Neuropeptidomics of the Bed Bug Cimex lectularius - Journal of

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Neuropeptidomics of the bed bug Cimex lectularius Reinhard Predel, Susanne Neupert, Christian Derst, Klaus Reinhardt, and Christian Wegener J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00630 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 18, 2017

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Journal of Proteome Research

Neuropeptidomics of the bed bug Cimex lectularius Reinhard Predela*, Susanne Neuperta, Christian Dersta, Klaus Reinhardtb*, Christian Wegenerc* a

Department for Biology, Institute for Zoology, University of Cologne, Zülpicher Straße 47b, D-50674 Cologne, Germany

b

Applied Zoology, Department of Biology, Technical University of Dresden, Zellescher Weg 20b, D01062 Dresden, Germany

c

Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany

* address for correspondence: Prof. Dr. Reinhard Predel Institute for Zoology, University of Cologne Zülpicher Straße 47b, D-50674 Cologne, Germany [email protected] Phone: +49-221-4705817 * address for correspondence: Prof. Dr. Klaus Reinhardt Applied Zoology, Department of Biology, Technical University of Dresden Zellescher Weg 20b, D-01062 Dresden, Germany [email protected] Phone: +49-351-463-37534 * address for correspondence: Prof. Dr. Christian Wegener, Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, University of Würzburg Am Hubland , D-97074 Würzburg, Germany [email protected] Phone: +49-931-31-85380 Fax: +49-931-31-844500

1 ACS Paragon Plus Environment


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email addresses for other authors: [email protected] [email protected]

ORCID: Klaus Reinhardt: 0000-0001-7200-4594 Christian Wegener: 0000-0003-4481-3567


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Abstract The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signalling is especially interesting since it regulates feeding, diuresis, digestion as well as reproduction, and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones as evidenced by the occurence of respective mass signals and immunoreactivity in neurohemal structures. Our data provides the most comprehensive peptidome of a Heteropteran species so far, and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.

key words: neuropeptides, mass spectrometry, peptide prediction, bed bug genome, blood-sucking insect, neurohormones, central nervous system, neuromodulation



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1. Introduction The bed bug Cimex lectularius is a globally distributed human ectoparasite with a fascinating biology. This includes an unusually long starvation resistance1,2, an obligate reliance on blood as a food resource1,2, combined with the ability to rapidly process blood meals several times their body weight and excrete excess water in females to avoid superfluous, costly matings3, as well as the unusual form of reproduction of traumatic insemination1,2, and a uniquely diverse response in females to tolerate the resulting copulatory damage4. Most of the molecular and regulatory mechanisms underlying these behavioural and physiological processes are unknown. Moreover, in the wild, bed bugs can also live on bats1,2, and are genetically separated from human-associated bed bugs5. 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 interest since the late 1990ies. The worldwide resurgence in the number of infestations observed since then is at least in part due to evolution of resistance to a broad range of insecticides6, increased human travel and perhaps increasing temperatures. Infestations can be massive and can occur in all kind of human settlements6. Skin reactions to bed bug bites are common among humans but sometimes represent as severe responses7. Due to these features, the bed bug was selected as a pilot species in the i5k arthropod genomes project8; the annotated bed bug genome was recently released9,10. Our group had participated in the bed bug genome annotation, focussing on the identification of genes for neuropeptides and their receptors9. Neuropeptides are key intercellular signalling molecules that regulate and orchestrate most developmental, physiological and behavioural processes. With regard to the biology of bed bugs, neuropeptides are especially interesting since they induce and terminate post-feeding diuresis and control feeding, digestion as well as reproductive behaviour11,12. Moreover, neuropeptides and their receptors provide potential targets for next generation insecticides that could be exploited to control insecticide-resistant bed bug strains13,14. Our genomic analysis identified 47 genes encoding putative prepropeptides9 which can posttranslationally processed by a specific set of enzymes15 to give rise to more than 100 bioactive neuropeptides. Though processing of prepropeptides can in principle be well predicted by bioinformatic tools16,17, differential and alternative processing can take place and in well-studied species such in Drosophila melanogaster, mismatches between predicted and produced peptides were found18. We therefore performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. By a combination of direct tissue profiling by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and Q Exactive Orbitrap MS/MS, we confirmed most predicted


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peptides are produced in the nervous system of the bed bug, and also confirmed the genomically deduced peptide sequences. To identify species-specific peptide features in bed bugs, we compare our results with peptidomic data from the kissing bugs R. prolixus and T. infestans. These bugs are also obligate bloodfeeders but belong to the Reduvioidea, a phylogenetically more basal group of the Heteropteran infraorder Cimicomorpha than the Cimicoidea19 comprising C. lectularius. Both taxa have been separated in the Triassic around 200 Mya ago20. To illustrate the distribution of identified peptides in the CNS, we also immunolocalised selected peptides in the CNS. The results represent the most comprehensive peptidomic description of a Heteropteran insect, and suggest that the peculiar biology of the bed bug is not reflected by a peculiar neuropeptide complement, at least not on the qualitative level.

2. Experimental section

2.1 Prediction of prepropeptide genes and peptide processing in the bed bug genome The general procedures of automated gene annotation and community 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 available peptide sequences from other species, including the predicted sequences from the related reduviid bug R. prolixus22 and brown leafhopper Nilaparvata lugens23. Signal peptide sequences, cleavage sites, posttranslational modifications and monoisotopic masses were predicted

by

SignalP

4.024

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

and

NeuroPred16

(http://neuroproteomics.scs.illinois.edu/cgi-bin/neuropred.py). Peptide alignments were performed using JalView2.925.

2.2. Animals The bedbug populations used in this study originate from two locations. Population 1, also called F4, was originally collected from a human dwelling in London in 2003. The population originally showed resistance to several insecticides, including pyrethroids and carbamates. The population was maintained in a large (>1000 individuals) outbred population and, as expected, gradually lost insecticide resistance in the laboratory. Current population size is several hundred and insecticide susceptibility is most likely. Population 2 consisted of individuals originally collected in two bat roosts in Serbia in 2013 (collector: Milan Paunovic, Serbian Natural History Museum Belgrade). This population is, therefore, likely not to be resistant against insecticides. This population has been maintained at a smaller size (ca. 100 individuals).



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Bed bugs were kept in large colonies at 25 °C, ca. 60% RH, and regularly fed, as previously described26.

2.3 Anatomy Anatomical descriptions and dissection for tissue sampling of the nervous system and retrocerebral complex of C. lectularius were 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 CNS of last instar nymphs and males (n=5) from both populations was dissected in ice-cold saline and then transferred into 30 µl extraction solution (50% methanol, 49% H2O, 1% formic acid [FA]). Tissues samples were disintegrated using an ultrasonic bath (Transonic 660/H, Elma Schmidbauer GmbH, Hechingen, Germany) for 5 minutes on ice, and an ultrasonic-probe three times for 5 seconds (BandelinSono HD 200, Bandelin electronic GmBH, Berlin, Germany), respectively. Afterwards, the samples were centrifuged for 15 minutes at 13,000 rpm at 4 °C. The supernatants were transferred to new tubes and methanol was evaporated in a vacuum concentrator. Extracts were stored at -20 °C until use.

2.5 Direct MALDI-TOF MS Corpora cardiaca, corpora allata, perisympathetic organs, peripheral nerves, smaller parts of the brain and subesophageal ganglion from males were separately dissected as described for cockroach samples27 and directly transferred into a drop of water on the sample plate for MALDI-TOF MS. Immediately after transfer, water was removed and the tissue samples were dried prior to deposition of matrix.

2.6 Quadrupole Orbitrap MS coupled to nanoflow HPLC Before injecting the samples into the nanoLC system, extracts were desalted using self-packed Stage Tip C18 (IVA Analysentechnik e.K., Meerbusch,Germany) spin columns28. Peptides were then separated on an EASY nanoLC 1000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) using in-house packed RPC18-columns 50 cm (fused Silica tube with ID 50µm ± 3µm, OD 150 µm ± 6 µm; Reprosil 1.9µm, pore diameter 60A°; Dr. Maisch GmbH, 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 2 to 62% B in 110 min, 62 to 75% B in 30 min, and final washing from 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 (Thermo Scientific, Bremen, Germany) mass spectrometer. MS data were acquired in a top-10 data-dependent method dynamically choosing the most abundant peptide ions from


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the respective survey scans in a mass range of 300–3000 m/z for HCD fragmentation. Full MS1 acquisitions ran with 70,000 resolution; with automatic gain control target (AGC target) at 3e6 and maximum injection time at 80 ms. HCD spectra were measured with a resolution of 35,000, AGC target at 3e6, maximum injection time at 240 ms, 28 eV normalized collision energy, and dynamic exclusion set at 25 s. The instrument was run with peptide recognition mode (i.e., from two to eight charges), singly charged and unassigned precursor ions were excluded. Raw data were analyzed with PEAKS 8.0 (PEAKS Studio 8.0, BSI, ON, Canada). Neuropeptides were searched against an internal database comprising Cimex neuropeptide precursor sequences (see Supporting Information1) with parent error mass tolerance of 0.1 Da and fragment mass error tolerance of 0.2 Da. None enzyme mode was selected because samples were not digested. The False Discovery Rate (FDR) was determined by a decoy database search as implement in PEAKS 8.029 and set below 1%. Conversion of C-terminal glycine was selected as fixed post-translational modification (PTM) while variable PTMs included in the searches were oxidation at methionine, n-terminal acetylation, pyroglutamate from glutamine, pyroglutamate from glutamic acid, sulfation of tyrosine, serine and threonine, and disulfide bridges. In each run a maximum of 6 variable PTMs per peptide were allowed. Fragment spectra with a peptide score (-10lgP) greater than 20, which is equivalent to a P-value of about 1%, were manually reviewed.

2.7. MALDI-TOF MS 10 mg/ml 2,5–dihydroxybenzoic acid (Sigma-Aldrich, Steinheim, Germany) dissolved in 20 % acetonitrile/1 % formic acid or alternatively 10mg/ml α-cyano-4- hydroxycinnamic acid dissolved in 60 % ethanol, 36 % acetonitrile, 4 % Fluka water were used as matrix salts and loaded onto the dried samples using a 0.1-10 µl pipette (about 0.3 µl for direct tissue profiling). Mass fingerprint spectra were acquired in positive ion mode with an UltrafleXtreme TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). All MS acquisitions were made under manual control in reflector positive mode in a detection range of 600–10,000 Da. The instrument settings were optimized for the mass ranges of m/z 600–3,500 and 3,000–10,000 and calibrated using Bruker peptide and protein standard kits, respectively. Laser fluency was adjusted to provide the optimal signal-to-noise ratio. The data obtained in these experiments were processed with a FlexAnalysis3.4 software package. MS/MS was performed with LIFT technology. LIFT acceleration was set at 1 kV. The number of laser shots used to obtain a spectrum varied from 5,000 to 25,000, depending on signal quality. For MS/MS experiments, we also used an ABI 4800 proteomics analyzer (Applied Biosystems, Framingham, USA). MS/MS fragment spectra were acquired manually in gas-off mode. Peptide identities were verified by (1) comparison of theoretical and experimentally obtained ion signals of neuropeptides ([M+H]+) and (2) comparison of theoretical (http://prospector.ucsf.edu) and experimentally obtained fragments following MS/MS experiments.



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2.8 Peptide derivatisation using 4-sulfophenyl isothiocyanate (SPITC) Based on a protocol by Wang et al.30, tissue extracts were transferred in 40 µl SPITC dissolved in 20 mM NaHCO3 (pH 9.0) at a concentration of 10 mg/ml. The sulfonation reaction was carried out in a 0.5 ml Eppendorf tube for 1 h at 55 °C and 300 rpm. Samples were then acidified by adding 2.5 µl 10% acetic acid followed by 50 µl 0.5% acetic acid, and loaded onto an activated and equilibrated StageTip C18 column28. The column was rinsed with 50 µl 0.5% acetic acid and peptides were eluted from the column with solutions of 10/20/30/40/50/80% acetonitrile (0.5% acetic acid) and spotted onto a MALDI target. Before drying, 0.3 µl matrix solution was added to each sample, respectively. MALDI-TOF mass spectrometry was performed using the UlrafleXtreme as described in 2.7. First, MS1 spectra were manually searched for the presence of signals of predicted Cimex neuropeptides with derivatization ([M+H]+ + 215). Such ions were then fragmented (MS/MS) using the same samples. Resulting fragments were compared with theoretical fragments of the predicted neuropeptides to confirm the identity of the substances.

2.9 Immunostaining Nervous tissue of adult males was dissected in 0.1 M phosphate-buffered saline (PBS, pH 7.2) fixed for 3 hours in 4% paraformaldehyde in PBS at room temperature, washed in PBS with 0.5% TritonX (PBT0.5) and incubated for at 48-72 h in PBT containing 5% normal goat serum in combination with rabbit polyclonal anti-allatostatin A31 (Jena Bioscience, Jena, Germany), anti-corazonin32 (kind gift of Jan A Veenstra, Bordeaux, France) antiserum (diluted 1:1000) or anti-Pea-PVK-233 (kind gift of Manfred Eckert, Jena, Germany diluted 1:4000). Then, preparations were washed 5 times during a day with PBT and incubated for 48-72 h in PBT0.5 containing 5% normal goat serum with DyLight488-conjugated AffiniPure goat anti-rabbit IgG (H+L; Jackson ImmunoResearch, Germany) at a dilution of 1:1000 or with Cy3-coupled goat anti-rabbit antisera (Jackson ImmunoResearch, Germany) at a concentration of 1:3000. Preparations were subsequently washed for 24 h, and then mounted in 80% glycerol diluted in PBS or embedded in Mowiol (Merck KGaA, Darmstadt, Germany). For the in situ CAPA peptide immunostaining, the abdominal tergites and subsequently the intestine of adult males were removed using fine scissors, and the remaining preparation was fixed with microneedles. Preparations were then rinsed with ice-cold insect saline and treated with Histofix (SigmaAldrich, St. Louis, MO, USA) at 4 °C for 12 hours. After washing with PBS containing 1% TritonX-100 (PBT1.0) for 24 hours, the samples 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 bovine serum and 10% normal goat serum. Subsequently, the


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samples were washed in PBT1.0 for 24 hours at 4 °C. Then, the samples were incubated with Cy3-coupled goat anti-rabbit antisera diluted 1:3000 (Jackson ImmunoResearch) in PBT1.0 for 5 days at 4 °C. After washing in PBT1.0 for 24 hours at 4 °C, the preparations were embedded in Mowiol (Merck KGaA, Darmstadt, Germany). Confocal stacks were acquired on a confocal laser scanning microscope (SPE, Leica Microsystems, Wetzlar, Germany) with a 20x objective (ACS APO 20x N.A. 0.6 IMM) or 40x objective (ACS APO 40x N.A. 1.15 oil) or a LSM 510 Meta confocal laser scanning system (Zeiss, Jena, Germany) equipped with a C-Apochromat 10x/0.45W and a Plan-Apochromat 20x/0.75 objectives. Pictures were analysed using the Fiji image processing package34 (http://fiji.sc/About) based on the public domain software ImageJ 1.46 (NIH, public domain:http://rsb.info.nih.gov/ij/) or Zeiss LSM 5 image browser v.3 software. Figures were generated with the help of Adobe Photoshop CS6 (Adobe Systems Inc.) using only brightness and contrast adjustments.

Results & Discussion In silico prediction of prepropeptide sequences During the bed bug genome annotation, we previously predicted 47 different prepropeptide genes for C. lectularius9, 45 full and 2 partial prepropeptide sequences (Supporting Information S1). One partial sequence, that for CNMamide, could now be completed by a BLAST search against updated NCBI reference sequences (Supporting information S1). In addition, we found genes for neuropeptide-like precursor NPLP3 and NPLP4 homologs previously identified in a neuropeptidomic analysis in Drosophila35. Based on their sequence, in C. lectularius they appear to represent cuticular peptides rather than neuropeptides in C. lectularius9. This view is supported by the lack of NPLP3 and NPLP4 peptides in CNS extracts during this study. From the predicted propeptides, more than 100 different putative neuropeptides can potentially be derived. C. lectularius possesses the “core set” of 20 genes coding for insect regulatory peptides, and there are no unexpected or unique peptidomic features in comparison with other true bugs9. Yet, the genomic annotation predicts very unusual sequences of myosuppressin and corazonin for the bed bug9.

Identification of neuropeptides in the central nervous system (CNS) and neurohemal release sites To identify which of the predicted neuropeptides are expressed in the CNS, and to confirm the unusual myosuppressin and corazonin sequences of C. lectularius, we biochemically characterised the peptidome of larval and male adult bed bugs in the CNS and attached neurohemal release sites. For our study we used two populations of C. lectularius which originate from human dwellings and bat caves, respectively.



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Human- and bat-associated bed bugs are known to show some degree of genetic differentiation 36. In our study, however, we did not observe a single difference in the sequences of neuropeptides between the two populations. Therefore all given information on neuropeptides is valid for both populations. Since the gross anatomy of the bed bug CNS is not well described, we provide an overview in Figure 1 A-B. Major neurohemal release sites along the CNS of the bed bug are the very compact retrocerebral complex (see Figure 1 C-E) which is closely fused with the anterior aorta, and the abdominal segmental nerves 2-4 (Figure 2) which are the equivalent of perisympathetic organs in hemimetabolic insects with unfused abdominal ganglia. By a combination of direct tissue profiling by MALDI-TOF MS and Q Exactive Orbitrap MS/MS, we biochemically identified a total of 144 peptides from 29 of the identified precursor genes. For 27 of these precursors, peptides were sequence-confirmed by MS/MS fragmentation, while we have peptide mass matches only for the proctolin and CCAP precursor. Based on sequence similarities to peptides in other insect species with known functions, at least 67 of the identified peptides represent bioactive and mature neuropeptides. 63 of these peptides were confirmed by MS/MS fragmentation, four peptides were mass matches only. In addition to the bioactive neuropeptides, we identified 23 peptides (21 confirmed by MS/MS) from the NPLP1 and NVP precursors. These precursors show key features of preproneuropeptides (signal peptide, cleavage sites, expression in the CNS) and are usually annotated as such in diverse insect genomes, yet bioactivity and function of NPLP1 and NVP peptides have not been tested in any insect so far. 31 peptides (27 sequence-confirmed by MS/MS) represent additional precursor (“spacer”) peptides which likely do not have any signalling function. All identified peptide sequences are summarized in Table 1.


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Figure 1. Gross morphology of the adult bed bug CNS and immunostaining of selected neuropeptides in neurohemal organs. A) Dorsal and B) lateral view of the CNS, showing a high degree of ganglion fusion typical of Heteroptera. The brain is fused with the subesophageal ganglion (SEG) and prothoracic ganglion (PRO), the remaining thoracic and all abdominal ganglions are fused to the mesothoracic ganglionic mass (MTGM). In B), part of the esophagus (eso) is visible that projects through a foramen in the midline approximately at the brain-SEG border. C) AstA immunoreactivity in the brain of an adult bed bug. In each brain hemisphere, primary neurites of two large secretory neurons in the pars lateralis of the protocerebrum (arrows) project towards the midline, then bifurcate (asterisk). One small branch of each neurite projects to the contralateral side, while the other projects through the nervus corporis cardiaci II (NCC II, arrowheads) to the neurohemal retrocerebral-aorta complex (arrowhead) where it forms a dense net of terminal varicosities. D) Close-up of AstA-immunoreactive varicosities in the retrocerebral complex comprising the corpora cardiaca (CC) in a different preparation. E) Close-up of corazonin-immunoreactive varicosities in the retrocerebral complex comprising the CC and corpora allata (CA). Arrowheads mark the entry of neurites from the protocerebrum via NCC II.

In the following, we describe and discuss specific details of the identified peptides. This includes a



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comparison with available data for the reduviid bugs R. prolixus and T. infestans, the other blood-feeding heteropteran species well-described in terms of neuropeptides and their receptors37,38. Known physiological functions of neuropeptides in these species are summarized in Ons37 and are not repeated here.

Figure 2. In situ immunostaining against PRXamide which labels CAPA peptides, adult male preparation. In the subesophageal ganglion which is fused to the prothoracic ganglion (SEG+Th1), a pair of strongly stained neurons is visible. Further immunoreactive neurons in the protocerebral part of the brain (BR) provide arborisations mostly in the superior brain. Two descending varicose axons (#) of unknown origin innervate the mesothoracic ganglionic mass (MTGM). Three secretory neurons (Va) in the abdominal portion of the MTGM project via the abdominal segmental nerves 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 in more detail. Numbers mark the segmental nerves 2-4.

AKH/corazonin-related peptide (ACP): We predicted one ACP from the prepropeptide, which could be biochemically identified and sequence-confirmed from preparations of the bed bug CNS. As for AKH and corazonin, the N-terminal Gln is fully modified to pyroglutamate and no trace of unblocked ACP was detectable. In R. prolixus immunostainings revealed three pairs of ACP-immunoreactive neurons in the protocerebrum which send descending projections through the ventral ganglia39. Adipokinetic hormone (AKH): The prepropeptide gene codes for one AKH, which was biochemically identified in preparations of the corpora cardiaca (Figure 3A) and sequence-confirmed. Based on the


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genomic sequence, it contains Ile at position 2, while all other MS-identified heteropteran AKHs are reported with a Leu at this position40–42. Although these peptidomic studies did not biochemically distinguish between the isobaric Ile and Leu, genomic and transcriptomic data confirmed Leu2 at least for R. prolixus54 and T. infestans30. AstA: In R. prolixus, several predicted AstA peptides could not be found in peptidomic approaches41,43. In the bed bug, we did not find ion signals corresponding to the predicted AstA-1 peptide (LYDFGLa) located most N-terminally on the propeptide. The remaining AstA peptides could be sequence-confirmed as predicted; including LPKQYSFGLa and PEGKMYSFGLa. In the propeptide, these two peptides are separated

by

an

internal

prohormone

convertase

KR cleavage

site. Their

long

product

LPKQYSFGLGKRPEGKMYSFGLa could also be sequenced-confirmed. Though putatively bioactive, this long form likely represents a processing intermediate since only weak MS signals were detected; a similarly long form was not detected in the R. prolixus brain41,43. Masses corresponding to additional precursor peptides were found as well but could only partially be fragmented (Table 1). The presence of densely arborised AstA immunoreactive varicosities in the retrocerebral-aorta complex (Figure 1C-D) and the identification of AstA peptides in mass spectra from this complex (Figure 3A) suggest a hormonal role for AstA in bed bugs, similar to the situation in R. prolixus44. AstC: Ion signals indicating the presence of AstC were found with direct tissue profiling of brain but also anterior aorta tissue using MALDI TOF MS (Figure 3B) as well as in Orbitrap mass spectra. In addition, partial sequences of the N-terminal precursor peptide have been identified in Orbitrap analyses of CNS tissue.The occurrence of AstC in the R. prolixus CNS43 was supported by the MS/MS identification of putative AstC propeptide-processing products but not bioactive AstC, though the AstC prepropeptide sequence remains unidentified in this species.



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Figure 3. Direct MALDI-TOF MS tissue profiling of tissue samples from C. lectularius; only more prominent ion signals are labelled. The spectra verify the tissue-specific presence of products of several neuropeptide precursors. Ion signals which could not be assigned to products of known neuropeptide precursors are marked with question marks. A) Retrocerebral complex (RC) containing the corpora cardiaca, corpora allata and a small piece of anterior aorta. B) Piece of aorta adjacent to the RC that stores peptide hormones. Low ion signal intensities suggest a local release of the peptides rather than a release as classical hormones. C) A single abdominal segmental nerve 2 shows prominent ion signals for products of the CAPA precursor. Note the presence of C-terminally and C- and Nterminally extended sequences of CAPA-tryptoPK which likely are not bioactive. Only traces of the canonical trypto-PK (see arrow) are detectable in preparations of abdominal segmental nerves. AKH, adipokinetic hormone; AstA, allatostatin A; AstC, allatostatin C; cor, corazonin; CRF-DH, corticotropin releasing factor-like diuretic hormone; MIP, myoinhibitory peptide; MS, myosuppressin; NPLP1, neuropeptide-like precursor 1; PK, pyrokinin; PP, precursor peptide; PVK, CAPA-periviscerokinin; RYa, RYamide; sNPF, short neuropeptide F; tPK, CAPAtryptoPK.

Allatotropin: Allatotropin was sequence-confirmed in its predicted amidated form in CNS samples, where it was commonly detected. Whereas the sequence of allatotropin is highly conserved and mostly identical in different families of true bugs45, the C. lectularius allatotropin differs at three amino acid positions in


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the middle part of its sequence from other heteropteran species which have been analysed so far. Calcitonin-like diuretic hormone (CT-like DH): CT-like DH could be sequence-confirmed in the predicted full-length form, 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. prolixus46, where CT-like DH was found in its full-length form and a C-terminally truncated form (CT-like DH(1-16))41. CAPA: The capa gene of the bed bug codes for two periviscerokinins (PVKs) plus one tryptophancontaining pyrokinin9 (CAPA-tryptoPK). C. lectularius PVK-1 lacks the complete N-terminal motif of the consensus sequence typical of basal hexapods (LIPFPRVa47). This peptide is therefore a good candidate to test the specificity of PVK receptors. The two predicted PVKs were biochemically confirmed and represent the most abundant peptides in mass spectra from preparations of abdominal segmental nerves 24 (Figure 3C) which are the major CAPA hormone release sites in true bugs. In addition to the predicted PVK-1 (EVRPFEFPRVa), the prominent ion signal of a truncated version of this peptide (PVK-13-10) was observed in all mass spectra from these nerves (n>10, Fig 3C) which also revealed the presence of low amounts of pyroglutamyl forms of both PVKs. A C-terminally extended CAPA-tryptoPK (NGGNGNGGLWFGPRLGKIQ), but not the predicted tryptoPK itself was abundant in preparations of abdominal segmental nerves (Figure 3C). This peptide originates from C-terminal processing at a dibasic KR site and can certainly be associated with a loss of bioactivity of tryptoPK. Whereas traces of the predicted tryptoPK were observed in few preparations only, we could identify an N- and C-terminally extended CAPA-tryptoPK (Figure 3C), which results from incomplete propeptide processing at a dibasic KR cleavage site ((SGPKRNGGNGNGGLWFGPRLGKIQ)), possibly due to proline in position -3. The occurrence of this peptide suggests a preferential cleavage of the N-terminally situated monobasic R cleavage site and a similar processing product was already reported for R. prolixus47. Different from the processing of likely not bioactive extended forms of tryptoPK in abdominal ganglia, CAPA-tryptoPK was identified in mass spectra from the brain/subesophageal ganglion (SEG) and retrocerebral complex where the C-terminally extended forms of this peptide could not be detected. This finding suggests that tryptoPK is differentially processed in abdominal ganglia and brain/SEG tissue. The differential processing of the CAPA prepropeptide is corroborated by the complete absence of PVKs in mass spectra of brain/SEG samples which was earlier reported for R. prolixus48 and also for Drosophila melanogaster49. In addition to

CAPA-tryptoPK,

only

ion

signals

(SGPKRNGGNGNGGLWFGPRLa)

were

of

an

N-terminal

detectable

in

extended

mass

spectra

form of of

this

peptide

preparations

of

brain/subesophageal ganglion (SEG) and retrocerebral complex. The different CAPA-peptides identified in preparations of CNS, retrocerebral complex and CAPA-release sites of abdominal ganglia are summarized in Table 1.



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The R. prolixus genome contains two capa genes50,51, each of which contains only one canonical and bioactive CAPA-PVK51 plus a tryptoPK. The presence of only one capa gene in the bed bug and the confirmed colocalization of products from both R. prolixus capa genes in the same secretory neurons48 suggest that the unusual duplication of capa in R. prolixus compensates for the loss of one functional PVK rather than representing an adaptation to a blood-sucking life style. As in R. prolixus50 and other true bugs52, CAPA peptides of the bed bug are expressed in three pairs of secretory ventral abdominal ganglia neurons (Va neurons, with homology to Drosophila and other insect species53) which release CAPA peptide hormones via the abdominal segmental nerves 2-4 (Figure 2). Different from the other hemipteran species analysed so far, the release sites 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 not found in peptidomic screens from R. prolixus41,43 or T. infestans38 although CCAP mRNA is known to be expressed in the brain and CCAP-immunoreactivity is rather widespread54. In the bed bug brain, we found peaks mass-matching the predicted CCAP. Though we were unable to confirm the identity by MS/MS, the occurrence of low intensity signals mass-matching also the predicted precursor peptide FYFPDEVSEPIDPKI lends support to the chemical presence of processed CCAP because this precursor peptide results from predicted CCAP processing. CCHamide-1: CCHamide-1 could be identified by nanoLC-MS in brain extracts, while CCHa-2, the only predicted and mass-spectrometrically identified CCHamide in the reduviids R. prolixus55545354535251,41 and T. infestans38 was not found38,5537,5436,5337,5436,5335,5235,51. CNMamide: CNMamide is a recently identified insect neuropeptide; the corresponding gene was also reported from R. prolixus56. We were now able to confirm the presence of an amidated and likely bioactive CNMamide by Orbitrap MS/MS analysis in a true bug. Corazonin: Most insects possess either [Arg7]- or [His7]-corazonin though other variants exist with variable amino acids in positions 3, 4 and 1057 ( Figure 5). [Arg7]-corazonin is also found in pentatomid and reduviid heteropterans40,41. For the cimiciid bed bug, the genomic prepropeptide sequence infers an unusual and unique [Met2, Arg10]-corazonin. We confirmed this sequence biochemically in CNS extracts and by direct tissue profiling, and thereby confirm the unusal sequence is not an artefact of genomic sequencing errors. Arg10 confers a negative charge in the C-terminal part of the peptide instead of the uncharged polar T or Q at this position. This is remarkable, as the C-terminal part is usually considered to be important for receptor binding and activation. In R. prolixus39, T. infestans58, the linden bug Pyrrhocoris apterus59 and indeed most other insects, corazonin is expressed in secretory neurons of the pars lateralis of the protocerebrum. These neurons release the peptide as a hormone at the neurohemal retrocerebral-aorta complex. To obtain a first indication whether the unusual C. lectularius corazonin is


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released as a hormone, we performed immunostainings of the bed bug CNS with an antiserum raised against [Arg7]-corazonin. We observed immunoreactivity within the brain and ventral ganglion, it was very weak and mostly confined to a few neuronal somata in the brain and neurites in the ventral ganglion, indicating that the antiserum only weakly recognises [Met2, Arg10]-corazonin. We nevertheless observed distinct corazonin-immunoreactive staining of densely arborising neurite endings in the retrocerebralaorta complex (Figure 1E), similar to the situation in other Heteroptera39,58,59. We also observed ion signals of corazonin in MALDI TOF mass spectra from preparations of the retrocerebral complex (Figures 2)) and Orbitrap MSMS spectra from CNS extract (Figure 4B) which confirmed that the unusual C. lectularius corazonin is released as a hormone.

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

Corticotropin releasing factor-like diuretic hormone (CRF-like DH): We were able to sequence-confirm the predicted full-length amidated peptide in preparations from the brain and retrocerebral complex/aorta. This is in line with the presence of a similar full-length amidated peptide in R. prolixus, demonstrated by mass spectrometry of bioactive purified CRF-like DH. In addition to the amidated peptide, we identified three further precursor peptides, altogether covering the complete propeptide sequence (Table 1). Extended FMRFamide peptides (FMRFamide): Out of the eight predicted extended FMRFamides in the propeptide, six were sequence-confirmed by Orbitrap MS/MS fragmentation (FMRFamides 1, 3, 4, 6-8), while the presence of FMRFamide 5 was indicated by mass-match. Although extended FMRFamides are usually easy detectable by MALDI-TOF MS due to the presence of Arg, our MALDI-TOF mass spectra failed to show the respective ion signals in most cases. The absence of extended FMRFamides in these mass spectra suggests a very low quantity in the tissue samples analysed. In R. prolixus, two out of the



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nine predicted extended FMRFamides have been biochemically confirmed41,43, yet our results 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 and sequence-confirmed by MS/MS. Interestingly, the third PK in the yet incomplete propeptide shows the unusual C-terminal PRFNH2 motif which was found already in R. prolixus54 but not in other true bugs33. In addition to the predicted PKs, an extended form of PK-2 (EEDIIFTETSRSPPFAPRLamide) was observed, indicating an incomplete processing at the N-terminal cleavage site. In R. prolixus, an extended form of the sequenceidentical homolog SPPFAPRLamide also seems to exist because the predicted propeptide in R. prolixus harbors an identical sequence motif just N-terminal of that peptide55. Hugin-PKs of C. lectularius were found in mass spectra from corpora cardiaca preparations (Figure 3A) and are likely produced in neurosecretory cells of the SEG (Figure 5A); as it is known from other insects as well.

Figure 5. Direct MALDI-TOF MS profiling of tissue samples from the central nervous system of C. lectularius; only more prominent ion signals are labelled. Ion signals which could not be assigned to products of known neuropeptide precursors are marked with question marks. A) Ventral portion of the subesophageal ganglion (SEG), containing secretory neurons that synthesise the hugin-encoded pyrokinins (PK) and release their products via the retrocerebral-aorta complex (see Figures 2, 3A). Ion signals of three predicted PKs are marked. B) Portion of the brain containing antennal lobe (AL) tissue. Ion signals of products from 10 neuropeptide precursors are labelled; the asterisk marks sNPF. AstA; allatostatin A; CT-DH, calcitonin-like diuretic hormone; MIP, myoinhibitory peptide;


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MS, myosuppressin; NPLP1, neuropeptide-like precursor 1; NVP, NVP-containing precursor peptide; Orc, Orcokinin; PK, pyrokinin; PP, precursor peptide; sNPF, short neuropeptide F; tPK, tryptoPK; TKRP, tachykininrelated peptide.

Kinin: Seven of the nine predicted bed bug kinins, including the longest one (DSPGPKMRFSSWamide) were mass-spectrometrically confirmed. In addition, we identified several structurally unrelated precursor peptides. Direct peptide profiling of small areas of the mesothoracic ganglionic mass of R. prolixus biochemically identified most of the 12 predicted kinins in this species60. The high number of kinin paracopies in C. lectularius and R. prolixus cannot be attributed to a hematophagous life style because kinin precursors of many other insects also contain numerous paracopies. Myoinhibing peptide (MIP)/Allatostatin B: All of the eleven predicted MIPs were sequence-confirmed by Orbitrap MS/MS fragmentation, with MIP5-7 sharing the identical sequence GWQDLNSGGWamide. The Arg-containing MIP-10 was the only MIP which was regularly identified by direct tissue profiling and was observed in different CNS samples and neurohemal tissues. In R. prolixus, MIP immunoreactivity is widespread throughout the CNS61, and eight out of the eleven predicted MIPs could be mass-spectrometrically confirmed in brain extracts43,55. Myosuppressin: By tandem mass spectrometry, the predicted myosuppressin was confirmed in an Nterminally unprocessed and a more abundant pyroglutamyl form (Figures 5B, 6B), similar to the situation in R. prolixus41 and other insects. A highly derived myosuppressin sequence as found in C. lectularius (Figure 6) appears not to be a general feature of the Heteroptera, as pentatomid myosuppressin shows the typical insect consensus sequence40. Myosuppressin of reduviid bugs38,41 contains two amino acid substitutions which are not intermediate between the consensus sequence and C. lectularius myosuppressin. The presence of myosuppressin receptors in the bed bug genome9 suggests that C. lectularius myosuppressin is biologically active despite its unusual sequence. Whether and how this unusual sequence affects receptor activation kinetics or peptide stability in the hemolymph has to be tested in future experiments.



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Figure 6. A) Alignment of the unusual myosuppressin sequence of C. lectularius with other known myosuppressin sequences of various insect taxa and selected crustacean species. Amino acids are coloured 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 prominent b- and y-type fragments are labelled. Fragments confirm the predicted unusal sequence from genome data of this species.

Natalisin: The natalisin precursor of the bed bug contains one paracopy for each of the natalisin consensus sequences FXPXRa and FWAHRa, as well as a putative amidated peptide with a moderately similar Cterminus (DVLDQPLWVRa). Orthologs of these peptides were also predicted from the natalisin precursor of R. prolixus62. In the bed bug, we identified the three peptides, which represent the first biochemically characterised natalisins from hemimetabolic insects. Neuropeptide F: We identified peptides corresponding to parts of the predicted full-length NPF1-42 (see Table 1). This suggests that full-length NPF1-42 is processed as predicted, yet further endogenously cleaved by unknown enzymes. It is unclear whether the C-terminal fragment YAVAGRPRFa represents the major bioactive form of NPF or whether it is a degradation product. We note, however, that the identical short peptide but not the full length NPF was also found in T. infestans38 and a similar Cterminal NPF sequence has been reported from R. prolixus41,55 and showed bioactivity in this species63. Neuropeptide-like precursor 1 (NPLP1): 18 NPLP1 peptides could be identified from the NPLP1 propeptide; four of them carrying a C-terminal amidation. These peptides encompass nearly the complete set of predictable precursor products and were detected in preparations of the CNS (e.g. in the antennal lobe, Figure 5B) and retrocerebral complex (Figure 3A). A comparable diversity of thirteen NPLP1 peptides was also found in R. prolixus41,43 where the nplp1 gene seems to be expressed also in ovaries, testes and salivary glands. Neither an NPLP1 receptor nor a consensus sequence of NPLP1 peptides is


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known so far. NVP-like peptides: We found four peptides from the NVP precursor of C. lectularius, including two longer peptides originating from processing of the first and last dibasic processing sites. An orthocopy of one of these peptides (AQEFIMFGNQQNRAINNFGSTKNE) was also observed by mass spectrometry in R. prolixus37. In the latter species, a single NVP-like peptide (EHVLNPFEEFLAL) was found to be significantly reduced 4 and 24h after a blood meal43. A similar sequence is, however, not contained in the bed bug NVP-like precursor. Orcokinin: In many insects, the orcokinin gene is alternatively spliced37. For the bed bug, we identified only one orcokinin A-type transcript9. A single orcokinin (NMDEIDRAGFDTFV) is contained within the orcokinin A precursor and the cleavage of this peptide could be mass-spectrometrically confirmed. Orcokinin A precursors of R. prolixus and T. infestans both contain three orcokinin paracopies. In addition to orcokinin, an orcomyotropin-like peptide64 is encoded in the orcokinin A transcript which was also identified by mass spectrometry, as were four unrelated precursor peptides (Table 1). Pigment-dispersing factor (PDF): Although PDF-encoding genes were earlier identified in the true bugs Riptortus pedestris65 and R. prolixus37, the biochemically identified C. lectularius PDF is the first confirmed mature PDF in Heteroptera. Immunostainings in R. prolixus located PDF-expression to clockgene expressing lateral neurons66,67 and the removal of homologous PDF neurons in R. pedestris impaired the photoperiodic regulation of diapause65. This suggest that PDF serves well-conserved circadian functions68,69 in heteropteran insects, amd may also play a role in regulating circadian and 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 various reduviid bugs38,70. Accordingly, proctolin could be biochemically identified by LC-MALDI MS/MS in CNS extracts of R. prolixus70. By MALDI-MS, we found ion signals matching proctolin in the CNS of C. lectularius, yet we were unable to obtain a tandem mass-spectrometric fragmentation. RYamide: Different from R. prolixus37, the bed bug RYamide precursor contains only one canonical RYamide C-terminus. This peptide could be mass-spectrometrically sequenced following SPITCderivatization (Figure 7) and represents the first biochemically identified RYamide in a hemimetabolous insect. Preparations of anterior aorta tissue yielded a particularly high relative signal intensity of RYamide (Figure 3B)



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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 labelled.

SIFamide: The bed bug SIFamide is a 12-amino acid peptide with the C-terminal canonical PFNGSIFa sequence71. It differs from R. prolixus SIFamide only in position 1 (Ser instead of Thr). In R. prolixus, SIFamide-immunoreactive cells in the CNS are restricted to two pairs of neurons in the pars intercerebralis of the protocerebrum with projections along the entire CNS55. In contrast to peptidomic studies in the kissing bug, we were unable to find N-terminally truncated SIFamide degradation products. Short neuropeptide F (sNPF): Typically, in hemimetabolic insects72 the sNPF gene codes for only one canonical sNPF peptide. Similar to the situation in various insect taxa72, we identified sNPF in a more abundant long (sNPF1-11: DNRTPQLRLRFa) and a short form (sNPF4-11) originating from internal processing at the monobasic Arg3 of the long form. A similar Arg3 in R. prolixus sNPF1-11 suggests that also reduviid bugs produce a short and a long form of sNPF, though only the long form has so far been biochemically identified in the kissing bug41. Besides the canonical sNPF, various precursor peptides originating from propeptide processing at mono- or dibasic cleavage sites could be detected in samples from the CNS and retrocerebral complex (Figure 3A). Sulfakinins (SKs): Using a combination of positive and negative ionisation mode in MALDI-TOF MS73 as well as Q Exactive mass spectra, both predicted sulfakinins were biochemically confirmed in CNS samples, including posttranslational pyroglutamate formation (SK-1) and Tyr-sulfation. SK-2 possesses two Tyr residues as potential targets for sulfation. MALDI-TOF mass spectra in negative mode yielded weak ion signals supporting sulfation of both Tyr but so far Q Exactive MS/MS spectra confirmed sulfation of only the first Tyr. Interestingly, the C-terminal sequence of SK-2 (GYLRFa) deviates even more strongly from the insect consensus sequence GHMRFa than the already unusual R. prolixus SK-2 (GYMRFa). It is thus conceivable that the two bed bug SKs each have a dedicated function and receptor,


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which is supported by the presence of two SK receptor genes in the bed bug genome9. Tachykinin-related peptides (TKRP): The Tkrp gene encodes eight amidated peptides, all of which were sequence-confirmed by mass spectrometric fragmentation in C. lectularius. In R. prolixus, where TKRPlike immunoreactivity is widely distributed throughout the CNS74, the thirteen predicted TKRPs of this species could be mass-spectrometrically confirmed as well43,55. These data suggest a high abundance of TKRPs in the CNS of these species which is corroborated by high signal intensity of TKRP signals in mass spectra from brain tissue such as the antennal lobe (Figure 5B). Ion signals of TKRPs were not detected in preparations of the retrocerebral complex in C. lectularius (Figure 3A).

Peptides not detected though respective genes identified Several of the neuropeptide precursor genes identified in the bed bug genome9 code for larger protein-like hormones, most of them forming dimers via cysteine bridges. These include bursicon, GPA2, GPB5, insulin-like peptides 1-2, ion transport peptide (ITP), neuroparsins 1-4 and prothoracicotropic hormone (PTTH). These hormones are too large to be detected in the peptidomic approaches employed here, and the lack of corresponding mass signals thus does not allow any conclusion regarding their presence or absence. In contrast, a few smaller predicted peptides that were not detected should, in principle, be well detectable by our peptidomic approach and are discussed below. Allatostatin CC (AstCC): An ion signal matching the theoretical mass ([M+H]+) of an N-terminally truncated AstCC (CYFNAVSCF) was observed in direct brain profiling, but could not be confirmed by MS/MS. This truncated form suggests unpredicted prohormone convertase processing at a single Arg preceding the N-terminal C. AstCC was also predicted from the R. prolixus genome75, but no mature products have been biochemically identified. CCHa-2: In the reduviids R. prolixus5554535453 and T. infestans, CCHa-2 is the only predicted CCHa37,38. The peptide could be mass-spectrometrically identified in the CNS of both species38,55. Since CCHamide2 is encoded in the C. lectularius genome9, the absence of CHHa-2 signals in our investigation hints towards an expression of CCHa-2 outside the CNS, possibly in the midgut and fat body76–78. Ecdysis-triggering hormone (ETH): Throughout the insects, ETH peptides are expressed outside the nervous system in specialised gland cells79. As we only extracted nervous 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 annelid species80–82. Elevenin was also predicted in C. lectularius9 and R. prolixus18. Curiously, to the best of our knowledge, a bioactive form of elevenin has never been identified in any insect. We obtained a single

Orbitrap MS/MS spectrum that revealed an unpredicted amidated peptide sequence

pQTEYVTGRGa, a sequence just C-terminal of the elevenin signal peptide that is C-terminally flanked



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by a putative monobasic prohormone convertase cleavage site. A similar sequence is however not contained in the elevenin precursors from various molluscs and the nematod Caenorhabditis elegans80, as well as R. prolixus37, and thus is unlikely to represent a bioactive peptide. SEFLamide: SEFLamides were only recently identified in arthropods83, and –to the best of our knowledge- bioactive SEFLamide peptides have not been biochemically characterised in any arthropod so far. Thus it remained unclear if this peptide is expressed in the CNS.

Peptidergic signalling pathways in the bed bug as possible targets for chemical control With the pertinent problem of acquired resistances to organic or genetically expressed insecticides, peptides and their receptors are often suggested as alternative target structures for pest control14,84,85. The resistance problem is especially virulent for bed bugs6, and thus peptide signalling pathways involved in feeding, diuresis, host location or ecdysis are potentially well suited targets for chemical control. Peptides can be modified to increase their stability, oral availability or cuticle permeability. For example, a biostable kinin analog was developed that could be fed and lead to reduced blood meals and disrupted ecdysis in R. prolixus86. Similar strategies could also be developed for the bed bug. Based on their effects in other insect species, we suggest the following peptidergic signalling 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.

Acknowledgment Funding was provided by Deutsche Forschungsgemeinschaft (PR766/11-1 to RP) and intramural

funds of the University of Würzburg (to CW). KR was supported by the Zukunftskonzept of the Technische Universität Dresden awarded by the Deutsche Forschungsgemeinschaft through the Excellence Initiative. The authors would like to thank Christian Frese, Corinna Klein, Astrid Wilbrand-Hennes, Ursula Cullman (CECAD Cologne Proteomics Facility) and Lapo Ragionieri (Institute for Zoology, Cologne) for the valuable help in optimizing Orbitrap analyses, and Susanne Klühspies (Würzburg) for excellent support with immunostainings. References

(1) (2) (3)

Usinger, R. L. Monograph of Cimicidae (Hemiptera, Heteroptera); Entomological Society of America, 1966. Reinhardt, K.; Siva-Jothy, M. T. Biology of the Bed Bugs (Cimicidae). Annu. Rev. Entomol. 2007, 52 (1), 351–374. Reinhardt, K.; Naylor, R. A.; Siva-Jothy, M. T. Situation exploitation: higher male mating 24 ACS Paragon Plus Environment

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reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector, Rhodnius prolixus. Peptides 2016, 80, 108–113.


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Tables Table 1: Summary of neuropeptide precursor products of C. lectularius which were identified by mass spectrometry analyses. (+), mass match only; +*, partial sequence confirmed by MS/MS; underlined, half of disulfide bridge. peptide name

calculated [M+H]+

peptide sequence

MALDITOF MS

Q Exactive

ACP

pQVTFSRDWSA-NH2

1178.58

+

+

AKH

pQITFSTGW-NH2

921.47

+

+

LPVYNFGL-NH2 AAGEKTYSFGL-NH2 GRQYAFGL-NH2 LPKQYSFGLGKRPEGKMYSFGL-NH2 LPKQYSFGL-NH2 PEGKMYSFGL-NH2 TKSGKDLRYMFGI-NH2 DVDEDERARQERSMHYNFGL-NH2 DYDMDEEYDDEMRDDFN-OH SQMAESDDYDSIENGRGMEE-OH IANMQEKDTTQSALVN-OH

921.52 1142.58 910.49 2502.33 1051.59 1127.55 1514.81 2466.12 2216.75 2262.88 1762.86

(+) (+) + + + (+) + + (+) (+) (+)

+ + + + + + +* +

SYWKQCAFNAVSCF-NH2 pQPVDKEKLLNELELVDDDGSIETAVMNYLFAKQIV NRLRSQGDISDLQ-OH

1650.73

(+)

+

5442.80

(+)

-

GFKNGPLNTARGF-NH2 GRSTRGFKNGPLNTARGF-NH2

1377.74 1935.04

+ -

+ +

GLDLGLSRGFSGSQAAKHLMGLAAANFAGGP-NH2

2970.54

+

+*

EVRPFEFPRV-NH2 pQVRPFEFPRV-NH2 RPFEFPRV-NH2 EGGLIPFPRI-NH2 pQGGLIPFPRI-NH2 NGGNGNGGLWFGPRL-NH2

1274.70 1256.72 1046.59 1097.65 1080.65 1514.76

+ + + + (+) +

+ + + + + +

SGPKRNGGNGNGGLWFGPRL-NH2

2040.06

(+)

+

SGPKRNGGNGNGGLWFGPRLGKIQ-OH

2467.31

+

-

NGGNGNGGLWFGPRLGKIQ-OH EDIPSHAPKL-OH DIPSHAPKL-OH SKNFPVTWGLQEDKY-OH

1942.00 1106.58 977.54 1811.89

+ + + +

+ + + +

SKNFPVTWGLQEDKYKREGGLIPFPRI-NH2

3174.72

+

-

PFCNAFTGC-NH2

956.39

(+)

-

AstA AstA-2 AstA-3 AstA-4 AstA-5+6 AstA-5 AstA-6 AstA-7 AstA-8 AstA-PP3 AstA-PP4 AstA-PP5 AstC AstC-PP1

Allatotropin AT extended AT CT-DH

CAPA PVK-1 PVK-1 (pQ) PVK-13-10 PVK-2 PVK-2 (pQ) tryptoPK N-term. ext.tryptoPK N- + C-term. ext.tryptoPK C-term.ext.tryptoPK CAPA-PP1 CAPA-PP12-10 CAPA-PP2 CAPAPP2+PVK-2 CCAP



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CCAP-PP1

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FYFPDEVSEPIDPKI-OH

1795.88

(+)

-

SGSCLSYGHSCWGAH-NH2

1548.63

(+)

+

AGYMALCHFKICNM-NH2

1598.72

-

+

pQMFQYSRGWRN-NH2

1454.70

+

+

SSPSLSVANPIDVLRSRLMLEINRRWMNKVDEGQVQ ANRQFLQTI-NH2 TLQEVPRDPETTWPL-OH

5208.76

(+)

+

1781.91

(+)

+

TLQEVPRDPETTWPLRKL-OH

2179.19

(+)

+

YVDVQPRMTAELQDLLEDLEKV-OH YIMPQRALYQDKSDSPTDYNNSGDLNWDSS-OH

2604.32 3480.53

(+) (+)

+* +*

SALEKNFMRF-NH2 AKGNFIRF-NH2 NADNFMRF-NH2 ARNNFIRL-NH2 NTENFIRF-NH2 NDNFMRF-NH2 GRGFVGFARQKEMDKGGGFIRF-NH2 NRDVVLSPWT-NH2 STNENILRKN-OH

1241.65 951.55 1013.46 1002.59 1039.53 942.43 2459.29 1185.63 1188.63

(+) (+) -

+ + + + + +* + +

HuginPyrokinin PK-1 PK-2 ext. PK-2 PK-3

NTVNFRPRL-NH2 SPPFAPRL-NH2 EEDIIFTETSRSPPFAPRL-NH2 MVPFSPRF-NH2

1115.64 883.51 2204.13 979.52

+ + + +

+ + + +

Kinin K-1 K-2 K-3 ext. K-3 K-4 K-6 K-7 K-9 K-PP21-11 K-PP6 K-PP7

DSPGPKMRFSSWG-NH2 SKFSSWG-NH2 KFSSWG-NH2 KFSSWGGKRLPADYEE-OH AKFNSWG-NH2 STFNSWG-NH2 TKFNSWG-NH2 RGPDFYAWG-NH2 GASGLDEILEE-OH EDETWRDMI-OH AINSTADMLIF-OH

1450.69 797.39 710.36 1869.91 808.41 797.36 838.42 1067.51 1132.54 1194.51 1195.60

+ (+) + -

+ + (+) + + + + + + + +

AWKDLTTAW-NH2 GWKDLPATGW-NH2 GWQDLQSAW-NH2 GWTDLNSGGW-NH2 GWQDLNSGGW-NH2 GWQDLQAPAW-NH2 SWVSLHSGW-NH2 AADWGSFRGSW-NH2

1090.57 1129.58 1089.51 1091.49 1118.50 1170.57 1057.52 1238.57

(+) +

+ + + + + + + +

CCHamide-1 CNMamide Corazonin CRF-DH CRF-DH-PP1 ext. CRF-DHPP1 CRF-DH-PP2 CRF-DH-PP3

ext. FMRFamides FMRFa-1 FMRFa-3 FMRFa-4 FMRFa-5 FMRFa-6 FMRFa-7 FMRFa-8 FMRFa-PP3 FMRFa-PP6

MIP/AST-B MIP-1 MIP-2 MIP-3 MIP-4 MIP-5,6,7 MIP-8 MIP-9 MIP-10


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MIP-11

DPAWQNLKGLW-NH2

1326.69

-

+

pQDQMTDHIFLRF-NH2 QDQMTDHIFLRF-NH2

1532.76 1549.76

+ +

-

pQPMPADAMARPA-OH PKTFDSPDDLRTYLNQLGQY-OH YAVAGRPRF-NH2 TGFSPRLHLALDGLSSYRYPLSDPSELYELLFPQAD -OH

1238.59 2371.16 1035.58

+ (+) +

+ +

4068.04

(+)

+*

SDVRAVLGAETEPGFWPTR-NH2 GGSSEEQPPPFWAHR-NH2 DVLDQPLWVR-NH2 YAQEPKYLLIQ-OH

2087.07 1680.79 1239.68 1365.74

+ + (+) -

+ + + +

2660.37 2239.02 1290.62 1757.79 1230.68 1328.66 1943.82 1358.75 771.48 1085.39 1708.93 1707.86 1967.09 1692.91 833.35 1008.59

(+) + + + + + + + + + + (+)

+ + + +* + + +* + +* +* + + + + +

4403.01

+

+*

NPLP1-20 NPLP1-21

pQANIIARMAHLQSYPRYYSNIAP-OH DEKSSLDELAERLEEEEFA-OH SGDIRVTGDLEE-OH DELEGLVHDLASAEEM-OH SFAALARVDALP-OH GISSIARNGYYQ-OH MDDDLEQLMSEVYGIGE-OH NVASLARGFSLPQ-NH2 NIASIVR-NH2 DSFEETNED-OH NLPSIIRDRTEPLGE-NH2 HIGAFVANHGIPFVND-OH NVGVLAKNRDYPYALKF-NH2 NVGVLAKNRDYPYAL-OH DVDEEIN-OH HVATLLRQA-OH LSDHPTETDDISLKETAQDDLKEEMSQITKDDMEHT RT-OH MAREELE-OH GNRNENS-OH

877.41

(+)

+ -

NVP-like NVP-1 NVP-2 NVP-570-77 NVP-7

LPTSLLEDMKDAQFHTPVRTNKV-OH AQEFIMFGNQQNRAINNFGSTKNE-OH EVAWPRQH-OH FGRDSIPPVMPFQEEYADEIPL-OH

2640.38 2758.30 1022.52 2550.22

(+) + +

+ + + +*

1311.61 1629.72

+

+ +

5077.61

(+)

-

3090.66 2005.96 848.36 1300.73

(+) (+) +

+ + + -

Myosuppressin MS (pQ) MS NPF NPF1-12 NPF114-33 NPF34-42 NPF-PP2

Natalisin Nat-1 Nat -2 Nat -3 Nat-PP NPLP1 NPLP1-2 NPLP1-3 NPLP1-4 NPLP1-5 NPLP1-7 NPLP1-9 NPLP1-10 NPLP1-11 NPLP1-12 NPLP1-13 NPLP1-14 NPLP1-15 NPLP1-16 NPLP1-161-15 NPLP1-17 NPLP1-18 NPLP1-19

Orcokinin A NLDSLDGVTFGSS-OH Orcomyotropin-like NMDEIDRAGFDTFV-OH Orc-1 LPRPEPAENSLFKGERNLYGLGGGHLLRDLESALRE Orc-PP1 NPMVYERPS-OH LPRPEPAENSLFKGERNLYGLGGGHLLR-OH Orc-PP11-28 DLESALRENPMVYERPS-OH Orc-PP129-45 NSVDPSET-OH Orc-PP2 pQLYNVGLADRVA-NH2 Orc-PP3 (pQ)



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Orc-PP3 (Q) Orc-PP5

QLYNVGLADRVA-NH2 NFVPSHYEMYGNYNPVPFF-OH

1317.73 2322.03

+ (+)

+ +

PDF

NSEIINSLLGLPKVLNDA-NH2

1909.07

-

+

Proctolin

RYLPT-OH

649.37

(+)

-

RYamide

AESRFVIGSRY-NH2

1283.68

(+)

+

SYKKPPFNGSIF-NH2

1383.74

+

+

DNRTPQLRLRF-NH2 TPQLRLRF-NH2 AAYPERVYKMEE-OH SVPLFPLEE-OH LDQSRMDYFSN-OH SVPLFPLEERRLDQSRMDYFSN-OH

1414.80 1029.63 1485.70 1030.55 1375.59 2699.32

+ + + + +

+ +* + + +*

pQFNDYGHMRF-NH2 pQFNDY(SO3H)GHMRF-NH2 QFNDYGHMRF-NH2 QFNDY(SO3H)GHMRF-NH2 EGTDEKFEDYGYLRF-NH2 EGTDEKFEDY(SO3H)GYLRF-NH2

1296.58 1376.58 1313.58 1393.58 1867.85 1947.85

+ (+) (+) + -

+ + + + +* +*

pQERRQLGFLGVR-NH2 APTLGFQGLR-NH2 APPMGFQGVR-NH2 GPTMGFVGMR-NH2 APASGFFGMR-NH2 GPSNAGFFGMR-NH2 GPSGFLGLR-NH2 DGTDEEFKRAPTLGFQGLR-NH2

1440.84 1058.61 1058.56 1051.52 1039.51 1139.54 902.52 2136.08

+ + + + + + + -

+ +* + + + + + +

SIFamide sNPF sNPF sNPF4-11 sNPF-PP2 sNPF-PP3 sNPF-PP4 sNPF-PP3+4 Sulfakinin SK-1 (non-sulf.) SK-1 (sulf.) SK-1 (Q, non-sulf.) SK-1 (Q, sulf.) SK-2 (non-sulf.) SK-2 (sulf.) Tachykinin-RP TKRP-1 TKRP-2 TKRP-3 TKRP-4 TKRP-5,6 TKRP-7 TKRP-8 TKRP-PP1 + TKRP-2


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1

Supporting Information S1-S2 available: This material is available free of charge via the Internet at

2

http://pubs.acs.org

3 4

S1: Prepropeptide sequences identified from the bed bug genome9. Predicted neuropeptides incl. cleavage

5

and amidation sites are indicated.

6 7

S2: Q Exactive orbitrap MS/MS spectra of C. lectularius peptides listed in Table 1.

8 9 10

S3: List of the peptides with Q Exactive Orbitrap MSMS confirmation shown in Supporting Information 2; with page designations, precursor masses, charge states, and mass errors.

11 12 13 14



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15

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Abstract graphic – for TOC only

16

17


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Figure 1. Gross morphology of the bed bug CNS and immunostaining of selected neuropeptides in neurohemal organs. A) Dorsal and B) lateral view of the CNS, showing a high degree of ganglion fusion typical of Heteroptera. The brain is fused with the subesophageal ganglion (SEG) and prothoracic ganglion (PRO), the remaining thoracic and all abdominal ganglions are fused to the mesothoracic ganglionic mass (MTGM). In B), part of the esophagus (eso) is visible that projects through a foramen in the midline approximately at the brain-SEG border. C) AstA immunoreactivity in the brain of an adult bed bug. In each brain hemisphere, primary neurites of two large secretory neurons in the pars lateralis of the protocerebrum (arrows) project towards the midline, then bifurcate (asterisk). One small branch of each neurite projects to the contralateral side, while the other projects through the nervus corporis cardiaci II (NCC II, arrowheads) to the neurohemal retrocerebral-aorta complex (arrowhead) where it forms a dense net of terminal varicosities. D) Close-up of AstA-immunoreactive varicosities in the retrocerebral complex comprising the corpora cardiaca (CC) in a different preparation. E) Close-up of corazonin-immunoreactive varicosities in the retrocerebral complex comprising the CC and corpora allata (CA). Arrowheads mark the entry of neurites

ACS Paragon Plus Environment


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from the protocerebrum via NCC II. 153x284mm (300 x 300 DPI)


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Figure 2. In situ immunostaining against PRXamide which labels CAPA peptides. In the subesophageal ganglion which is fused to the prothoracic ganglion (SEG+Th1), a pair of strongly stained neurons is visible. Further immunoreactive neurons in the protocerebral part of the brain (BR) provide arborisations mostly in the superior brain. Two descending varicose axons (#) of unknown origin innervate the mesothoracic ganglionic mass (MTGM). Three secretory neurons (Va) in the abdominal portion of the CG project via the abdominal segmental nerves 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 the Va neurons and segmental branchings in more detail. Numbers mark the segmental nerves 2-4. 177x105mm (300 x 300 DPI)



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Figure 3. Direct MALDI-TOF MS tissue profiling of tissue samples from C. lectularius; only more prominent ion signals are labelled. The spectra verify the tissue-specific presence of products of several neuropeptide precursors. Ion signals which could not be assigned to products of known neuropeptide precursors are marked with question marks. A) Retrocerebral complex (RC) containing the corpora cardiaca, corpora allata and a small piece of anterior aorta. B) Piece of aorta adjacent to the RC that stores peptide hormones. Low ion signal intensities suggest a local release of the peptides rather than a release as classical hormones. C) A single abdominal segmental nerve 2 shows prominent ion signals for products of the CAPA precursor. Note the presence of C-terminally and C- and N-terminally extended sequences of CAPA-tryptoPK which likely are not bioactive. Only traces of the canonical trypto-PK (see arrow) are detectable in preparations of abdominal segmental nerves. AKH, adipokinetic hormone; AstA, allatostatin A; AstC, allatostatin C; cor, corazonin; CRF-DH, corticotropin releasing factor-like diuretic hormone; MIP, myoinhibitory peptide; MS, myosuppressin; NPLP1, neuropeptide-like precursor 1; PK, pyrokinin; PP, precursor peptide; PVK, CAPAperiviscerokinin; RYa, RYamide; sNPF, short neuropeptide F; tPK, CAPA-tryptoPK.


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82x147mm (300 x 300 DPI)



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Figure 4. A) Alignment of the unusual corazonin sequence of C. lectularius with other known insect corazonin sequences. Amino acids are coloured according to the Taylor scheme. B) Orbitrap TOF/TOF fragment spectrum of corazonin from C. lectularius. Ion signals with prominent b- and y-type fragments are labelled. Fragments confirm the predicted unusual sequence from genome data of this species 172x61mm (300 x 300 DPI)


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Figure 5. Direct MALDI-TOF MS profiling of tissue samples from the central nervous system of C. lectularius; only more prominent ion signals are labelled. Ion signals which could not be assigned to products of known neuropeptide precursors are marked with question marks. A) Ventral portion of the subesophageal ganglion (SEG), containing secretory neurons that synthesise the hugin-encoded pyrokinins (PK) and release their products via the retrocerebral-aorta complex (see Figures 2, 3A). Ion signals of three predicted PKs are marked. B) Portion of the brain containing antennal lobe (AL) tissue. Ion signals of products from 10 neuropeptide precursors are labelled; the asterisk marks sNPF. AstA; allatostatin A; CT-DH, calcitonin-like diuretic hormone; MIP, myoinhibitory peptide; MS, myosuppressin; NPLP1, neuropeptide-like precursor 1; NVP, NVP-containing precursor peptide; Orc, Orcokinin; PK, pyrokinin; PP, precursor peptide; sNPF, short neuropeptide F; tPK, tryptoPK; TKRP, tachykinin-related peptide.



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82x108mm (300 x 300 DPI)


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Figure 6. A) Alignment of the unusual myosuppressin sequence of C. lectularius with other known myosuppressin sequences of various insect taxa and selected crustacean species. Amino acids are coloured 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 prominent b- and y-type fragments are labelled. Fragments confirm the predicted unusal sequence from genome data of this species. 172x85mm (300 x 300 DPI)



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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 labelled. 82x65mm (300 x 300 DPI)


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TOC 77x63mm (300 x 300 DPI)


Neuropeptidomics of the Bed Bug Cimex lectularius - Journal of

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