Mating-induced differential peptidomics of neuropeptides and protein

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Mating-induced differential peptidomics of neuropeptides and protein hormones in Agrotis ipsilon moths Max Diesner, Aurore Gallot, Hellena Binz, Cyril Gaertner, Simon Vitecek, Jörg Kahnt, Joachim Schachtner, Emmanuelle Jacquin-Joly, and Christophe Gadenne J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00779 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Journal of Proteome Research

Mating-induced differential peptidomics of neuropeptides and protein hormones in Agrotis ipsilon moths

a

,c

,d

Max Diesner†, , Aurore Gallot‡,b, Hellena Binz¶, Cyril Gaertner‡ , Simon Vitecek‡ , Jörg ,*

Kahnt°, Joachim Schachtner†, Emmanuelle Jacquin-Joly‡ and Christophe Gadenne§

†

Department of Biology – Animal Physiology, Philipps University Marburg, D-35032

Marburg, Germany ‡

INRA, Institut d’Ecologie et des Sciences de l’Environnement de Paris (UMR iEES-Paris),

Route de Saint-Cyr, 78026 Versailles Cedex, France ¶

Institute of Zoology, University of Mainz, Johann-Joachim-Becher-Weg 6, 55128 Mainz,

Germany °Max-Planck-Institute für terrestrische Mikrobiologie, Marburg, Germany §

INRA, Institut de Génétique, Environnement et Protection des Plantes (UMR IGEPP),

Agrocampus Ouest, rue Le Nôtre, 49054 Angers cedex 01, France

KEYWORDS : neuropeptides, mass spectrometry, peptide prediction, transcriptome, moth, plasticity, mating, sex pheromone, olfaction, Agrotis ipsilon

ABSTRACT

ACS Paragon Plus Environment

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In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used nextgeneration RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed post-mating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

INTRODUCTION Insect neuropeptides are short signaling peptides produced by endocrine cells or neurons that play key roles in many physiological processes including development and growth, metabolism, reproduction and behavior

1, 2

. They are typically encoded as prepropeptides

that contain single or multiple copies of active peptides and a signal peptide. Maturation via post-translational processing, such as proteolytic cleavages including e.g., amidation

5

3, 4

and other modifications

or pyroglutamate formation 6, leads to biologically active

molecules. Secreted neuropeptides can have autocrine, paracrine and hormonal effects and


2

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commonly are activated by G protein-coupled receptors (GPCRs) 7. Recently, genomic, transcriptomic and peptidomic data acquisition and analyzes have led to the discovery of complete or partial repertoires of neuropeptides and their associated GPCRs in many insect species, including holometabolous insects such as the fruit fly

8-9

, the honey bee 10, the red

flour beetle 11-13, a parasitic wasp 14, the yellow fever mosquito 15,16, the carpenter ant 17, the tse-tse fly 18, and several hemimetabolous insects such as the pea aphid 19, the body louse 20, locusts 21,22, the brown planthopper 23 and the kissing bug 24. In Lepidoptera, such neuropeptide repertoires are limited to species with a sequenced genome or brain transcriptome such as the silkworm moth Bombyx mori butterfly Danaus plexippus

26

, the sphinx moth Manduca sexta

27

25

, the Monarch

and the rice stem borer

Chilo suppressalis 28. Lepidoptera, however, comprise a large and diverse group of species, many of which are important crop pests as voracious herbivorous caterpillars. A better knowledge of Lepidoptera neuropeptides and their receptors could help in the establishment of new pest control strategies based on peptidomimetics or pseudopeptides, aiming at disrupting important physiological or behavioral processes. For instance, many studies focused on the moth-specific pheromone biosynthesis activating neuropeptide (PBAN), a neurohormone triggering sex pheromone production in females, as a promising target for sex pheromone biosynthesis inhibition and in consequence limiting reproduction

29, 30

.

Successful mating also relies on simultaneous sex pheromone recognition by conspecific males. This recognition involves peripheral detection of the sex pheromone by receptor neurons located on the antennae, central processing of the information in the primary olfactory center, the antennal lobes (ALs), and transfer of the integrated information to higher processing centers in the protocerebrum

31-34

, leading in fine to the attraction

behavior. It is now clearly established that this olfactory response is plastic: behavioral


3


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response to the sex pheromone can be switched on or off depending on the age, the physiological state or even a pre-exposure to the pheromone or a predator noise 35-37. In the noctuid moth Agrotis ipsilon, the modulation of the pheromone response occurs through central nervous system plasticity

38

. First, there is an age-dependent olfactory

plasticity: newly emerged males are sexually immature and do not respond behaviorally to the female-produced sex pheromone. Three to five days after emergence, males become sexually mature and are highly attracted to the sex pheromone

39

. This increase in

pheromone response with age is paralleled by an increase in the sensitivity of central neurons in the ALs

40

. Second, there is also a mating-dependent olfactory plasticity in this

species: newly-mated A. ipsilon males are no longer attracted to the sex pheromone, and the response to the pheromone is restored during the next night

41

. This plasticity occurs not

only at the behavioral level, it is also accompanied by a decrease in the sensitivity of pheromone-specific neurons within the ALs: most neurons have a much higher pheromone response threshold after mating

41, 42

. Thus, these two forms of olfactory plasticity (a slow

switch-on, and a fast switch-off) are in large parts driven by the central nervous system, and the involvement of neuroregulatory peptides, biogenic amines or hormones, through interaction with their receptors, is probable. For instance, we showed that steroid and juvenile hormones, and possibly biogenic amines, regulate the age-dependent olfactory maturation plasticity

43-46

, and that ecdysteroids may be involved in the mating-dependent olfactory

47

. However, most neuropeptides are still undescribed in A. ipsilon and the

literature only reports the molecular cloning of a PBAN cDNA precursor in this species 48. In this study, we used both transcriptomic and peptidomic approaches to identify a large array of candidate neuropeptides and neurohormones in the brain of A. ipsilon. In particular, we speculated that some neuropeptides could be involved in the rapid post-mating olfactory switch in males. Peptidomics has been recently proven to be efficient in establishing


4

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comprehensive neuropeptidomic analyzes in insect disease vectors including the yellow fever mosquito Aedes aegypti

15

and the Chagas disease vector Rhodnius prolixus

24

.

Expressed sequence tag (EST) strategies were also efficient in identifying candidate neuropeptide encoding genes, for example in the migratory locust 22 and the desert locust 21. Recently, next-generation sequencing technologies greatly improved the efficiency of new gene discovery: 47 genes encoding candidate neuropeptides or peptide hormones and 57 putative neuropeptide GPCR genes were found via RNAseq analyzes in Nilaparvata lugens 23

. Here, we combined different next-generation sequencing technologies to construct a de

novo brain transcriptome in A. ipsilon and comparison with peptide sequences identified by MALDI-TOF mass spectrometry (MS) allowed us to identify 54 putative bioactive neuropeptides (from a total of 70 identified sequences) from 23 neuropeptide precursors in different areas of the nervous system including the ALs, gnathal ganglion (GNG, following the novel nomenclature 49) and the corpora cardiaca-corpora allata (CC-CA). We moreover compared virgin and mated female and male samples, revealing differences in peptide occurrence according to sex and physiological state.

EXPERIMENTAL SECTION Insect rearing and tissue preparation Adult males and females of A. ipsilon Hufnagel (Lepidoptera: Noctuidae) originated from a laboratory colony. Wild insects were introduced into the colony each spring. The animals were reared on a semi-artificial diet in individual cups until pupation 50. Pupae were sexed and males and females were kept separately in an inversed light/dark cycle (16 h light: 8 h dark photoperiod) at 22°C, 50% relative humidity. Newly emerged adults were collected every day and were given access to a 20% sucrose solution ad libitum. The day of emergence was considered as day-0. Sexually mature virgin males (VM, 5-day-old) and


5


females (VF, 3-day-old) were paired in plastic containers, allowing them to copulate

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41

.

Every 30 min, couples were checked to record the start and the end of copulation, which lasts for more than 30 min in this species. Mated males (MM) were quickly removed from the pairing box and their brain tissues were collected not later than one hour after the end of copulation. All mated females (MF) were checked for the presence of the spermatophore, in order to confirm that mating was successful. For transcriptomic analyzes, whole brains (brain + GNG + CC-CA) were dissected at mid-scotophase from a total of fifty 5-day-old sexually mature virgin males (when male response to the sex pheromone reaches its maximum 42) and fifty 5-day-old mated males (1 h after the end of mating). Tissues from both virgin and mated males were blended to enrich the transcriptome in potential peptides involved in olfactory plasticity. After dissection, brains were immediately flash frozen in liquid nitrogen and then stored at -80°C until RNA extraction. For MS direct tissue profiling analyzes, tissues were dissected as described earlier

51

.

ALs, GNGs and CC-CAs were individually collected with a pair of fine scissors in ice cold 0.01M NH4CL-phosphate-buffer (Na2HPO4 7.89 nM, NaH2PO4 1.9 nM, NH4Cl 164.52 nM), washed in a drop of ice cold aqua bidest and transferred with the help of a glass capillary on a stainless steel sample plate and left to dry at room temperature. Subsequently, tissues were covered with 0.1 µl saturated α-cyano-4-hydroxycinnamic acid solution (Sigma-Aldrich GmbH, Munich, Germany), dissolved in ethanol/methanol/aqua bidest/trifluoracetic acid (30/30/39/1) and left to dry at room temperature and stored in darkness until further usage. For peptide extracts, tissues (AL, GNG, CC-CA; 20 of each tissue) of virgin and mated males and females were collected in a 0.5 ml Protein-LowBind tube (Eppendorf, Hamburg, Germany), filled with 100 µl extraction buffer (50 methanol/1 formic acid/ 49 aqua bidest) and stored on ice until collection completion. Extracts were sonicated for 1 min in a


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sonication bath filled with ice and centrifuged for 5 min at 4°C and 13,400 rpm. The procedure was repeated three times and resulting extracts were concentrated in a vacuum centrifuge and stored at -20°C until usage.

Transcriptome sequencing, assembly and annotation Total RNAs from A. ipsilon brains were extracted using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. RNA quantity was determined on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and RNA quality was checked on a Bioanalyzer (Agilent) before sequencing. Two different sequencing technologies were used: 454 and Illumina. A pool of RNAs from both virgin and mated male brains (20 µg each) was used as a template to construct a normalized cDNA library for 454 sequencing (454 Roche GS FLX Titanium, 1 Pico Titer Plate, GATC Biotech SARL, Mulhouse, France). In parallel, RNAs from virgin and mated male brains (10 µg each) were used as templates for Illumina sequencing (one channel, single read 51 base pair length, HiSeq2000; GATC Biotech). The generated data have been deposited in the NCBI SRA database (Bioproject ID PRJNA310836). After sequencing, 454 and Illumina sequences were submitted to preprocessing steps as follows: First,

data

were

analyzed

with

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)

FastQC that

provided

v.0.10.0 general

information on sequence quality and identified over-represented sequences. Then, adapters and other over-represented sequences were removed with Cutadapt 52. Lastly, all sequences were trimmed from the 3' extremity with PRINSEQ v. 0.17.3 53. Briefly, quality scores were estimated on the mean of 10 nucleotide quality scores on a two-nucleotides-sliding window. Sequences were trimmed for mean score inferior to 20. Finally, sequences shorter than 20


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nucleotides were removed. After these preprocessing steps, 719,042 sequences from 454 and 79,956,990 sequences from HiSeq were kept for further analyzes (Table 1).

Table 1. Data summary Sequencing technology

454 sequencing

Tissues Number of raw reads Number of processed reads Assembly Number of annotated contigs (BLASTX) Number of contigs with GO term

Illumina sequencing

Brains of virgin and mated 5-day-old males 759,071 93,787,265 719,042 79,956,990 50,750 contigs (mean: 880 nt, median: 618 nt) 17,608 7,874

De novo transcriptome assembly was performed in two steps. First, HiSeq sequences were preassembled using Trinity

54

with jellyfish method for k-mer counting. This first

assembly step permitted the reconstruction of 66,951 contigs (> 200bp). Second, the final transcriptome assembly was performed on the 66,951 contigs previously described and the 719,042 sequences from 454 with MIRA assembler v 3.2.1

55

using as parameters de novo

assembly method, EST assembly type, accurate quality, and Sanger sequencing technology. Contigs were compared to the NCBI non-redundant protein database (NR), using BLASTX, with a 1e-8 value threshold. BLAST2GO was used for the Gene Ontology (GO) annotation (GO association done by a BLAST against the NCBI NR database) were translated to peptides using FrameDP 1.2.0

57

56

. Contigs

with three training iterations and using

Swissprot as the reference protein database. Transcripts encoding candidate neuropeptides were searched within the assembly with a set of insect neuropeptide amino acid sequences 1, 25

that included B. mori and Drosophila melanogaster neuropeptide sequences as queries

using TBLASTN, with a 1e-1 value threshold. Resulting sequences were reversely compared to NCBI NR database using the BLASTX application to confirm annotation and their


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translation was manually verified or corrected. Signal peptides were identified using SignalP 4.0 58.

Liquid chromatography and Mass Spectrometry For primary neuropeptide identification, extracts were analyzed by nanoLC (Reposil-Pur C18-AQ nanocolumn, 3 µm, Dr. Maisch HPLC GmbH; UltiMate 3000 liquid chromatography system, Dionex, Sunnyvale, CA, USA) connected to a sample spotter (probot, Dionex, Sunnyvale, CA, USA) and automated MALDI-TOF MS (4800 plus Proteomics Analyzer, AB Sciex, Farmingham, MA, USA). The mass spectrometer was operated automatically in positive-ion reflector mode in a mass range from m/z 800 to 5,500 and an S/N minimum set to 110. For a mass spectrum, 70 subspectra on 15 positions were averaged. Close external calibration was performed with the calibration standard no. 206195 (Bruker Daltonics, Bremen, Germany), spotted onto 13 positions of the MALDI target plate. Ion signals were fragmented by post-source decay (PSD) with 60 subspectra on 12 positions per spectrum. All resulting MALDI-TOF MS spectra were analyzed with DataExplorer v4.10 (AB Sciex). For identification of peptides, precursors were subjected to NeuroPred 59 (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py) for in silico identification of putative cleavage sites and potential peptide products. Ion signals were mass matched to predicted peptide products and fragment spectra were subsequently matched to predicted peptide sequences. In silico prediction of fragments was performed using ProteinProspector (http://prospector.ucsf.edu/prospector/mshome.htm)

or

using

the

ion

fragmentation

calculator function in DataExplorer. A peptide was considered identified at a sequence coverage ≥ 50% and if major ion fragments were detected (e.g. breakage at Pro residues). New identified peptides were submitted to UniProt (http://www.uniprot.org/).


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For comparison of mated/virgin tissue neuropeptide composition, direct profiling of samples was conducted on the same MALDI-TOF mass spectrometer in manual mode. The mass spectrometer was operated in positive-ion reflector mode in a mass range from m/z 800-3,600. For one spectrum 50 subspectra on 20 positions were averaged. Neuropeptide ion signals were fragmented by PSD.

Statistics Mass spectrometric profiles as obtained through MALDI-TOF were used to construct presence/absence matrices using only unequivocally identified neuropeptide-related masses. Ion signal presence/absence was assessed based on automatically computed mass lists: An ion signal was defined as present if the corresponding S/N ratio was ≥ 10. All resulting mass lists were cross-checked manually against the corresponding mass spectrum to prevent falsepositive data entries. Furthermore, ion signals affected by masking from other ion signal isotopes (e.g. m/z 1374.72 NPLP-51-13, m/z 1376.66 CAPA-tryptoPK) were only recorded if clearly identifiable as a distinct signal. To assess similarities of neuropeptide composition between samples, we calculated Jaccard’s similarity between single samples. Samples were grouped based on tissue-type, sex, and mating status. Similarity of samples was visualized in a non-metric multidimensional scaling (nMDS) plot

60

. To detect global patterns, we ran nested

PERMANOVA with 999 random permutations to detect differences between tissue types, sexes, and mating status. Moreover, we introduced terms describing interaction effects between tissue type and sex (tissue:sex), tissue types and mating status (tissue:mating status), sex and mating status (sex:mating status), and tissue types, sex, and mating status (tissue:sex:mating status). Subsequently, we performed pairwise comparisons via analysis of similarities (ANOSIM) with 999 random permutations between the following a priori


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groups: (1) tissue types — 3 groups (testing for differences between ALs, CC-CAs, and GNGs), 3 combinations, (2) sexes and mating status — 4 groups (testing for differences between virgin males [VM], mated males [MM], virgin females [VF], mated females [MF]), 6 combinations, (3) tissue types, sexes and mating status — 12 groups (VM-AL, VM-CCCA, VM-GNG, MM-AL, MM-CC-CA, MM-GNG, VF-AL, VF-CC-CA, VF-GNG, MF-AL, MF-CC-CA, MF-GNG), 66 combinations. A similarity percentage analysis (SIMPER) was performed to identify neuropeptide candidates that contribute to significant differences between physiological states (sex:mating status) within each tissue. For this, the raw data was pretreated a second time by quantifying ion similarities between tissue samples by Bray-Curtis similarities. Only putative biologically active peptides were evaluated from the list generated by SIMPER analysis. Detection ratios of these neuropeptides in a specific tissue were analyzed using generalized linear models (GLMs) with binomial distributions. ANOSIM and SIMPER were calculated with the software Primer v7 (PRIMER-E Ltd., Luton, UK, trial version) and PERMANOVA, nMDS ordination and GLMs were calculated and visualized in R 3.4.1 (R Development Core Team 2017) using the packages vegan

61

,

ggplot2 62, MASS 63, and sciplot 64.

RESULTS AND DISCUSSION De novo generation and annotation of an Agrotis ipsilon brain transcriptome We generated a de novo transcriptome assembly using transcriptomic data from 454 and Illumina sequencing. These sequencing platforms were combined to enhance the quality of the final assembly since they produce substantially different read length and transcript representation (454 sequences were obtained from a normalized library, supposed to enrich the dataset in rare and low expressed genes) and they are supposed to recover different


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sequence types from a sample. The obtained 454 dataset contained 759,071 raw sequences, while the Illumina dataset contained 93,787,265 raw sequences (Table 1). The two-step assembly provided the final transcriptome of A. ipsilon male brains, composed of 50,750 contigs (length from 42 to 12,955 bp, mean: 880 bp, median: 618 bp). It has to be noticed that these contigs do not represent unigenes, since their assembly include possible splice variants, polymorphism or reverse transcriptase errors. Among these 50,750 contigs, only 21,254 (42%) could be translated in a putative open reading frame (ORF) (19,495 unique ORFs). Thus, a majority of resulting contigs corresponded likely to noncoding sequences. 17,629 (83%) predicted proteins translated from these regions showed similarity to known proteins when compared to the non-redundant protein database using BLASTX. Among transcripts with BLAST-hits, 11,390 (64.6%) corresponded to at least one Gene Ontology (GO) term. Among those associated to a GO term, 10,065 were assigned to a molecular function (88.3%), 7,563 were assigned to a biological process (66.4%), and 5,549 were assigned to a cellular component (48.7%) (Supporting Information Figure S1). In the molecular function category, level 2, the terms “binding” and “catalytic activities” were the most represented (67.9% and 50.6%, respectively). In the biological process category, the terms “cellular process” and “metabolic process” were the most represented (76.8% and 75.6%, respectively). In the cellular component category, the terms “cell” and “organelle” were the most represented (76.3% and 47.6%, respectively).

Identification of transcripts encoding putative neuropeptides Within the A. ipsilon brain transcriptome, at least one copy of 37 contigs encoding putative neuropeptides were identified (Table 2). Eight neuropeptides were represented by 2 sequences or, more likely, corresponding to different splicing variants or different gene duplicates. Single amino acid polymorphism was considered as allelic or inter-individual


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polymorphism rather than two different sequences. In total, 56 contigs encoding putative neuropeptides including duplicated/splicing variants were identified (Table 2). Most were complete and exhibited a signal peptide, a hallmark of secreted proteins.


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Table 2. List of contigs encoding putative prepropeptides and hormones identified in the brain transcriptome from A. ipsilon males. Signal peptides are in bold italics (SignalP 4.0

58

). Predicted consensus cleavage sites are highlighted in light grey.

Amidation sites are highlighted in dark grey. Corresponding peptides identified by peptidomics are underlined. * : the stop codon was found in the contig. Neuropeptides and peptide hormones Adipokinetic hormone 1

A. ipsilon contig names

Complete sequence

Deduced amino acid sequence of the preprohormone MNYVSIFVLIVACLCVLADAQLTFTSSWGGGKRGAVAATMSCR SEETIA--AIYKLIQNEAERLLLCQKP* MNKIFFLFVFVACLCLFAEAQLTFTSSWGG-KRGGVAP-ISCKN EEGVT--TIFKLIQNEAERFIICQQKS* MNKIYFVIVFVACFCLFAEAQLTFTSSWGGGKRSGVAP-MSCK NEEAVA--TIFKLIQNEAERFIICQQKS*

Agrotis_qualite_rep_c44757

Yes


Yes


Yes

Adipokinetic hormone 2


Yes

MCRIFIVLLVVAALAIIIEGQLTFSSGWGNGKRSISSEQINDDCN PEEAIFQIY--KLIVSEGERIRACQRDGKM*

Adipokinetic hormone 3


Yes

MTPVRCSRCVAVVAACVLAAALVSAQITFSRDWTGGKRSAPQ LALDCGQFTRLCRHFVHELKAALTSEMASKHHSDLDKPPLYDE E*

Allatostatin A


Yes

MLSTSLPVCFLVIGAALCAPERMQNDPDPHDSTAQGSDNHSD HIAPLAKRSPHYDFGLGKRAYSYVSEYKRLPVYNFGLGKRSRP YSFGLGKRSVDEDQTNDDQQQIMNNDLDQAALAEFFDQYDDA GYEKRARPYSFGLGKRFADDDTSEEKRARAYDFGLGKRLPLYN FGLGKRARSYNFGLGKRLASKFNFGLGKRERDMHRFSFGLGK RSADDASTEDSDNYFDV*

Allatostatin C


No

MRRALDGPGSSSLDTRQ--ADKRQVRFRQCYFNPISCFRK*

Allatotropin

Agrotis_qualite_rep_c1029/ Agrotis_qualite_rep_c12163

Yes

MNFSMHLVLAVAAAACLCVVTAAPEGRLTRTKQQRPTRGFKN VEMMTARGFGKRDRPHTRAE-LYGLDNFWEMLEAAPEREGQE STDEKTLESIPLDWFVNEMLNNPDFARSVVRKFIDLNQDGMLSS EELLRNVA*

Apis-ITG-like


Yes

MHRTMAVTAVLVLSAAGAAHAWGGLFNRFSSDMLANLGYGR SPYRHYPYGQ-EPEEVYAEALEGNRLDDVIDEPGHCYSAPCTT NGDCCRGLLCLDTEDGGRCLPAFAGRKLGEICNRENQCDAGL VCEEVVPGEMHVCRPPTAGRKQYNEDCNSSSECDVTRGLCCI MQRRHRQKPRKSCGYFKEPLVCIGPVATDQIREFVQHTAGEKR IGVYRLH*

Yes

MKSFMVFVLIFACFSCYYAQESTNFYCGRTLS-RALAVLCYGAE SKRDAGWWIPQHGHHALAGVRGKRGPVDECCEKACSIQELMT YC*


Yes

MKFYIVFALILACAACVSSQEGTNFYCGRQLS-RTLALVCWGAE —KRDAGWWVPPQSARALGGGRGKRGPVDECCLKPCSIEEML TYC*


Yes

MKFYIVFALILACAACVSSQEGTNFYCGRQLS-RTLALVCWGAE --KRDAGWWVPPQSARGSGRRSW*


Yes

MKLTLIILLVVAYSWCSEAQNEARVFCGRVLSERLAAL-CWGPN

Bombyxin/Insulin-related Agrotis_qualite_rep_c8024 peptide

SVKRDAGWWLTRGAARSLGGVRGKRGLATECCDKACTVEELL SYC* Agrotis_qualite_c16607

?

MRLIIMSC?LVLAYVAADQTPVI?--CGRQ?ANARVVLCYGAEYV NSKRSRTMTTADFLPGDK??EVDWPWSGRRGSLTANWSRY-KREGLVDECCLKPCTTDVILNYC*

Agrotis_qualite_c33828

Yes

MKLTLILIFVVACFWCCEAQNGANFYCGRRLSQVLAAICWGAE EKRSTGWWMPEDSSEGLSEDSGASEAWLTSAATNPALWMNC SPTRLVFDHLRLIRLLQIVTCFLNC*


?

MNPQQSILLVTLCTITLCAAHIGGGTPYQDTNPQVYCGRSLARA LAFLCFEDGGSESKRSDSGSMYNAILSPYYKDQEGQFGWPWM TPHKARGLSLPSRGKRFVVNECCDKACSLSEL?SYC*


14

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No

?GASIRVCGRNLPVLVAFYCDKYGMAGKRNGVSVPRYGGVNG WPWLDTKRVLDFGTAKR-GIVDDCCYNTCSLDTILSYC*

Bursicon alpha subunit


Yes

MFRFEIISLCLVFMGFFLYHDKPVRAQSVEVPLSPGQECQMTP VIHILKHPGCIPKAIPSFACIGKCTSYVQVSGSKIWQMERTCNCC QESGEREATVILLCPKAKTEDKKLRKITTKAPLECMCRPCGNIDE SSIIPQEVAGYADDGPLYNHFRKSF*

CAPA (PVKs and tryptoPK)


Yes

MQPTMRIIVSMALLAYAVASAYHSNVKLRRDGKMVLYPFPRVG RASGNTWQLPLNDLYPEYEPAQVKRQLYAFPRVGRDPVMSRL GRSDLSRVESHEFQPMAVRRTESPGMWFGPRLGRAFKNDDD EITIQNESNDHSEPEQTELI-HEDRRKRQTLN*

CCHamide2


Yes

MAQMYLAVTIIALLAISHGVSAKRGCSAFGHSCFGGHGKRSGD TSAMDQLSNQDGVLMARHQLGQEETPPHPVYPHSGYNVLASG DDIIPIRDGGVYDREDGGAAREVMKMKLRNIFKHWMDNYRRSQ QNPDDGYYIESL*

Corazonin


Yes

MSANVTLLLIFVTLASVTAQTFQYSRGWTNGKRDQGHLRPELK ELINNMDKILSPCQKNKLKYLLEGKPVTERLLIPCDILDTEEYPRA LTERNLNAMMDAFY*


Yes

MSANVTLLLIFVTLASVTAQTFQYSRGWTNGKRDQGHLRPELK ELINNMDKILSPCQKNKLKYLLEGKPVTERLLIPCDILDTEEYPRA IN*


Yes

MSANVTLLLIFVTLASVTAQTFQYSRGWTNGKRDQGHLRPELK ELINNMDKILSPCQKNKLKYLLEGKPVTERLLIPCDILDTSQ*

Crustacean cardioactive peptide


Yes

MSVYRVSVCIVIALLYLQCCYTATIPRNFDSRLSEEMIMTPKKRP FCNAFTGCGRKRSQQAPGMPSQELLRQKQYVDEDTFGSFLDS ?SAIEELSRQILSEAKLWEAIQEASAEIARRKQKEAY*---

Diapause hormone /PBAN/Pyrokinin


Yes

Diuretic hormone 31/Calcitonin-like peptide


Yes

MVRATCLLASCVLFALLLIVPASAYPRYPSNYFREEGQYEPEEI MDMLNRLGNLIQMERKMENYKEDITSEKRALDLGLSRGYSGAL QAKHLMGLAAANYAGGPGRRRRDAH*-----------


Yes

MVRATCLLASCVLFALLLIVPASAYPRYPSNYFREEGQYEPEEI MDMLNRLGNLIQMERKMENYKEDITSEKRALDLGLSRGYSGAL QAKHLMGLAAANYAGGPGRRRTRCPLRSE*


Yes

MVRATCLLASCVLFALLLIVPASAYPRYPSNYFREEGQYEPEEI MDMLNRLGNLIQMERKMENYKEDITSEKRALDLGLSRGYSGAL QAKHLMGLAAANYDRRPRKEATRCPLIFSISTRILH*


?

MVRATCLLASCVLFALLLIVPASAYPRYPSNYFREEGQYEPEEI MDMLNRLGNLIQMERKMENYKEDITSEKRALDLGLSRGYSGAL QAKHLMGLAAANYA-EAPKEATRCPFAVSDR?


?

MVRATCLLASCVLFALLLIVPASAYPRYPSNYFREEGQYEPEEI MDMLNRLGNLIQMERKMEN-------EKRALDLGLSR GYSGALQAKHLMGLAAANYAGGPGRRRRDAHFAVSDR?-----

MY—GAVLPGLFFIFISCVVASSNDVKDGGADRGAHSDRGGM WFGPRIGKRSLRMATEDNRQAFFKLLEAADALKYYYDQLPYEM QADEPEARVTKKVIFTPKLGRSLSYEDKMFDNVEFTPRLGRRLA DDTPATPADQEMYRPDPEQIDSRTKYFSPRLGRTMNFSPRLGR ELAYEMLPSKVRVVRSTNKTQST*

Diuretic hormone 41/Corticotropin releasing factor (CRFDH)


Yes

MMWWALWCAVVVAAGSGVAAAPAPDSLSPLDMVQMDSSAP DDETLYAMSPMAARYSAGAPWLYLLADMPRDSQTGSGRVKRR MPSLSIDQPMSVLRQKLSQEMERKQQAFRAAVNRNFLNDIGKR GFQWTPSVQAVRYI*---

Diuretic hormone 34 /splicing variant of CRFDH


Yes

MMWWALWCAVVVAAGSGVAAAPAPDSLSPLDMVQMDSSAP DDETLYAMSPMAARYSAGAPWLYLLADMPRDSQVASSDAEMR TRRSFSVNPAVELLQRGAYNNYMERVAHN--NRNFLDRVGKRGSWRSQN*------------

Diuretic hormone 45/splicing variant of CRF-DH


No

Eclosion hormone


No

?VACNPAIATIYDPMQVCIENCAQCKKMLGAWFEGPLCAESCIT FKGKL?PDCENIASISPFLNKL*


No

?KITAAFVIFITVAFLANLGHVACNPAIATINDPMQVCIENCAQCK EMLGDWFNGPFCADACIKLRGKLIPDCENIASIKPFLNINQL*

------------------------------?LYAMSPMAARYSAGAPWLYLLADMPRD SQRLVDPADLHEGRARPKRKMPSLSINNP ME----VLRQRLILEV ARKQMREANQRQAVANRLFLQNVGKRGFWANSAPTRYDN*-


15


Page 16 of 62

FMRFamide


Yes

MSCSRTVALLAALWLVVGATSSPVRRSPDLEARRRSAIDRSMI RFGRSYPPEPSAADIREAFERPTRRGNSFLRFGRSQPLTLSTD DLVSLLRAYEEDYDTPMTKKSASFVRFGRDPNFIRLGRSADDD KSAFEQNSELVVSGYPQRKSRARDHFIRLGRDSEEVNENEFEE T---EESRRKRSADSCHDCQS*

IMFamide


Yes

MMRFTIGVV---CLVAVLLSLAEVSEANYKNAPMNGIMFGKRGP TEYDQRGKTFTALCEIATEACQAWFPSTENK*

Ion transport peptide/Crustacean hyperglycemic hormone


Yes

Insect kinin


Yes

MLHHWLLITVSWLGSVRPQYISSGGPGNDYVLDVLNANPQFPL PYYYNVPSTRHDYVTTLNNPIASFNNGQQADLQRRRRTTDQKE H----RWLPNLLDIDRTMYLQNEEQVGSPFVSNFEGTVDENATNF LDPKDKKYFSPWGGKRDKNNIENMWTWKRATNIREPSMPKRV RFSPWGGKRSGQMIYKPGNKNSKIIFSTTMPELTRILSNYNGNG NGNERVNLAGFQFIPTLDKRHPIKILALSTNTNERTLREALPFNS FLESLPKLFKPGHPYLDVNLKKDGKRKVKFSAWGGKRSPPIIGP IWTPASQNLKESTLDTILLIRNSQNRDDPTTMAL*

Myosuppressin


Yes

MALGNGYYCAVVCVVLACASVVLCAPAQLCAGAADDDPRAAR FCQALNTFLELYAEAAGEQVPEYQALVRDYPQLLDTGMKRQDV VHSFLRFGRRR*

Neuropeptide F 1/splicing variant a


Yes

GVYDAAIFARLDRICDDCYNLFREPQLYTLCREKCFTSPYFKGC MESLYLYDEKEQIDQMIDFVGKR*

MLNKNIVIAIAVV-LAVVCLAEAREEGP?DMADALRMLQELDRYY TQAARPRIDRRERCGWRPRRSRLVKSRCAAPRAAEIRQHLLLP HPTKVRQTL*

Agrotis_qualite_rep_c584 Neuropeptide F 2


Neuropeptide-like precursor 1


Yes

MLNKNIVIAIAVV-LAVVCLAEAREEGPHDMADALRMLQELDRY YTQAARPRFGKRSDVYTNWAKDLEKPDLPAWLSYARRR* MRFLLSAILLLSAILSCATQAQYPRPRRPERFDTAEQISNYLKEL QEYYSVHGRGRYGKRQMHIADASVIFRESPFFEHNLNDEPAFK NLGYK*

Yes

------MNDAGTSIGRHRGCLLLFVALAVAFSSYVEQVESMPAEPV SQWPTFPRRNIAALARDGYLRNSASRAYKRGISTLAKNGLLPTY RSPYVETDKEDQNQDESQEKRNMASIARLRSYAAMKRNIQALA RDGYRVGRGQYNPSNDKRNIAALARNGLLHKKDEATENEYYFP FYQNPIAPLSEIDHPLDVNEMYDLQQSMNPDMFPSMSQVYKRS L-YEPYYDYYSPKDYDNSYFKRSSAGSPVHGLY RPN

Agrotis_qualite_rep_c10355/

Yes

MNDAGASIGRHRGCLLLFVALAVAFSSYVEQVESMPAEPVSQ WPTFPRRNIAALARDGYLRNSASRAYKRGISTLAKNGLLPTYRS PYVETDKEDQNQDESQEKRNMASIARLRSYAAMKRNIQALARD GYRVGRGQYNPSNDKRNIAALARNGLLHKKDEATENEYYFPFY QNPIAPLSEIDHPLDVNEMYDFQQSMNPDMFPSMSQVYKRSLY EPYYDYYSPKDY-DNSYFKRSSAGSPVHGLYRPNYLEPNTRTK RYVMPYPDILEGDEMIEQNDIDGE-KRSVDDDDDDDDDDGEVH ENFQKRHIGSLARLGLLPSFRYSGGRYSRSGRARLLLPSQELY RKHSPDENFGIREYLSSPEVESPVDPDDADIPPPPVPAHSHPTG RLLHRPLSNDLPHTSPLPLPPIAPTNYADTFTKNRWQSYTKETP KFYYFRSLKVPYHTSGKRYLLLPAVDNILLRKGYRNSSLPSRRK NQ*

Yes

-MRGAGGALAVAVAALLVCCSADPHQEGDVSGSDHNTAERY


Orcokinin A+B

MHLSSIQLACAALVAVAL—GAAAPPSSSHHVARRSFFTLECK


DAAGQKTRTLDTLGG-NLVREVREIRHSGYVPYLISRRFDFASP YGGKREPWPLAPIEFSGYYGDGLPKRNFDEIDRSGLDTFVKKR NFDEIDRSSMPFPYATKRFYHLSSFDKKRYRADYPMDEIDLSHF PIGSKRSQDSYPLLPRNLL*-----------------------Orcokinin B


Yes

MMRGAGGALAVAVAALLVCCSADPHQEGDVSGSDHNTAERY DAADYKQKEDLEEFIRLLLHYDERNLNGFGRNTNGLGGSSLLG RNLGNLGGSSLVGRNTHGLGGSSLAGRSFNGIGGRDIEGDKRL YSRFVDSLGGGNFVRNLDSIGGGNFVRNLDSIGGSNFVKKNLD QLGGPNLVKRNLDSLGGGNFVRNLDSIGGGNFVRELDSIGGSN FV*

Partner of bursicon/Bursicon beta subunit


No

?THCYNPDGVRLEDEDSAIMEVRLREPDDCKCYKCGDFSR*


16

Page 17 of 62 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


Pigment dispersing factor


Yes

MLNLIRRGVFLFMALVLTCEIFASDYVEEPELESYGEEELAEIIPY EENDKYIRHIHSMMNPYRDYTNALADIKYKMWKRNGDLINSLLT LPKGMNDAGR*

Myoinhibitory peptide/ Allatostatin-B/ Prothoracicostatic peptide


Yes

MRKSARGQVCTEAGAGASGDWQDMSSAWGKRAWQDLNSA WGKRGWNDMSSAWGKRAWQDLNSAWGKRGWQDLNSAWG KRAWRDMSQSPWGKRGWNDMSSAWGKRGWNDMSSAW GKRGWNDMSSAWGKRGWNDMSSAWGKRGPEKWANFHGSW GKRAAEPDYEEIDAAIEQLIPI QQLSDNERMEV-PEKKAWSALH GAWGKRPVKQAQYNSGSYYWKREPAWTNLRGMWGKRSAP DADAVDDDHESSARDEA*------------------------

Prothoracicotropic hormone


Yes

MITRPLVFVIVCFGLIILIQSLVPKVMAMKNSNVDEYMLEDQRTR KRKNYVVRLARDSEILGNSGNLGTNYDTDSVQPDPANPEELSA FIVDYANMIRNDVVLLDKSVETRTRKRGNIKVKKHHNQAIPEPPC ACNPNNGTEDFGENTIPRIVNTYNCNKQACPWPYQCMENIYNL KIIKRKETVNQVSRFELPDDLHRKWVGDYRPISVGCICSRDYYG TTN*

Short Neuropeptide F


Yes

MGRARRTVRAPAQHDALGGHALARKSVRSPSRRLRFGRRSDP DMPPQAPLDEMNELLSLREVRTPVRLRFGRRSEERAVPHIFPQ EFLTQEQDRAVRAPSIRLRFGRRSDNNMFLLPYESALPQEVKA NGSVEDDRQQE*

SIFamide


Yes

MRFIVALCLFAIVMCIIHK----AEGTYRKPPFNGSIFGKRGVVEYD

Sulfakinin


Yes

MRLVTIVAMAITVALAMLVRCCEGANLHHFALPEEDEEFRHRPL YRDYSLIR-RAVRADDAFDDYGHLRFGRSDD*

Tachykinin


Yes

MGAPRTCLIFITIQLVSLAYAQEVSKRVPQGFLGMRGKKYFDEE GIEQFYKRKPQFFVGVKGKKSLQDILEAPEEYYKRAPMGFMGM RGKKDLGDSQST---ELFPKRDGSLIGKIDYSSKEENADPDFPILN ELLLQYLSQLDAPRNTYMQSSESMEPEQSNDLDKRAANFNQFY GVRGKKSINNKRPYDLTFRGKFIGVRGKKDLKNSNAHEIKFLVD QNGPLPKRKAQMGFFGMRGKKWTDEPSLEMDMPN*

Trissin


Yes

MFXITAXVSLMLIGSTLWAASLSCDXGSEXAXACGTRHFRSCC FNYLRKKRGPDTLKFLTPNQDIKENQQRTNKLPMFVVENIPMPE QWISNMLANENHPYSIDDMVENRMYDA*

TTGRALSALCEIASETCQAWYQTLENK*


17


Page 18 of 62

We could annotate three contigs encoding adipokinetic hormone, AKH-1, AKH-2, and AKH-3, which are well conserved with the triplet of paralogous AKH genes in B. mori. This triplet has not been found in other insect orders

25

. In Spodoptera frugiperda, another

noctuid species, 4 AKHs closely related to B. mori AKH-1 and AKH-2 have been identified 65

, and it has been proposed that this gene family evolved differently after the split between

the Bombycoid complex and Noctuoidea

25

. Here, we demonstrated that at least some

noctuids express an AKH-3 homolog. Interestingly, we assembled 3 forms of A. ipsilon AKH-1 (named a, b, c; Table 2, Figure 1) that could represent different variants.

Figure 1. Alignment of AKH neuropeptides from Bombyx mori (Bmor, AB298930, AB298931, AB298938), Spodoptera frugiperda (Sfru, AJ871195, AJ871196, AJ871197, AM269886) and Agrotis ipsilon (Aips, this study).

Two contigs encoding different neuropeptide Fs (NPF-1 and 2) were found, but out of the two splicing variants of NPF-1 that exist in B. mori and C. suppressalis, only one could be identified in A. ipsilon, the NPF-1 variant a. In B. mori, the NPF-1 splicing variant b results from incomplete splicing of NPF-1a 25.


18

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While insect genomes usually contain two CCHamide genes (CCHa-1 and CCHa-2), of which CCHa-1 has two splicing variants in C. suppressalis 28, only one transcript (CCHa-2) was found in the brain transcriptome of A. ipsilon. Eight different variants of diuretic hormones (DHs) were observed that could represent splicing variants, as the occurrence of such DH splicing variants was evidenced in B. mori and C. suppressalis

28

. Only one contig related to CAPA genes and encoding

periviscerokinins (CAPA-PVKs, C-terminal PRVamide) and CAPA-tryptopyrokinin (CAPA-tryptoPK, C-terminal WFGPRLamide) could be identified in A. ipsilon, as in C. suppressalis 28, whereas B. mori has two different splicing variants of CAP2b (CAPAa and CAPAb)

25

. Natalisin, which was recently identified and characterized in D. melanogaster,

Tribolium castaneum, B. mori 66 and in C. suppressalis 28 was not detected in A. ipsilon. In total, we failed to annotate 12 neuropeptides usually observed in insects: CNMamide (this was expected since, although conserved in Arthropods, this peptide is absent in Lepidoptera), one of the CAPA splicing variants found in B. mori (also absent in C. suppressalis), glycoprotein hormones alpha2 and beta5, neuroparsin, NPF1 splicing variant b, RYamide (whereas all these last peptides are present in other Lepidoptera), neuropeptidelike precursors 2-4 (NPLP2-4), vasopressin-like peptide and proctolin. These peptides were also absent in our mass spectrometry analyses (see below). It is possible that we missed these peptides in the A. ipsilon transcriptome. However, no gene encoding proctolin, vasopressin-like peptide nor NPLP2-4 could be identified in the B. mori genome 25 nor in C. suppressalis 28, further suggesting that these neuropeptides are not present in Lepidoptera, as it is the case for CNMamide 28. The A. ipsilon transcriptome contained both the SIFamide (SIFa) (IMFa)

25

67

and IMFamide

neuropeptides. IMFa neuropeptide encoding genes have been identified only in


19


Page 20 of 62

Lepidoptera (Figure 2). As suggested by analysis of the B. mori genome, the IMFa gene originated from duplication of the SIFa gene 25.

Figure 2. Alignment of IMFamide neuropeptides from Lepidoptera. Harm: Helicoverpa armigera (AGH25559), Dple: Danaus plexippus (EHJ78184), Bmor: Bombyx mori (NP_001124359), Csup: Chilo suppressalis (KT005973), Aips: Agrotis ipsilon (this study).

Identification of A. ipsilon neuropeptides by mass spectrometry Our analysis of extracts prepared from AL, CC-CA and GNG by nanoLC/MALDI-TOF MS yielded a total of 70 neuropeptide precursor-related peptide sequences from 25 neuropeptide precursors (Table 3). All corresponding neuropeptide masses were verified via tandem mass spectrometry (Figure 3, and Supporting Information Table S1 and Figure S2). From the 70 sequences, 54 were related to putative bioactive mature neuropeptide sequences. The remaining 16 identified peptides were matched to spacer peptides originating from neuropeptide precursors (precursor peptides, PP; Table 3).


20

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Figure 3. Exemplary sequence confirmation of putative neuropeptide ion signals by post source decay of corresponding ion precursors; (A) m/z 1246.73 (Myosuppressin), (B) m/z 1369.63 (Corazonin) and (C) m/z 1486.61 (Allatotropin). Only b- and y-fragments are labelled. Recorded ion signals are labelled as monoisotopic masses [M+H]+.


21


Page 22 of 62

Table 3. Overview of identified neuropeptide sequences recorded from A. ipsilon antennal lobe (AL), corpora cardiaca-corpora allata (CC-CA) and gnathal ganglion (GNG) by nLC-MALDI-TOF, and their occurrence (expressed as % of sample group) in mass spectrometric fingerprints from direct-profiled tissue preparations. Peptide name

Sequence

Adipokinetic Hormone-1 AKH-1 pQLTFTSSWGGamide Adipokinetic Hormone-2 AKH-2 pQLTFSSGWGNamide Allatostatin-A AST-A-1 SPHYDFGLamide AST-A-3 SRPYSFGLamide AST-A-4 ARPYSFGLamide AST-A-5 ARAYDFGLamide AST-A-6 LPLYNFGLamide AST-A-7 ARSYNFGLamide AST-A-8 LASKFNFGLamide AST-A-9 ERDMHRFSFGLamide Allatostatin-C AST-C QVRFRQCYFNPISCF-OH AST-C [pQ] pQVRFRQCYFNPISCF-OH Allatotropin AT GFKNVEMMTARGFamide AT-PP-1 APEGRLTRTKQQRPT-OH Insulin-related peptide DAGWWIPQHGHHALAGV IRP-1 Ramide DAGWWVPPQSARALGGG IRP-2 Ramide DAGWWLTRGAARSLGGV IRP-4 Ramide CAPA CAPA-PVK-1 DGKMVLYPFPRVamide CAPA-PVK-2 pQLYAFPRVamide [pQ] CAPA-PVK-2 QLYAFPRVamide CAPA-PVK-3 DPVMSRLamide SDLSRVESHEFQPMAVCAPA-PP-3 OH CAPA-tryptoPK TESPGMWFGPRLamide CCHamide CCHa GCSAFGHSCFGGHamide Corazonin Cor pQTFQYSRGWTNamide Diuretic hormone 31 ALDLGLSRGYSGALQAKH DH-31 LMGLAAANYAGGPamide Diuretic hormone 41 DH-41-PP GFQWTPSVQAVRYI-OH Diuretic hormone 45 DH-45-PP GFWANSAPTRYDN-OH FMRFamide-related peptides RFa-1 SAIDRSMIRFamide SYPPEPSAADIREAFERPTRFa-PP-2 OH RFa-3 SASFVRFamide

[M+H]+ Accession no. Uniprot calculated

[M+H]+ measured

MS² nLCMS

AL (n=49)

CA-CC GNG (n=37) (n=32)

MALDI-TOF MS direct profiling

C0HKR0

1087.49*

1087.48*

+

18.37

100

43.75

C0HKR1

1100.48*

1100.47*

+

n.d.

100

18.75

C0HKR2 C0HKR3 C0HKR4 C0HKR5 C0HKR6 C0HKR7 C0HKR8 C0HKR9

934.44 925.49 909.49 911.47 935.54 926.48 995.57 1393.68

934.44 925.51 909.47 911.48 935.53 926.48 995.56 1393.66

+ + + + + + + +

81.63 100 100 100 32.65 100 100 100

40.54 89.19 100 100 n.d. 78.38 86.49 45.95

78.13 96.88 100 100 12.5 90.63 93.75 93.75

C0HKS0 C0HKS0

1905.89 1888.89

1905.88 1888.89

+ +

85.71 10.20

18.92 8.11

6.25 n.d.

C0HKS1 C0HKS2

1486.73 1738.97

1486.73 1783.93

+ +

100 100

13.51 29.73

96.85 71.88

C0HKS3

2007.02

2007.02

+

10.20

94.6

25

C0HKS4

1879.97

1879.94

+

26.53

97.3

6.25

C0HKS5

1928.04

1928.03

+

4.08

83.78

6.25

C0HKS6

1420.78

1420.78

+

69.39

78.38

71.88

C0HKS7

975.54

975.51

+

42.86

83.78

68.75

C0HKS8 C0HKS8

992.57 816.44

992.55 816.47

+ +

100 32.65

91.89 86.49

93.75 34.38

C0HKS9

1831.86

1831.87

+

6.12

54.05

21.88

C0HKT0

1376.68

1376.70

+

36.74

97.3

96.88

C0HKT1

1263.48

1263.49

+

4.8

10.81

5.13

C0HKT2

1369.63

1369.62

+

26.53

100

59.38

C0HKT3

3042.59

3042.65

+

87.76

78.38

6.25

C0HKT4

1651.86

1651.88

+

55.10

100

87.5

C0HKT5

1498.67

1498.71

+

28.57

5.41

53.13

C0HKT6

1194.64

1194.62

+

91.84

70.27

46.88

C0HKT7

2133.02

2132.99

+

61.23

18.92

8.38

C0HKT8

812.44

812.42

+

100

81.08

75


22

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RFa-5 ARDHFIRLamide IMFamide IMFa NYKNAPMNGIMFamide ITG-like peptide ITG-like peptide IGVYRLH-OH Myosuppressin MS [pQ] pQDVVHSFLRFamide MS QDVVHSFLRFamide Neuropeptide-like precursor-1 NPLP1-1 MPAEPVSQWPTFPR-OH NIAALARDGYLRNSASRAY NPLP1-2 -OH GISTLAKNGLLPTYRSPYV NPLP1-3 ETDKEDQNQDESQE-OH NPLP1-4 NMASIARLRSYAAM-OH YRVamide NIQALARDGYRVamide YRVamideNIQALARDGYRVGRGQYN extended PSND-OH NPLP1-6 NIAALARNGLLH-OH NPLP1-61-11 NIAALARNGLL-OH SSAGSPVHGLYRPNYLEP NPLP1-9 NTRT-OH Orcokinin Orcokinin-likeNFDEIDRSSMPFPYAT-OH peptide Orcokinin-likeSQDSYPLLPRNLL-OH peptide-PP Pyrokinin/PBAN Diapause SSNDVKDGGADRGAHSD hormone RGGMWFGPRIamide SLRMATEDNRQAFFKLLEA PBAN-PP-1 ADALKY-OH YYDQLPYEMQADEPEARV PBAN-PP-2 T-OH α-SGNP VIFTPKLamide SLSYEDKMFDNVEFTPRLa β-SGNP mide γ-SGNP TMNFSPRLamide PBAN-PP-3 ELAYEMLPSKVRVV-OH MIP/AstB/PTSP Prothoracicostatic peptide PTSP-5 AWRDMSQSPWamide PTSP-6 GPEKWANFHGSWamide PTSP-7 AWSALHGAWamide PTSP-PP-2 PVKQAQYNSGSYYW-OH PTSP-8 EPAWTNLRGMWamide SAPDADAVDDDHESSARD PTSP-PP-3 EA-OH SIFamide SIFa TYRKPPFNGSIFamide Short Neuropeptide F sNPF-1 SVRSPSRRLRFamide sNPF-2 TPVRLRFamide sNPF-3 APSIRLRFamide Tachykinin related peptides TK-1 VPQGFLGMRamide TK-2 KPQFFVGVKamide TK-4 AANFNQFYGVRamide TK-5 PYDLTFRGKFIGVRamide TK-6 KAQMGFFGMRamide

C0HKT9

1026.60

1026.63

+

100

56.76

62.5

C0HKU0

1398.67

1398.67

+

14.29

21.62

15.63

C0HKU1

857.46

857.46

+

100

16.22

96.88

C0HKU2 C0HKU2

1229.65 1246.67

1229.64 1246.66

+ +

51.02 95.92

100 97.3

100 100

C0HKY1

1642.81

1642.81

+

100

16.22

96.88

C0HKU3

2082.08

2082.05

+

100

21.62

68.75

C0HKU4

3725.78

3725.79

+

n.d.

n.d.

n.d.

C0HKU5 C0HKU6

1554.79 1374.76

1554.78 1374.76

+ +

93.88 97.96

n.d. 8.11

53.13 96.88

C0HKU6

2464.21

2464.21

+

85.71

n.d.

53.13

C0HKU7 C0HKU7

1262.76 1125.67

1262.76 1125.68

+ +

95.92 95.92

5.41 n.d.

100 90.63

C0HKU8

2416.20

2416.21

+

100

n.d.

87.5

C0HKU9

1889.84

1889.85

+

12.25

35.14

43.75

C0HKV0

1515.82

1515.82

+

44.9

29.73

87.5

C0HKV1

2844.33

2844.31

+

n.d.

83.78

68.75

C0HKV2

2901.49

2901.54

+

n.d.

n.d.

n.d.

C0HKV3

2318.03

2318.01

+

n.d.

n.d.

n.d.

C0HKV4

816.53

816.47

+

6.12

83.78

75

C0HKV5

2190.05

2190.06

+

14.29

94.6

96.88

C0HKV6 C0HKV7

964.50 1633.90

964.50 1633.90

+ +

59.18 28.57

100 97.3

100 93.75

C0HKW7 C0HKW8 C0HKW9 C0HKX0 C0HKX1

1262.57 1414.67 997.50 1690.79 1359.66

1262.58 1414.66 997.47 1690.78 1359.67

+ + + + +

100 100 83.67 18.37 100

86.49 37.84 81.08 2.7 45.95

96.88 87.5 43.75 n.d. 84.38

C0HKX2

2072.83

2072.80

+

22.45

n.d.

n.d.

C0HKV8

1425.76

1425.77

+

95.92

n.d.

90.63

C0HKV9 C0HKW0 C0HKW1

1359.81 887.56 958.60

1359.81 887.54 958.58

+ + +

100 100 100

45.95 97.3 100

90.63 100 100

C0HKW2 C0HKW3 C0HKW4 C0HKW5 C0HKW6

1003.55 1048.61 1285.64 1667.94 1171.59

1003.55 1048.62 1285.64 1667.92 1171.58

+ + + + +

100 18.37 97.96 6.12 100

n.d. 5.41 18.92 27.5 n.d.

78.13 n.d. 93.75 5.13 68.75

Masses labeled with an asterisk are given as [M+Na]+; PP = Precursor-related Peptide. n.d. = not detected. Colors indicate occurrence range of detected peptides: Red : 0.1-25% ; Yellow : 25.1-75% ; Green : 75.1-100%.


23


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Mass spectrometric profiling analysis of single AL, CC-CA and the GNG preparations revealed numerous mass peaks corresponding to prior identified neuropeptide sequences. Identification of the recorded mass signals was either confirmed by fragmentation patterns of ion signals or by mass match. Obtained spectra were nearly identical for each tissue type, respectively, providing typical mass spectrometric fingerprints for each region of the nervous system (Figure 4, Supporting Information Figures S3-9). Nested PERMANOVA revealed significant differences in peptide composition between tissue types (p < 0.001, R² = 0.603) and mating status (p = 0.021, R² = 0.012) but not between sexes (p = 0.378, R² = 0.003) (Figure 5 and Supporting Information Table S1). Furthermore, testing for interaction effects between tissue types and sex as well as for interactions effects between mating status and sex did not indicate significant influences of either combination (tissue:sex, p=0.56, R² = 0.005; sex:mating status, p=0.24, R² = 0.004). However, we found that the interaction term between tissue type and mating status significantly explained variation in our dataset (p=0.04, R² = 0.014). We thus conclude that different tissues, as expected, exhibit distinct neuropeptide composition patterns — likely related to their function in the CNS — while there seem to be no differences between sexes (sexual dimorphism). Furthermore, our analyses clearly indicate that neuropeptide composition in single tissue types varies also in function of the mating status, but not as much as between tissues. Pairwise comparison through one-way ANOSIMs showed that ion signal compositions differed significantly between all tissue types (one-way ANOSIM: global R = 0.81, p < 0.001; AL~CC-CA, R = 0.99, p < 0.001; AL~GNG, R = 0.65, p < 0.001; CC-CA~GNG, R = 0.86, p < 0.001) (Figure 5 and Supporting Information Table S1), corroborating results from PERMANOVA. Likewise, one-way ANOSIM indicated no difference in overall neuropeptide composition between the four physiological states when disregarding tissue type (sex and


24

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mating status combined; VF, MF, VM, MM; one-way ANOSIM: global R = 0.01, p = 26.6) (Figure 5). Within specific tissue types one-way ANOSIMs identified significantly different neuropeptide composition between physiological states. All groups representing combinations of physiological states from two different tissue types showed significant variation in their neuropeptide composition (one-way ANOSIM: all p < 0.0014), corroborating and elucidating PERMANOVA results. Interestingly, when analyzing tissue types separately, significant differences in neuropeptide ion signal composition were found only between some groups. Results and elaborated analysis of these findings are discussed in tissue-specific chapters.


25


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Figure 4. Representative mass spectra obtained by direct tissue profiling of virgin male A. ipsilon antennal lobe (AL) (A), corpora cardiaca-corpora allata (CC-CA) (B) and gnathal ganglion (GNG) (C). Left y-axis: relative signal intensity, right y-axis: signal intensity in absolute counts. X-axis: mass/charge. Unlabeled ion signals or signals without a peptide-name represent unidentified signals. Masses marked by an asterisk represent [M+Na]+, masses with a tilde represent [M+K]+, all other masses are


26

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labelled as monoisotopic masses [M+H]+. CC-CA (A2) mass spectrum was cropped to m/z 3010 for better visualization.

Figure 5. Non-metric multidimensional scaling (nMDS) ordination plot (based on Jaccard similarities) for analyzed tissue types (light blue: antennal lobe (AL); yellow: corpora cardiaca-corpora allata (CC-CA); red: gnathal ganglion (GNG)) and their corresponding sexes and physiological states. Tissue types differed highly significantly between each other (nested PERMANOVA: p < 0.001, R² = 0.603; oneway ANOSIM: global R = 0.81, p < 0.001; AL~CC-CA, R = 0.99, p < 0.001; AL~GNG, R = 0.65, p < 0.001; CC-CA~GNG, R = 0.86, p < 0.001).

Antennal lobes MALDI-TOF mass spectra of ALs of 17 females and 28 males revealed 52 ion signals that could be assigned to putative mature neuropeptides (64 sequences in total) encoded by 21 different precursors (24 in total) (Table 3). The majority of the detected ion signals (36 peptides from 13 precursors) were regularly detected in most spectra (75.1% to 100%). Fourteen peptide sequences (from 10 precursors) occurred in 25.1% to 75% of all AL spectra


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with nine of the sequences (from 6 precursors) belonging to putative functional mature neuropeptides. Fourteen of the detected 64 ion signals (from 11 precursors) were typically only detected in several or a few spectra (0.1% to 30%), with fluctuating signal intensity. Eleven of the sequences (from 9 precursors) belong to putative functional mature neuropeptides. In some spectra (n=9) we found a small ion signal of AKH-1, which we conclude is an artifact from the dissection procedure. A comparison with available neuropeptidome data of ALs from D. melanogaster (22 peptides, 7 precursors 68), A. aegypti (28 peptides, 10 genes

69

) and T. castaneum (28 peptides, 11 precursors

70

) reveals that the

moth ALs, with 52 peptides and 21 precursors, contain by far the highest diversity of neuropeptides. Peptide families occurring in all 4 species are the allatotropins (ATs), the NPLP1s, the SIFas, and the tachykinin-related peptides (TKRPs). Allatostatin-A (AST-As), FMRFamide-like-peptides (FaLP), and short Neuropeptide Fs (sNPFs) occur in three of the species but not in T. castaneum. Insulin-related peptide-1 (IRP-1) and diuretic hormone-31 (DH-31) in contrast have only been found in A. ipsilon and T. castaneum

70

. The peptide

composition of A. ipsilon ALs is different to that of the other three species, as CAPA, corazonin (Cor), IRP-2, DH-41, DH-45 and PBAN are missing in the fly, the mosquito and the beetle. Although PBAN was detected in a few spectra (Table 3), its presence in the AL is surprising, as it was always detected only in the GNG and retrocerebral complex of all insect species studied so far, including A. ipsilon

30, 71

. Several studies have shown the presence of

some neuropeptides in ALs of Lepidopteran species, using immunocytochemistry or direct tissue profiling, and focusing on AST, AT, FMRF-amide related peptides, TKRP and MIP 34, 72-76

. In summary, the large number of neuropeptides in the moth ALs may reflect a more

pronounced involvement in the regulation of the activity of the AL. To our knowledge, this is the first study comparing the AL neuropeptide composition of different physiological states in an adult insect. In ALs of the moth M. sexta, the neuropeptide


28

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composition was found to be changing during development between late pupae and adults 76. Statistical analysis by ANOSIM revealed four pairings with no significant difference in their neuropeptide composition (VM~MM, MM~MF, VF~MF and VF~MM, p > 0.05, R = 0.09). However, significant differences were found for two pairs (VM~VF, p = 0.005, R = 0.22; VM~MF, p = 0.002, R = 0.23) (Figure 5). A detailed analysis with SIMPER, revealing the most important ion signals contributing to the dissimilarity between the states (> 5% contribution, 8 signals), showed that three of the major contributors were ion signals related to precursor-related peptides, which should have no bioactive function. To evaluate potential differences among the remaining contributors, we tested their occurrence between the different states using GLMs. Four potentially bioactive neuropeptides from four different precursors showed significant differences in their occurrence in the ALs between different physiological states; γ-SGNP (subesophageal neuropeptide) showed a significant lower occurrence in virgin males compared to all other states (VM~VF, p = 0.008; VM~MM, p = 0.005; VM~MF, p = 0.013) (Figure 6). Two additional products of the PBAN precursor were detected in the ALs: α-SGNP and PBAN-PP-3. However, α-SGNP was discarded from the analysis due to a potential masking from CAPA-PVK-3 (m/z 816.44) and the corresponding ion signal for PBAN-PP-3 showed no significant difference between physiological states or sexes. Although PBAN acts in females to induce pheromone production in the pheromone gland 77, its presence has been detected in male brains from many moth species including A. ipsilon, but its role in this sex is so far unclear 71. In A. ipsilon, newly-mated males show an inhibition of behavioral and neuronal responses to sex pheromone 41. The higher occurrence of γ-SGNP in ALs of mated males could participate in this post-mating olfactory plasticity. IRP-1 showed significant elevated occurrence in mated males compared to all other states (MM~VM, p = 0.009; MM~VF, p = 0.009; MM~MF, p = 0.012) (Figure 6). IRP-2 revealed a significant higher occurrence in mated males compared to virgin males (VM~MM, p = 0.01)


29


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(Figure 6). So far, insulin has only been detected in ALs of T. casteneum 70. Its presence in mated A. ipsilon males could reflect a possible role of these peptides in the initiation of pheromone sensing switch-off. Moreover, the possible involvement of insulin in the switchoff of A. ipsilon males is opposite to that found in Drosophila, in which insulin signaling was found to increase sexual behavior

78

. In Drosophila, insulin signaling modulates olfactory

sensitivity through the action of sNPF receptors

79

. Indeed, olfactory sensory neurons

projecting to a specific AL glomerulus express insulin receptors and therefore could be subject to insulin modulation 79. Finally, CAPA-PVK-2 [pQ] showed a significantly lower detection ratio in virgin males in contrast to mated males and virgin females (VM~MM, p = 0.01; VM~VF, p = 0.03) (Figure 6). In insects, CAPA has different roles including myotropic, diuretic or anti-diuretic effects 80

. Recently, mature peptides from PBAN and CAPA genes were also detected in the moths

Heliothis peltigera and Spodoptera littoralis 81. Its differential mating-induced occurrence in ALs of A. ipsilon males remains unexplained. In contrast, the second analyzed CAPA product, CAPA-PVK-1, showed no significant differences between the compared states. The remaining two detected CAPA products, CAPA-PVK-3 (m/z 816.44) and CAPAtryptopyrokinin (CAPA-tryptoPK) (m/z 1376.68), were excluded from the detailed analysis due to potential masking from α-SGNP (m/z 816.53) and YRVamide (m/z 1374.76). Products of all three precursors (CAPA, PBAN and NPLP) were detected in AL spectra, which hampered a robust recording of CAPA-PVK-3 and CAPA-tryptoPK ion signal occurrences from these samples.


30

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Figure 6. Significant changes in ion signal occurrences as recorded by direct MALDI-TOF MS profiling of mated/virgin, male and female antennal lobes (AL), corpora cardiacacorpora allata (CC-CA) and gnathal ganglion (GNG) dissections. Occurrences were compared using generalized linear models (GLMs). Y-axis median normalized detection ratio of corresponding ion signal, x-axis sex and physiological state; VM =


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virgin male, VF = virgin female, MM = mated male, MF = mated female. Significance level, * = p 3.5% contribution, 10 signals) originated from a precursor-related peptide, which are hypothesized to have no function in the CNS. Only one precursor product showed significant changes in occurrence;


34

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Cor was significantly less detected in virgin males compared to all other states (VM~MM, p = 0.002; VM~VF p = 0.02; VM~MF p = 0.002) (Figure 6). It was the only product detected in any sample of the cor-precursor. In Drosophila, it has been shown that Cor is involved in the regulation of feeding 87, nutrient-sensing 88, sperm transfer and copulation as well as fecundity 89

. Three additional products of three neuropeptide precursors were found to show significant

difference in occurrence when states were compared to MF samples (for which sample set was not as consistent as the remaining sets and was only represented by four samples); SIFa showed a reduced occurrence in MF preparations compared to all other states (MF~VF, p = 0.003; MF~MM, p = 0.003; MF~VM p = 0.025) (Figure 6). It was the only product detected from the corresponding precursor in any sample. RFa-3 was significantly less often detected in MF samples compared to VF and MM preparations (MF~VF p = 0.013; MF~MM p = 0.032) while in comparison to VM only a trend was observed (MF~VM p = 0.057) (Figure 6). Two additional products of the FMRF-precursor, RFa-1 and 5, were detected in GNG dissections but showed no significant difference in their occurrence between states or sexes. PTSP-6 showed significant changes in occurrence between different states. In MF preparations PTSP-6 showed a significant lower occurrence compared to VF and MM (MF~VF, p = 0.009; MF~MM p = 0.009), while VM showed trends compared to VF and MM preparations (VM~MM, p = 0.09; VM~VF p = 0.09) (Figure 6). Three additional products of the MIP/AstB/PTSP prothoracicostatic peptide-precursor were detected in GNG samples, PTSP-5, 7 and 8. PTSP-7 and 8 (m/z 997.50, m/z 1359.67) were excluded from a detailed analysis due to a possible overlap with sNPF-1 (m/z 1359.81) and AST-A8 (m/z 995.57). Detected ion signals of PTSP-5 showed no significant differences between states. Products of all three precursors were present in GNG dissections.


35


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CONCLUSIONS The combinatorial approach of transcriptomics, mass spectrometry and well-defined dissections of parts of the nervous system opens the opportunity to study neuropeptidergic signaling systems in great detail, even in organisms with unknown genome. Moreover, it allows comparison of neuropeptide content between different physiological states and sexes. By using next generation-sequencing transcriptomics we were able to identify most of the regularly detected neuropeptide precursors from B. mori 43

28

25

and C. suppressalis (total genes:

) in A. ipsilon (total genes: 37). From these precursors we were able to identify 54

putative bioactive neuropeptides (from 23 neuropeptide precursors) by nLC-MALDI-TOF MS/MS and direct MALDI-TOF MS, representing a large part but not the full mature peptide repertoire. Some neuropeptides usually observed in Lepidoptera could not be identified in this study, neither in the brain transcriptome nor in the tissue peptidomes. Although higher coverage of the transcriptome may lead to the identification of additional peptide precursors, it has to be noticed that we only investigated brain tissues in a restricted time window of the photoperiod and of the insect life, focusing only on adults and of specific ages at a specific time (5-day-old males, 3-day-old females during mid-scotophase). Thus we cannot exclude that the unidentified peptides would indeed be expressed in different conditions or in different tissues, such as the ventral nerve cord, the midgut or other non-neuronal tissues. Furthermore, by comparing recorded MALDI-TOF MS fingerprints of virgin/mated male and female specimens we revealed that mating causes differential detection of products of a few neuropeptide precursors in the analyzed tissues, likely corresponding to differential expression levels of genes. These findings will help to understand single peptide function in physiological processes and will also support the development of pseudopeptides, peptidomimetics and other targeted pesticides.


36

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AUTHOR INFORMATION Corresponding author : *

E-mail : [email protected]

Phone: +33 2 41 22 56 55

Present Addresses a

Institut für Zoologie, Universität zu Köln, D-50674 Cologne, Germany

b

Laboratoire de biométrie et biologie évolutive, CNRS, Université Claude Bernard, F-69622

Villeurbanne cedex, France c

INRA, Institut Jean-Pierre Bourgin, AgroParisTech, CNRS, Université Paris-Saclay, F-

78000 Versailles, France d

Senckenberg Research Institute and Natural History Museum, Senckenberganlage 25, 60325,

Frankfurt am Main, Germany

Funding Sources This work was supported by the Plant Health and Environment Department at INRA.

ACKNOWLEDGMENTS We thank Martina Kern and Corinne Chauvet for technical assistance and insect rearing.

Supporting Information Table S1: Overview of identified neuropeptide sequences recorded from virgin and mated males and females A. ipsilon antennal lobe (AL), corpora cardiaca-corpora allata (CC-CA) and gnathal ganglion (GNG) by nLC-MALDI-TOF and their occurrence


37


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(expressed as % of sample group) in mass spectrometric fingerprints from directprofiled tissue preparations. Figure S1: GO analyses of Agrotis ipsilon male brain transcriptome data, as predicted for their involvement in biological processes, molecular functions, and cellular components. Figure S2: Sequence confirmation of putative neuropeptide ion signals by post source decay of corresponding ion precursors; Only b- and y-fragments are labelled. Recorded ion signals are labelled as monoisotopic masses [M+H]+. Figure S3 : Representative fingerprint mass spectrum of a virgin female A. ipsilon dissection. Figure S4 : Comparison between recorded mass fingerprints of mated and virgin female A. ipsilon AL dissections. Figure S5 : Comparison between recorded mass fingerprints of mated and virgin male A. ipsilon AL dissections. Figure S6 : Comparison between recorded mass fingerprints of mated and virgin female A. ipsilon CC-CA dissections. Figure S7 : Comparison between recorded mass fingerprints of mated and virgin male A. ipsilon CC-CA dissections. Figure S8 : Comparison between recorded mass fingerprints of mated and virgin female A. ipsilon GNG dissections. Figure S9 : Comparison between recorded mass fingerprints of mated and virgin male A. ipsilon GNG dissections.

REFERENCES (1)

Nässel, D. R., Neuropeptides in the nervous system of Drosophila and other insects:

multiple roles as neuromodulators and neurohormones. Prog. Neurobiol. 2002, 68, (1), 1-84.


38

Page 39 of 62 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


(2)

Hull, J. J.; Matsumoto, S., Molecular mechanisms underlying insect behaviors:

receptors, peptides, and biosynthetic pathways. Front. Endocrinol. 2013, 4, 120. (3)

Veenstra, J. A., Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine

peptide precursors. Arch. Insect Biochem. Physiol. 2000, 43, (2), 49-63. (4)

Rholam, M.; Fahy, C., Processing of peptide and hormone precursors at the dibasic

cleavage site. Cell. Mol. Life Sci. 2009, 66, 2075. (5)

Han, M.; Park, D.; Vanderzalm, P. J.; Mains, R. E.; Eipper, B. A.; Taghert, P. H.,

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2. J. Neurochem. 2004, 90, (1), 129-141. (6)

Garden, R. W.; Moroz, T. P.; Gleeson, J. M.; Floyd, P. D.; Li, L. J.; Rubakhin, S. S.;

Sweedler, J. V., Formation of N-pyroglutamyl peptides from N-Glu and N-Gln precursors in Aplysia neurons. J. Neurochem. 1999, 72, (2), 676-681. (7)

Hewes, R. S.; Taghert, P. H., Neuropeptides and neuropeptide receptors in the

Drosophila melanogaster genome. Genome Res. 2001, 11, (6), 1126-42. (8)

Predel, R.; Wegener, C.; Russell, W. K.; Tichy, S. E.; Russell, D. H.; Nachman, R. J.,

Peptidomics of CNS-associated neurohemal systems of adult Drosophila melanogaster: A mass spectrometric survey of peptides from individual flies. J. Comp. Neurol. 2004, 474, (3), 379-392. (9)

Yew, J. Y.; Wang, Y.; Barteneva, N.; Dikler, S.; Kutz-Naber, K. K.; Li, L.; Kravitz, E.

A., Analysis of Neuropeptide Expression and Localization in Adult Drosophila melanogaster Central Nervous System by Affinity Cell-Capture Mass Spectrometry. J. Proteome Res. 2009, 8, 1271-1284. (10)

Hummon, A. B.; Richmond, T. A.; Verleyen, P.; Baggerman, G.; Huybrechts, J.;

Ewing, M. A.; Vierstraete, E.; Rodriguez-Zas, S. L.; Schoofs, L.; Robinson, G. E.; Sweedler,


39


Page 40 of 62

J. V., From the genome to the proteome: uncovering peptides in the Apis brain. Science 2006, 314, (5799), 647-9. (11)

Li, B.; Predel, R.; Neupert, S.; Hauser, F.; Tanaka, Y.; Cazzamali, G.; Williamson, M.;

Arakane, Y.; Verleyen, P.; Schoofs, L.; Schachtner, J.; Grimmelikhuijzen, C. J.; Park, Y., Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum. Genome Res. 2008, 18, (1), 113-22. (12)

Hauser, F.; Cazzamali, G.; Williamson, M.; Park, Y.; Li, B.; Tanaka, Y.; Predel, R.;

Neupert, S.; Schachtner, J.; Verleyen, P.; Grimmelikhuijzen, C. J. P., A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum. Frontiers in Neuroendocrinology 2008, 29, (1), 142-165. (13)

Consortium, T. G. S.; leader, P.; investigators, P.; paper, W.; leaders, A.; sequencing,

D. s. a. g. a. D.; sequencing, E.; assembly, g.; content, G. C.; repetitive DNA, t. a. t.; set, g. p. a. c. g.; analysis, g. g. c.; pathways, D. p. a. s.; Pest biology, s., Medea and RNAi: ligand gated ion channels; phosphorylation, o.; genes, P.; proteins, c. a. c.; proteinases, d.; immunity; GPCRs, n. a.; enzymes, n. p.; opsins; receptors, o. r. a. g.; proteins, o. b. a. c.; Medea; RNAi, s.; Richards, S.; Gibbs, R. A.; Weinstock, G. M.; Brown, S. J.; Denell, R.; Beeman, R. W.; Gibbs, R.; Beeman, R. W.; Brown, S. J.; Bucher, G.; Friedrich, M.; Grimmelikhuijzen, C. J. P.; Klingler, M.; Lorenzen, M.; Richards, S.; Roth, S.; Schröder, R.; Tautz, D.; Zdobnov, E. M.; Muzny, D.; Gibbs, R. A.; Weinstock, G. M.; Attaway, T.; Bell, S.; Buhay, C. J.; Chandrabose, M. N.; Chavez, D.; Clerk-Blankenburg, K. P.; Cree, A.; Dao, M.; Davis, C.; Chacko, J.; Dinh, H.; Dugan-Rocha, S.; Fowler, G.; Garner, T. T.; Garnes, J.; Gnirke, A.; Hawes, A.; Hernandez, J.; Hines, S.; Holder, M.; Hume, J.; Jhangiani, S. N.; Joshi, V.; Khan, Z. M.; Jackson, L.; Kovar, C.; Kowis, A.; Lee, S.; Lewis, L. R.; Margolis, J.; Morgan, M.; Nazareth, L. V.; Nguyen, N.; Okwuonu, G.; Parker, D.; Richards, S.; Ruiz, S. J.; Santibanez, J.; Savard, J.; Scherer, S. E.; Schneider, B.; Sodergren, E.; Tautz, D.; Vattahil, S.; Villasana,


40

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D.; White, C. S.; Wright, R.; Park, Y.; Beeman, R. W.; Lord, J.; Oppert, B.; Lorenzen, M.; Brown, S.; Wang, L.; Savard, J.; Tautz, D.; Richards, S.; Weinstock, G.; Gibbs, R. A.; Liu, Y.; Worley, K.; Weinstock, G.; Elsik, C. G.; Reese, J. T.; Elhaik, E.; Landan, G.; Graur, D.; Arensburger, P.; Atkinson, P.; Beeman, R. W.; Beidler, J.; Brown, S. J.; Demuth, J. P.; Drury, D. W.; Du, Y.-Z.; Fujiwara, H.; Lorenzen, M.; Maselli, V.; Osanai, M.; Park, Y.; Robertson, H. M.; Tu, Z.; Wang, J.-j.; Wang, S.; Richards, S.; Song, H.; Zhang, L.; Sodergren, E.; Werner, D.; Stanke, M.; Morgenstern, B.; Solovyev, V.; Kosarev, P.; Brown, G.; Chen, H.-C.; Ermolaeva, O.; Hlavina, W.; Kapustin, Y.; Kiryutin, B.; Kitts, P.; Maglott, D.; Pruitt, K.; Sapojnikov, V.; Souvorov, A.; Mackey, A. J.; Waterhouse, R. M.; Wyder, S.; Zdobnov, E. M.; Zdobnov, E. M.; Wyder, S.; Kriventseva, E. V.; Kadowaki, T.; Bork, P.; Aranda, M.; Bao, R.; Beermann, A.; Berns, N.; Bolognesi, R.; Bonneton, F.; Bopp, D.; Brown, S. J.; Bucher, G.; Butts, T.; Chaumot, A.; Denell, R. E.; Ferrier, D. E. K.; Friedrich, M.; Gordon, C. M.; Jindra, M.; Klingler, M.; Lan, Q.; Lattorff, H. M. G.; Laudet, V.; von Levetsow, C.; Liu, Z.; Lutz, R.; Lynch, J. A.; da Fonseca, R. N.; Posnien, N.; Reuter, R.; Roth, S.; Savard, J.; Schinko, J. B.; Schmitt, C.; Schoppmeier, M.; Schröder, R.; Shippy, T. D.; Simonnet, F.; Marques-Souza, H.; Tautz, D.; Tomoyasu, Y.; Trauner, J.; van der Zee, M.; Vervoort, M.; Wittkopp, N.; Wimmer, E. A.; Yang, X.; Jones, A. K.; Sattelle, D. B.; Ebert, P. R.; Nelson, D.; Scott, J. G.; Beeman, R. W.; Muthukrishnan, S.; Kramer, K. J.; Arakane, Y.; Beeman, R. W.; Zhu, Q.; Hogenkamp, D.; Dixit, R.; Oppert, B.; Jiang, H.; Zou, Z.; Marshall, J.; Elpidina, E.; Vinokurov, K.; Oppert, C.; Zou, Z.; Evans, J.; Lu, Z.; Zhao, P.; Sumathipala, N.; Altincicek, B.; Vilcinskas, A.; Williams, M.; Hultmark, D.; Hetru, C.; Jiang, H.; Grimmelikhuijzen, C. J. P.; Hauser, F.; Cazzamali, G.; Williamson, M.; Park, Y.; Li, B.; Tanaka, Y.; Predel, R.; Neupert, S.; Schachtner, J.; Verleyen, P.; Raible, F.; Bork, P.; Friedrich, M.; Walden, K. K. O.; Robertson, H. M.; Angeli, S.; Foret, S.; Bucher, G.; Schuetz, S.; Maleszka, R.; Wimmer, E. A.; Beeman, R. W.; Lorenzen, M.; Tomoyasu, Y.; Miller, S.


41


Page 42 of 62

C.; Grossmann, D.; Bucher, G., The genome of the model beetle and pest Tribolium castaneum. Nature 2008, 452, (7190), 949-955. (14)

Hauser, F.; Neupert, S.; Williamson, M.; Predel, R.; Tanaka, Y.; Grimmelikhuijzen, C.

J. P., Genomics and Peptidomics of Neuropeptides and Protein Hormones Present in the Parasitic Wasp Nasonia vitripennis. J. Proteome Res. 2010, 9, (10), 5296-5310. (15)

Predel, R.; Neupert, S.; Garczynski, S. F.; Crim, J. W.; Brown, M. R.; Russell, W. K.;

Kahnt, J.; Russell, D. H.; Nachman, R. J., Neuropeptidomics of the Mosquito Aedes aegypti. J. Proteome Res. 2010, 9, (4), 2006-2015. (16)

Christ, P.; Hill, S. R.; Schachtner, J.; Hauser, F.; Ignell, R., Functional characterization

of the dual allatostatin-A receptors in mosquitoes. Peptides 2018, 99, 44-55. (17)

Schmitt, F.; Vanselow, J. T.; Schlosser, A.; Kahnt, J.; Roessler, W.; Wegener, C.,

Neuropeptidomics of the Carpenter Ant Camponotus floridanus. J. Proteome Res. 2015, 14, (3), 1504-1514. (18)

Caers, J.; Boonen, K.; Van Den Abbeele, J.; Van Rompay, L.; Schoofs, L.; Van Hiel,

M. B., Peptidomics of Neuropeptidergic Tissues of the Tsetse Fly Glossina morsitans morsitans. J. Am. Soc. Mass Spectrom. 2015, 12, 2024-2038. (19)

Huybrechts, J.; Bonhomme, J.; Minoli, S.; Prunier-Leterme, N.; Dombrovsky, A.;

Abdel-Latief, M.; Robichon, A.; Veenstra, J. A.; Tagu, D., Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisum. Insect Mol. Biol. 2010, 19 Suppl 2, 87-95. (20)

Kirkness, E. F.; Haas, B. J.; Sun, W.; Braig, H. R.; Perotti, M. A.; Clark, J. M.; Lee, S.

H.; Robertson, H. M.; Kennedy, R. C.; Elhaik, E.; Gerlach, D.; Kriventseva, E. V.; Elsik, C. G.; Graur, D.; Hill, C. A.; Veenstra, J. A.; Walenz, B.; Tubio, J. M.; Ribeiro, J. M.; Rozas, J.; Johnston, J. S.; Reese, J. T.; Popadic, A.; Tojo, M.; Raoult, D.; Reed, D. L.; Tomoyasu, Y.; Kraus, E.; Mittapalli, O.; Margam, V. M.; Li, H. M.; Meyer, J. M.; Johnson, R. M.; RomeroSeverson, J.; Vanzee, J. P.; Alvarez-Ponce, D.; Vieira, F. G.; Aguade, M.; Guirao-Rico, S.;


42

Page 43 of 62 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


Anzola, J. M.; Yoon, K. S.; Strycharz, J. P.; Unger, M. F.; Christley, S.; Lobo, N. F.; Seufferheld, M. J.; Wang, N.; Dasch, G. A.; Struchiner, C. J.; Madey, G.; Hannick, L. I.; Bidwell, S.; Joardar, V.; Caler, E.; Shao, R.; Barker, S. C.; Cameron, S.; Bruggner, R. V.; Regier, A.; Johnson, J.; Viswanathan, L.; Utterback, T. R.; Sutton, G. G.; Lawson, D.; Waterhouse, R. M.; Venter, J. C.; Strausberg, R. L.; Berenbaum, M. R.; Collins, F. H.; Zdobnov, E. M.; Pittendrigh, B. R., Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle. Proc. Natl. Acad. Sci. U S A 2010, 107, (27), 12168-73. (21)

Badisco, L.; Huybrechts, J.; Simonet, G.; Verlinden, H.; Marchal, E.; Huybrechts, R.;

Schoofs, L.; De Loof, A.; Vanden Broeck, J., Transcriptome analysis of the desert locust central nervous system: production and annotation of a Schistocerca gregaria EST database. PLoS One 2011, 6, (3), e17274. (22)

Kang, L.; Chen, X.; Zhou, Y.; Liu, B.; Zheng, W.; Li, R.; Wang, J.; Yu, J., The

analysis of large-scale gene expression correlated to the phase changes of the migratory locust. Proc. Natl. Acad. Sci. U S A 2004, 101, (51), 17611-5. (23)

Tanaka, Y.; Suetsugu, Y.; Yamamoto, K.; Noda, H.; Shinoda, T., Transcriptome

analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens. Peptides 2014, 53, 125-133. (24)

Ons, S.; Sterkel, M.; Diambra, L.; Urlaub, H.; Rivera-Pomar, R., Neuropeptide

precursor gene discovery in the Chagas disease vector Rhodnius prolixus. Insect Mol. Biol. 2011, 20, (1), 29-44. (25)

Roller, L.; Yamanaka, N.; Watanabe, K.; DaubnerovA, I.; Zitňan, D.; Kataoka, H.;

Tanaka, Y., The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 2008, 38, (12), 1147-1157.


43


(26)

Page 44 of 62

Zhan, S.; Merlin, C.; Boore, J. L.; Reppert, S. M., The monarch butterfly genome

yields insights into long-distance migration. Cell 2011, 147, (5), 1171-85. (27)

Kanost, M. R.; Arrese, E. L.; Cao, X.; Chen, Y.-R.; Chellapilla, S.; Goldsmith, M. R.;

Grosse-Wilde, E.; Heckel, D. G.; Herndon, N.; Jiang, H.; Papanicolaou, A.; Qu, J.; Soulages, J. L.; Vogel, H.; Walters, J.; Waterhouse, R. M.; Ahn, S.-J.; Almeida, F. C.; An, C.; Aqrawi, P.; Bretschneider, A.; Bryant, W. B.; Bucks, S.; Chao, H.; Chevignon, G.; Christen, J. M.; Clarke, D. F.; Dittmer, N. T.; Ferguson, L. C. F.; Garavelou, S.; Gordon, K. H. J.; Gunaratna, R. T.; Han, Y.; Hauser, F.; He, Y.; Heidel-Fischer, H.; Hirsh, A.; Hu, Y.; Jiang, H.; Kalra, D.; Klinner, C.; Konig, C.; Kovar, C.; Kroll, A. R.; Kuwar, S. S.; Lee, S. L.; Lehman, R.; Li, K.; Li, Z.; Liang, H.; Lovelace, S.; Lu, Z.; Mansfield, J. H.; McCulloch, K. J.; Mathew, T.; Morton, B.; Muzny, D. M.; McCulloch, K. J.; Mathew, T.; Morton, B.; Muzny, D. M.; Neunemann, D.; Ongeri, F.; Pauchet, Y.; Pu, L.-L.; Pyrousis, I.; Rao, X. J.; Redding, A.; Roesel, C.; Sanchez-Gracia, A.; Schaack, S.; Shukla, A.; Tetreau, G.; Wang, Y.; Xiong, G.H.; Traut, W.; Walsh, T. K.; Worley, K. C.; Wu, D.; Wu, W.; Wu, Y.-Q.; Zhang, X.; Zou, Z.; Zucker, H.; Briscoe, A. D.; Burmester, T.; Clem, R. J.; Feyereisen, R.; Grimmelikhuijzen, C. J. P.; Hamodrakas, S. J.; Hansson, B. S.; Huguet, E.; Jermiin, L. S.; Lan, Q.; Lehman, H. K.; Lorenzen, M.; Merzendorfer, H.; Michalopoulos, I.; Morton, D. B.; Muthukrishnan, S.; Oakeshott, J. G.; Palmer, W.; Park, Y.; Passarelli, A. L.; Rozas, J.; Schwartz, L. M.; Smith, W.; Southgate, A.; Vilcinskas, A.; Vogt, R.; Wang, P.; Werren, J.; Yu, X.-Q.; Zhou, J.-J.; Brown, S. J.; Scherer, S. E.; Richards, S.; Blissard, G. W., Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta. Insect Biochem. Mol. Biol. 2016, 76, 118-147. (28)

Xu, G.; Gu, G.-X.; Teng, Z.-W.; Wu, S.-F.; Huang, J.; Song, Q.; Ye, G.-Y.,

Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis. Sci. Rep. 2016, 6, 28976.


44

Page 45 of 62 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


(29)

Altstein, M., Novel insect control agents based on neuropeptide antagonists: The

PK/PBAN family as a case study. J. Mol. Neurosci. 2004, 22, (1-2), 147-57. (30)

Rafaeli, A., Pheromone biosynthesis activating neuropeptide (PBAN): regulatory role

and mode of action. Gen. Comp. Endocrinol. 2009, 162, (1), 69-78. (31)

Anton, S.; Homberg, U., Antennal lobe structure. In Insect olfaction, Hansson, B. S.,

Ed. Springer: Berlin, 1999; pp 98-125. (32)

Hansson, B. S., Olfaction in Lepidoptera. Experientia 1995, 51, 1003-1027.

(33)

Koontz, M. A.; Schneider, D., Sexual dimorphism in neuronal projections from the

antennae of silkmoths (Bombyx mori, Antheraea polyphemus) and the gypsy moth (Lymantria dispar). Cell Tissue Res. 1987, 249, 39-50. (34)

Schachtner, J.; Schmidt, M.; Homberg, U., Organization and evolutionary trends of

primary olfactory brain centers in Tetraconata (Crustacea plus Hexapoda). Arthropod Struct. Dev. 2005, 34, (3), 257-299. (35)

Anderson, P.; Hansson, B. S.; Nilsson, U.; Han, Q.; Sjöholm, M.; Skals, N.; Anton, S.,

Increased Behavioral and Neuronal Sensitivity to Sex Pheromone after Brief Odor Experience in a Moth. Chem. Senses 2007, 32, 483–491. (36)

Anton, S.; Evengaard, K.; Barrozo, R. B.; Anderson, P.; Skals, N., Brief predator

sound exposure elicits behavioral and neuronal long-term sensitization in the olfactory system of an insect. Proc. Natl. Acad. Sci. U S A 2011, 108, (8), 3401-5. (37)

Gadenne, C.; Barrozo, R. B.; Anton, S., Plasticity in Insect Olfaction: To Smell or Not

to Smell? Annu. Rev. Entomol. 2016, 61, (1), 317-333. (38)

Anton, S.; Dufour, M. C.; Gadenne, C., Plasticity of olfactory-guided behaviour and

its neurobiological basis: lessons from moths and locusts. Entomol. Exp. Appl. 2007, 123, 111.


45


(39)

Page 46 of 62

Gadenne, C.; Renou, M.; Sreng, L., Hormonal control of sex pheromone

responsiveness in the male black cutworm, Agrotis ipsilon. Experientia 1993, 49, 721-724. (40)

Anton, S.; Gadenne, C., Effect of juvenile hormone on the central nervous processing

of sex pheromone in an insect. Proc. Natl. Acad. Sci. U S A 1999, 96, (10), 5764-5767. (41)

Gadenne, C.; Dufour, M. C.; Anton, S., Transient post-mating inhibition of

behavioural and central nervous responses to sex pheromone in an insect. Proc. R. Soc. London B 2001, 268, (1476), 1631-5. (42)

Barrozo, R. B.; Gadenne, C.; Anton, S., Switching attraction to inhibition: mating-

induced reversed role of sex pheromone in an insect. J. Exp. Biol. 2010, 213, (Pt 17), 2933-9. (43)

Duportets, L.; Barrozo, R. B.; Bozzolan, F.; Gaertner, C.; Anton, S.; Gadenne, C.;

Debernard, S., Cloning of an octopamine/tyramine receptor and plasticity of its expression as a function of adult sexual maturation in the male moth Agrotis ipsilon. Insect. Mol. Biol. 2010, 19, (4), 489-99. (44)

Duportets, L.; Bozzolan, F.; Abrieux, A.; Maria, A.; Gadenne, C.; Debernard, S., The

transcription factor Kruppel homolog 1 is linked to the juvenile hormone-dependent maturation of sexual behavior in the male moth, Agrotis ipsilon. Gen. Comp. Endocrinol. 2012, 176, (2), 158-66. (45)

Duportets, L.; Maria, A.; Vitecek, S.; Gadenne, C.; Debernard, S., Steroid hormone

signaling is involved in the age-dependent behavioral response to sex pheromone in the adult male moth Agrotis ipsilon. Gen. Comp. Endocrinol. 2013, 186, 58-66. (46)

Jarriault, D.; Barrozo, R. B.; de Carvalho Pinto, C. J.; Greiner, B.; Dufour, M. C.;

Masante-Roca, I.; Gramsbergen, J. B.; Anton, S.; Gadenne, C., Age-dependent plasticity of sex pheromone response in the moth, Agrotis ipsilon: combined effects of octopamine and juvenile hormone. Horm. Behav. 2009, 56, (1), 185-91.


46

Page 47 of 62 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


(47)

Vitecek, S.; Maria, A.; Blais, C.; Duportets, L.; Gaertner, C.; Dufour, M.-C.; Siaussat,

D.; Debernard, S.; Gadenne, C., Is the rapid post-mating inhibition of pheromone response triggered by ecdysteroids or other factors from the sex accessory glands in the male moth Agrotis ipsilon? Horm. Behav. 2013, 63, (5), 700-708. (48)

Duportets, L.; Gadenne, C.; Couillaud, F., A cDNA, from Agrotis ipsilon, that encodes

the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides. Peptides 1999, 20, (8), 899-905. (49)

Ito, K.; Shinomiya, K.; Ito, M.; Armstrong, J. D.; Boyan, G.; Hartenstein, V.; Harzsch,

S.; Heisenberg, M.; Homberg, U.; Jenett, A.; Keshishian, H.; Restifo, L. L.; Rössler, W.; Simpson, J. H.; Strausfeld, N. J.; Strauss, R.; Vosshall, L. B.; Group, I. B. N. W., A Systematic Nomenclature for the Insect Brain. Neuron 2014, 81, (4), 755-765. (50)

Poitout, S.; Buès, R., Elevage de chenilles de vingt-huit espèces de Lépidoptères

Noctuidae et de deux espèces d'Arctiidae sur milieu artificiel simple. Particularités de l'élevage selon les espèces. Ann. Zool. Ecol. anim. 1974, 6, (3), 431-441. (51)

Schachtner, J.; Wegener, C.; Neupert, S.; Predel, R., Direct peptide profiling of brain

tissue by MALDI-TOF mass spectrometry. Methods Mol. Biol. 2010, 615, 129-135. (52)

Martin, M., Cutadapt removes adapter sequences from high-throughput sequencing

reads. EMBnet. J. 2011, 17, 10-12. (53)

Schmieder, R.; Edwards, R., Fast Identification and Removal of Sequence

Contamination from Genomic and Metagenomic Datasets. PLoS One 2011, 6, (3), e17288. (54)

Grabherr, M. G.; Haas, B. J.; Yassour, M.; Levin, J. Z.; Thompson, D. A.; Amit, I.;

Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.; Chen, Z.; Mauceli, E.; Hacohen, N.; Gnirke, A.; Rhind, N.; di Palma, F.; Birren, B. W.; Nusbaum, C.; Lindblad-Toh, K.; Friedman, N.; Regev, A., Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotech. 2011, 29, (7), 644-652.


47


(55)

Page 48 of 62

Chevreux, B.; Pfisterer, T.; Drescher, B.; Driesel, A. J.; Müller, W. E. G.; Wetter, T.;

Suhai, S., Using the miraEST Assembler for Reliable and Automated mRNA Transcript Assembly and SNP Detection in Sequenced ESTs. Genome Res. 2004, 14, (6), 1147-1159. (56)

Conesa, A.; Gotz, S.; Garcia-Gomez, J. M.; Terol, J.; Talon, M.; Robles, M.,

Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, (18), 3674-6. (57)

Gouzy, J.; Carrere, S.; Schiex, T., FrameDP: sensitive peptide detection on noisy

matured sequences. Bioinformatics 2009, 25, (5), 670-1. (58)

Petersen, T. N.; Brunak, S.; von Heijne, G.; Nielsen, H., SignalP 4.0: discriminating

signal peptides from transmembrane regions. Nat. Methods 2011, 8, (10), 785-6. (59)

Southey, B. R.; Amare, A.; Zimmerman, T. A.; Rodriguez-Zas, S. L.; Sweedler, J. V.,

NeuroPred: a tool to predict cleavage sites in neuropeptide precursors and provide the masses of the resulting peptides. Nucleic Acids Res. 2006, 34, (Web Server issue), W267-72. (60)

Clarke, K. R., Nonparametric Multivariate Analyses of Changes in Community

Structure. Aust. J. Ecol. 1993, 18, (1), 117-143. (61)

Oksanen, J.; Blanchet Guillaume, F.; Friendly, M.; Kindt, R.; Legendre, P.; McGlinn,

D.; Minchin, P.; O'Hara, R.; Simpson, G.; Solymos, P.; Stevens, M.; Szoecs, E.; Wagner, H. vegan: Community Ecology Package. R Package, Version 2.4-4. https://CRAN.Rproject.org/package=vegan, 2017. (62)

Wickham, H., ggplot2: Elegant Graphics for Data Analysis. Springer: Verlag New

York, 2009; p 260. (63)

Venables, W. N.; Ripley, B. D., Modern Applied Statistics with S. Springer: 2002; p

495. (64)

Morales, M. Sciplot: Scientific Graphing Functions for Factorial Designs., R package

version 1.1-1 https://CRAN.R-project.org/package=sciplot, 2017.


48

Page 49 of 62 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


(65)

Abdel-Latief, M.; Hoffmann, K. H., The adipokinetic hormones in the fall armyworm,

Spodoptera frugiperda: cDNA cloning, quantitative real time RT-PCR analysis, and gene specific localization. Insect Biochem. Mol. Biol. 2007, 37, (10), 999-1014. (66)

Jiang, H.; Lkhagva, A.; DaubnerovA, I.; Chae, H.-S.; Simo, L.; Jung, S.-H.; Yoon, Y.-

K.; Lee, N.-R.; Seong, J. Y.; Zitňan, D.; Park, Y.; Kim, Y.-J., Natalisin, a tachykinin-like signaling system, regulates sexual activity and fecundity in insects. Proc. Natl. Acad. Sci. U S A 2013, 110, (37), E3526-E3534. (67)

Verleyen, P.; Huybrechts, J.; Baggerman, G.; Van Lommel, A.; De Loof, A.; Schoofs,

L., SIFamide is a highly conserved neuropeptide: a comparative study in different insect species. Biochem. Biophys. Res. Commun. 2004, 320, (2), 334-341. (68)

Carlsson, M. A.; Diesner, M.; Schachtner, J.; Nässel, D. R., Multiple neuropeptides in

the Drosophila antennal lobe suggest complex modulatory circuits. J. Comp. Neurol. 2010, 518, (16), 3359-3380. (69)

Siju, K. P.; Reifenrath, A.; Scheiblich, H.; Neupert, S.; Predel, R.; Hansson, B. S.;

Schachtner, J.; Ignell, R., Neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti. J. Comp. Neurol. 2014, 522, (3), 592-608. (70)

Binzer, M.; Heuer, C. M.; Kollmann, M.; Kahnt, J.; Hauser, F.; Grimmelikhuijzen, C.

J. P.; Schachtner, J., Neuropeptidome of Tribolium castaneum antennal lobes and mushroom bodies. J. Comp. Neurol. 2014, 522, (2), 337-357. (71)

Duportets, L.; Gadenne, C.; Dufour, M.; Couillaud, F., The pheromone biosynthesis

activating

neuropeptide

(PBAN)

of

the

black

cutworm

moth,

Agrotis

ipsilon:

immunohistochemistry, molecular characterization and bioassay of its peptide sequence. Insect Biochem. Mol. Biol. 1998, 28, (8), 591-599.


49


(72)

Page 50 of 62

Berg, B. G.; Schachtner, J.; Utz, S.; Homberg, U., Distribution of neuropeptides in the

primary olfactory center of the heliothine moth Heliothis virescens. Cell Tissue Res. 2007, 327, (2), 385-398. (73)

Kromann, S. H.; Hansson, B. S.; Ignell, R., Distribution of neuropeptides in the

antennal lobes of male Spodoptera littoralis. Cell Tissue Res. 2013, 354, (2), 431-440. (74)

Utz, S.; Huetteroth, W.; Vömel, M.; Schachtner, J., Mas-allatotropin in the developing

antennal lobe of the sphinx moth Manduca sexta: distribution, time course, developmental regulation, and colocalization with other neuropeptides. Dev. Neurobiol. 2008, 68, (1), 123142. (75)

Zhao, X. C.; Xie, G.-Y.; Berg, B. G.; Schachtner, J.; Homberg, U., Distribution of

tachykinin-related peptides in the brain of the tobacco budworm Heliothis virescens. J. Comp. Neurol. 2017, 525, (18), 3918-3934. (76)

Utz, S.; Huetteroth, W.; Wegener, C.; Kahnt, J.; Predel, R.; Schachtner, J., Direct

peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development. Dev. Neurobiol. 2007, 67, (6), 764-777. (77)

Raina, A. K.; Klun, J. A., Brain factor control of sex pheromone production in the

female corn earworm moth. Science 1984, 225, 531-532. (78)

Kuo, T.-H.; Fedina, T. Y.; Hansen, I.; Dreisewerd, K.; Dierick, H. A.; Yew, J. Y.;

Pletcher, S. D., Insulin signaling mediates sexual attractiveness in Drosophila. PLoS Genet. 2012, 8, (4), e1002684. (79)

Root, C. M.; Ko, K. I.; Jafari, A.; Wang, J. W., Presynaptic Facilitation by

Neuropeptide Signaling Mediates Odor-Driven Food Search. Cell 2011, 145, (1), 133-144. (80)

Predel, R.; Wegener, C., Biology of the CAPA peptides in insects. Cell. Mol. Life Sci.

2006, 63, (21), 2477-2490.


50

Page 51 of 62 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


(81)

Ragionieri, L.; Özbagci, B.; Neupert, S.; Salts, Y.; Davidovitch, M.; Altstein, M.;

Predel, R., Identification of mature peptides from pban and capa genes of the moths Heliothis peltigera and Spodoptera littoralis. Peptides 2017, 94, 1-9. (82)

Ma, P. W. K.; Roelofs, W. L., Sites of synthesis and release of PBAN-like factor in

the female european corn borer, Ostrinia nubilalis. J. Insect Physiol. 1995, 41, 339-350. (83)

Christensen, T. A.; Itagaki, H.; Teal, P. E. A.; Jasensky, R. D.; Tumlinson, J. H.;

Hildebrand, J. G., Innervation and neural regulation of the sex pheromone gland in female Heliothis moths. Proc. Natl. Acad. Sci. U S A 1991, 88, 4971-4975. (84)

Krajniak, K. G., Invertebrate FMRFamide Related Peptides. Protein Peptide Lett.

2013, 20, (6), 647-670. (85)

Raina, A. K., Male-induced termination of sex pheromone production and receptivity

in mated females of Heliothis zea. J. Insect Physiol. 1989, 35, 821-826. (86)

Nagalakshimi, V. K.; Applebaum, S. W.; Azrielli, A.; Rafaeli, A., Female sex

pheromone suppression and the fate of sex-peptide-like peptides in mated moths of Helicoverpa armigera. Arch. Insect Biochem. Physiol. 2007, 64, (3), 142-155. (87)

Kubrak, O. I.; Lushchak, O. V.; Zandawala, M.; Nässel, D. R., Systemic corazonin

signalling modulates stress responses and metabolism in Drosophila. Open Biol. 2016, 6, (11). (88)

Miyamoto, T.; Amrein, H., Diverse roles for the Drosophila fructose sensor Gr43a. Fly

2014, 8, (1), 19-25. (89)

Bergland, A. O.; Chae, H.-S.; Kim, Y.-J.; Tatar, M., Fine-Scale Mapping of Natural

Variation in Fly Fecundity Identifies Neuronal Domain of Expression and Function of an Aquaporin. PLoS Genet. 2012, 8, (4), 215-222.


51


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Figure legends Figure 1. Alignment of AKH neuropeptides from Bombyx mori (Bmor, AB298930, AB298931, AB298938), Spodoptera frugiperda (Sfru, AJ871195, AJ871196, AJ871197, AM269886) and Agrotis ipsilon (Aips, this study).

Figure 2. Alignment of IMFamide neuropeptides from Lepidoptera. Harm: Helicoverpa armigera (AGH25559), Dple: Danaus plexippus (EHJ78184), Bmor: Bombyx mori (NP_001124359), Csup: Chilo suppressalis (KT005973), Aips: Agrotis ipsilon (this study).

Figure 3. Exemplary sequence confirmation of putative neuropeptide ion signals by post source decay of corresponding ion precursors; (A) m/z 1246.73 (Myosuppressin), (B) m/z 1369.63 (Corazonin) and (C) m/z 1486.61 (Allatotropin). Only b- and y-fragments are labelled. Recorded ion signals are labelled as monoisotopic masses [M+H]+.

Figure 4. Representative mass spectra obtained by direct tissue profiling of virgin male A. ipsilon antennal lobe (AL) (A), corpora cardiaca-corpora allata (CC-CA) (B) and gnathal ganglion (GNG) (C). Left y-axis relative signal intensity, right y-axis signal intensity in absolute counts. X-axis mass/charge. Unlabeled ion signals or signals without a peptide-name represent unidentified signals. Masses marked by an asterisk represent [M+Na]+, masses with a tilde represent [M+K]+, all other masses are labelled as monoisotopic masses [M+H]+. CCCA (A2) mass spectrum was cropped to m/z 3010 for better visualization.

Figure 5. Non-metric multidimensional scaling (nMDS) ordination plot (based on Jaccard similarities) for analyzed tissue types (light blue: antennal lobe (AL); yellow: corpora cardiaca-corpora allata (CC-CA); red: gnathal ganglion (GNG)) and their corresponding sexes


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and physiological states. Tissue types differed highly significantly between each other (nested PERMANOVA: p < 0.001, R² = 0.603; one-way ANOSIM: global R = 0.81, p < 0.001; AL~CC-CA, R = 0.99, p < 0.001; AL~GNG, R = 0.65, p < 0.001; CC-CA~GNG, R = 0.86, p

Mating-induced differential peptidomics of neuropeptides and protein

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