Leucokinin 1MVVNKSLLLILLQVFAGFFLTCSGQGGCNGACNNMETFEDQSLIDSEPIEEKRKFSPWAGKRGGPVLPSYIIRTRTNSKPNVITHKKSRFSPWHGKRSAAGISDPELSDNLLERLLLQYTDSDIGRLQKLLEKRGFNPWAGK* 

Leucokinin 2MQRNKRILLIVVQVVAGLLVSCSAQIGCQGSTDCHNGGDEQSLINSESTEDKRKFNPWGGKRAGPVFSVYDESQSDGTSEYSAESKRSFSPWAGKRTAQSSLGYVVRARDARADRKKTRFHPWHGKRSGSPLGSEVPENLLRQLVFEYIDLDSNKLSDKRGFNPWAGK* 

Luqin or cardioexcitatory peptideMKISELILSITAVLLIALTIADGSPAPKWRPQGRFGKRLSELNDDPLWVLLSSESEKREDILPYGVANKPVHFENLLCVPVGVKNAYKCTRPES*

MyomodulinMNLTLTLICVLLCLQQLRQGACEDNESNNNNNNAVETGATPALRERRAVGMLRLGRGVQMLRLGKRAPYDDLKTIVATILGRQEQQFNRQAPLPRYGKEEGLLVDAYPADSPSQVSQILRRSYPSYFEDEDLLHQEAPLPHLGYLQKRSAEIRHAPLPRYGKGPDYEDLTNEVSSEEGGDDHEDENVSDTVSSEMFERQPPLPRYGKDEIAGIDCEAYDESGNCLRFEDIEKKDVRMLRMGRQVNMLRMGKRALSMLRMGRNGGDKRAVSMLRLGRSDNVEDSKRALAMLRLGRSGEKRAVSMLRLGRSGLDEESMPSEEQKRALAMLRLGRSGSEEQKRAVSMLRLGRSGEGDQKRAVSMLRLGRSGPDGQKRAVSMLRLGRNFPVSDAEKRAVSMLRLGRSGSDEEKRAVSMLRLGRSAEEEAEKRAVSMLRLGRSGADKRAVSMLRLGRRNQGADDEKRAVSMLRLGRSGPESDESKRAVSMLRLGRSGPETDESKRAVSMLRLGRSGPETEESKRAVSMLRLGRSDEKFGADKRAVSMLRLGRSDKNTDADKRAVSMLRLGRSAEEEAEKRAVSMLRLGRSGADKRAVSMLRLGRRNQGADEEKRAVSMLRLGRSGPESDESKRAVSMLRLGRSGPETDESKRAVSMLRLGRSG…… 

NKY 1MQNLTSHIVIFALCCIGLTISGDLWQGNRPHADKNLLSLITRATAARDNALMMPPSGYQGRLPSYYDRPLSKREPLWIWMPAQGYVPVPRTSNINNSDGSGSSVIRYG* 

NKY 2MAKVVFFMLLSAMVAILSPFCRASSEQMNQPQAVASFRTDHEKEALASLLLHVLIQRSIAPVYSSHPHWSNLASKAEMPSQLKKKDTRYRYRGIDSRVPAFGSFFSPSPSDNSDTSKIFRYG* 

NKY 3MTVNAVHVLCIFALLFACAHSLPKRTDHASTLRYLQQSGLSDSDSRALLQAYLLGKLSNGDGSIGKELETSEYPTIKRKAFWRPMGYLPFENHVGSGASSSNDNAAGTGSASAVFRYG*

Characterization of the neuropeptidome of the cuttlefish Sepia officinalis: identification of

neuropeptides and neurohormones involved in the regulation of egg-laying.Céline Zatylny-Gaudin1,2, Valérie Cornet1,2, Alexandre Leduc1,2, Bruno Zanuttini3, Erwan Corre4, Gildas Le Corguillé4, Benoît Bernay5, Alexandra Kraut6 and Joël Henry1,2,5

Like many other cephalopods, the cuttlefish Sepia officinalis exhibits a wide variety of behaviors such as prey capture, communication, camouflage and reproduction, thanks to a complex central nervous system (CNS) divided into several functional lobes that express a wide range of neuropeptides. However, the diversity of these neuropeptides is crucial to modulate behavior and physiological mechanisms associated with the main stages of their life cycle.This work focuses on the neuropeptidome expressed during egg-laying, the last step of reproduction. We first identified neuropeptide transcripts through de novo construction of the CNS transcriptome using an RNASeq approach (Illumina sequencing). Then we completed the in silico analysis of the transcriptome by characterizing and tissue-mapping neuropeptides by mass spectrometry. To identify neuropeptides involved in the egg-laying process, we determined (1) the neuropeptide contents of the neurohemal area, haemolymph (blood) and nerve endings in mature females, and (2) the expression levels of these peptides. Among the 38 neuropeptide families identified from 54 transcripts, 30 were described for the first time in Sepia officinalis, 5 were described for the first time in the animal kingdom, and 14 were strongly over-expressed in egg-laying females as compared to mature males.Mass spectrometry screening of haemolymph and nerve ending contents allowed us to clarify the functional status of many neuropeptides: neuromodulators and/or neurohormones. Besides the data concerning egg-laying regulation in cephalopods, this work brings very new and important structural and expression data about the neuropeptidome of S. officinalis.

1Normandy University, Caen, France E-mail: joel.henry@unicaen.fr, 2Université de Caen Basse-Normandie, UMR BOREA MNHN, UPMC, UCBN, CNRS-7208, IRD-207, F-14032 Caen, France, 3Normandy University, GREYC, UMR CNRS 6072, F-14032 Caen, France, 4UPMC, CNRS, FR2424, ABiMS, Station Biologique, 29680, Roscoff, France, 5Post Genomic platform PROTEOGEN, Université de Caen Basse-Normandie, SF ICORE 4206, F-14032 Caen, France, 6Proteomics platform EDyP / BGE / iRTSV / CEA / INSERM U1038 / UJF, F-38054 Grenoble, France.

Sepia officinalis

Phylum: MolluscaClass: CephalopodaOrder: SepiidaFamily: SepiidaeGenus: Sepia

In silico data mining usingthe homemade software PEPTRAQ

38 neuropeptide families expressed by 58 protein precursors

5 novel neuropeptides families notyet described in the animal kingdom

14 neuropeptides families over expressed in egg-laying females

8 neuropeptides families detected in the nerve endingsof female accessory sex glands

Large stocks of neuropeptide mRNAs at the levelof ovary and female accessory sex glands

Achatin 2MVKVTSVCLCVLIGLVVLFDSWTDASCAKPCLINFFKCVRGENDGCCSVYGGCMKESCGSATVQCDDSMGKRGSWNKRGSWNKRGSWDKRGSWNKRDAAEEKRGSWNKRAEDIEISQRGSWNKRADANAEDYSEAILRRLLLENYGARL*

Allatostatin A1 or buccalin 1MTSVGVWSRALLCSLFISVHMVDCADSLKTNLSDEQILSDSSDNEEHKVEKKRSADPTLFTDTLKKRNVPERILTNGKDIHVISRGMDPMMFGHLGKRPDPQMFDNINQRMDPFMFGNLGKRFDPMLYGNLGKREHSIALGPLNKKMDPMMFGGLGKKMDPLMFGGLGKKMDPMMFGGLGKKMDPMMF……

Allatostatin A2 or buccalin 2MTSVGVWSRALLCSLFISVHMVDCADSLKTNLSDEQILSDSSDNEEHKVEKKRSADPTLFTDTLKKRNVPERILTNGKDIHVISRGMDPMMFGHLGKRPDPQMFDNINQRMDPFMFGNLGKRFDPMLYGNLGKREHSIALGPLNKKMDPMMFGGLGKKMDPMMFGSLGKRDVSEDTLDESTD*

AllatotropinMNVSGARGLCTLCCLGLLVLLASSDAHASVVVPNRPQRGFKDNVSNRIAHGFGKRTFQDTYDLAPSLDDSKNLITPRKLAELIMYDRNLAYIVALKLDSNGDGVISMNELILKDYV*

Cholecystokinin MNHHFVEIGMSFALLAIIVTHLTTAAPSPYVESDMTHSLNNYKQQLMNALRKRSEDEATNHVLTASARHFMSSAFHKKRSGLAAQKKRDLDLAGRDDDASKRRGAWYYDYGLGGGRFGKRKDYGYTDDYGIGGGRFGRDVDHVDLLDA*

FaRPsMRCWSPCSLLVVIAIYCLSSHTSEAFDLAQACVESQRLSLLPICDTIFAVQQEGAQQSADDGLRSKRFIRFGRALSGDAFLRFGKNVPDLPFEDKRFLRFGRAAPQLDDLLKQALQRVESLQKSDDTSVRRKRSTDAAPQSNTDSAEQKNDSAKITKRYVDDVEDSDVKRFMRFGKRFMRFGRNPSDVGSKLTEKRFMRFGRDPEKRFMRFGKSDDKRFMRFGRNPGDAEDELEEDKRFMRFGRGDEEDEEEAEKRFMRFGRDPEKKFMRFGKNGEEKRFMRFGRNPEEPEADKRFMRFGRGGEEDDVNTEEKRFMRFGRSAEKCKGCLEG*

FLGamideMAFSQILVLLLGVSYVMTAPKSAADEAPVDLKKRGAESGEAHVFDSLGGGHVPYYKRYLEDNDVKRVFDTLGGGHVPYYKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFL……… ……KRSFDSLGGGSFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRTFDSLGGGSFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRGFDSLGGGSFIGVKRIHELDNMDGGSGDN* ……KRSFDSLGGGSFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRTFDSLGGGSFLGGKRSFDSLGGGAFLGGKRSFDSLGGGAFLGGKRGFDSLGGGSFIGVKRIHELDNMDGGSGHNPPCCSGTS*

FVRIamide MVSFWKILILGTIGVLVLMTQWASFVRAESPSNGEDLVNAAGAAVESADEPSGRSVSDSPYDIKRTNQFLRIGRGSHFIRIGRGGASSFLRIGRNPLSQFVRIGKAPSSMFLRIGKSSAAGNPELGDLAAGPSSLGDNSIDEDEVLKRASSFLRIGRSNPSTFLRIGKSAGNLDEETAANEDIVTDDIDVPSESMEKRANAFLRIGKIPASSFVRIGRGPYGIDNRSNPRGFLSVGSRFVRIGKREAIPSETGPTHARLLPNLHDQAQ*

GnRHMSTSALSSNLRKMAFLTCAILLLSFCMQIQAQNYHFSNGWHPGGKRSGLPDMQCHFRPQTKALIEKLLDEEITRIITTCTNTVNDIADLQ*

Insulin MKSSTVCMGTFLLATLLSIVNWQVVNAGLEHTCNEETIRQGPAQGAHCGVEIPNILQLLCAPAGYNERMSDRQRRNLVPTSRAIGRRGNGLRDIIISKRQAKSYLTKRDRNWTGIVCECCYNKCILEELLDYCKDPSYFKSQKLRS*

LASGLXamideMWKLGIFLLGIWLVPQSVLCKDHIEKRSLDPLASGLIGKKRDIDDEKEEIDQKSKRQFDHLASGLIGKRPF…………

…………SLASGLIGKRPFDHLASGLIGKRPFDPLASGLIGKRPFDPLASGLIGKRPFDSLASGLIGKRPFDHLASGLIGKRPFDSLASGLIGKRPFDSLASGLIGKRPFDHLASGLIGKRPFDPLASGLIGKRSFDPLASGLIGKKSFDPLASGLIGKRR*

……………RPFDSLASGLIGKRELDTLASGLIGKRDNDSEMEDKRSFDPLASGLIGKKSFDPLASGLIGKRR*

LFRFamideMETKVMSLLATVLTVFIVQINCEDLHKIQTDTSGISNFIGLPDGEEGELVRSPIVDESALGIDDVDKRNSLFRFGKRGNLFRFGKRGNLFRFGKRGNLFRFGRGGNKDDPENEGLKRTIFRFGKRDGLEDLYDYEDPSVQQVAPTAGDKRGSFFRYGRSRTFFRYGRSTDKNAEKRPHTPFRFGREEE*

Neuropeptide Y3 (NPF3 or NPY3)MQKSFFVILLIAVMFTGQVFSQEGLLAAPKIPGELSEYLKALSDYYAIAARPRFGRSLKQRTSYRTLADDA*

Neuropeptide L11 or eleveninMLKLRSSTFQKFLIWTFVLLLLNLHVNAQNKFELTKKLNCRKFIFAPRCRGVAAKRSLNMATSYPPVNTQMADERNYITGNHDSTIREILLNYILSRLEARSLFQNDANTDSAEDLTSYR*

Pedal Peptide 2MISFHNRLFVVILSLLGQNLLISTYADIQGTSHVSYVHENPEEKRQLDSIGSGLIKKKNIDSIGSGLIKKNLDSIGAGLIKKNLDSIGAGLIKKNLDSIGSGLVRRNFDSIGSGLIKKNFDSIGSGLIKKNLDLIGSGLVRRNFDLIGSGLIKKNFDTVGSGLVRRNFDSVGSGLIKKNLDAIGSGLVRRNFDSIGSGLIKKNLDRIGSGLVRRNFDSIGSGLIKKNLDSIGSGLVRRNFDSIGSGLIKKNLDSIGSG……………………………DFDSIGSGLIKKNLDSIGSGLVRRNFDSIGSGLIKKNLNLDSIGSGLVDSVGAGLIKKNLDTIGMGLIRRQYENGMEMGDDNGYEGDKRHLDYIGSGLIKRSM*

Pro-SepiatocinMGSGRFLFSSTKCQVACVLFNFCVFLICTTDACFFRNCPPGGKRAVAMNDGVAHKQCMACGPEGKGRCAGPNICCQKEGCIIGDMAKECMQEDEGTTVCEVKGIPCGAEGQGRCVAAGVCCDTSACSTNSHCGSALPRTSSRRQELFSLLKRLINKVN*

PRQFVamideMRIYWPMYVVALLCASGQDFNAAEPDAVKNLVKDTVLKPLADSSYLNLPDEHTQDNALSEIVSGTEDGDIPEPLIGYSEEDLPKRPMEFLGKRAMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLG…………………

MRIYWPMYVVALLCASGQGQGKGKNAQHFNAAEPDAVKNLVKDTVLKPLADSSYLNLPDEHTQDNALSEIVSGTEDGDIPEPLIGYSEEDLPKRPMEFLGKRAMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLG……

……………………RPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRSDESEDKRRMEFLGKRRMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRGMEFLGKRPMEFLGKRRMEFLGKRPMEFLGKTSLNSLDPRPWHNSSETLAGQADREHHDFLDDSLNALEPDRKHHFLDEMIDKGALGRYLQEDMSILDQGDKPLPEGHDINKVSDEEIPTRMHLGSNPVSVKHKREIPIFVGKREQSISLNKRNVDPVLEEMLTIKPSERDREKENIMSNSVREILKNIQMNEKTDNQKADTDFEGTAGESDFHDEQFGEKPSIYEKKAYQMFVGKKNKPMFVGKKNKPMFVGKKNNPMFVGKRDNPMFVGKKTNPMFEEEMANPMFAGKRDDPMFVGKRNNLMIFDEKARPVDVTKKAKPLFVGKKEKIGPENKKDSMFPLNSNLILAEKKDPMFVGRKEDPILFSINGNPLLMDDTKNPTFTDENDKPVSVSKKDDPMFVGKKSKPMFVGKKNHPISISKKYSPRFVSKKEFPLVVNRERISLVGDNRNTPIFVSKVYSKKVLGKKNNPAFIVANEDPMSVDKKYLKVSTESRNPTNTNGYKIPLAEKDYPQPSLHDYGVFSSSPIYVDHSLMKNKKNKPMFVGKRKLPRLSTNWNESMTFKDHFDEPSPTHKRFSKRSIMLYRLRTLPPHHRRLNYLDVLHFILAREELIKKDGMKWHNQQTPIFVGKREDNPMFV………

………GKKSKPMFVGKKSKPMFVGKKSKPMFVGKKNNPMFVGKKSKPMFVGKKNNPMFVGKKSNPIFLDNPMLTGKRSKPMFVGKKNNLMFLALKDNPRFVGKKSKPMFVGKKNHPISISKKYSPRFVSKKEFPLVVNRERISLVGDNRNTPIFVSKVYSKKVLGKKNNPAFIVANEDPMSVDKKYLKVSTESRNPTNTNGYKIPLAEKDYPQPSLHDYGVFSSSPIYVDHSLMKNKKNKPMFVGKRKLPRLSTNWNESMTFKDHFDEPSPTHKRFSKRSIMLYRLRTLPPHHRRLNYLDVLHFILAREELIKKDGMKWHNQQTPIFVGKREDNPMFV… …

……KREFQPMLMGKRDDNPMFVGKRDLQPMFVGKRDDNPMFVGRRVDNPIFLGNRGLQPMFAVEKELQPMLVGKRDDNPMFLRTGVLREIGKRDNKPMFVG……

……KRDDNPMFIGKRDPSPMFLGKRTDNPMISSKAGLQPMFVGRRMDNPMFVGKRNPQVMFVGKRVHNPMFVGKRDPQPIFVGKKNNKPMFVGKRENNQMFAGKRKDHQIFVGQRIAQPLTLERTYSRRMFVGERGPKIMSVRRGGDNPMFIGKKVPEPMFVGKKENKPMFVGKRDFLPMFVGKMDPQPMFMGKREDKPMFIGKREPQTMFLKISEPEPMFVGKRQTQTMFVEKRGTFPSFVEKTDTQPMLIWKRGLQPLFVQRRISMPRFFFGSYKNMKYWHKRQPIPLFVGKRETESLFRGNAERQSLFVGKKEVMPMFVGKKNNQPSAWGKHLSQPMFIRNRNVPMFVGKRGGILAPETKRLPNQSLFDVADFDSSIEEIDNSLNKIQHRRDTSGVQDRLTNDISHKELDQPIVKASFKEKHMNDNFNQGIVSNNSSQSILRNRNDLSTDGNSRQVIDFLNPNKIERNIVKRSTGKYYNQMPVSKESNVSKRSRYSEENFIGQRYLHDDAPSEILSINKRNTPLTFGKSNFPHRSQQRSNSLSLVLDQSSPHKSRFVHSEMALSGPYPADEQTHFLKRNDLPNYEFRLAAAGTKPEPMIETGQTKNDHFGKMESEDIPFGSKHQSTNKSFRANGSDTIVVASRYWKPAGVAVNVRDFRNSAVSAPASQVR*

……RPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRPMEFLGGKRSDESEDKRRMEFLGKRRMEFLGKRPMEFLGKRPMEFLGKRPMEFLGKRGMEFLGKRPMEFLGKRRMEFLGKRPMEFLG*

SamideMTRNLLVVVLVAILVSTLANGRYIADTKLRSKRQTGDLKAAAYQAWLALGRTLPPDCPEVACGVVDVEASGKKKRTDTNSVDFRRSLLVERLLRLAAEGLVNSV*

TAamideMWSSQIVVFVLTVCVCGSLCLTRTKRAVLTGDENPYDLREALRVLERERRRLELRTLDPVSQREVRFEQQPVIVDSENDDPDFLARAVAEKGDSVFYSSDQANDVENLLREIEDQKEAENEASEDDLQTVYGRPLPDTEMDVDVDNYPSVVEKRTPTKKAVSRKRVTIKGLDLTAGKRSAETLKTVFNSLSPEEIHKLLLLEGRLQTKEMNKRQRKARPLVVETRAQSDTGLEDAMEVEDAAAIPITKEELKKLIQGQGEADQIEDPNDINNFNQMNEIIQAESAPADPSLDQPVDSDIFKSALQDLASLQMLQKQENDGRSEEQEALTQWGPAAMTKVVPVLLRPVPVLTQSRTAPVRVDEGRKMVKGIAAKPEPSLTTTPSLAEMMAKLWIKHKLEGIEIDYLADALNAATLTQSGVSPGVNNIPVEISSLQKAIRIEKLMKELGGNDADIEEDGMLDQLALKYLQRHQLQAEDSGIALSKKSFFDDEIEAEARKEKEEEEEESAAAAAAAAARQQQEEEEEEEEEEEEYRDNLLRGDVNRQIPLLYEIPDNKINQIPSLPRPDQQNRIPYQLMDIPDDSDREEDKESDEIPLSPDIRLLNTEDFY*

Tachykinin MHTVRVSTFQLLNCVCYVVLISGGLGNTAWGVLASYLPQTPESQQDKTAQVDESPVEKGFIDNNDILKSLSKLHMLAIGEPNDASSDPSSGLVLPIYEQEQEETADLVDDIAGDESPDEMKRFSPYAFQGSRGKKMMTSIEDKKAHASLGFVGSRGKRQPASLGFVGSRGKKQLNFIPSRGRKQMSTAFVGSRGRRISAEAFAPSRGRRLSSQAFFGSRGKKYSALGFMGSRGKKDSDFENLLLKNEDGWTQDNYNRRAAPFYHGFVASRGKRSAETNSA*

Achatin 1MAGQCKCLSSLVVILLITLSARGGFSNYVQNYNSRQLLSLHSFNDDQMNKDRLTALEESFKSDGAGKRGFGDKRSIHVPHIIVETRGFGDKRDEKNDESIDTGKFRPIPRGFGDKRGFGDKRGFGDKKSLAQMDNYEPKWIHLVNRGFGDKRGFGDKRYFLDRRGFGDKRGFGDKRGFGDKRGFGDKRGFGDKRAFDDDTGFDDKRGFGDK* 

Allatostatin A3MNAYILSAILLFVNAQHIKCDSKNLADFYPKLRDEYVKPYMQLDGGKYFPWKLLDLVPSREKFSRTAFPIIAANKKIDPMLFKLGIGKRNSGSVDPFLFKIGLGKKSVRRVGEDGEQMWQMKDRRKGIESFQNGFGTEARPHVFLSSKNKEDINAENADIFSDVNM* 

APGWamideMKNQELFCKGTSLCLSRHSLLIVLLLVLPVFSTNSGQSSRNPADNEASSLVQESISRLLEASVDGSLSDEDDSDDDDYGSLTNLDDRTVKRAPGWGKRAPGWGKRAPGWGKRAPGWGKRAPGWGKRTSPKLDSDEIATMLFSLENADNGEDTNAFEKRAPGWGKRAPGWGKRTVMNEDLASFLASLNAAETAEASSDTDKRAPGWGKRAPGWGKRAPGWGKRAPGWGKRDQEKRAPGWGKRAPGWGKRDTEKRAPGWGKRDTQGHNIAATN* 

Bursicon AMMERNSNLAMYNSQFVTTCLFIFVLLHPIASIRRFSTSPSLGIISEGDVFDDTNQWCHLRKFIHTINFRRCLPQNFRSYVCSGKCHSYTGADPNITTYGNSEMSLMLRACRCCQKLSVMSTRVVLKCRDDETGIYRRMQVKLQVPTRCICRHCSI* 

Bursicon BMHLIPLASLLMFAIRHVSSQDCETLGSEIRVNKFLNVEHHGRQMNVRCGVQLSLNKCEGTCWSYESPSVIDPRGFRKKCNCCRERELVDRSVVLDECHDAVSGLIVTGLHPVIVIKEPMNCECRECANFI*

CerebrinMQKAIIIFAALTLFSTAFVTDVFASQEEISEIVSLASRILKIAMLMDKPRRIDKRNHGLIDTIINLPDLDKIGKK*

ClioninMVKPTAFFTVVLVIAAVFMKPANLYPTPSVPLEEMDPRDACLFQCSNCFGDQYQPLLDCANDVCPKVKSFSEVGYECSTILRHPNLGRRSLFKF* 

Crustacean Cardioactive Peptides (CCAPs)MQSVSSSLSTGLFFLTVNVLCVSTLIRVTSSQPQENTFQMKPSESDILNMKIRHLIRRAVQRQPTANDFSNEELASLPDKRVFCNSFGGCTNIKRLFDEENDFKLVASDSIQPMEQETAIDKRVFCNSYGGCKSFKRVSANDIKKPEMIKNKQRHLIRRLKLQPIKISRRVFCNSFGGCQN* 

FFamideMSGRLAFHSSLVLLICFALFFGQDSAVEAVYAPTRGQQNPHSYGRRGLNPNVNSLFFGKRAGAEQEALSNTDMGRKCMAAMSMCNMYFESNNVNES* 

GGNamideMGRHFILFVLTSALLFCISNAVKCKWYDHICLGGNGKRSAIGQSEQDQQLLKILTQEFIRQPHKRDSFITDVNDDDDDDAIFPHGKPFDQDYGQTNLSPTIKRLAKLMLPDERQN*

Neuropeptide Y1 (NPY1 or NPF1)MQKATIILLLLVAMFSADAYSQNNGGAAPQSPEELTNYLKALNEYYAIVARPRFGRSIIQKRSFLGSLADAA*

Neuropeptide Y2 (NPY2 or NPF2)MLSPMLTIFLIAVMLAANVSGQNGLLGPPNRPGDLKNPGVLNNYLKALNEYQDALSRPRFGRSSSKRFSFNEFANNLPERIE* 

Neuropeptide Y4 (NPY4 or NPF4)MRKSFVIVFVIAVVLVIQISSQEIMLSPPSRPAEFRNPKELREYMKALNEYYAIVGRPRFGRSIFNKRFGSNSFNEDLKSDENKE* 

Neuropeptide Y5 (NPY5 or NPF5)MQKIVIASLLVVLFTLNVSSQDSLLAPPSRPSEFRTPEELRQYLKALNEYYAIVGRPRFGRSAVNRFTRTAIAKARTDP* 

NdWFamide-likeMKPACICLIVILAASIFQTNANYYGKRDEKLDGFREFLSKRMGELNEQEIEARDVLRTISSLVQAWEARQRKYDTLQKAA* 

Orcokinin BMRYWFCACFVLLQNSLISVSGVEKVNKKSHHSENEATLAHHGGNDGHGSTISEKRSFDSIDGGMFRTMGKRPFDSISDSAFGGMGKRQFDSISHSSFRQMGKRSFDSIDGSAFGGMGKKSFDSIASSGFGGMGKRPFDSIDSSAFGGMGKKSFDSIASSGFGGMGK…… 

PKYMDT (Proctolin)MDSRLLAFVSVCLFLLTSPVFSAPAADPKPHLEQSKDLPISKRPKYMDTREPQDIFKDLVFLTLQQLVSDGKVNPEAITDTDAGVPNKRGYQGLCLRRTANQRYIAYPCWRTGSK* 

PTSP-like PeptideMASIFSHFLVIMILALTQTRRLIAEEKNDIGSSKSLKPESVVKSENLKSWSKRSTTNGKALNAIRQAVARGAFSGPANPLDSSDERYWNLMMLWLKENGYPSTTVNAGGRRTGLRSRVARETDGTDEELLEKKDRPDTWNSMNTWGKRSPNTWDSMAAWGKRNPNTWDSMAAWGKRNGD……… 

……NPDTWDSMSAWGKRSPDTWDSMSAWGKRGADTWDSMSAWGKRGADTWDSMSAWGKRNGDSKDKRDWDSLQAWGKRANAKNKKDWDSLAAWGKRDIAGDGNDEIGSQVQSLMKRSSGKSSKSRR* 

……KRNPNTWDSMAAWGKRNPNTWDSMAAWGKRNGDTWDSMSAWGKRNPDTWDSMSAWGKRGADTWDSMSAWGKRGADTWDSMSAWGKRNGDSKDKRDWDSLQAWGKRANAKNKKDWDSLAAWGKRDIAGDGNDEIGSQVQSLMKRSSGKSSKSRR* 

PXXXamideMTRNLLVVVLVAILVSTLANGRYIADTKLSSYKRTSSDQRIAELQALIALSNTIGHGQVNPEEIGKKKRTDTNSVDFRRSLLVERLLRLAAEGLVNSV* 

Small Cardioactive Peptide (SCP)MFSQNLSVLAFSVCILLTMANTSYGYLVLPRQGRSDDRAEPSCCGMPLMKATGLCPIGMECCPGLKKVLQKSGQKTVYSICIADLY* 

Sepiatocin MASYRWGSWALLLLIVVLPLVSLVEGCFWTTCPIGGKRSASEFRECMACGPEGKGRCAGPNICCQKEGCIIGDMAKECMQEDEGTTVCEVKGIPCGAEGQGRCVAAGVCCDTSACSTNSHCGSALPRTSSRRQELFSLLKRLINKVN* 

SPamide 1MTRVVLLLLMFIQLVQIRANYPFLEQVEKVEEKNLENLLLRMLMERTNKFGRPVKRTCQIEATGECRSEEAAEVADKYHYLLSSKSPGRKRNLFS* 

SPamide 2MAPLQYILPLLLVLPIIAAWNPVLRSNEINSIRRAALLKHNEDSSDFGVYQRNGRDASDLQRAFSDYLKSSVEDSKSAWSDPCRLNLGGRCATEIASDLVKAWHYLNSSNSPGRKRRDVREALRTILRHSAAAAAAAAADNR* 

Urotensin IIMDSQLQTKQFLTLFCFCLCFVAIAKAMPAPQDPSQEEILAKRWLYRMLDREGRATYPLNLAALRELESNLRLGMSNVKRGSNVPSRASRGGMGLCLWKVCPTAPWMRST*

Transcripto-peptidomic strategy

neurohormones neuromodulators

XXXXXX: Signal peptideRR KK RK KR: Convertase cleavage sites

Q: Predicted N-terminal pyroglutamic acidG: Predicted C-terminal amidation

PMEFLG: C-terminal amidated neuropeptide detected by mass spectrometry

Subesophagal massSupesophagal massOptic lobesOptic glands

Previtellogenic folliclesVitellogenic folliclesMature oocytes

Oviduct gland (OG)Accessory nidamental glandsMain nidamental glands

Ventral view of egg-laying female

16 transcriptomes

Posterior salivary glands

CNS(M/F)

Ovary

ASGs(F)

(M/F)

RNAseq de novo

- Allatostatin 2 and 3- Crustacean Cardio Active Peptides (CCAPs)- FaRPs- FLGamide- LFRFamide- Myomodulin- PTSP-like peptide neurotransmitter- Small Cardioactive Peptide

- Allatostatin 3- FaRPs- FLGamide- Myomodulin

Oviduct Gland

Main Nidamental Glands

Ovarian stroma- FLGamide

Neuropeptides detected in the nerveendings of ASGs of egg laying females

Neuropeptides over expressed in the sub-esophagal mass of egg laying females versus mature males

Neuropeptide mRNAs recovered in ovary and ASGsexpressed as a ratio of CNS mRNAs