Neuropeptidomics of the Carpenter Ant Camponotus floridanus

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Neuropeptidomics of the carpenter ant Camponotus floridanus Franziska Schmitt, Jens T. Vanselow, Andreas Schlosser, Jörg Kahnt, Wolfgang Rössler, and Christian Wegener J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5011636 • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 10, 2015

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Journal of Proteome Research

Neuropeptidomics of the carpenter ant Camponotus floridanus Franziska Schmitta, Jens T. Vanselowb, Andreas Schlosserb, Jörg Kahntc, Wolfgang Rösslera, Christian Wegenerd*

a

Behavioral Biology and Sociobiology, Theodor-Boveri-Institute, Biocenter, University of Würzburg,

Am Hubland, D-97074 Würzburg, Germany b

Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-

Strasse 2, D-97080 Würzburg, Germany c

Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg,

Germany d

Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, University of Würzburg, Am

Hubland, D-97074 Würzburg, Germany

* 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: +49-931-31-84452

ORCIDs:

CW: orcid.org/0000-0003-4481-3567 email addresses for other authors: [email protected] [email protected] [email protected] [email protected] [email protected]

1 ACS Paragon Plus Environment


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Abstract Ants show a rich behavioral repertoire and a highly complex organization, which is attracting behavioral and sociobiological researchers since a long time. The neuronal underpinnings of ant behavior and social organisation is, however, much less understood. Neuropeptides are key signals that orchestrate animal behavior and physiology, and it is thus feasible to assume that they play an important role also for the social constitution of ants. Despite the availability of different ant genomes and in silico prediction of ant neuropeptides, a comprehensive biochemical survey of the neuropeptidergic communication possibilities of ants is missing.

We therefore combined different mass spectrometric methods to characterise the neuropeptidome of the adult carpenter ant Camponotus floridanus. We also characterised the local neuropeptide complement in different parts of the nervous and neuroendocrine system, including the antennal and optic lobes. Our analysis identifies 39 neuropeptides encoded by different prepropeptide genes, and in silico predicts new prepropeptide genes encoding CAPA peptides, CNMamide as well as homologs of the honey bee IDLSRFYGHFNT and ITGQGNRIF-containing peptides.

Our data provides basic information about the identity and localisation of neuropeptides that is required to anatomically and functionally address the role and significance of neuropeptides in ant behavior and physiology.

key words: neuropeptides, mass spectrometry, peptide prediction, ant genome, social hymenopterans, social behaviour, neurohormones, brain plasticity, central nervous system, neuromodulation


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Introduction Ants belong to the most successful eukaryotes in terms of number and biomass. Their rich behavioral repertoire and highly complex social organization allows them to colonize different habitats ranging from tundra to desert, and has attracted significant scientific interest. Not surprisingly, genomes have been sequenced for a variety of ant species from different families1,2, including the carpenter ant Camponotus floridanus3. A major interest in social insect research is in the regulation of behavioural plasticity. Individual members of ant colonies perform different tasks ranging from duties inside the nest like brood care, nest building activities to sometimes long-ranging outdoor foraging trips, either related to different morphs or associated with age4. This polyethism represents a fundamental prerequisite for task allocation and division of labour in ant colonies. In addition to age-related changes in behavioural tasks, social insect colonies are also able to respond in an adaptive manner to changing environmental and social conditions (e.g. Gronenberg et al.5 for C. floridanus). Interestingly, the behavioural transitions are associated with structural neuronal plasticity in the brain, in particular in the mushroom bodies, learning and memory centres in the insect brain5–9. The transition from nursing to foraging is associated with a rise in juvenile hormone (JH)10,11 and changes in biogenic amines12, but their causal contributions to behavioural and neuronal plasticity still remain unclear9. Neuropeptides are important regulators of social behavior in mammals13–15 and key signals that orchestrate various other behaviors as well as physiology and development16. Neuropeptides, therefore, represent important and unexplored further candidate signals involved in the regulation of behavioural plasticity that could promote the transitions between behavioural tasks and physiological states. Consequently, these intercellular messengers have been in focus during the annotation of the honey bee17,18 and ant genomes, and have been in silico predicted for several ant species including C. floridanus3,19. However, bioactive neuropeptides arise from larger precursor proteins (prepropeptides) from which they are sequentially cleaved out by a set of specific enzymes. Further, insect neuropeptides are typically C-terminally amidated or carry further modifications20. Due to these post-translational modifications, it is very difficult to precisely predict in silico which peptides are produced. Even if correctly predicted, it is not clear whether a given prepropeptide is expressed in a given insect stage and which bioactive peptides are made out of it. A required biochemical peptidome analysis is, however, missing for ants; only the products of one capa prepropeptide are mass spectrometrically characterised so far21.

We therefore combined different mass spectrometric methods to biochemically identify the neuropeptidome of adult carpenter ants Camponotus floridanus. We also characterised the local neuropeptide complement in different parts of the nervous and neuroendocrine system, providing first hints to which neuropeptide is used as a neuromodulator or neurohormone by the carpenter ant.



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Although ants are difficult to manipulate by genetic means, first successful approaches using gene knockdown by dsRNA in ants have been conducted, again including C. floridanus3,22 and neuropeptide genes23,24. Our identification of expressed neuropeptides together with the predicted prepropeptide sequences thus provides solid ground to interfere with neuropeptide signaling by RNAi, and to dissect the role of neuropeptides in regulating (social) behavior, physiology or development in C. floridanus.

Experimental section Genome searches To re-evaluate already predicted3,19 and to identify new prepropeptide genes in the C. floridanus genome, peptide sequences from the parasitic wasp Nasonia vitripennis25, the honey bee Apis mellifera17, and missing or recently discovered new insect peptide families were blasted against the C. floridanus

scaffold

assembly

version

3.326

in

the

Hymenopteran

Genome

database

(http://hymenopteragenome.org), using tblastn and the BLOSUM62 and PAM30 matrix. To predict peptide sequences, nucleotide sequences obtained from blast searches were translated using EMBOSS (http://www.ebi.ac.uk/Tools/st/emboss_transeq27). GENSCAN28

Prepropeptide

(http://genes.mit.edu/GENSCAN.html)

or

genes

were

Augustus29

predicted

using

(http://bioinf.uni-

greifswald.de/augustus/). Signal peptide sequences, cleavage sites, posttranslational modifications and monoisotopic masses were predicted by SignalP 4.030 (http://www.cbs.dtu.dk/services/SignalP/) and NeuroPred31 (http://neuroproteomics.scs.illinois.edu/cgi-bin/neuropred.py)

Animals C. floridanus were kept at a 12:12 hour light:dark cycle at 25 °C, and fed with dead cockroaches and honey water (1:2) with free access to water. All animals for the analysis were adult (female) workers of nonspecified age taken from queen-less colonies. Before dissecting neuronal tissues, the ants were anesthetized on ice.

Tissue preparation For mass spectrometric analyses, the C. floridanus nervous system was dissected in fresh, ice-cold

ant-saline solution (127 mM NaCl, 7 mM KCl, 1.5 mM CaCl2, 0.8 mM Na2HPO4, 0.4 mM KH2PO4, 4.8 mM TES, 3.2 mM trehalose, pH 7.0). The ants were fixed in a preparation dish with pins crossing over the neck. Ants were laying on the back while the cuticle plates were gently pulled apart at the intersegmental membrane to gain access to the abdominal ganglia. Abdominal ganglion 3 (A3), situated in the petiole, was carefully drawn out at the connectives to either abdominal or thoracic ganglia. The thoracic ganglia were dissected by cutting off the ant’s legs and subsequently opening the


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cuticle along the beginning of the legs. The brain and gnathal ganglia (GNG, following new nomenclature32) were dissected by cutting a square window into the bottom side of the head capsule, thereby ensuring that the (neuro)endocrine glands corpora cardiaca and corpora allata remained attached. Trachea, fat bodies and glands were gently removed and the GNG with corpora cardiaca, corpora allata and the brain were drawn out. Dissected tissues were immediately transferred to a low protein-binding Eppendorf tube and frozen at -20 °C until the peptides were extracted.

For direct MALDI-TOF MS profiling, ventral ganglia were separated at the connectives and singularly put on a stainless-steel MALDI target. GNG, corpora cardiaca and corpora allata were separated from the central brain as well as the antennal lobes (AL) and optic lobes (OL) and were also put singularly onto the MALDI plate. Additionally, the retrocerebral complex (corpora cardiaca and corpora allata) was separated from the GNG using a glass pipette connected to a silicone hose. The retrocerebral complex was first sucked into the sharp glass pipette and then gently detached from the GNG. Tissue was then placed on the MALDI plate.

For peptide extraction, the entire dissection took place on ice and 10 samples of each part of the nervous system (brain, corpora cardiaca/corpora allata, and ventral ganglia) were pooled and frozen at -20 °C in a low protein-binding Eppendorf tube. Peptides were either extracted in a MeOH-based solution or in H2O. For the MeOH-based extraction, the tissue samples were incubated on ice for 30 min and ultrasonicated (Labsonic L, Broch, Göttingen, Germany) for 10 sec in 50 µl extraction solution containing 90 % Methanol, 9 % H2O and 1 % formic acid (v/v/v). After centrifugation of the homogenisate at 15,000 x g (Hettich Mikro 200, Tuttlingen, Germany) for 15 min at room temperature (RT) the supernatant was transferred into a new tube. The extraction was repeated once to increase peptide yield. Subsequently the samples were vacuum-dried (UNIVAPO H100 Vacuum Concentrator) and either purified by C18 reversed phase solid phase extraction (stage tips, see below) or stored dry and dark until further processing. To extract the peptides in H2O, 99 µl MilliQ water were added to the tissue samples, followed by homogenization in an ice-cold ultrasonic bath. To denaturate proteins, the homogenized tissue was boiled for 10 min in cooking water. After cooling the samples for 20 min on ice, they were acidified by adding 1 µl 1mM HCl and centrifuged at 15,000 x g.

For stage-tip clean-up, C18 material (3M Empore, High performance extraction Disks, Chrom Tech Inc, Apple Valley, MN, USA) was punched out to fit into a pipette tip which was then put into the lit of an Eppendorf cup33. The C18 material was washed with 30 µl of 100 % ACN and equilibrated with 30 µl of 10 mM HCl in MilliQ water. The supernatant or dry extract dissolved in 30 µl of 10 mM HCl, respectively, were given onto the equilibrated stage-tips and centrifuged at 8,000 x g for 1 min. The



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centrifugation step was repeated until no liquid remained on the C18 filter. The samples were subsequently stored dry and dark until elution with 20 µl 40-60 % ACN.

Capillary reversed-phase high pressure liquid chromatography (capHPLC) for LC-MALDIMS/MS To separate and clean up peptides for MALDI-TOF MS, capHPLC was performed for extracts of brains, ventral ganglia and retrocerebral complex on a capillary UltiMate 3000 system (Dionex, Idstein, Germany) connected to a spotter (SunCollect MALDI spotter, SunChrom GmbH, Friedrichsdorf, Germany). Dried unpurified samples were dissolved in 50 µl Eluent A (98 % H2O, 2 % ACN, 0.05 % TFA) in an ultrasonic bath for 15 min and then centrifuged at 13,000 x g for 15 min. The supernatant was then manually injected and loaded onto a reversed phase C18 trap column (Acclaim PepMap100 C18, 5 µm particles, 100 Ǻ pore size) using eluent A at a flow rate of 20 µl/min. Then the trap column was switched on-line with the analytical reversed phase column (Acclaim PepMap100 C18, 3 µm particles, 100Ǻ pore size). Peptides were eluted by running a linear gradient from 0 % to 100 % eluent B (80 % acetonitrile / 0.04 % TFA in water, v/v) in 30 minutes at a flow rate of 2 µl/min. Spotting position on the MALDI target was changed every 30 seconds. 1 µl sample fraction and 1 µl of matrix solution (half-saturated α-cyano-4-hydroxycinnamic acid in 60 % acetonitrile / 40 % water / 0.05 % TFA, v/v/v) were mixed per spot and left to dry. HPLC gradient grade water and acetonitrile were used throughout.

MALDI-TOF mass spectrometry Nervous tissue samples for direct MALDI-TOF peptide profiling34 were put and dried on the MALDI target. Before matrix application, dried samples were washed with 4 µl pure ice-cold water immediately removed. Subsequently, a saturated solution of α-cyano-4-hydroxy cinnamic acid in 30 % MeOH, 30 % EtOH and 40 % H2O was added. For ventral ganglia, optic and antennal lobes 0.2 µl matrix solution was sufficient, while 0.4 µl were used for GNG and central brain.

MS analysis for both direct peptide profiling and capLC-MALDI-TOF MS/MS was accomplished using a 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems/MDS Sciex,Framingham, MA, USA) and 4800 Series Explorer software version 3.5.1. A Nd:YAG laser was used for peptide ionization. Mass spectra were acquired in positive ion reflector mode in a mass range of 800 - 4000 Da. Laser intensity and the number of laser shots per sample was adjusted to tissue and instrument conditions. Peptides were fragmented by PSD with 80 shots per subsectrum and 25 subspectra per sample. MALDI TOF-MS data were analyzed with Data Explorer 4.10 (Applied Biosystems/MDS Sciex, Framingham, MA, USA). For direct peptide profiling, all mass spectra were baseline corrected, a noise filter with the


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correlation factor 0.7 was applied, and a manual peptide sequence confirmation by MALDI TOF/TOF fragmentation considering b and y ions was performed. A peptide was considered identified if b/y fragment coverage was above 50 % and the most likely ion fragments (e.g. breakage at Pro residues) could be detected. A mass tolerance of +/-0.5 Da was accepted for peptide assignment when no MS/MS fragmentation was performed.

NanoLC-ESI-MS/MS NanoLC-ESI-MS/MS analyses were performed on an LTQ-Orbitrap Velos Pro (Thermo Scientific) equipped with an EASY-Spray Ion Source and coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded on a trapping column (2 cm x 75 µm ID. PepMap C18 3 µm particles, 100 Å pore size) and separated on an EASY-Spray column (25 cm x 75 µm ID, PepMap C18 2 µm particles, 100 Å pore size) with a 30 minute linear gradient from 3 % to 30 % acetonitrile and 0.1 % formic acid applying a flow rate of 200 nL/min. Alternatively, a monolithic column (EASY-Spray column, 25 cm x 200 µm ID, PepSwift™) without trapping column was used for peptide separation with a 45 minute linear gradient from 3 % to 30 % acetonitrile and 0.1 % formic acid with a flow rate of 1500 nL/min. MS scans were acquired in the Orbitrap analyzer with a resolution of 30,000 at m/z 400, MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 7,500 at m/z 400 using HCD fragmentation with 30 % normalized collision energy. A TOP5 data-dependent MS/MS method was used; dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 30 seconds; singly charged precursors were excluded from selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with AGC target a value of 1e6 for MS scans and 5e4 for MS/MS scans. Lock mass option was applied for internal calibration using background ions from protonated decamethylcyclopentasiloxane (m/z 371.10124).

Data analysis was performed with PEAKS 7.0. The data refinement step was performed with precursor mass correction enabled, de novo sequencing was performed with the following parameters: parent mass tolerance: 8 ppm, fragment mass tolerance: 0.02 Da, enzyme: none, variable modifications: oxidation (M), pyro-glutamate (N-term. Q), amidation (C-terminal), maximum number of PTMs per peptide: 6. Database searching with PEAKS was performed with the same settings using a custom Formicinae database containing all protein entries from UniProt with the taxonomy Formicinae (18219 protein sequences). New peptide sequences are being submitted to the Uniprot database (http://www.uniprot.org/)

Anatomy For anatomical analyses of the nervous system and retrocerebral complex of C. floridanus, tissues were



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dissected as described and pictures were taken with a stereo microscope (M165 FC) equipped with a DFC 450C CCD-camera (Leica, Wetzlar, Germany).

Results & Discussion In silico prediction of new neuropeptide genes The first genome-wide annotations of neuropeptide genes in the C. floridanus genome3 predicted 16 prepropeptide genes. Shortly after, Nygaard et al.19 added further 15 predicted prepropeptide genes, and suggested the absence of several peptides that were found in other insect species. A gene for one of these missing peptides, allatotropin with a deviating C-terminal sequence, could later on be identified35. By homology search against the current assembly, we now identified the missing gene capa that encodes two periviscerokinins (PVK) with the uncommon C-terminus PRIamide, plus one pyrokinin (PK, consensus sequence FXPRLa) for which the typical phenylalanine residue at position -5 from the Cterminus36 is substituted by tyrosine, another aromatic amino acid (Fig. 1). This capa gene and peptide products are highly similar to the recently reported capa gene and encoded peptides in the fire ant21. We also found a homolog for the recently identified CNMamide37 (Fig. 1), and could predict the so far unidentified signal peptide and missing N-terminal cleavage sequence for CCHa 2 (Fig. 1). Two further new neuropeptide genes encode IDLSRFYGHINT (Fig. 1) and an ITG-like peptide (Fig. 1), neuropeptides with high similarity to the enigmatic IDLSRFYGHFNT and ITGQGNRIF-containing peptides from the honeybee17. This brings the total number of predicted C. floridanus prepropeptide genes up to 37.

Identification of neuropeptides in the central nervous system (CNS) By a combination of LC-ESI-MS/MS, LC-MALDI-MS/MS and direct MALDI peptide profiling, we were able to identify by MS/MS fragmentation a total of 39 neuropeptides encoded by 18 different prepropeptide genes within the CNS. The results are summarized in Table 1; respective fragment ion spectra are shown in Supporting information S1. The predicted preprohormone sequences for the detected neuropeptides are given in Supporting information S2. We additionally identified by MS/MS fragmentation (putatively non-bioactive) processing products of two further predicted prepropeptides of which the predicted bioactive peptides (Allatostatin C (AST C), CCHamide 2) were not detected (see below); and found a mass peak matching the predicted sulfakinin which could not be fragmented. While this additional data indicates the expression of the respective prepropeptides and most likely also the predicted peptide, it does not confirm the predicted sequence of the bioactive peptides. In our analysis, water extraction combined with LC-ESI-MS/MS yielded the highest number of identified peptides (Table 1). CAPA PVK1 was, however, only detected and fragmented by direct MALDI peptide profiling.


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To assign the identified peptides to specific parts of the CNS, we performed direct peptide profiling of single tissues, or performed LC-ESI-MS/MS on extracts from separated ganglia or brain parts. The results are summarized in Table 2 and 3, respectively. Micrographs of the brain and retrocerebral complex are shown in Supporting information S3. Typical direct mass profiles from the central brain containing proto- and deutocerebrum, retrocerebral complex containing corpora cardiaca and corpora allata, optic lobes, antennal lobes or gnathal, thoracic or abdominal ganglia are given in Fig. 2.

The information gained on the different neuropeptides is summarised below. Note that in this summary and Tab. 1 all peptides or precursor processing products as suggested by mass match are listed, while Tab. 2-3 list only peptides unambigously identified from peptide fragment ion spectra.

Allatotropin: We could identify allatotropin in the brain and VG by LC-ESI-MS/MS with preceding water extraction. Though an allatotropin gene is contained in the genome of all sequenced Hymenoptera, the processed bioactive peptide had so far been elusive in peptidomic studies35. Our data now shows that at least in C. floridanus, allatotropin is produced as a Hymenopteran neuropeptide. Allatostatin A (AST A): All five predicted AST A peptides could be identified by either PSD in LCMALDI-MS/MS or LC-ESI-MS/MS. Direct profiling and LC-MS show a distribution of the peptides throughout the CNS of C. floridanus (see Table 2). AST C: The predicted peptide for AST C (SYWKQCAFNAVSCFa) could not be detected in any part of the CNS. Yet a fragment with the mass 1256.67 Da could be assigned to the sequence LRSQLDIGDLQ via fragmentation in the LC-ESI-MS/MS . This fragment corresponds to a part of the AST C precursor framed by N- and C-terminal cleavage sites (Supporting information S2). CAPA: The predicted C. floridanus CAPA prepropeptide contains two periviscerokins (PVK) and one pyrokinin (PK, Supporting information S2). Both CAPA PVKs could be detected by direct profiling, and CAPA PVK 2 was additionally identified by LC-ESI-MS/MS (see Table 1). With exception of the optic lobes and the retrocerebral complex, both CAPA-PVK peptides were detected throughout the CNS. We nevertheless failed to detect CAPA PK in any tissue which -in other insects- is very well detectable in the retrocerebral complex and abdominal neurohemal organs36. Remarkably, a similar situation is found in the fire ant S. invicta21. Like in C. floridanus, the fire ant CAPA PK has a Cterminal YXPRLa motif, and is produced in very low amounts compared to the CAPA-PVKs though an equimolar amount is expectable from the prepropeptide structure. Our results suggest that not just the fire ant but ants in general have uniquely lost the CAPA PKs of the WFGPRLa type (for an in-depth discussions see Choi et al.21). CCHamide 2: We identified the complete genomic prepropeptide sequence, including the signal peptide



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(Fig. 1). Although we were unable to detect the bioactive CCHamide 2 sequence by MS, the identification

of

prepropeptide

processing

FDSTIRHNTLQESISQKFQESEIPSPNDELF

products –

(spacer 3635.73

peptides: Da;

FDSTIRHNTLQESISQKFQESEIPSPNDEL – 3488.66 Da; YVFPNDEFRERSDNSLPFIRT – 2601.26 Da) suggests the expression of CCHamide 2 in the C. floridanus CNS. Corazonin: Both nanoLC-ESI-MS and MALDI MS revealed corazonin. Corazonin was found in the central brain, antennal lobes as well as in the neurohemal retrocerebral complex. Calcitonin-like (DH31-like) diuretic hormone: calcitonin-like DH was detected and sequence-confirmed by ion trap MS, but could not be detected by MALDI-TOF MS. It was found in the central brain, antennal lobes and thoracic and abdominal ganglia (Tab. 2), and was also detected in the corpora cardiaca/corpora allata (Tab. 3). Corticotropin releasing factor-like (CRF-like (DH44-like)) diuretic hormone: Although we were unable to detect the predicted full-length peptide, we identified a N-terminally shortened form pQIEENRRFLENIa by LC-ESI-MS/MS throughout the nervous system (Table 3). Due to the pyroglutamination and cleavage consensus sequences framing QIENNRRFLENIG in the prepropeptide, it does not appear that this shorter form is a degradation product. Rather, it may represent the major active form of DH44 originating from proper propeptide processing. FMRFamide-like peptides: All three predicted FMRFamide-like peptides could be identified by fragmentation (Table 1), and were detected in the brain and ventral ganglia by LC-ESI-MS/MS (Tables 2, 3). By direct profiling, only FMRFa 2 was found throughout the CNS; FMRFa 1 and 3 were exclusively found in ventral ganglia (see Table 2). As the peptides are likely to be produced in equimolar amounts (Supporting information S2), this may indicate that the MALDI properties of FMRFa 2 are more favorable than that of FMRFa 1 and 3. IDLSRF-like peptide: This peptide could be detected with and without C-terminal T in all tissues by LC-ESI-MS/MS and was also confirmed by MALDI MS/MS (Table 1). It is present in all parts of the nervous system (Tables 2, 3). ITG-like peptide: The ITG-like peptide could be confirmed by both MS/MS methods (Tab. 1). The peptide was found in all parts of the CNS (Tables 2, 3). In many direct peptide profiling spectra, ITGlike peptide gave the most dominant mass peak (Fig. 2). Myosuppressin: Myosuppressin was identified by both MS/MS methods (Table 1), and could be found throughout the CNS (see Tab. 2-3). Neuropeptide-like precursor 1(NPLP1): All seven predicted NPLP1 peptides could be identified. They are distributed throughout the CNS. Direct profiling only detected NPLP1 5 in the retrocerebral complex, while all NPLP1 peptides are absent from this part of the CNS in the LC-MS studies. Orcokinin: Orcokinin 3 was found throughout the CNS and also in the corpora cardiaca/corpora allata


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(Tables 2, 3) and could be identified by direct peptide profiling and LC-ESI-MS/MS (Table 1). In contrast, the predicted orcokinin 2 was not detectable, and orcokinin 1 and 4 were only found in whole CNS homogenates (Table 1). In the brain, a partial sequence of orcokinin 1 (predicted full sequence: NFDEIDRSVFDRFP) with the sequence EIDRSVFDRFPK was found, possibly representing a degraded form of orcokinin 1. In addition, also orcokinin 1 sequences with extended N- or C-terminus have

been

identified:

DEIDRSVFDRFP,

FDEIDRSVFDRFPK,

GNFDEIDRSVFDRFPK,

IRPGNFDEIDRSVFDRFP, IRPGNFDEIDRSVFDRFPK. From genome data, we and Nygaard et al.19 were unable to predict the N-terminus including the signal peptide sequence of the prepropeptide. Thus, one of the N-terminally extended orcokinin-1 sequences may represent a properly processed form. In addition, LNNYLADRQ (Supporting information S2) from the orcokinin prepropeptide was detected by LC-ESI-MS/MS. It is unclear whether this peptide has a biological function or simply is a nonbioactive spacer. HUGIN/PBAN pyrokinins: Pyrokinins are chemically classified by the C-terminal sequence FXPRLamide. Insect genomes typically contain at least two genes encoding pyrokinins38. One of the genes is capa (see above). The other gene is named either hugin referring to Drosophila39, or pheromone biosynthesis-activating neuropeptide (pban) gene referring to moths38, though there is good evidence that hugin and pban are actually orthologs40. The HUGIN/PBAN prepropeptide of C. floridanus contains four different predicted pyrokinins. HUGIN/PBAN-PK 1-3 possess the typical FXPRLa motif at their C-terminus, while HUGIN/PBAN-PK 4 has an unusal proline residue instead of phenylalanine. All 4 HUGIN/PBAN-PKs were identified by LC-ESI-MS/MS. The masses of HUGIN/PBAN-PK 1-3 could also be detected by direct profiling in parts of the nervous system (Table 2). An N-terminally shorter form of HUGIN/PBAN-PK 3 (RNEIDEDDPLFTPRLa) was detected in the brain by LC-ESI-

MS/MS. HUGIN/PBAN-PK 1 and 2 have a tryptophane-containing WFXPRLa C-terminus which is unusual for HUGIN/PBAN-PKs but typical for CAPA-PKs with an obvious exception of ants incl. C. floridanus (see above). In most if not all insects, both HUGIN/PBAN-PKs as well as CAPA-PKs are stored and released from the neurohemal corpora cardiaca, though they are expressed by different secretory neurons projecting to the corpora cardiaca36,41. We found HUGIN/PBAN-PK 1- 4 in the retrocerebral complex of C. floridanus (Tables 2, 3), but were unable to detect CAPA-PK. The presence of the WFGPRLa-type motifs on the C. floridanus HUGIN/PBAN prepropeptide may thus compensate for the biochemical loss of a CAPA-PK WFGPRLa. Pigment dispersing factor (PDF): PDF was only identifiable in the brain by LC-ESI-MS/MS. Prothoracicotropic hormone (PTTH): We could identify a fragment of the predicted full-length PTTH by LC-ESI-MS/MS in the corpora cardiaca/ corpora allata extracts (Table 1). Interestingly, this fragment is flanked by processing sites in the prepropeptide (see Supporting information S2), opening the possibility



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that this small fragment may be bioactive by itself. SIFamide: Direct profiling detected SIFamide in the brain, the TG and AG (Table 2). The sequence could be confirmed by LC-ESI-MS/MS (Table 1) which also revealed the presence of SIFa in the brain, VG and corpora cardiaca/corpora allata (Table 3). short NPF (sNPF): The short NPF was confirmed by LC-ESI-MS/MS (see Table 1) and could be found in all parts of the nervous system including the neurohemal retrocerebral complex by both MS methods (Table 1-2). Sulfakinin: A mass peak indicative of sulfakinin (1471.57 Da, pQQLDDYGHMRFa) was found in direct peptide profilings of the central brain, GNG and VG, but was too small for fragmentation. Tachykinin-related peptides (TK): All four predicted TKs could be identified by LC-ESI-MS/MS (Table 1). TK 1 was a highly dominant mass peak in direct peptide profiles (Fig. 2), which corresponds to the presence of multiple copies of the TK 1 sequence in the TK prepropeptide. TKs are present throughout the nervous system (Tables 2, 3), but only TK1 was detectable in the corpora cardiaca/corpora allata by direct peptide profiling. A further TK-related mass could be assigned to the sequence AAMGFQDMRGNKNLIPTSLEHNKLS which, in the prepropeptide sequence, is flanked by cleavage sites (Supporting information S2). Due to its position in the prepropeptide and the N-terminal resemblance to TK1 (APMGFQGMRa), it is possible that this peptide represents a TK paracopy lost by mutation of the C-terminal cleavage site.

Absence of adipokinetic hormone (AKH) and kinin signals While the absence of peptide signals in peptidomic studies does not prove the non-expression of these peptides, it is nevertheless interesting to note that we did not find mass signals indicative of kinins or AKH. This supports previous studies that were unable to find a gene for kinin (leucokinin) or kinin receptor in ant genomes19,35. In contrast, an AKH gene is present in the C. floridanus and other ant and hymenopteran genomes17,19,25. AKH is typically produced in large amounts by intrinsic endocrine cells in the glandular part of the corpora cardiaca and is thus usually easy to characterise by MS. Intriguingly, an AKH peptide has nevertheless not been biochemically detected in the honey bee17 and also not here in C. floridanus. For the honey bee, the presence of a second TATA box in the promotor region of the prepropeptide gene has been suggested to account for a low expression and hence the non-detectability of bioactive AKH35. This second TATA box leads to the production of an alternative mRNA encoding a prepropeptide from which no AKH can be processed. Future transcriptomic studies will show whether the same applies for C. floridanus; the functional consequences remain enigmatic.

Prediction of peptide function based on tissue distribution In contrast to the central brain and gnathal/thoracic/abdominal ganglia containing different neuropils


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subserving a variety of functions, the optic (OL) and antennal (AL) lobes are specialized structures dedicated to the processing of visual and olfactory information. It can be assumed that those neuropeptides detected by direct profiling in the OL and AL (Table 2) subserve a modulatory function in the processing of visual and olfactory input. Our results show that the majority of the peptides found in the central brain occur in these two highly specialized sensory lobes. This is especially pronounced for the AL which lack only corazonin and SIFa compared to the central brain. While a dedicated analysis of OL peptides in social insects is missing, there are several detailed direct peptide profiling studies on the AL peptidome in other insect species (fly Drosophila melanogaster42, cockroach Periplaneta americana43, beetle Tribolium castaneum44, mosquito Aedes aegypti45). In all these species, a comparable high diversity of AL peptides have been found (Supporting information S4). C. floridanus ALs share a “core set” of tachykinin-like peptides, AST A, sNPF and myosuppressin with the cockroach, fly and mosquito. Of this “core set” of AL neuropeptides, Tribolium only has tachykinin-like peptides. On the other hand, SIFamide occurs in the AL of all species but C. floridanus. Interestingly, we found the masses of orcokinins, HUGIN/PBAN-PK and IDLSRF-like peptide. These peptides were not found in the AL of the other investigated insects.

The corpora cardiaca are the major cephalic neuroendocrine organ. Together with the non-peptide producing endocrine corpora allata, they form the so-called retrocerebral complex. In ants, the small retrocerebral complex is situated at the transition from GNG to the central brain and laterally of the esophageal foramen46–48. Direct profiling of the retrocerebral complex identified corazonin, myosuppressin, NPLP 1-5, HUGIN/PBAN-PKs and sNPF as bona fide neurohormones in C. floridanus. Myosuppressin, the HUGIN/PBAN-PKs and sNPF were also found in the retrocerebral complex by LCESI-MS/MS, along with larger peptide hormones that are very difficult to detect by direct MALDI-TOF profiling: the CRF and calcitonin-like DHs and PTTH. The presence of HUGIN/PBAN-PKs in the retrocerebral complex and gnathal ganglia is cross-confirmed by earlier immunostainings using an antiserum against a moth PBAN48. Somewhat surprisingly, TK-1 was only found by direct peptide profiling of the retrocerebral complex, while AST A, FMRFa-like peptide 2, IDLSRF-like peptide, ITG-like peptide, orcokinin 3 and SIFamide were found in pooled corpora cardiaca/corpora allata extracts by LC-ESI-MS/MS but could not be detected by direct peptide profiling. In contrast to the larger peptide hormones, this result is unlikely to be due to a general MALDI-TOF detection problem due to a low MALDI efficiency as these smaller peptides were readily detected with the same solvent-matrix combination during direct profiling of the CNS. Given the higher sensitivity of the orbitrap LC-ESI-MS/MS, these peptides appear to occur in low amounts in the retrocerebral complex and are thus unlikely to be stored and secreted as neurohormones. Instead, these peptides may rather modulate neurohormone release as shown for TK, CCAP, proctolin



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and myosuppressin in locusts16. We can also not fully exclude small brain tissue contaminations in the pooled corpora cardiaca/corpora allata extracts, as these organs are very closely situated to the central brain (Supporting information S4). Future immunostainings will have to confirm whether these peptides are indeed contained within the retrocerebral complex.

Conclusions In total, we identified 39 neuropeptides from 18 different prepropeptides in the CNS of the carpenter ant C. floridanus. This number is somewhat higher than that reported in previous peptidomic studies of the CNS of other Hymenoptera like Nasonia vitripennis25 and Apis mellifera17 using MALDI-TOF and ESIQ-TOF MS; peptidomic data from other ant species are missing as to date. The higher number of peptides is likely to be attributed to an improved detection rate by our LC-ESI-MS/MS instrumentation, as this method resulted in a larger number of sequenced peptides than LC-MALDI-MS/MS or direct MALDI-TOF peptide profiling in this study. Based on the knowledge from the peptidomically wellstudied fruit fly, locust and cockroach20,49,50, and the identification of so far 37 prepropeptide genes encoding putative ( neuro)peptides in the C. floridanus genome, we assume that the 39 detected neuropeptides represent the better part but clearly not the full C. floridanus neuropeptidome. Some of the undetected peptides such as crustacean cardioactive peptide (CCAP), insulin-like peptides or very large peptides such as full-length PTTH, neuropeptide F or ion-transporting peptides (ITP) are notoriously difficult to detect by peptidomics due to their chemical characteristics (here: disulfide bridges, larger size). For example, the exact sequence of the bioactive Drosophila insulin-like peptides is still unclear despite a vast literature on the function and expression of this peptide family (see Pauls et al.20). Other peptides may occur in low amounts in the CNS and may become identifiable in other tissues such as the midgut, as exemplified by CCHamides and myoinhibiting peptides in the fruit fly51. A few of the predicted yet undetected peptides such as the ecdysis-triggering hormones are by homology to the situation in other insects52 inferred to be exclusively expressed outside the CNS. In the future, further neuropeptides are thus likely to be biochemically characterised with advancing LC-MS technology, or by looking into non-neural tissue, different developmental stages and casts. Nevertheless, our data on the biochemical identity and distribution of neuropeptides provides an ample basis for studies interfering with neuropeptide signaling and using MALDI imaging53 to study development and plasticity of peptidergic systems in the ant brain.

Acknowledgment Funding

was

provided

by

the

German

Research

Foundation

(DFG),


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collaborative research center SFB 1047 "Insect timing", collaboration between project B2 (to CW) and B6 (to WR).

References

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Tables Tab. 1: Complete list of neuropeptides and non-peptide processing products detected and identified from peptide fragment ion spectra in the CNS of C. floridanus. Non-peptide processing products are marked by “pp”.

Peptide

Mass (M+H)+ [Da]

Sequence

Sequence confirmed by direct MALDI TOF profiling

Allotropin Allatostatin A AST A1 AST A2 AST A3 AST A4 AST A5 Allatostatin C AST C pp CAPA Capa PVK1 Capa PVK2 CCHamide 2 CCHamide 2 pp1 CCHamide 2 pp2 CCHamide 2 pp3 Corazonin DH31/calcitonin-like DH44/CRF-like FMRFamide-like peptides FMRFa 1

GFKPEYISTAIGFa

1427.74

LPLYNFGIa TRPFSFGIa LRDYRFGIa GGQPFSFGIa GWKLPMGEMAVSa

935.54 923.51 1038.58 908.46 1306.65

LRSQLDIGDLQ

1257.68

SAGLVPYPRIa ALGLIHQPRIa

1071.63 1116.70

FDSTIRHNTLQESISQKFQESEIPSPNDELF FDSTIRHNTLQESISQKFQESEIPSPNDEL YVFPNDEFRERSDNSLPFIRT pQTFQYSRGWTNa GLDLGLSRGFSGSQAAKHLMGLAAANYAGGPa pQIEENRRFLENIa

3635.73 3488.66 2601.26 1369.62 2986.53 1542.80

STMGSSFIRFa

1131.56

√

Orbi trap (Stage tip MeOH)

Orbi trap (Stage tip H2O) √

√ √

√ √ √

√ √ √ √ √

√

√

√ √

√ √

√ √ √ √ √ √

√

√

√ √

18

ACS Paragon Plus Environment

Orbi trap (MeOH extraction)

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FMRFa 2 FMRFa 3 HUGIN/PBAN pyrokinins HUGIN/PBAN PK 1 HUGIN/PBAN PK 2 HUGIN/PBAN PK 3 HUGIN/PBAN PK 4 IDLSRF-like ITG-like Myosuppressin Neuropeptide-like precursor 1 NPLP 1-1 NPLP 1-2 NPLP 1-3 NPLP 1-4 NPLP 1-5 NPLP 1-6 NPLP 1-7 Orcokinin Orcokinin 1 Orcokinin 1 pp1 Orcokinin 1 pp2 Orcokinin 1 pp3 Orcokinin 1 pp4 Orcokinin 1 pp5 Orcokinin 3 Orcokinin 4 Pigment-dispersing-factor (PDF) PTTH (fragment) SIFamide

WKSPDIVIRFa GKNDLNFIRFa

1259.73 1222.59

TTTTQESGISSGMWFGPRLa pQPTWFTPRLa GSEEKFIYSDATDRNEIDEDDPLFTPRLa VPWIPSPRLa IDLSRFYGHINT ITGQGNRLF pQDVDHVFLRFa

2069.98 1069.57 3275.55 1065.64 1435.73 1005.55 1257.63

NVGSLARDFALPTa HIASVARDHGLPSa NVGSLARQSMLPLSa NVASLARYYMLPQSa NVAALARDSSLPYa YLGSLARSGSYPTRDYDEa GRMTSGRIMARVLNRRYa

1359.73 1358.72 1471.80 1611.83 1375.73 2048.96 2036.12

EIDRSVFDRFPK DEIDRSVFDRFP FDEIDRSVFDRFPK GNFDEIDRSVFDRFPK IRPGNFDEIDRSVFDRFP IRPGNFDEIDRSVFDRFPK NIDEIDRVGWNGFV LNNYLADRQ NSELINSLLGLPKNMHNAa

1508.78 1496.62 1771.97 1943.12 2181.41 2309.59 1633.80 1104.56 1964.04 1538.85 1367.75

IAGAEDVGLQPRLVT AYKKPPFNGSIFa

√ √

√ √

√ √ √ √ √ √

√ √ √

√ √ √

√

√ √

√

√

√ √

√ √ √ √ √ √ √

√ √ √ √ √ √ √ √ √ √ √ √ √ √

√ √

√

19


√ √

√

√ √ √


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

sNPF Tachykinin-related peptides Tachykinin 1 Tachykinin 2 Tachykinin 3 Tachykinin 4 Tachykinin pp

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SPSLRLRFa

974.58

√

√

√

APMGFQGMRa TIMGFQGMRa SPFRYFEMRa NPRWELRGMFVGVRa AAMGFQDMRGNKNLIPTSLEHNKLS

993.48 1039.52 1231.60 1715.93 2774.19

√

√ √ √ √

√ √ √ √

20


√ √ √ √

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Tab. 2: Distribution of neuropeptides in different parts of the CNS of C. floridanus based on direct MALDI-TOF peptide profiling of individual tissues (central brain (n= 18), antennal lobes (n= 8), optic lobes (n= 6), gnathal ganglia (n= 10), retrocerebral complex (RC, consisting of corpora cardiaca/corpora allata, n= 2), thoracic ganglia (n= 25), abdominal ganglia (n= 23). n.d. = not detected.

Central brain

Antennal lobes

Optic lobes

Gnathal ganglia

RC

Thoracic ganglia Abdominal ganglia

AST A1-3

AST A1-3

AST A 1-3

AST A1-3

n.d.

AST A1-3

AST A1-3

Capa PVK 1, 2

Capa PVK 1, 2

n.d.

Capa PVK 1, 2

Capa PVK 2

Capa PVK 1, 2

Capa PVK 1, 2

Corazonin

n.d.

n.d.

n.d.

Corazonin

n.d.

n.d.

DH31-like

DH31-like

n.d.

DH31-like

DH31-like

DH31-like

DH31-like

FMRFa-like 2

FMRFa-like 2

FMRFa-like 2

FMRFa-like 1-3

n.d.

FMRFa-like 1-3

FMRFa-like 1, 2

HUGIN/PBAN PK 1

HUGIN/PBAN PK 1

HUGIN/PBAN PK 2, 3

HUGIN/PBAN PK 2

HUGIN/PBA N PK 1, 2

HUGIN/PBAN PK 2, 3

HUGIN/PBAN PK 1-3

IDLSRF-like

IDLSRF-like

IDLSRF-like

IDLSRF-like

n.d.

IDLSRF-like

IDLSRF-like

ITG-like

ITG-like

ITG-like

ITG-like

n.d.

ITG-like

ITG-like

Myosuppressin

Myosuppressin

Myosuppressin

Myosuppressin

Myosuppressin

Myosuppressin

Myosuppressin

NPLP1 1-7

NPLP1 1-7

NPLP1 1-7

NPLP1 1-7

NPLP1 5

NPLP1 1-7

NPLP1 1-7

Orcokinin 3

Orcokinin 3

Orcokinin 3

Orcokinin 3

n.d.

Orcokinin 3

Orcokinin 3

SIFamide

n.d.

n.d.

n.d.

n.d.

SIFamide

SIFamide

sNPF

sNPF

sNPF

sNPF

sNPF

sNPF

sNPF

TK 1-3

TK 1, 2

TK 1-3

TK 1-3

TK 1

TK 1-3

TK 1, 2

21



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Tab. 3: Distribution of neuropeptides in different parts of the CNS of C. floridanus based on LCESI-MS/MS measurements of an extract of pooled tissues from 10 animals. n.d. = not detected. Brain

Ventral ganglia

RC

Allatotropin

Allatotropin

n.d.

AST A 1 - 5

AST A 1- 3

AST A 2, 3

Capa PVK2

n.d.

n.d.

Corazonin

n.d.

n.d.

DH31

DH31

DH31

DH44

DH44

DH44

FMRF1-3

FMRF1-3

FMRF 2

n.d.

n.d.

HUGIN/PBAN-PK 1-4

IDLSRF-like

IDLSRF-like

IDLSRF-like

n.d.

ITG-like

ITG-like

Myosuppressin

n.d.

Myosuppressin

NPLP1 1-7

NPLP1 1-7

n.d.

Orcokinin 3, 4

Orcokinin 3

Orcokinin 3

PDF

n.d.

n.d.

n.d.

n.d.

PTTH

SIFamide

SIFamide

SIFamide

sNPF

sNPF

sNPF

TK 1-4

TK 1-4

n.d.


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Figure legends Fig. 1: Newly predicted genomic prepropeptide sequences. Signal peptide is boxed, bioactive peptides are underlaid in grey, amidation signal is underlaid in turquoise, basic processing sites are underlined.

Fig. 2: Typical direct MALDI-TOF MS single tissue profiles for the different tissues shown in Tab. 2.



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Supporting Information S1-S4 available: This material is available free of charge via the Internet at http://pubs.acs.org

S1: LC-ESI-MS/MS or direct MALDI-TOF MS/MS profiling fragment spectra for all identified peptides. The left fragment spectra represent LC-ESI MS/MS, fragment spectra on the right show MALDI TOF MS/MS data. Red = y-fragments, blue: b- or a-fragments. Pre = precursor = unfragmented mother ion.

S2: Genomic prepropeptide sequences for the 20 prepropeptides with detected peptides. Signal peptide is boxed, the bioactive peptide is underlaid in grey, amidation signal is underlaid in turquoise, basic processing sites are underlined.

S3: Morphology and anatomical position of the retrocerebral complex in adult C. floridanus. A) Posterior view of the brain, with mushroom bodies (MB) and optic lobes (OL) visible. The retrocerebral complex comprising the neurohemal corpora cardiaca and the endocrine corpora allata (encirclement) is situated behind the brain, dorsal to the gnathal ganglion. B) shows a dorsal view onto the retrocerebral complex. Anterior is at the top. The bubble-like structures represent the juvenile hormone-producing corpora allata (CA). The corpora cardiaca (CC) are situated ventrally and are connected to the CA and the brain. Scale bars: A =100 µm, B = 50 µm.

S4: Comparison of peptides detected by direct MALDI-TOF peptide profiling in the antennal lobes of insects. Green: peptide was detected, Red: peptide was not detected. S5: Excel list of the ion mass peaks for the fragmentation spectra in S1. The ion masses represent all peaks above an adjusted threshold of 2-10% intensity based on the spectrum’s signal-to-noise ratio.


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Figure 1


CAPA (Cflo_023009, gene model is incomplete) MQDNRFFILMILLVFSTSLNQGQKLKANDRRSAGLVPYPRIGRNSEISSFSRSERALGLIH QPRIGRSDVSSFDNLNRFHDLSSDADIEFYAMDMDPLLSPDYEDYASKPIALKYADKLQK DNSWLMPDHIHGYKDFHFAQKINDPRLYYSILRDSRNTQGQGGYTPRLGRENERDTANF L CNMamide (no gene predicted in the genome v3.3, scaffold2593:72738..74693 (+ strand)) MTERASRRGAARTSFCLLVAFVLSCVISGSEKVLAFPQPEPLPPGFYGDNLAYDAQSSNL LNRLKQLAEHKHRVDEQIEREIADEREITDEQIEIQAMLEANARERAAGRSQPSDYSEEPE PEALPIPAAMVHHEQHPGKRHGMNGVSYHMSLCHFKICNMGRKRQSK CCHamide 2 (Cflo_13756) MRTKLPATSGPIFSLYLSIVVLLVLISVSDNVHAKPREERSTNIRRNTNYVRKGGCASFGH SCFGGHGKRFDSTIRHNTLQESISQKFQESEIPSPNDELFYVFPNDEFRERSDNSLPFIRTRK DTQQFDPYPLSSLVDQWIASHRRQHRTDIDVTNK IDLSRFYGHINT (Cflo_18422) MVRRFCNGAVALGIALTACAAFPRAIMAIDLSRFYGHINTKRSDACHPYEPFKCPGDGL CISIQYLCDGAPDCQDGYDEDSRLCTAAKRPPVEETATFLQSLLASHGPNYLEKLFGNKA RDTLKPLGGVEKVAIALSESQTIEDFGAALHLMRSDLEHLRSVFMAVENGDLGMLKSIGI KDSELGDVKFFLEKLVKTGFLD ITG-like (Cflo_14492, gene model is incomplete) MRVYAAITLVLVANTAYIGVEAWGGLFNRFSPEMLSNLGYGGHGSYMNRPGLLQEGY DGIYGEGAEPTEEPCYERKCMYNDHCCPGSICMNFNGDEECAMSSECDISRGLCCQLQR RHRQAPRKVCSYFKDPLVCIGPVATDQIKSVIQYTSGEKRITGQGNRLFKRMPFA


Figure 2


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 ACS Paragon Plus Environment

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Figure 2 continued 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 ACS Paragon Plus Environment


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

Abstract graphic 90x44mm (300 x 300 DPI)


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Neuropeptidomics of the Carpenter Ant Camponotus floridanus

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