Neuropeptidomics of the Bed Bug Cimex lectulariusReinhard Predel,*,† Susanne Neupert,†,∥ Christian Derst,†,∥ Klaus Reinhardt,*,‡and Christian Wegener*,§

†Department for Biology, Institute for Zoology, University of Cologne, Zulpicher Straße 47b, D-50674 Cologne, Germany‡Applied Zoology, Department of Biology, Technical University of Dresden, Zellescher Weg 20b, D-01062 Dresden, Germany§Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, University of Wurzburg, Am Hubland, D-97074 Wurzburg,Germany

*S Supporting Information

ABSTRACT: The bed bug Cimex lectularius is a globally distributed humanectoparasite with fascinating biology. It has recently acquired resistance against abroad range of insecticides, causing a worldwide increase in bed bug infestations.The recent annotation of the bed bug genome revealed a full complement ofneuropeptide and neuropeptide receptor genes in this species. With regard to thebiology of C. lectularius, neuropeptide signaling is especially interesting because itregulates feeding, diuresis, digestion, as well as reproduction and also providespotential new targets for chemical control. To identify which neuropeptides aretranslated from the genome-predicted genes, we performed a comprehensivepeptidomic analysis of the central nervous system of the bed bug. We identified intotal 144 different peptides from 29 precursors, of which at least 67 likely presentbioactive mature neuropeptides. C. lectularius corazonin and myosuppressin areunique and deviate considerably from the canonical insect consensus sequences.Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of respective mass signals andimmunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species sofar and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.KEYWORDS: neuropeptides, mass spectrometry, peptide prediction, bed bug genome, blood-sucking insect, neurohormones,central nervous system, neuromodulation

1. INTRODUCTIONThe bed bug Cimex lectularius is a globally distributed humanectoparasite with a fascinating biology. This includes anunusually long starvation resistance,1,2 an obligate reliance onblood as a food resource,1,2 combined with the ability to rapidlyprocess blood meals several times their body weight and excreteexcess water in females to avoid superfluous, costly matings,3 aswell as the unusual form of reproduction of traumaticinsemination1,2 and a uniquely diverse response in females totolerate the resulting copulatory damage.4 Most of themolecular and regulatory mechanisms underlying thesebehavioral and physiological processes are unknown. Moreover,in the wild, bed bugs can also live on bats1,2 and are geneticallyseparated from human-associated bed bugs.5

The bed bug is not a disease vector6 as its Trypanosoma cruzi-transmitting relatives Rhodnius prolixus or Triatoma infestans(kissing bugs) but has regained clinical and societal interestsince the late 1990s. The worldwide resurgence in the numberof infestations observed since then is at least in part due toevolution of resistance to a broad range of insecticides,6

increased human travel, and perhaps increasing temperature.Infestations can be massive and can occur in all kind of humansettlements.6 Skin reactions to bed bug bites are commonamong humans but sometimes represent as severe responses.7

Because of these features, the bed bug was selected as a pilotspecies in the i5k arthropod genomes project;8 the annotatedbed bug genome was recently released.9,10

Our group had participated in the bed bug genomeannotation, focusing on the identification of genes forneuropeptides and their receptors.9 Neuropeptides are keyintercellular signaling molecules that regulate and orchestratemost developmental, physiological, and behavioral processes.With regard to the biology of bed bugs, neuropeptides areespecially interesting because they induce and terminatepostfeeding diuresis and control feeding, digestion, as well asreproductive behavior.11,12 Moreover, neuropeptides and theirreceptors provide potential targets for next-generationinsecticides that could be exploited to control insecticide-resistant bed bug strains.13,14

Our genomic analysis identified 47 genes encoding putativeprepropeptides,9 which can be post-translationally processed bya specific set of enzymes15 to give rise to more than 100bioactive neuropeptides. Although processing of prepropep-tides can, in principle, be well predicted by bioinformatictools,16,17 differential and alternative processing can take place,

Received: September 4, 2017Published: November 17, 2017

Article

pubs.acs.org/jprCite This: J. Proteome Res. XXXX, XXX, XXX−XXX

© XXXX American Chemical Society A DOI: 10.1021/acs.jproteome.7b00630J. Proteome Res. XXXX, XXX, XXX−XXX

and in well-studied species such as Drosophila melanogaster,mismatches between predicted and produced peptides werefound.18 We therefore performed a comprehensive peptidomicanalysis of the central nervous system of the bed bug. By acombination of direct tissue profiling by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) and Q Exactive Orbitrap MS/MS, weconfirmed that most predicted peptides are produced in thenervous system of the bed bug and also confirmed thegenomically deduced peptide sequences. To identify species-specific peptide features in bed bugs, we compare our resultswith peptidomic data from the kissing bugs R. prolixus and T.infestans. These bugs are also obligate blood-feeders but belongto the Reduvioidea, a phylogenetically more basal group of theHeteropteran infraorder Cimicomorpha than the Cimicoidea19

comprising C. lectularius. Both taxa have been separated in theTriassic around 200 Mya ago.20 To illustrate the distribution ofidentified peptides in the CNS, we also immunolocalizedselected peptides in the CNS.The results represent the most comprehensive peptidomic

description of a Heteropteran insect and suggest that thepeculiar biology of the bed bug is not reflected by a peculiarneuropeptide complement, at least not on the qualitative level.

2. EXPERIMENTAL SECTION

2.1. Prediction of Prepropeptide Genes and PeptideProcessing in the Bed Bug Genome

The general procedures of automated gene annotation andcommunity curation of the bed bug genome via Web Apollo21

are described in Benoit et al.9 We extensively also used BLAST(tblastn) to search for peptide sequences based on availablepeptide sequences from other species, including the predictedsequences from the related reduviid bug R. prolixus22 andbrown leafhopper Nilaparvata lugens.23 Signal peptide sequen-ces, cleavage sites, posttranslational modifications, andmonoisotopic masses were predicted by SignalP 4.024

(http://www.cbs.dtu.dk/services/SignalP/) and NeuroPred16

(http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py). Pep-tide alignments were performed using JalView2.9.25

2.2. Animals

The bedbug populations used in this study originate from twolocations. Population 1, also called F4, was originally collectedfrom a human dwelling in London in 2003. The populationoriginally showed resistance to several insecticides, includingpyrethroids and carbamates. The population was maintained ina large (>1000 individuals) outbred population and, asexpected, gradually lost insecticide resistance in the laboratory.The current population size is several hundred, and insecticidesusceptibility is most likely. Population 2 consisted ofindividuals originally collected in two bat roosts in Serbia in2013 (collector: Milan Paunovic, Serbian Natural HistoryMuseum Belgrade). This population is therefore likely not tobe resistant against insecticides. This population has beenmaintained at a smaller size (ca. 100 individuals). Bed bugswere kept in large colonies at 25 °C, ca. 60% RH, and regularlyfed, as previously described.26

2.3. Anatomy

Anatomical descriptions and dissection for tissue sampling ofthe nervous system and retrocerebral complex of C. lectulariuswere carried out using a stereo microscope (M165 FC)

equipped with a DFC 450C CCD-camera (Leica, Wetzlar,Germany).

2.4. Tissue Preparation

Just before and during dissection, bed bugs were kept on ice.For Q Exactive Orbitrap mass spectrometry analyses, the CNSof last instar nymphs and males (n = 5) from both populationswas dissected in ice-cold saline and then transferred to 30 μL ofextraction solution (50% methanol, 49% H2O, 1% formic acid[FA]). Tissue samples were disintegrated using an ultrasonicbath (Transonic 660/H, Elma Schmidbauer, Hechingen,Germany) for 5 min on ice and an ultrasonic-probe threetimes for 5 s (BandelinSono HD 200, Bandelin electronic,Berlin, Germany), respectively. Afterward, the samples werecentrifuged for 15 min at 13 000 rpm at 4 °C. The supernatantswere transferred to new tubes and methanol was evaporated ina vacuum concentrator. Extracts were stored at −20 °C untiluse.

2.5. Direct MALDI-TOF MS

Corpora cardiaca, corpora allata, perisympathetic organs,peripheral nerves, smaller parts of the brain, and subesophagealganglion from males were separately dissected as described forcockroach samples27 and directly transferred to a drop of wateron the sample plate for MALDI-TOF MS. Immediately aftertransfer, water was removed and the tissue samples were driedprior to deposition of matrix.

2.6. Quadrupole Orbitrap MS Coupled to Nanoflow HPLC

Before injecting the samples into the nanoLC system, extractswere desalted using self-packed Stage Tip C18 (IVAAnalysentechnik e.K., Meerbusch, Germany) spin columns.28

Peptides were then separated on an EASY nanoLC 1000 UPLCsystem (Thermo Fisher Scientific, Waltham, MA) using in-house packed RPC18-columns 50 cm (fused Silica tube with ID50 μm ± 3 μm, OD 150 μm ± 6 μm; Reprosil 1.9 μm, porediameter 60 Å; Dr. Maisch, Ammerbuch-Entringen, Germany)and a binary buffer system (A: 0.1% FA; B: 80% ACN, 0.1%FA). Running conditions were as follows: linear gradient from 2to 62% B in 110 min, 62 to 75% B in 30 min, and final washingfrom 75 to 95% B in 6 min (45 °C, flow rate 250 nL/min).Finally, the gradients were re-equilibrated for 4 min at 5% B.The HPLC was coupled to a Q-Exactive Plus (ThermoScientific, Bremen, Germany) mass spectrometer. MS data wereacquired in a top-10 data-dependent method dynamicallychoosing the most abundant peptide ions from the respectivesurvey scans in a mass range of 300−3000 m/z for HCDfragmentation. Full MS1 acquisitions ran with 70 000resolution, with automatic gain control target (AGC target)at 3e6 and maximum injection time at 80 ms. HCD spectrawere measured with a resolution of 35 000, AGC target at 3e6,maximum injection time at 240 ms, 28 eV normalized collisionenergy, and dynamic exclusion set at 25 s. The instrument wasrun with peptide recognition mode (i.e., from two to eightcharges); singly charged and unassigned precursor ions wereexcluded. Raw data were analyzed with PEAKS 8.0 (PEAKSStudio 8.0, BSI, ON, Canada). Neuropeptides were searchedagainst an internal database comprising Cimex neuropeptideprecursor sequences (see Supporting Information S1) withparent mass error tolerance of 0.1 Da and fragment mass errortolerance of 0.2 Da. Setting enzyme: none was selected becausesamples were not digested. The false discovery rate (FDR) wasdetermined by a decoy database search as implement in PEAKS8.029 and set below 1%. Conversion of C-terminal glycine was

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selected as fixed post-translational modification (PTM),whereas variable PTMs included in the searches were oxidationat methionine, n-terminal acetylation, pyroglutamate fromglutamine, pyroglutamate from glutamic acid, sulfation oftyrosine, serine, and threonine, and disulfide bridges. In eachrun a maximum of six variable PTMs per peptide were allowed.Fragment spectra with a peptide score (−10lgP) greater than20, which is equivalent to a P-value of ∼1%, were manuallyreviewed.

2.7. MALDI-TOF MS

Ten mg/mL 2,5-dihydroxybenzoic acid (Sigma-Aldrich,Steinheim, Germany) dissolved in 20% acetonitrile/1% formicacid or alternatively 10 mg/mL α-cyano-4-hydroxycinnamicacid dissolved in 60% ethanol, 36% acetonitrile, and 4% Flukawater were used as matrix salts and loaded onto the driedsamples using a 0.1−10 μL pipet (∼0.3 μL for direct tissueprofiling). Mass fingerprint spectra were acquired in positiveion mode with an ultrafleXtreme TOF/TOF mass spectrom-eter (Bruker Daltonik, Bremen, Germany). All MS acquisitionswere made under manual control in reflector positive mode in adetection range of 600−10 000 Da. The instrument settingswere optimized for the mass ranges of m/z 600−3500 and m/z3000−10 000 and calibrated using Bruker peptide and proteinstandard kits, respectively. Laser fluency was adjusted toprovide the optimal signal-to-noise ratio. The data obtained inthese experiments were processed with a FlexAnalysis3.4software package. MS/MS was performed with LIFTtechnology. LIFT acceleration was set at 1 kV. The numberof laser shots used to obtain a spectrum varied from 5000 to25 000, depending on signal quality. For MS/MS experiments,we also used an ABI 4800 proteomics analyzer (AppliedBiosystems, Framingham, MA). MS/MS fragment spectra wereacquired manually in gas-off mode. Peptide identities wereverified by (1) comparison of theoretical and experimentallyobtained ion signals of neuropeptides ([M + H]+) and (2)comparison of theoretical (http://prospector.ucsf.edu) andexperimentally obtained fragments following MS/MS experi-ments.

2.8. Peptide Derivatization Using 4-SulfophenylIsothiocyanate (SPITC)

On the basis of a protocol by Wang et al.,30 tissue extracts weretransferred in 40 μL of SPITC dissolved in 20 mM NaHCO3(pH 9.0) at a concentration of 10 mg/mL. The sulfonationreaction was carried out in a 0.5 mL Eppendorf tube for 1 h at55 °C and 300 rpm. Samples were then acidified by adding 2.5μL of 10% acetic acid, followed by 50 μL of 0.5% acetic acid,and loaded onto an activated and equilibrated StageTip C18column.28 The column was rinsed with 50 μL of 0.5% aceticacid, and peptides were eluted from the column with solutionsof 10/20/30/40/50/80% acetonitrile (0.5% acetic acid) andspotted onto a MALDI target. Before drying, 0.3 μL of matrixsolution was added to each sample, respectively. MALDI-TOFmass spectrometry was performed using the ultrafleXtreme asdescribed in 2.7. First, MS1 spectra were manually searched forthe presence of signals of predicted Cimex neuropeptides withderivatization ([M + H]+ + 215). Such ions were thenfragmented (MS/MS) using the same samples. Resultingfragments were compared with theoretical fragments of thepredicted neuropeptides to confirm the identity of thesubstances.

2.9. Immunostaining

Nervous tissue of adult males was dissected in 0.1 Mphosphate-buffered saline (PBS, pH 7.2) fixed for 3 h in 4%paraformaldehyde in PBS at room temperature, washed in PBSwith 0.5% TritonX (PBT0.5), and incubated for at 48−72 h inPBT containing 5% normal goat serum in combination withrabbit polyclonal antiserum directed against allatostatin A31

(Jena Bioscience, Jena, Germany), corazonin32 (kind gift of JanA Veenstra, Bordeaux, France) (both diluted 1:1000), or Pea-PVK-233 (kind gift of Manfred Eckert, Jena, Germany; diluted1:4000). Then, preparations were washed five times during aday with PBT and incubated for 48−72 h in PBT0.5 containing5% normal goat serum with DyLight488-conjugated AffiniPuregoat antirabbit IgG (H+L; Jackson ImmunoResearch, Ger-many) at a dilution of 1:1000 or with Cy3-coupled goatantirabbit sera (Jackson ImmunoResearch, Germany) at aconcentration of 1:3000. Preparations were subsequentlywashed for 24 h and then mounted in 80% glycerol dilutedin PBS or embedded in Mowiol (Merck KGaA, Darmstadt,Germany).For the in situ CAPA peptide immunostaining, the

abdominal tergites and subsequently the intestine of adultmales were removed using fine scissors, and the remainingpreparation was fixed with microneedles. Preparations werethen rinsed with ice-cold insect saline and treated with Histofix(Sigma-Aldrich, St. Louis, MO) at 4 °C for 12 h. After washingwith PBS containing 1% TritonX-100 (PBT1.0) for 24 h, thesamples were incubated for 5 days at 4 °C in the anti-Pea-PVK-2 serum diluted 1:4000 in PBT1.0 containing 2,5 mg/mL bovineserum and 10% normal goat serum. Subsequently, the sampleswere washed in PBT1.0 for 24 h at 4 °C. Then, the samples wereincubated with Cy3-coupled goat antirabbit sera diluted 1:3000(Jackson ImmunoResearch) in PBT1.0 for 5 days at 4 °C. Afterwashing in PBT1.0 for 24 h at 4 °C, the preparations wereembedded in Mowiol (Merck KGaA, Darmstadt, Germany).Confocal stacks were acquired on a confocal laser scanning

microscope (SPE, Leica Microsystems, Wetzlar, Germany) witha 20× objective (ACS APO 20× N.A. 0.6 IMM) or 40×objective (ACS APO 40× N.A. 1.15 oil) or a LSM 510 Metaconfocal laser scanning system (Zeiss, Jena, Germany)equipped with a C-Apochromat 10×/0.45W and a Plan-Apochromat 20×/0.75 objectives. Pictures were analyzed usingthe Fiji image processing package34 (http://fiji.sc/About) basedon the public domain software ImageJ 1.46 (NIH, publicdomain:http://rsb.info.nih.gov/ij/) or Zeiss LSM 5 imagebrowser v.3 software. Figures were generated with the help ofAdobe Photoshop CS6 (Adobe Systems) using only brightnessand contrast adjustments.

3. RESULTS AND DISCUSSION

3.1. In Silico Prediction of Prepropeptide Sequences

During the bed bug genome annotation, we previouslypredicted 47 different prepropeptide genes for C. lectularius,9

45 full and 2 partial prepropeptide sequences (SupportingInformation S1). One partial sequence, that for CNMamide,could now be completed by a BLAST search against updatedNCBI reference sequences (Supporting Information S1). Inaddition, we found genes for neuropeptide-like precursorNPLP3 and NPLP4 homologues previously identified in aneuropeptidomic analysis in Drosophila.35 On the basis of theirsequence, they appear to represent cuticular peptides ratherthan neuropeptides in C. lectularius.9 This view is supported by

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the lack of NPLP3 and NPLP4 peptides in CNS extracts duringthis study. From the predicted propeptides, more than 100different putative neuropeptides can potentially be derived. C.lectularius possesses the “core set” of 20 genes coding for insectregulatory peptides, and there are no unexpected or uniquepeptidomic features in comparison with other true bugs.9 Yetthe genomic annotation predicts very unusual sequences ofmyosuppressin and corazonin for the bed bug.9

3.2. Identification of Neuropeptides in the Central NervousSystem (CNS) and Neurohemal Release Sites

To identify which of the predicted neuropeptides are expressedin the CNS and to confirm the unusual myosuppressin andcorazonin sequences of C. lectularius, we biochemicallycharacterized the peptidome of larval and male adult bedbugs in the CNS and attached neurohemal release sites. For ourstudy we used two populations of C. lectularius, which originatefrom human dwellings and bat caves, respectively. Human- andbat-associated bed bugs are known to show some degree ofgenetic differentiation.5,36 In our study, however, we did notobserve a single difference in the sequences of neuropeptidesbetween the two populations. Therefore, all given informationon neuropeptides is valid for both populations. Because thegross anatomy of the bed bug CNS is not well described, weprovide an overview in Figure 1A,B. Major neurohemal releasesites along the CNS of the bed bug are the very compactretrocerebral complex (see Figure 1C−E), which is closelyfused with the anterior aorta, and the abdominal segmentalnerves 2−4 (Figure 2), which are the equivalent ofperisympathetic organs in hemimetabolic insects with unfusedabdominal ganglia. By a combination of direct tissue profilingby MALDI-TOF MS and Q Exactive Orbitrap MS/MS, webiochemically identified a total of 144 peptides from 29 of theidentified precursor genes. For 27 of these precursors, peptideswere sequence-confirmed by MS/MS fragmentation, while wehave peptide mass matches only for the proctolin and CCAPprecursor. On the basis of sequence similarities to peptides inother insect species with known functions, at least 67 of theidentified peptides represent bioactive and mature neuro-peptides. 63 of these peptides were confirmed by MS/MSfragmentation, and 4 peptides were confirmed by mass matchesonly. In addition to the bioactive neuropeptides, we identified23 peptides (21 confirmed by MS/MS) from the NPLP1 andNVP precursors. These precursors show key features ofpreproneuropeptides (signal peptide, cleavage sites, expressionin the CNS) and are usually annotated as such in diverse insectgenomes, yet bioactivity and function of NPLP1 and NVPpeptides have not been tested in any insect so far. 31 peptides(27 sequence-confirmed by MS/MS) represent additionalprecursor (“spacer”) peptides, which likely do not have anysignaling function. All identified peptide sequences aresummarized in Table 1.In the following, we describe and discuss specific details of

the identified peptides. This includes a comparison withavailable data for the reduviid bugs R. prolixus and T. infestans,the other blood-feeding heteropteran species well-described interms of neuropeptides and their receptors.37,38 Knownphysiological functions of neuropeptides in these species aresummarized in Ons37 and are not repeated here.AKH/Corazonin-Related Peptide (ACP). We predicted

one ACP from the prepropeptide, which could be biochemi-cally identified and sequence-confirmed from preparations ofthe bed bug CNS. As for AKH and corazonin, the N-terminal

Gln is fully modified to pyroglutamate, and no trace ofunblocked ACP was detectable. In R. prolixus immunostainings

Figure 1. Gross morphology of the adult bed bug CNS andimmunostaining of selected neuropeptides in neurohemal organs.(A) Dorsal and (B) lateral view of the CNS, showing a high degree ofganglion fusion typical of Heteroptera. The brain is fused with thesubesophageal ganglion (SEG) and prothoracic ganglion (PRO), andthe remaining thoracic and all abdominal ganglions are fused to themesothoracic ganglionic mass (MTGM). In panel B, part of theesophagus (eso) is visible that projects through a foramen in themidline approximately at the brain-SEG border. (C) AstA immunor-eactivity in the brain of an adult bed bug. In each brain hemisphere,primary neurites of two large secretory neurons in the pars lateralis ofthe protocerebrum (arrows) project toward the midline, then bifurcate(asterisk). One small branch of each neurite projects to thecontralateral side, while the other projects through the nervus corporiscardiaci II (NCC II, arrowheads) to the neurohemal retrocerebral−aorta complex (arrowhead), where it forms a dense net of terminalvaricosities. (D) Close-up of AstA-immunoreactive varicosities in theretrocerebral complex comprising the corpora cardiaca (CC) in adifferent preparation. (E) Close-up of corazonin-immunoreactivevaricosities in the retrocerebral complex comprising the CC andcorpora allata (CA). Arrowheads mark the entry of neurites from theprotocerebrum via NCC II.

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revealed three pairs of ACP-immunoreactive neurons in theprotocerebrum that send descending projections through theventral ganglia.39

Adipokinetic Hormone (AKH). The prepropeptide genecodes for one AKH, which was biochemically identified inpreparations of the corpora cardiaca (Figure 3A) and sequence-confirmed. On the basis of the genomic sequence, it containsIle at position 2, while all other MS-identified heteropteranAKHs are reported with a Leu at this position.40−42 Althoughthese peptidomic studies did not biochemically distinguishbetween the isobaric Ile and Leu, genomic and transcriptomicdata confirmed Leu2 at least for R. prolixus and T. infestans.Allatostatin A (AstA). In R. prolixus, several predicted AstA

peptides could not be found in peptidomic approaches.41,43 Inthe bed bug, we did not find ion signals corresponding to thepredicted AstA-1 peptide (LYDFGLa) located most N-terminally on the propeptide. The remaining AstA peptidescould be sequence-confirmed as predicted, including LPKQYS-FGLa and PEGKMYSFGLa. In the propeptide, these twopeptides are separated by an internal prohormone convertaseKR cleavage site. Their long product LPKQYSFGLGKRPEGK-MYSFGLa could also be sequenced-confirmed. Althoughputatively bioactive, this long form likely represents aprocessing intermediate because only weak MS signals weredetected; a similarly long form was not detected in the R.prolixus brain.41,43 Masses corresponding to additionalprecursor peptides were found as well but could only partiallybe fragmented (Table 1). The presence of densely arborisedAstA immunoreactive varicosities in the retrocerebral−aortacomplex (Figure 1C,D) and the identification of AstA peptidesin mass spectra from this complex (Figure 3A) suggest a

hormonal role for AstA in bed bugs, similar to the situation inR. prolixus.44

Allatostatin C (AstC). Ion signals indicating the presence ofAstC were found with direct tissue profiling of brain but alsoanterior aorta tissue using MALDI TOF MS (Figure 3B) aswell as in Orbitrap mass spectra. In addition, partial sequencesof the N-terminal precursor peptide have been identified inOrbitrap analyses of CNS tissue. The occurrence of AstC in theR. prolixus CNS43 was supported by the MS/MS identificationof putative AstC propeptide-processing products but notbioactive AstC, although the AstC prepropeptide sequenceremains unidentified in this species.

Allatotropin. Allatotropin was sequence-confirmed in itspredicted amidated form in CNS samples, where it wascommonly detected. Whereas the sequence of allatotropin ishighly conserved and mostly identical in different families oftrue bugs,45 the C. lectularius allatotropin differs at three aminoacid positions in the middle part of its sequence from otherheteropteran species that have been analyzed so far.

Calcitonin-like Diuretic Hormone (CT-like DH). CT-likeDH could be sequence-confirmed in the predicted full-lengthform and represented a common signal throughout the CNS.This is in agreement with the widespread distribution of CT-like DH-expressing neurons in the CNS in R. prolixus,46 whereCT-like DH was found in its full-length form and a C-terminally truncated form (CT-like DH1−16).41

CAPA. The Capa gene of the bed bug codes for twoperiviscerokinins (PVKs) plus one tryptophan-containingpyrokinin9 (CAPA-tryptoPK). C. lectularius PVK-1 lacks thecomplete N-terminal motif of the consensus sequence typical ofbasal hexapods (LIPFPRVa47). This peptide is therefore a good

Figure 2. In situ immunostaining against PRXamide, which labels CAPA peptides, adult male preparation. In the subesophageal ganglion that isfused to the prothoracic ganglion (SEG+Th1), a pair of strongly stained neurons is visible. Further immunoreactive neurons in the protocerebral partof the brain (BR) provide arborisations mostly in the superior brain. Two descending varicose axons (#) of unknown origin innervate themesothoracic ganglionic mass (MTGM). Three secretory neurons (Va) in the abdominal portion of the MTGM project via the abdominal segmentalnerves 2−4 (arrows) into a large territorium in the periphery. The varicose nature of the segmental nerve branches in the periphery (asterisks)suggest a hormonal release into the abdomen. The insert (top left) shows a dissected MTGM with the Va neurons and segmental branchings inmore detail. Numbers mark the segmental nerves 2−4.

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Table 1. Summary of Neuropeptide Precursor Products of C. lectulariusWhich Were Identified by Mass Spectrometry Analysesa

peptide name peptide sequence calculated [M+H]+MALDI-TOF

MS Q Exactive

ACP pQVTFSRDWSA−NH2 1178.58 + +AKH pQITFSTGW−NH2 921.47 + +AstAAstA-2 LPVYNFGL−NH2 921.52 (+) +AstA-3 AAGEKTYSFGL−NH2 1142.58 (+) +AstA-4 GRQYAFGL−NH2 910.49 + −AstA-5+6 LPKQYSFGLGKRPEGKMYSFGL−NH2 2502.33 + +AstA-5 LPKQYSFGL−NH2 1051.59 + +AstA-6 PEGKMYSFGL−NH2 1127.55 (+) +AstA-7 TKSGKDLRYMFGI−NH2 1514.81 + +AstA-8 DVDEDERARQERSMHYNFGL−NH2 2466.12 + +*AstA-PP3 DYDMDEEYDDEMRDDFN−OH 2216.75 (+) −AstA-PP4 SQMAESDDYDSIENGRGMEE−OH 2262.88 (+) −AstA-PP5 IANMQEKDTTQSALVN−OH 1762.86 (+) +AstC SYWKQCAFNAVSCF−NH2 1650.73 (+) +AstC-PP1 pQPVDKEKLLNELELVDDDGSIETAVMNYLFAKQIVNRLRSQGDISDLQ−OH 5442.80 (+) −AllatotropinAT GFKNGPLNTARGF−NH2 1377.74 + +extended AT GRSTRGFKNGPLNTARGF−NH2 1935.04 − +CT-DH GLDLGLSRGFSGSQAAKHLMGLAAANFAGGP−NH2 2970.54 + +*CAPAPVK-1 EVRPFEFPRV−NH2 1274.70 + +PVK-1 (pQ) pQVRPFEFPRV−NH2 1256.72 + +PVK-13−10 RPFEFPRV−NH2 1046.59 + +PVK-2 EGGLIPFPRI−NH2 1097.65 + +PVK-2 (pQ) pQGGLIPFPRI−NH2 1080.65 (+) +tryptoPK NGGNGNGGLWFGPRL−NH2 1514.76 + +N-term.ext.tryptoPK SGPKRNGGNGNGGLWFGPRL−NH2 2040.06 (+) +N- + C-term.ext.tryptoPK SGPKRNGGNGNGGLWFGPRLGKIQ−OH 2467.31 + −C-term.ext.tryptoPK NGGNGNGGLWFGPRLGKIQ−OH 1942.00 + +CAPA-PP1 EDIPSHAPKL−OH 1106.58 + +CAPA-PP12−10 DIPSHAPKL−OH 977.54 + +CAPA-PP2 SKNFPVTWGLQEDKY−OH 1811.89 + +CAPA-PP2+PVK-2 SKNFPVTWGLQEDKYKREGGLIPFPRI−NH2 3174.72 + −CCAP PFCNAFTGC−NH2 956.39 (+) −CCAP-PP1 FYFPDEVSEPIDPKI−OH 1795.88 (+) −CCHamide-1 SGSCLSYGHSCWGAH−NH2 1548.63 (+) +CNMamide AGYMALCHFKICNM−NH2 1598.72 − +Corazonin pQMFQYSRGWRN−NH2 1454.70 + +CRF-DH SSPSLSVANPIDVLRSRLMLEINRRWMNKVDEGQVQANRQFLQTI−NH2 5208.76 (+) +CRF-DH-PP1 TLQEVPRDPETTWPL−OH 1781.91 (+) +ext. CRF-DH-PP1 TLQEVPRDPETTWPLRKL−OH 2179.19 (+) +CRF-DH-PP2 YVDVQPRMTAELQDLLEDLEKV−OH 2604.32 (+) +*CRF-DH-PP3 YIMPQRALYQDKSDSPTDYNNSGDLNWDSS−OH 3480.53 (+) +*ext. FMRFamidesFMRFa-1 SALEKNFMRF−NH2 1241.65 (+) +FMRFa-3 AKGNFIRF−NH2 951.55 − +FMRFa-4 NADNFMRF−NH2 1013.46 − +FMRFa-5 ARNNFIRL−NH2 1002.59 (+) −FMRFa-6 NTENFIRF−NH2 1039.53 − +FMRFa-7 NDNFMRF−NH2 942.43 − +FMRFa-8 GRGFVGFARQKEMDKGGGFIRF−NH2 2459.29 − +*FMRFa-PP3 NRDVVLSPWT−NH2 1185.63 − +FMRFa-PP6 STNENILRKN−OH 1188.63 − +Hugin-PyrokininPK-1 NTVNFRPRL−NH2 1115.64 + +PK-2 SPPFAPRL−NH2 883.51 + +ext. PK-2 EEDIIFTETSRSPPFAPRL−NH2 2204.13 + +PK-3 MVPFSPRF−NH2 979.52 + +KininK-1 DSPGPKMRFSSWG−NH2 1450.69 + +K-2 SKFSSWG−NH2 797.39 − +

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Table 1. continued

peptide name peptide sequence calculated [M+H]+MALDI-TOF

MS Q Exactive

K-3 KFSSWG−NH2 710.36 − (+)ext. K-3 KFSSWGGKRLPADYEE−OH 1869.91 +K-4 AKFNSWG−NH2 808.41 − +K-6 STFNSWG−NH2 797.36 − +K-7 TKFNSWG−NH2 838.42 − +K-9 RGPDFYAWG−NH2 1067.51 (+) +K-PP21−11 GASGLDEILEE−OH 1132.54 − +K-PP6 EDETWRDMI−OH 1194.51 + +K-PP7 AINSTADMLIF−OH 1195.60 − +MIP/AST-BMIP-1 AWKDLTTAW−NH2 1090.57 − +MIP-2 GWKDLPATGW−NH2 1129.58 (+) +MIP-3 GWQDLQSAW−NH2 1089.51 − +MIP-4 GWTDLNSGGW−NH2 1091.49 − +MIP-5,6,7 GWQDLNSGGW−NH2 1118.50 − +MIP-8 GWQDLQAPAW−NH2 1170.57 − +MIP-9 SWVSLHSGW−NH2 1057.52 − +MIP-10 AADWGSFRGSW−NH2 1238.57 + +MIP-11 DPAWQNLKGLW−NH2 1326.69 − +MyosuppressinMS (pQ) pQDQMTDHIFLRF−NH2 1532.76 + −MS QDQMTDHIFLRF−NH2 1549.76 + −NPFNPF1−12 pQPMPADAMARPA−OH 1238.59 + −NPF114−33 PKTFDSPDDLRTYLNQLGQY−OH 2371.16 (+) +NPF34−42 YAVAGRPRF−NH2 1035.58 + +NPF-PP2 TGFSPRLHLALDGLSSYRYPLSDPSELYELLFPQAD−OH 4068.04 (+) +*NatalisinNat-1 SDVRAVLGAETEPGFWPTR−NH2 2087.07 + +Nat-2 GGSSEEQPPPFWAHR−NH2 1680.79 + +Nat-3 DVLDQPLWVR−NH2 1239.68 (+) +Nat-PP YAQEPKYLLIQ−OH 1365.74 − +NPLP1NPLP1-2 pQANIIARMAHLQSYPRYYSNIAP−OH 2660.37 (+) +NPLP1-3 DEKSSLDELAERLEEEEFA−OH 2239.02 + +NPLP1-4 SGDIRVTGDLEE−OH 1290.62 + +NPLP1-5 DELEGLVHDLASAEEM−OH 1757.79 − +*NPLP1-7 SFAALARVDALP−OH 1230.68 + +NPLP1-9 GISSIARNGYYQ−OH 1328.66 + +NPLP1-10 MDDDLEQLMSEVYGIGE−OH 1943.82 − +*NPLP1-11 NVASLARGFSLPQ−NH2 1358.75 + +NPLP1-12 NIASIVR−NH2 771.48 + +*NPLP1-13 DSFEETNED−OH 1085.39 − +*NPLP1-14 NLPSIIRDRTEPLGE−NH2 1708.93 + +NPLP1-15 HIGAFVANHGIPFVND−OH 1707.86 + +NPLP1-16 NVGVLAKNRDYPYALKF−NH2 1967.09 + +NPLP1-161−15 NVGVLAKNRDYPYAL−OH 1692.91 + −NPLP1-17 DVDEEIN−OH 833.35 − +NPLP1-18 HVATLLRQA−OH 1008.59 (+) +NPLP1-19 LSDHPTETDDISLKETAQDDLKEEMSQITKDDMEHTRT−OH 4403.01 + +*NPLP1-20 MAREELE−OH 877.41 − +NPLP1-21 GNRNENS−OH (+) −NVP-likeNVP-1 LPTSLLEDMKDAQFHTPVRTNKV−OH 2640.38 (+) +NVP-2 AQEFIMFGNQQNRAINNFGSTKNE−OH 2758.30 + +NVP-570−77 EVAWPRQH−OH 1022.52 − +NVP-7 FGRDSIPPVMPFQEEYADEIPL−OH 2550.22 + +*Orcokinin AOrcomyotropin-like NLDSLDGVTFGSS−OH 1311.61 − +Orc-1 NMDEIDRAGFDTFV−OH 1629.72 + +Orc-PP1 LPRPEPAENSLFKGERNLYGLGGGHLLRDLESALRENPMVYERPS−OH 5077.61 (+) −Orc-PP11−28 LPRPEPAENSLFKGERNLYGLGGGHLLR−OH 3090.66 − +

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candidate to test the specificity of PVK receptors. The twopredicted PVKs were biochemically confirmed and representthe most abundant peptides in mass spectra from preparationsof abdominal segmental nerves 2−4 (Figure 3C), which are themajor CAPA hormone release sites in true bugs. In addition tothe predicted PVK-1 (EVRPFEFPRVa), the prominent ionsignal of a truncated version of this peptide (PVK-13−10) wasobserved in all mass spectra from these nerves (n > 10, Figure3C), which also revealed the presence of low amounts ofpyroglutamyl forms of both PVKs. A C-terminally extendedCAPA-tryptoPK (NGGNGNGGLWFGPRLGKIQ), but notthe predicted tryptoPK itself, was abundant in preparations ofabdominal segmental nerves (Figure 3C). This peptideoriginates from C-terminal processing at a dibasic KR siteand can certainly be associated with a loss of bioactivity oftryptoPK. Whereas traces of the predicted tryptoPK wereobserved in few preparations only, we could identify an N- andC-terminally extended CAPA-tryptoPK (Figure 3C), whichresults from incomplete propeptide processing at a dibasic KRcleavage site (SGPKRNGGNGNGGLWFGPRLGKIQ), possi-bly due to proline in position −3. The occurrence of thispeptide suggests a preferential cleavage of the N-terminallysituated monobasic R cleavage site, and a similar processingproduct was already reported for R. prolixus.47 Different fromthe processing of likely not bioactive extended forms oftryptoPK in abdominal ganglia, CAPA-tryptoPK was identifiedin mass spectra from the brain/subesophageal ganglion (SEG)

and retrocerebral complex, where the C-terminally extendedform of this peptide could not be detected. This findingsuggests that tryptoPK is differentially processed in abdominalganglia and brain/SEG tissue. The differential processing of theCAPA prepropeptide is corroborated by the complete absenceof PVKs in mass spectra of brain/SEG samples, which waspreviously reported for R. prolixus48 and also for Drosophilamelanogaster.49 In addition to CAPA-tryptoPK, only ion signalsof an N-terminal extended form of this peptide (SGPKRN-GGNGNGGLWFGPRLa) were detectable in mass spectra ofpreparations of brain/SEG and retrocerebral complex. Thedifferent CAPA-peptides identified in preparations of CNS,retrocerebral complex, and CAPA-release sites of abdominalganglia are summarized in Table 1.The R. prolixus genome contains two Capa genes,50,51 each

of which contains only one canonical and bioactive CAPA-PVK51 plus a tryptoPK. The presence of only one Capa gene inthe bed bug and the confirmed colocalization of products fromboth R. prolixus Capa genes in the same secretory neurons48

suggest that the unusual duplication of Capa in R. prolixuscompensates for the loss of one functional PVK rather thanrepresenting an adaptation to a blood-sucking life style. As in R.prolixus50 and other true bugs,52 CAPA peptides of the bed bugare expressed in three pairs of secretory ventral abdominalganglia neurons (Va neurons, with homology to Drosophila andother insect species53), which release CAPA peptide hormonesvia the abdominal segmental nerves 2−4 (Figure 2). Different

Table 1. continued

peptide name peptide sequence calculated [M+H]+MALDI-TOF

MS Q Exactive

Orc-PP129−45 DLESALRENPMVYERPS−OH 2005.96 (+) +Orc-PP2 NSVDPSET−OH 848.36 (+) +Orc-PP3 (pQ) pQLYNVGLADRVA−NH2 1300.73 + −Orc-PP3 (Q) QLYNVGLADRVA−NH2 1317.73 + +Orc-PP5 NFVPSHYEMYGNYNPVPFF−OH 2322.03 (+) +PDF NSEIINSLLGLPKVLNDA−NH2 1909.07 − +Proctolin RYLPT−OH 649.37 (+) −RYamide AESRFVIGSRY−NH2 1283.68 (+) +SIFamide SYKKPPFNGSIF−NH2 1383.74 + +sNPFsNPF DNRTPQLRLRF−NH2 1414.80 + +sNPF4−11 TPQLRLRF−NH2 1029.63 + −sNPF-PP2 AAYPERVYKMEE−OH 1485.70 + +*sNPF-PP3 SVPLFPLEE−OH 1030.55 − +sNPF-PP4 LDQSRMDYFSN−OH 1375.59 + +sNPF-PP3+4 SVPLFPLEERRLDQSRMDYFSN−OH 2699.32 + +*SulfakininSK-1 (nonsulf.) pQFNDYGHMRF−NH2 1296.58 + +SK-1 (sulf.) pQFNDY(SO3H)GHMRF−NH2 1376.58 (+) +SK-1 (Q, nonsulf.) QFNDYGHMRF−NH2 1313.58 (+) +SK-1 (Q, sulf.) QFNDY(SO3H)GHMRF−NH2 1393.58 − +SK-2 (nonsulf.) EGTDEKFEDYGYLRF−NH2 1867.85 + +*SK-2 (sulf.) EGTDEKFEDY(SO3H)GYLRF−NH2 1947.85 − +*Tachykinin-RPTKRP-1 pQERRQLGFLGVR−NH2 1440.84 + +TKRP-2 APTLGFQGLR−NH2 1058.61 + +*TKRP-3 APPMGFQGVR−NH2 1058.56 + +TKRP-4 GPTMGFVGMR−NH2 1051.52 + +TKRP-5,6 APASGFFGMR−NH2 1039.51 + +TKRP-7 GPSNAGFFGMR−NH2 1139.54 + +TKRP-8 GPSGFLGLR−NH2 902.52 + +TKRP-PP1 + TKRP-2 DGTDEEFKRAPTLGFQGLR−NH2 2136.08 − +

a(+), mass match only; +*, partial sequence confirmed by MS/MS; underlined, half of disulfide bridge.

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from the other hemipteran species analyzed so far, the releasesites of CAPA peptides along the segmental nerves of C.lectularius are mainly located in the periphery (Figure 2).Crustacean Cardioactive Peptide (CCAP). CCAP is

notoriously difficult to detect by mass spectrometry. It was notfound in peptidomic screens from R. prolixus41,43 or T.infestans,38 although CCAP mRNA is known to be expressed

in the brain and CCAP-immunoreactivity is rather wide-spread.54 In the bed bug brain, we found an ion signal mass-matching the predicted CCAP. Although we were unable toconfirm the identity by MS/MS, the occurrence of low intensitysignals mass-matching also the predicted precursor peptideFYFPDEVSEPIDPKI lends support to the chemical presence ofprocessed CCAP because this precursor peptide results frompredicted CCAP processing.

CCHamide-1. CCHamide-1 could be identified bynanoLC−MS in brain extracts, while CCHa-2, the onlypredicted and mass-spectrometrically identified CCHamide inthe reduviids R. prolixus41 and T. infestans,38 was not found.38,51

CNMamide. CNMamide is a recently identified insectneuropeptide; the corresponding gene was also reported fromR. prolixus.56 We were now able to confirm the presence of anamidated and likely bioactive CNMamide by Orbitrap MS/MSanalysis in a true bug.

Corazonin. Most insects possess either [Arg7]- or [His7]-corazonin, although other variants exist with variable aminoacids in positions 3, 4, and 1057 (Figure 5). [Arg7]-corazonin isalso found in pentatomid and reduviid heteropterans.40,41 Forthe cimiciid bed bug, the genomic prepropeptide sequenceinfers an unusual and unique [Met2, Arg10]-corazonin. Weconfirmed this sequence biochemically in CNS extracts and bydirect tissue profiling and thereby confirmed that the unusualsequence is not an artifact of genomic sequencing errors. Arg10

confers a negative charge in the C-terminal part of the peptideinstead of the uncharged polar T or Q at this position. This isremarkable, as the C-terminal part is usually considered to beimportant for receptor binding and activation. In R. prolixus,39

T. infestans,58 the linden bug Pyrrhocoris apterus,59 and indeedmost other insects, corazonin is expressed in secretory neuronsof the pars lateralis of the protocerebrum. These neuronsrelease the peptide as a hormone at the neurohemalretrocerebral-aorta complex. To obtain a first indication ofwhether the unusual C. lectularius corazonin is released as ahormone, we performed immunostainings of the bed bug CNSwith an antiserum raised against [Arg7]-corazonin. We observedimmunoreactivity within the brain and ventral ganglion, it wasvery weak and mostly confined to a few neuronal somata in thebrain and neurites in the ventral ganglion, indicating that theantiserum only weakly recognizes [Met2, Arg10]-corazonin. Wenevertheless observed distinct corazonin-immunoreactive stain-ing of densely arborising neurite endings in the retrocerebral−aorta complex (Figure 1E), similar to the situation in otherHeteroptera.39,58,59 We also observed ion signals of corazoninin MALDI TOF mass spectra from preparations of theretrocerebral complex (Figure 3A) and Orbitrap MSMS spectrafrom CNS extract (Figure 4B), which confirmed that theunusual C. lectularius corazonin is released as a hormone.

Corticotropin Releasing Factor-Like Diuretic Hor-mone (CRF-like DH). We were able to sequence-confirmthe predicted full-length amidated peptide in preparations fromthe brain and retrocerebral complex/aorta. This is in line withthe presence of a similar full-length amidated peptide in R.prolixus, demonstrated by mass spectrometry of bioactivepurified CRF-like DH. In addition to the amidated peptide, weidentified three further precursor peptides, altogether coveringthe complete propeptide sequence (Table 1).

Extended FMRFamide Peptide (FMRFamide). Out ofthe eight predicted extended FMRFamides in the propeptide,six were sequence-confirmed by Orbitrap MS/MS fragmenta-tion (FMRFamides 1, 3, 4, 6−8), while the presence of

Figure 3. Direct MALDI-TOF MS tissue profiling of tissue samplesfrom C. lectularius; only more prominent ion signals are labeled. Thespectra verify the tissue-specific presence of products of severalneuropeptide precursors. Ion signals that could not be assigned toproducts of known neuropeptide precursors are marked with questionmarks. (A) Retrocerebral complex (RC) containing the corporacardiaca, corpora allata, and a small piece of anterior aorta. (B) Piece ofaorta adjacent to the RC that stores peptide hormones. Low ion signalintensities suggest a local release of the peptides rather than a releaseas classical hormones. (C) A single abdominal segmental nerve 2shows prominent ion signals for products of the CAPA precursor.Note the presence of C-terminally and C- and N-terminally extendedsequences of CAPA-tryptoPK, which likely are not bioactive. Onlytraces of the canonical trypto-PK (see arrow) are detectable inpreparations of abdominal segmental nerves. AKH, adipokinetichormone; AstA, allatostatin A; AstC, allatostatin C; cor, corazonin;CRF-DH, corticotropin releasing factor-like diuretic hormone; MIP,myoinhibiting peptide; MS, myosuppressin; NPLP1, neuropeptide-likeprecursor 1; PK, pyrokinin; PP, precursor peptide; PVK, CAPA-periviscerokinin; RYa, RYamide; sNPF, short neuropeptide F; tPK,CAPA-tryptoPK.

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FMRFamide 5 was indicated by mass-match. Althoughextended FMRFamides are usually easy detectable byMALDI-TOF MS due to the presence of Arg, our MALDI-TOF mass spectra failed to show the respective ion signals inmost cases. The absence of extended FMRFamides in thesemass spectra suggests a very low quantity in the tissue samplesanalyzed. In R. prolixus, two out of the nine predicted extendedFMRFamides have been biochemically confirmed,41,43 yet ourresults suggest that also the others likely are produced.Hugin-pyrokinin (Hugin-PK). The three predicted hugin-

PKs are all exclusively flanked by monobasic processing sites(Arg) in the propeptide sequence and were identified andsequence-confirmed by MS/MS. Interestingly, the third PK inthe yet incomplete propeptide shows the unusual C-terminalPRF−NH2 motif, which was found already in R. prolixus54 butnot in other true bugs.33 In addition to the predicted PKs, anextended form of PK-2 (EEDIIFTETSRSPPFAPRLamide) wasobserved, indicating an incomplete processing at the N-terminal cleavage site. In R. prolixus, an extended form of thesequence-identical homologue SPPFAPRLamide also seems toexist because the predicted propeptide in R. prolixus harbors anidentical sequence motif just N-terminal of that peptide.55

Hugin-PKs of C. lectularius were found in mass spectra fromcorpora cardiaca preparations (Figure 3A) and are likelyproduced in neurosecretory cells of the SEG (Figure 5A); as itis known from other insects as well.Kinin. Seven of the nine predicted bed bug kinins, including

the longest one (DSPGPKMRFSSWamide) were mass-spectro-metrically confirmed. In addition, we identified severalstructurally unrelated precursor peptides. Direct peptideprofiling of small areas of the mesothoracic ganglionic massof R. prolixus biochemically identified most of the 12 predictedkinins in this species.60 The high number of kinin paracopies inC. lectularius and R. prolixus cannot be attributed to ahematophagous life style because kinin precursors of manyother insects also contain numerous paracopies.Myoinhibiting Peptide (MIP)/Allatostatin B. All 11 of

the predicted MIPs were sequence-confirmed by Orbitrap MS/MS fragmentation, with MIP 5−7 sharing the identicalsequence GWQDLNSGGWamide. The Arg-containing MIP-10 was the only MIP that was regularly identified by directtissue profiling and was observed in different CNS samples andneurohemal tissues. In R. prolixus, MIP immunoreactivity is

Figure 4. (A) Alignment of the unusual corazonin sequence of C. lectularius with other known insect corazonin sequences. Amino acids are coloredaccording to the Taylor scheme. (B) Q Exactive fragment spectrum of corazonin from C. lectularius. Ion signals with prominent b- and y-typefragments are labeled. Fragments confirm the predicted unusual sequence from genome data of this species.

Figure 5. Direct MALDI-TOF MS profiling of tissue samples from thecentral nervous system of C. lectularius; only more prominent ionsignals are labeled. Ion signals that could not be assigned to productsof known neuropeptide precursors are marked with question marks.(A) Ventral portion of the subesophageal ganglion (SEG), containingsecretory neurons that synthesize the hugin-encoded pyrokinins (PK)and release their products via the retrocerebral-aorta complex (seeFigures 2 and 3A). Ion signals of three predicted PKs are marked. (B)Portion of the brain containing antennal lobe (AL) tissue. Ion signalsof products from 10 neuropeptide precursors are labeled; the asteriskmarks sNPF. AstA, allatostatin A; CT-DH, calcitonin-like diuretichormone; MIP, myoinhibiting peptide; MS, myosuppressin; NPLP1,neuropeptide-like precursor 1; NVP, NVP-containing precursorpeptide; Orc, Orcokinin; PK, pyrokinin; PP, precursor peptide;sNPF, short neuropeptide F; tPK, tryptoPK; TKRP, tachykinin-relatedpeptide.

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widespread throughout the CNS,61 and 8 out of the 11predicted MIPs could be mass-spectrometrically confirmed inbrain extracts.43,55

Myosuppressin. By tandem mass spectrometry, thepredicted myosuppressin was confirmed in an N-terminallyunprocessed and a more abundant pyroglutamyl form (Figures5B and 6B), similar to the situation in R. prolixus41 and otherinsects. A highly derived myosuppressin sequence as found inC. lectularius (Figure 6) appears not to be a general feature ofthe Heteroptera, as pentatomid myosuppressin shows thetypical insect consensus sequence.40 Myosuppressin of reduviidbugs38,41 contains two amino acid substitutions that are notintermediate between the consensus sequence and C. lectulariusmyosuppressin. The presence of myosuppressin receptors inthe bed bug genome9 suggests that C. lectularius myosuppressinis biologically active despite its unusual sequence. Whether andhow this unusual sequence affects receptor activation kinetics orpeptide stability in the hemolymph has to be tested in futureexperiments.Natalisin. The natalisin precursor of the bed bug contains

one paracopy for each of the natalisin consensus sequencesFXPXRa and FWAHRa as well as a putative amidated peptidewith a moderately similar C-terminus (DVLDQPLWVRa).Orthologs of these peptides were also predicted from thenatalisin precursor of R. prolixus.62 In the bed bug, we identifiedthe three peptides, which represent the first biochemicallycharacterized natalisins from hemimetabolic insects.Neuropeptide F. We identified peptides corresponding to

parts of the predicted full-length NPF1−42 (see Table 1). Thissuggests that full-length NPF1−42 is processed as predicted yetfurther endogenously cleaved by unknown enzymes. It isunclear whether the C-terminal fragment YAVAGRPRFarepresents the major bioactive form of NPF or whether it is adegradation product. We note, however, that the identical shortpeptide but not the full-length NPF was also found in T.infestans,38 and a similar C-terminal NPF sequence has beenreported from R. prolixus41,55 and showed bioactivity in thisspecies.63

Neuropeptide-like Precursor 1 (NPLP1). EighteenNPLP1 peptides could be identified from the NPLP1propeptide, four of them carrying a C-terminal amidation.These peptides encompass nearly the complete set ofpredictable precursor products and were detected in prepara-tions of the CNS (e.g., in the antennal lobe, Figure 5B) andretrocerebral complex (Figure 3A). A comparable diversity of13 NPLP1 peptides was also found in R. prolixus,41,43 where thenplp1 gene seems to be also expressed in ovaries, testes, andsalivary glands. Neither an NPLP1 receptor nor a consensussequence of NPLP1 peptides is known so far.

NVP-like Peptides. We found four peptides from the NVPprecursor of C. lectularius, including two longer peptidesoriginating from processing of the first and last dibasicprocessing sites. An orthocopy of one of these peptides(AQEFIMFGNQQNRAINNFGSTKNE) was also observed bymass spectrometry in R. prolixus.37 In the latter species, a singleNVP-like peptide (EHVLNPFEEFLAL) was found to besignificantly reduced 4 and 24 h after a blood meal.43 A similarsequence is, however, not contained in the bed bug NVP-likeprecursor.

Orcokinin. In many insects, the orcokinin gene isalternatively spliced.37 For the bed bug, we identified onlyone orcokinin A-type transcript.9 A single orcokinin (NMD-EIDRAGFDTFV) is contained within the orcokinin Aprecursor, and the cleavage of this peptide could be mass-spectrometrically confirmed. Orcokinin A precursors of R.prolixus and T. infestans both contain three orcokininparacopies. In addition to orcokinin, an orcomyotropin-likepeptide64 is encoded in the orcokinin A transcript, which wasalso identified by mass spectrometry, as were four unrelatedprecursor peptides (Table 1).

Pigment-Dispersing Factor (PDF). Although PDF-encod-ing genes were previously identified in the true bugs Riptortuspedestris65 and R. prolixus,37 the biochemically identified C.lectularius PDF is the first confirmed mature PDF inHeteroptera. Immunostainings in R. prolixus located PDF-expression to clock-gene expressing lateral neurons66,67 and theremoval of homologous PDF neurons in R. pedestris impaired

Figure 6. (A) Alignment of the unusual myosuppressin sequence of C. lectularius with other known myosuppressin sequences of various insect taxaand selected crustacean species. Amino acids are colored according to the Taylor scheme. T. infestans has the same myosuppressin sequence as R.prolixus. (B) MALDI TOF/TOF fragment spectrum of myosuppressin with N-terminal pyroglutamate from C. lectularius. Ion signals with prominentb- and y-type fragments are labeled. Fragments confirm the predicted unusal sequence from genome data of this species.

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the photoperiodic regulation of diapause.65 This suggests thatPDF serves well-conserved circadian functions68,69 in hetero-pteran insects and may also play a role in regulating circadianand seasonal rhythmicity in the bed bug.Proctolin. Proctolin is a sequence-invariant short insect

neuropeptide with myoregulatory effects, and proctolin-encoding genes have been identified in the genomes of variousreduviid bugs.38,70 Accordingly, proctolin could be biochemi-cally identified by LC−MALDI MS/MS in CNS extracts of R.prolixus.70 By MALDI-MS, we found ion signals matchingproctolin in the CNS of C. lectularius, yet we were unable toobtain a tandem mass-spectrometric fragmentation.RYamide. Different from R. prolixus,37 the bed bug

RYamide precursor contains only one canonical RYamide C-terminus. This peptide could be mass-spectrometricallysequenced following SPITC-derivatization (Figure 7) and

represents the first biochemically identified RYamide in ahemimetabolous insect. Preparations of anterior aorta tissueyielded a particularly high relative signal intensity of RYamide(Figure 3B)SIFamide. The bed bug SIFamide is a 12-amino acid

peptide with the C-terminal canonical PFNGSIFa sequence.71

It differs from R. prolixus SIFamide only in position 1 (Serinstead of Thr). In R. prolixus, SIFamide-immunoreactive cellsin the CNS are restricted to two pairs of neurons in the parsintercerebralis of the protocerebrum with projections along theentire CNS.55 In contrast with peptidomic studies in the kissingbug, we were unable to find N-terminally truncated SIFamidedegradation products.Short Neuropeptide F (sNPF). Typically, in hemi-

metabolic insects72 the sNPF gene codes for only one canonicalsNPF peptide. Similar to the situation in various insect taxa,72

we identified sNPF in a more abundant long (sNPF1−11:DNRTPQLRLRFa) and a short form (sNPF4−11) originatingfrom internal processing at the monobasic Arg3 of the longform. A similar Arg3 in R. prolixus sNPF1−11 suggests that alsoreduviid bugs produce a short and a long form of sNPF,although only the long form has so far been biochemicallyidentified in the kissing bug.41 Besides the canonical sNPF,various precursor peptides originating from propeptideprocessing at mono- or dibasic cleavage sites could be detected

in samples from the CNS and retrocerebral complex (Figure3A).

Sulfakinin (SK). Using a combination of positive andnegative ionization mode in MALDI-TOF MS73 as well as QExactive mass spectra, both predicted sulfakinins werebiochemically confirmed in CNS samples, including post-translational pyroglutamate formation (SK-1) and Tyr-sulfation. SK-2 possesses two Tyr residues as potential targetsfor sulfation. MALDI-TOF mass spectra in negative modeyielded weak ion signals supporting sulfation of both Tyr, butso far Q Exactive MS/MS spectra confirmed sulfation of onlythe first Tyr. Interestingly, the C-terminal sequence of SK-2(GYLRFa) deviates even more strongly from the insectconsensus sequence GHMRFa than the already unusual R.prolixus SK-2 (GYMRFa). It is thus conceivable that the twobed bug SKs each have a dedicated function and receptor,which is supported by the presence of two SK receptor genes inthe bed bug genome.9

Tachykinin-Related Peptide (TKRP). The Tkrp geneencodes eight amidated peptides, all of which were sequence-confirmed by mass-spectrometric fragmentation in C. lectular-ius. In R. prolixus, where TKRP-like immunoreactivity is widelydistributed throughout the CNS,74 the 13 predicted TKRPs ofthis species could be mass-spectrometrically confirmed aswell.43,55 These data suggest a high abundance of TKRPs in theCNS of these species, which is corroborated by high signalintensity of TKRP signals in mass spectra from brain tissue suchas the antennal lobe (Figure 5B). Ion signals of TKRPs werenot detected in preparations of the retrocerebral complex in C.lectularius (Figure 3A).

3.3. Peptides Not Detected though Respective GenesIdentified

Several of the neuropeptide precursor genes identified in thebed bug genome9 code for larger protein-like hormones, mostof them forming dimers via cysteine bridges. These includebursicon, GPA2, GPB5, insulin-like peptides 1−2, ion transportpeptide (ITP), neuroparsins 1−4, and prothoracicotropichormone (PTTH). These hormones are too large to bedetected in the peptidomic approaches employed here, and thelack of corresponding mass signals thus does not allow anyconclusion regarding their presence or absence. In contrast, afew smaller predicted peptides that were not detected should,in principle, be well detectable by our peptidomic approach andare discussed below.

Allatostatin CC (AstCC). An ion signal matching thetheoretical mass ([M + H]+) of an N-terminally truncatedAstCC (CYFNAVSCF) was observed in direct brain profilingbut could not be confirmed by MS/MS. This truncated formsuggests unpredicted prohormone convertase processing at asingle Arg preceding the N-terminal C. AstCC was alsopredicted from the R. prolixus genome,75 but no matureproducts have been biochemically identified.

CCHa-2. In the reduviids R. prolixus and T. infestans, CCHa-2 is the only predicted CCHa.37,38 The peptide could be mass-spectrometrically identified in the CNS of both species.38,55

Because CCHamide-2 is encoded in the C. lectularius genome,9

the absence of CHHa-2 signals in our investigation hintstoward an expression of CCHa-2 outside the CNS, possibly inthe midgut and fat body.76−78

Ecdysis-Triggering Hormone (ETH). Throughout theinsects, ETH peptides are expressed outside the nervous

Figure 7. MALDI TOF/TOF fragment spectrum of SPITC-derivatized RYamide from an extract of the central nervous system.Derivatization intensifies the generation of y-fragments. Assigned y-fragments are labeled.

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system in specialized gland cells.79 Because we only extractednervous tissue, these cells were not included in our analysis.Elevenin. An elevenin precursor was first predicted in

molluscs and later on also in various insect and annelidspecies.80−82 Elevenin was also predicted in C. lectularius9 andR. prolixus.18 Curiously, to the best of our knowledge, abioactive form of elevenin has never been identified in anyinsect. We obtained a single Orbitrap MS/MS spectrum thatrevealed an unpredicted amidated peptide sequence pQTE-YVTGRGa, a sequence just C-terminal of the elevenin signalpeptide that is C-terminally flanked by a putative monobasicprohormone convertase cleavage site. A similar sequence is,however, not contained in the elevenin precursors from variousmolluscs and the nematod Caenorhabditis elegans,80 as well as R.prolixus,37 and thus is unlikely to represent a bioactive peptide.SEFLamide. SEFLamides were only recently identified in

arthropods,83 and, to the best of our knowledge, bioactiveSEFLamide peptides have not been biochemically characterizedin any arthropod so far. Thus it remained unclear if this peptideis expressed in the CNS.3.4. Peptidergic Signaling Pathways in the Bed Bug asPossible Targets for Chemical ControlWith the pertinent problem of acquired resistances to organicor genetically expressed insecticides, peptides and theirreceptors are often suggested as alternative target structuresfor pest control.14,84,85 The resistance problem is especiallyvirulent for bed bugs,6 and thus peptide signaling pathwaysinvolved in feeding, diuresis, host location, or ecdysis arepotentially well suited targets for chemical control. Peptides canbe modified to increase their stability, oral availability, or cuticlepermeability. For example, a biostable kinin analog wasdeveloped that could be fed and lead to reduced blood mealsand disrupted ecdysis in R. prolixus.86 Similar strategies couldalso be developed for the bed bug. On the basis of their effectsin other insect species, we suggest the following peptidergicsignaling pathways may be well suited for bed bug control:ecdysis: ETH, kinin, PTTH; diuresis: calcitonin-like DH, CRF-like DH, CAPA-PVKs; feeding: sNPF, AstA, NPF, sulfakinins;mating: natalisins, MIPs, PDF; olfaction: CCHa-1.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jproteo-me.7b00630.

S1: Prepropeptide sequences identified from the bed buggenome.9 S2: Q Exactive orbitrap MS/MS spectra of C.lectularius peptides listed in Table 1. S3. List of thepeptides with Q Exactive Orbitrap MSMS confirmationshown in S2, with page designations, precursor masses,charge states, and mass errors. (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*R.P.: E-mail: rpredel@uni-koeln.de. Tel: +49-221-4705817.*K.R.: E-mail: klaus.reinhardt@tu-dresden.de. Tel: +49-351-463-37534.*C.W.: E-mail: christian.wegener@biozentrum.uni-wuerzburg.de. Tel: +49-931-31-85380. Fax: +49-931-31-844500.ORCID

Christian Wegener: 0000-0003-4481-3567

NotesThe authors declare no competing financial interest.∥C.D.: E-mail: christian.derst@t-online.de. S.N.: E-mail: mail@susanne-neupert.de.

■ ACKNOWLEDGMENTSFunding was provided by Deutsche Forschungsgemeinschaft(PR766/11-1 to R.P.) and intramural funds of the University ofWurzburg (to C.W.). K.R. was supported by the Zukunftskon-zept of the Technische Universitat Dresden awarded by theDeutsche Forschungsgemeinschaft through the ExcellenceInitiative. We thank Christian Frese, Corinna Klein, AstridWilbrand-Hennes, Ursula Cullman (CECAD Cologne Proteo-mics Facility), and Lapo Ragionieri (Institute for Zoology,Cologne) for the valuable help in optimizing Orbitrap analysesand Susanne Kluhspies (Wurzburg) for excellent support withimmunostainings.

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Journal of Proteome Research Article

DOI: 10.1021/acs.jproteome.7b00630J. Proteome Res. XXXX, XXX, XXX−XXX

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