray. The conserved cysteine residues forming cystine bridges are highlighted in orange with gray background. Lines above and below the sequences indicate the intramolecular cystine bridges, the star indicates the conserved cysteine residues making intermolecular cystine bridges, thus forming PTTH dimers.51 Note that Nasonia has one cystine bridge less than the other insects.

Y family and affects food intake and feeding behavior in the fruit fly.47 In Nasonia, we annotated a gene coding for a 39 amino acid residues long NPF orthologue with the C-terminal sequence KARYamide (Table 1 and Supporting Information, Figure S2), which resembles NPF from the honey bee.9 PDF. Pigment dispersing factor (PDF) was first isolated from crustaceans48 and got its name for its pigment cell dispersing activity. Similar peptides have later been found in insects,49 where they are involved in the regulation of circadian rhythm.50 In Nasonia, there is a PDF-like precursor gene coding for the PDF sequence NSELINSLLSLPKNMNNAamide (Supporting Information, Figure S2). The presence and structure of this peptide could be confirmed by MS (Figure 2, Table 1). Prothoracicotropic Hormone (PTTH). In insects, development and metamorphosis are coordinated by the steroid

hormones 20-hydroxyecdysone (20E). Production and release of 20E from prothoracic glands is regulated by the brain-derived prothoracicotropic hormone PTTH.51 The overall amino acid sequences of various insect PTTHs are not very well conserved, but they are all believed to form homodimers.52 We identified a gene coding for the Nasonia PTTH (Table 1 and Supporting Information, Figure S2). Interestingly, this Nasonia PTTH contains only 5 of the 7 characteristic cysteine residues present in PTTH sequences from other insects (Figure 8). Recently, it was found that the Drosophila tyrosine kinase named Torso was the receptor for Drosophila PTTH.53 In Nasonia, we could also identify a PTTH receptor ortholog (Table 2). Pyrokinins. Pyrokinins (PKs) are small neuropeptides characterized by the common C-terminal sequence FXPRLamide. They can regulate muscle activity, pheromone biosynthesis, Journal of Proteome Research • Vol. 9, No. 10, 2010 5303

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Figure 9. Alignment of the CCHamide preprohormones from N. vitripennis (Nv-CCHa1, annotated from WGS AAZX01006578, and Nv-CCHa2, annotated from WGS AAZX01016958), A. mellifera (Am-CCHa1, XM_625260, and Am-CCHa2, XM_001120020), D. melanogaster (Dm-CCHa1, NM_001104314, and Dm-CCHa2, NM_142028), A. gambiae (Ag-CCHa, XM_001237549), A. aegypti (Aa-CCHa, XM_001649897), T. castaneum (Tc-CCHa, annotated from WGS AAJJ01000164), B. mori (Bm-CCHa, NM_001130115), and I. scapularis (Is-CCHa, XM_002413842, manually corrected). Amino acid residues that are common in at least six sequences are highlighted in gray. The arrows indicate the cleavage positions of signal peptidase (SP) or prohormone convertase (PC). The immature CCHamide sequences are boxed. The two cysteine residues, forming a cystine bridge in each CCHamide peptide, are shown in orange; this cystine bridge is indicated above the sequences.

melanisation, pupariation, diapause, and feeding behavior in insects.11 There is a subgroup of pyrokinins characterized by the C-terminal sequence L/MWFGPRLamide, of which the specific function is largely unknown. In Drosophila, there are two precursor genes encoding pyrokinins: (i) the capability gene encoding one pyrokinin-1 (PK-1, which has the C-terminal sequence LWFGPRLamide) and two capa peptides (characterized by the C-terminal sequence FPRVamide, see below);54 and (ii) the hugin gene encoding one pyrokinin-2 (PK-2, SVPFKPRLamide).55 One receptor was identified in Drosophila that was preferentially activated by PK-1, whereas two other receptors were more specifically activated by PK-2.56,57 Most insects have, like Drosophila, two preprohormone genes encoding pyrokinins. In Nasonia, however, we only found one pyrokinin preprohormone gene that encodes three different pyrokinins (but no Capa peptides), of which one has the C-terminal sequence MWFGPRLamide, thus resembling Drosophila PK-1 in its structure (Table 1 and Supporting Information, Figure S2). The proposed structure of this peptide is unusual, because it is much longer than all other known pyrokinins and contains a potential cystine bridge. SIFamide. SIFamide is a highly conserved dodecapeptide AYRKPPFNGSIFamide that got its name after its C-terminal three amino acid sequence. It was first isolated from the gray flesh fly Neobellieria bullata and shortly after also found in various other insects.58 The physiological functions of SIFamide are still not fully understood, but targeted cell ablation and RNA interference experiments in Drosophila suggest that SIFamide might modulate sexual behavior.59 In Nasonia, we found a SIFamide precursor that contains one copy of SIFamide that is identical to the honey bee and Drosophila peptide (Support5304


ing Information, Figure S2). Furthermore, we could confirm the identity of this peptide by MS (Figure 3, Table 1). sNPF. Short neuropeptides F (sNPF) have the common C-terminal sequence RLRF/Wamide and are generally occurring in insects, where they control feeding and reproduction.60-62 In Nasonia, there is a sNPF precursor gene encoding a single amidated sNPF with the sequence SPSLRLRFamide (Supporting Information, Figure S2). The presence of this peptide and a less prominent N-terminally extended peptide sequence (AAERSPSLRLRFamide) could be verified by MS (Figures 1-3, Table 1). Tachykinins. Tachykinins stimulate visceral muscles, but also act as diuretic hormones on Malpighian tubules from insects.11,63,64 All known insect tachykinin precursors contain multiple tachykinin peptides. The annotated Nasonia tachykinin precursor gene contains 9 amidated peptides (7 different variants) having the common C-terminal sequence FXGM/ VRamide characteristic for tachykinins (Table 1 and Supporting Information, Figure S2). Five of these Nasonia tachykinins (TK1-5) showed remarkably high signal intensities in mass spectra from the antennal lobes (Figure 3). No signals from the remaining two tachykinins (TK-6 and -7), which are both located in the C-terminal sequence of the preprohormone, were detectable, which is amazing, because their sequences would suggest perfect ionization behavior. B. Neuropeptides with Unknown Receptors in Insects. We found Nasonia orthologs for 8 insect neuropeptide and protein hormone precursor genes, where there has not been identified a receptor in any insect species so far (Table 2, group B). These are two genes, each coding for a CCHamide peptide; two genes, each coding for an insulin-like peptide; and one gene each,


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Figure 10. Alignment of the RYamide preprohormone from N. vitripennis (Nv-RYa, annotated from WGS AAZX01010442 and AAZX01007154) with the corresponding preprohormones from A. mellifera (Am-RYa, annotated from WGS AADG05005050), A. gambiae (Ag-RYa, annotated from EST BM630458 and WGS AAAB01008807), A. aegypti (Aa-RYa, annotated from EST DV310014 and WGS AAGE02021324), C. pipiens (Cp-RYa, annotated from WGS AAWU01014648 and AAWU01014649), D. melanogaster (Dm-RYa, NM_001110912, manually corrected), D. grimshawi (Dg-RYa, XM_001987072), D. virilis (Dv-RYa, XM_002049998), D. willistoni (DwRYa, XM_002061063), B. mori (Bm-RYa, annotated from WGS AADK01014080), T. castaneum (Tc-RYa, annotated from WGS AAJJ01000008), P. humanus (Ph-RYa, XM_002423529), and A. pisum (Ap-RYa, annotated from WGS ABLF02038582). The putative signal peptides (predicted by SignalP) are highlighted in blue. The immature RYamide peptides are marked with yellow background. Green background indicates putative cleavage sites for prohormone convertases (PC). Glycine residues that are converted into C-terminal amide groups in the mature peptides are highlighted with a blue background.

coding for the ion transport peptide (ITP), neuroparsin, and a partial orcokinin precursor containing 10 orcokinin-like neuropeptides (Table 1 and Supporting Information, Figure S2). The presence of the orcokinin-like peptides could be confirmed by MS (Figure 1, Figure 2). In addition, we discovered a novel neuropeptide gene that codes for 7 peptides, of which 4 have the C-terminal sequence RYamide and which were named RYamide peptides (Table 1 and Supporting Information, Figure S2). All neuropeptides mentioned in this paragraph have been explained in a very recent review on Drosophila neuropeptides,11 except for CCHamide and RYamide peptides. These neuropeptides are discussed below. CCHamide. Recently, Roller et al. discovered the novel neuropeptide CCHamide in B. mori.14 The peptide contains two highly conserved cysteines and an amidated histidine residue at the C-terminus. The CCHamide preprohormone is expressed in several small neurons in the CNS and in endocrine midgut cells in B. mori larvae, but the biological function of

the peptide is unknown.14 One or two CCHamide precursor genes are found in all sequenced insect genomes, so far (Figure 9). Also in the Nasonia genome, we identified two paralog precursor genes (located on different scaffolds), each coding for a single, slightly different, CCHamide peptide (Table 1; Figure 9; Supporting Information, Figure S2). RYamide Peptides. In addition to the above-mentioned 7 neuropeptide and protein hormone genes that are orthologues of other known insect neuropeptide and protein hormone genes, we discovered a hitherto unknown insect neuropeptide gene in Nasonia. This gene codes for a preprohormone that contains 3 peptides with the C-terminal sequence RFamide and 4 peptides with the C-terminal sequence RYamide (therefore named RYamide peptides; Figure 10, Figure 11). All peptides are structurally clearly different from the insect FMRFamide,65 myosuppressin,11 NPF,11 and sNPF60 neuropeptides. Mass spectrometric analysis of the central nervous system of Nasonia revealed a mass-match with putative Nasonia RYamide-1 Journal of Proteome Research • Vol. 9, No. 10, 2010 5305

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Figure 11. Alignment of all mature insect RYamide peptides predicted, so far (see Figure 10). Residues that are conserved in at least 12 mature insect RYamide peptide sequences are highlighted in red. Conserved (similar, but not identical) residues (compared to the consensus sequence) are highlighted in green. Most insects have one shorter (8-13 residues) and one longer (27-43 residues) variant of the RYamide peptide. Note that Nasonia is the only insect that contains 7 copies of RYamide peptides.

(Figure 4) in a number of preparations. RYamide-1 is the shortest of the predicted Nasonia RYamides, which certainly favors detectability in mass spectra. Nevertheless, signal intensity was not sufficient for fragment analysis. Homology searches of the other sequenced arthropod genomes (various Drosophila species, A. aegypti, A. gambiae, C. pulex, B. mori, T. castaneum, A. mellifera, A. pisum, P. humanus, D. pulex, I. scapularis) revealed orthologs in most of them (Figure 10). In each arthropod, these novel neuropeptide genes code for at least two similar peptides with the C-terminal sequence RYamide or RFamide (Figure 10, Figure 11). For A. aegypti, we succeeded in the detection and subsequent fragmentation of Aedes RYamide-1 (Figure 11, Figure 12). Thus, we 5306


could confirm that the novel RYamide gene is indeed expressed and that it yields potentially bioactive neuropeptides. C. Absent Neuropeptides, for Which a Putative Receptor Could Be Annotated. We were unable to identify a precursor gene encoding FMRFamide or FMRFamide-like neuropeptides.65 However, there is a clear orthologue to the Drosophila FMRFamide receptor in Nasonia (Table 1, group C). D. Absent Ligand and Receptor Couples. We could not identify precursor genes encoding allatostatin-B, capa, the heterodimeric glycoprotein hormone subunits alpha2 (GPA2) and beta5 (GPB5), kinin, proctolin, sex peptide, and sulfakinin (Table 2, group D). Because we also were unable to identify their

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Figure 12. CID mass spectrum of a MALDI-TOF mass peak at ([M + H]+: 971.5) from the terminal ganglion of Aedes aegypti. Fragment ions are labeled and confirm the amino acid sequence of Aedes RYamide-1 (Figure 11), showing that the RYamide gene is indeed expressed and that its preprohormone is correctly processed into a neuropeptide.

corresponding receptors (deorphanized in other insects), these hormonal signaling systems are probably lacking in Nasonia. E. Absent Ligand/Orphan Receptor Couples. We could not identify the following neuropeptide or protein hormone precursors where there is no receptor known so far (orphans): allatotropin, antidiuretic factors, and the neuropeptide-like precursor genes NPLP1 to NPLP4 (Table 2, group E). These absent neuropeptides are discussed in a recent review.11

Discussion and Conclusions In our current paper, we could identify 51 neuropeptides and protein hormones from Nasonia (Table 1), which are encoded by 30 preprohormone genes (Table 2, group A and B; Supporting Information, Figure S2). One of these preprohomone genes is novel and has not been discovered, so far, in other insects or animal. This gene codes for 7 peptides, of which 3 peptides have the C-terminal sequence RFamide

and 4 peptides have the C-terminal RYamide sequence (Figure 11 and Supporting Information, Figure S2). These peptides are clearly different from NPF, which is much longer (36 residues), has the characteristic RVRFamide C-terminal sequence,andisonlypresentasonecopyinthepreprohormone.11,60 The RYamide peptides are also different from the sNPFs (short neuropeptides F), which are shorter, variable in size and have a conserved C-terminus, RLRF/Wamide11,60 or from the extended insect FMRFamides, which all have a Cterminus resembling FMRFamide.65 Finally, the insect myosuppressins are decapeptides only present as one copy in the preprohormone and have a rigid structure different from the RYamide peptides, being X1DVX2HX3FLRFamide (where X1 is pQ, P, T; X2 is D, G, V; X3 is V, S).11,60 Thus the Nasonia RYamide peptide gene appears to be a novel neuropeptide gene different from other genes coding for peptides containing the RFamide or RYamide C-terminal sequence. Screening of other arthropods with a sequenced genome revealed orthologs of the Nasonia RYamide peptide gene (Figure 10). These orthologs code for preprohormones containing mainly peptides with the C-terminal sequence RYamide (Figure 11), supporting our decision to name these peptides RYamides (and not RFamides). The identification and sequencing by MS of an Aedes RYamide in the central nervous system of Aedes aegypti (Figure 12) shows that the RYamide gene is indeed expressed and that its preprohormone is correctly processed into a mature neuropeptide. The novel RYamide genes from insects, therefore, should be regarded as genuine neuropeptide genes. Nasonia is, so far, the only insect, where the RYamide gene codes for 7 peptides. No FMRFamide precursor could be identified, although the corresponding putative FMRFamide receptor gene is present in Nasonia (Table 2, group C). This could mean that our genomics approach fails to detect these neuropeptides, either because of gaps in the sequenced genome or because the precursor sequence has diverged too much for homology-based searches. This last possibility might be the most likely reason, as FMRFamides are known to have highly variable peptide and precursor structures in insects.11 However, it can not be excluded that the putative FMRFamide receptor is, in fact, a receptor for the RYamide peptides. This interesting possibility has to be clarified in future studies. If both the peptide and the receptor genes are lacking in Nasonia (Table 2, group D), the likelihood that these hormonal

Table 3. Core Set of Neurohormone Precursors Found in Nasonia, Apis, Drosophila, Aedes, Bombyx, Tribolium and the Pea Aphid, Acyrthosiphon pisum Peptide

AKH Alllatostatin C Allatostatin CC Bursicon alpha Bursicon beta CCAP CCHamide DH (Calcitonin-like) DH (CRF-like) EH ETH ILP-B ITP Myosuppressin Pyrokinin RYamide SIFamide sNPF Tachykinin

Nasonia

Apis

Drosophila

Aedes

Bombyx

Tribolium

Pea Aphid

1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 20

1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 20

1 1 1 1 1 1 2 1 1 1 1 5 1 1 1 1 1 1 1 24

1 1 1 1 1 1 1 1 1 5 1 6 1 1 1 1 1 2 1 29

2 1 1 1 1 1 1 1 1 1 1 38 1 1 1 1 2 1 1 58

2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 21

1 1 1 1 1 1 2 1 1 3 1 7 1 1 1 1 1 1 1 28

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Table 4. Variable Set of Neurohormone Precursors Found in Nasonia, Apis, Drosophila, Aedes, Bombyx, Tribolium and the Pea Aphid, Acyrthosiphon pisum Peptide

ACP ADF-b Allatostatin A Allatostatin B Allatotropin Capa Corazonin FMRFamide GPA2 GPB5 ILP-A ILP-C Inotocin Kinin Neuroparsin NPF NPLP1 NPLP2 NPLP3 NPLP4 Orcokinin PDF Proctolin PTTH Sex peptide Sulfakinin

Nasonia

Apis

Drosophila

Aedes

Bombyx

Tribolium

Pea Aphid

1 nd 1 nd nd nd 1 nd nd nd nd 1 1 nd 1 1 nd nd nd nd 1 1 nd 1 nd nd 10

nd nd 1 nd nd 1 1 1 nd nd nd 1 nd nd 1 1 1 1 1 nd 1 1 nd nd nd 1 13

nd nd 1 1 nd 1 1 1 1 1 1 1 nd 1 nd 1 1 1 1 1 nd 1 1 1 2 1 21

1 nd 1 1 1 1 1 1 1 1 1 1 nd 1 1 1 1 nd nd nd 1 1 nd 1 nd 1 19

1 nd 1 1 1 1 1 1 1 1 nd nd nd 1 1 2 1 nd nd nd 1 1 nd 1 nd 1 18

1 5 nd 1 1 1 nd 1 1 1 1 1 1 nd 1 nd 1 nd nd nd nd nd 1 1 nd 1 20

nd nd 1 1 1 1 nd 1 1 1 nd 3 nd 1 nd 1 1 nd nd nd 1 nd 1 nd nd nd 15

systems are truly absent in the wasp strongly increases. Two orthologues of the previously proposed Drosophila allatostatin-B receptor CG3010666 are present in Nasonia, but the deorphanization of this receptor was questioned recently, as the Bombyx orthologue of CG30106 did not respond to Bombyx allatostatins-B.67 Surprisingly, it turned out that the Drosophila allatostatins-B are potent ligands for another receptor, the Drosophila sex peptide receptor CG16752,67,68 and it is this allatostatin-B/sex peptide receptor couple that got lost in Nasonia.67,69 Like the allatostatin-B/sex peptide receptor, also the receptors for capa, GPA2 and GPB5, kinin, proctolin, and sulfakinin are absent in Nasonia.8 We conclude, therefore, that the signaling systems for allatostatin-B, capa, GPA2 and GPB5, kinin, proctolin, sex peptide, and sulfakinin do not occur in Nasonia. The sets of neuropeptides identified in the wasp N. vitripennis (this study), the honey bee A. mellifera,9,10 the fruit fly D. melanogaster,12 the mosquito A. aegypti,13 the silkworm B. mori,14 the flour beetle T. castaneum15,16 and the pea aphid A. pisum17,36 can be subdivided into a basal set of 20 neuropeptide precursors that are present in all these seven arthropod genomes (Table 3) and into a variable set of 26 precursors that are not found in all of these genomes (Table 4). The basal set of peptides shown in Table 3 might contain the key regulators for common physiological processes such as development, metabolism, or reproduction. The peptides listed in Table 4, on the other hand, might regulate other more specialized processes not required for all insects and, therefore, can readily get lost during evolution. Nasonia contains, together with the honey bee, the lowest number of neurohormone precursor genes in the core set (Table 3). But also in the variable set (Table 4), this number is by far the lowest in Nasonia (10 compared to 13 in Apis, 15 in the pea aphid, 18 in Bombyx, 19 in Aedes, 20 in Tribolium and 21 in Drosophila), and its neuropeptide set is remarkably different even from the set identified in the honey bee, another hymenopteran. In Nasonia, there is ACP, inotocin, and PTTH, which are neuropeptides not found in Apis. On the other hand, in the honey bee there is capa, FMRFamide, three NPLP-like precursors, and sulfakinin; 5308


none of these neuropeptides are present in Nasonia. These differences might reflect differences in behavior (social vs parasitic), feeding (herbivores vs carnivores), or habitats of these two related insect species. We have previously noticed that insects can readily duplicate or abandon neuropeptide/GPCR genes.7,8,16,18,46 It is, therefore, one of the biggest challenges for insect endocrinologists to understand the basis for this phenomenon and eventually correlate the presence or absence of neuropeptide signaling systems with the ecological niche that is occupied by a certain insect.

Acknowledgment. We thank Professor Jack Werren (University of Rochester) for sending Nasonia, Professor Joachim Ruther (University of Regensburg) and Anders Illum (University of Copenhagen) for help with establishing and maintaining Nasonia cultures, Louise Lindbæk and Ida Signe Bohse Larsen (University of Copenhagen) for help with cDNA cloning of Allatostatin-C and Allatostatin-CC preprohormones, and the Danish Research Council for Nature and Universe, German Research Foundation, and Novo Nordisk Foundation for financial support. Supporting Information Available: Supplementary figures and table. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Saul, G. B.; Kayhart, M. Mutants and linkage in Mormoniella. Genetics 1956, 41, 930–937. (2) Beukeboom, L.; Desplan, C. Nasonia. Curr. Biol. 2003, 13, R860. (3) Werren, J. H. Sex ratio adaptations to local mate competition in a parasitic wasp. Science 1980, 208, 1157–1159. (4) Werren, J. H.; Richards, S.; Desjardins, C. A.; Niehuis, O.; et al. Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 2010, 327, 343–348. (5) Grimmelikhuijzen, C. J. P.; Cazzamali, G.; Williamson, M.; Hauser, F. Perspective. The promise of insect genomics. Pest Manag. Sci. 2007, 63, 413–416. (6) Weinstock, G. M.; Robinson, G. E.; Gibbs, R. A.; Worley, K. C.; Evans, J. D.; Maleszka, R.; Robertson, H. M.; Weaver, D. B.; Beye, M.; Bork, P.; Elsik, C. G.; Hartfelder, K.; Hunt, G. J.; et al. Insights

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PR100570J

Genomics and Peptidomics of Neuropeptides and Protein Hormones

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