peptides 28 (2007) 57–61 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides Structure-activity relationships for in vitro diuretic activity of CAP2b in the housefly Ronald J. Nachmana,*, Geoffrey M. Coastb aAreawidePestManagementResearch,SouthernPlainsAgriculturalResearchCenter,USDA,2881F/BRoad, CollegeStation,TX77845,USA bSchoolofBiologicalandChemicalSciences,Birkbeck(UniversityofLondon),LondonWC1E7HX,UK article info a bs t r ac t Articlehistory: A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b Received11September2006 (pELYAFPRV-NH )wereassayedfordiureticactivityonMalpighiantubulesofthehousefly 2 Receivedinrevisedform Muscadomestica(M.domestica).TheC-terminalhexapeptideprovedtobetheactivecore,the 15September2006 minimumsequencerequiredtoretainsignificantdiureticactivity.However,fullactivity Accepted15September2006 requiredtheC-terminalheptapeptide,whichwasequipotentwiththemostactiveofthe Publishedonline8December2006 nativehouseflyCAP2bpeptides.ReplacementofArg7andVal8withAlaledtoinactivity and a large 70-fold drop in potency, respectively, indicating that these were critical Keywords: residues. The Leu2 was semicritical, where a six-fold loss in potency was observed. CAPAgene Conversely,thereplacementofallotherresidueswithAlaledtomuchsmallereffectson Periviscerokinin potency and these positions were considered to be noncritical. This structure-activity CAP2b relationship data can aid in the design of mimetic agonist/antagonist analogs of this Perisympatheticorgans diuretic peptide family with enhanced biostability and bioavailability, as tools for Insectneuropeptide arthropodendocrinologistsandaspotentialpestmanagementagentscapableofdisrupting Malpighiantubule thewaterbalanceinpestflies. #2006ElsevierInc.Allrightsreserved. 1. Introduction bioassays on the bug Rhodnius prolixus with Manduca CAP2b [23,29] indicated that native CAPA peptides might reduce It is well established that neuropeptides are key factors in secretion by Malpighian tubules and thus show antidiuretic controlling primary urine production in Malpighian tubules effects. These peptides are putative hormones typical of the (CRF-like peptides, insect kinins, CAPA peptides) and fluid neurosecretory system in the abdominal ventral nerve cord reabsorption from the hindgut (ion transport peptide (ITP)) (VNC)ofinsects.TheCAPAgene,whichencodesfortwoormore [1,29].CAP2b(pELYAFPRVa),aCAPApeptide(alsoincludedin CAP2b/PVKs and a single pyrokinin (PK), is known from a the literature as a member of the periviscerokinin (PVK) number of holometabolous insects, including D. melanogaster family), was first identified as a cardioacceleratory factor in [12,13]andM.sexta[14].Expressedinmedianneurosecretory themothManducasexta[11].CAPApeptides,includingCAP2b, neurons of abdominal ganglia, these putative peptide hor- demonstrate diuretic effects on the Malpighian tubules of a mones may be releasedintothe hemolymph via perisympa- numberofflies,includingthefruitflyDrosophilamelanogaster, thetic organs (PSOs) which are segmentally reiterated thehouseflyMuscadomestica(M.domestica),andthestablefly neurohemalorgansoftheabdominalganglia[7,8].LarvalPSOs Stomoxyscalcitrans[1,2–5,9,10,15,18,21,22,24–26,28].Bycontrast, of cycloraphous Diptera, however, become incorporated into * Correspondingauthor.Tel.:+19792609315;fax:+19792609377. E-mailaddress:[email protected](R.J.Nachman). 0196-9781/$–seefrontmatter#2006ElsevierInc.Allrightsreserved. doi:10.1016/j.peptides.2006.09.020 Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 3. DATES COVERED 15 SEP 2006 2. REPORT TYPE 00-00-2006 to 00-00-2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Structure-activity relationships for in vitro diuretic activity of CAP2b in 5b. GRANT NUMBER the housefly 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION U.S. Department of Agriculture,Areawide Pest Management REPORT NUMBER Research,2881 F/B Road,College Station,TX,77845 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT see report 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE Same as 5 unclassified unclassified unclassified Report (SAR) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 58 peptides 28 (2007) 57–61 thedorsalganglionicsheath[20,30,31]duringmetamorphosis. Manse-CAP2b[4-8], 11.5min; Manse-CAP2b[5-8], 10.5min; DirectanalysisofabdominaldorsalsheathtissuesviaMALDI- Manse-CAP2b[Ala1], 15.5min; Manse-CAP2b[Ala2], 15.0min; TOF/TOFmassspectrometryledtotheidentificationofCAP2b/ Manse-CAP2b[Ala3], 13.5min; Manse-CAP2b[Ala5], 16.5min; PVKpeptidesnativetothehouseflyM.domestica[18,26,32]and Manse-CAP2b[Ala6], 11.0min; Manse-CAP2b[Ala7], 5.5min; three other species of flies [18,19]. The housefly CAP2b/PVK Manse-CAP2b[Ala8],15.0min.Aminoacidanalysiswascarried sequenceswererecentlydeterminedtobeAGGTSGLYAFPRVa out under previously reported conditions [17] and used to (Musdo-CAP2b/PVK-1)andASLFNAPRVa(Musdo-CAP2b/PVK-2) quantify the peptide and to confirm identity, leading to the [18]. The octapeptide CAP2b shares a common C-terminal following analyses: Manse-CAP2b[2-8]: A[1.0], F[1.0], L[1.0], heptapeptide sequence (LYAFPRVa) with the most active P[1.0], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[3-8]: A[1.0], F[1.0], houseflyCAP2bsequence,Musdo-CAP2b-1,revealingthelikely P[1.1], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[4-8]: A[0.8], F[1.0], reasonthattheManducapeptidedemonstratesrelativelypotent P[1.0], R[1.0], V[0.9]; Manse-CAP2b[5-8]: F[1.0], P[1.0], R[1.0], diureticactivityintheheterologoushouseflyMalpighiantubule V[1.0]; Manse-CAP2b[Ala1]: A[1.9], F[1.0], L[1.0], P[1.0], R[1.0], fluidsecretionbioasssay. Y[1.1], V[0.9]; Manse-CAP2b[Ala2]: A[2.0], E[0.9], F[1.0], P[0.9], Inthisstudy,weuseManducaCAP2basamodelofMusdo- R[1.0], Y[1.1], V[1.0]; Manse-CAP2b[Ala3]: A[2.0], E[0.9], F[1.0], CAP2b-1andevaluateaseriesoftruncatedandAla-replace- L[1.0], P[1.0], R[1.0], V[0.9]; Manse-CAP2b[Ala5]: A[2.0], E[0.8], ment CAP2b analogs in a housefly Malpighian tubule fluid L[1.0], P[0.9], R[1.0], Y[1.2], V[0.9]; Manse-CAP2b[Ala6]: A[2.0], secretion bioassay to identify the active core and those E[1.0], F[1.0], L[0.9], R[1.0], Y[1.0], V[1.0]; Manse-CAP2b[Ala7]: residuesmostcriticaltoactivity. A[2.1], E[0.7], F[1.0], L[1.1], P[1.1], Y[0.8], V[1.0]; Manse-CAP2- b[Ala8]: A[2.0], E[1.0], F[1.0], L[1.0], P[0.9], R[1.0], Y[1.0]. The identities of the peptide analogs were confirmed via MALDI- 2. Materials and methods TOF-MSonaKratosKompactProbeMALDI-TOFMSmachine (KratosAnalytical,Ltd.,Manchester,UK)withthepresenceof 2.1. Insects thefollowingmolecularions(M+H+):Manse-CAP2b[2-8],864.9 [M+H+];Manse-CAP2b[3-8],751.5[M+H+];Manse-CAP2b[4-8], Housefly(M.domestica)larvaewereobtainedfromacommercial 588.6[M+H+];Manse-CAP2b[5-8],517.6[M+H+];Manse-CAP2- fishbaitsupplier.Theywereheldinthelaboratoryuntilthey b[Ala1], 936.8 [M+H+]; Manse-CAP2b[Ala2], 933.8 [M+H+]; pupatedandwerethenmovedtoaconstanttemperatureroom Manse-CAP2b[Ala3], 883.9 [M+H+]; Manse-CAP2b[Ala5], 899.5 (288C;12L:12Dlight:darkcycle).Fliesthatemergedwithin12h [M+H+]; Manse-CAP2b[Ala6], 949.3 [M+H+]; Manse-CAP2- ofoneanotherwerekeptasseparateagecohortsandprovided b[Ala7],890.6[M+H+];Manse-CAP2b[Ala8],947.7[M+H+]. with water and sucrose ad libidum. All of the experiments describedinthispaperused3–4day-oldadultfemaleflies. 2.3. IsolatedhouseflyMalpighiantubulepreparations 2.2. Peptidesynthesis FluidsecretionfromisolatedhouseflyMalpighiantubuleswas measuredusingthe‘‘Ramsayassay’’aspreviouslydescribed TheCAP2banalogsweresynthesizedviaFmocmethodology [10]. Tubules were removed from 3 to 4 day post-emergent on Rink Amide resin (Novabiochem, San Diego, CA) using adultfemaleflies.FliesweredissectedunderMuscasaline[10] Fmocprotectedaminoacids(AdvancedChemtech,Louisville, and both anterior and posterior tubules were transferred to KY)onanABI433Apeptidesynthesizer(AppliedBiosystems, small(10ml)dropsofbathingfluid(a1:1mixtureofsalineand Foster City, CA) under previously described conditions [17]. Schneider’s Drosophila medium) beneath water-saturated CrudeproductswerepurifiedonaWatersC SepPakcartridge liquid paraffin in a SylgardTM lined Petri dish. The tubules 18 andaDeltaPakC reverse-phasecolumn(8mm(cid:2)100mm, were allowed to equilibrate for 1h before being challenged 18 15mm particle size, 100A˚ pore size) on a Waters 510 HPLC with test peptides. Urine escaped from the cut end of the controlledwithaMillennium2010chromatographymanager tubule,whichwaspulledoutintotheliquidparaffin.Dropsof system (Waters, Milford, MA) with detection at 214nm at urinewerecollectedat15minintervalsandtheirdiameter(d) ambienttemperature.SolventA=0.1%aqueoustrifluoroace- measuredastheyrestedontheSylgardbaseofthePetridish ticacid(TFA);SolventB=80%aqueousacetonitrilecontaining using a Wild digital (MMS235) eyepiece micrometer. Urine 0.1%TFA.Conditions:initialsolventconsistingof20%Bwas volume was calculated as pd3/6 and the rate of secretion followedbytheWaterslinearprogramto100%Bover40min; obtained by dividing the secreted volume by the collection flow rate, 2.0ml/min. Delta-Pak C-18 retention times: period. Data were normalized by expressing the increase in Manse-CAP2b[2-8], 9.0min; Manse-CAP2b[3-8], 7.5min; fluid secretion as a percentage of the response to Manse-CAP2b[4-8], 5.0min; Manse-CAP2b[5-8], 4.5min; 10nMMusdo-K [16], which was added to all tubules at the Manse-CAP2b[Ala1], 10.5min; Manse-CAP2b[Ala2], 9.0min; endofeachexperiment.Dose-responsecurveswereprepared Manse-CAP2b[Ala3], 4.5min; Manse-CAP2b[Ala5], 7.5min; using the computer program GraphPad Prism version 4.02 Manse-CAP2b[Ala6], 12.5min; Manse-CAP2b[Ala7], 10.5min; (GraphPadSoftware,SanDiego,CA). Manse-CAP2b[Ala8], 9.0min. The peptides were further pur- ified on a Waters Protein Pak I125 column (7.8mm(cid:2) 300mm)(Milligen Corp., Milford, MA). Conditions: flow rate: 3. Results 2.0ml/min;isocraticwithsolventconsistingof80%acetoni- trile made to 0.01% TFA. WatPro retention times: A list of the fluid secretion activity of the two CAP2b/PVK Manse-CAP2b[2-8], 7.0min; Manse-CAP2b[3-8], 8.0min; peptidesnativetothehousefly,Manse-CAP2b,Manse-CAP2b peptides 28 (2007) 57–61 59 Table1–FluidsecretionactivityofCAP2b/PVKpeptidesandanalogsonhousefly(M.domestica)Malpighiantubules Peptide Sequence Fluidsecretiona EC (nM)(95%CL) Maximalresponse(%) 50 Musdo-CAP2b/PVK-1 AGGTSGLYAFPRVa 9(7.2–11.1)[6] 100 Musdo-CAP2b/PVK-2 ASLFNAPRVa 102(96–109)[6] 100 Manse-CAP2b pELYAFPRVa 53(39–72)[6] Manse-CAP2b[2-8] LYAFPRVa 8(3–22) 100 Manse-CAP2b[3-8] YAFPRVa 428(298–613) 100 Manse-CAP2b[4-8] AFPRVa Tr Manse-CAP2b[5-8] FPRVa Tr Manse-CAP2b[Ala1] ALYAFPRVa 23(11–46) 100 Manse-CAP2b[Ala2] pEAYAFPRVa 373(255–544) 80 Manse-CAP2b[Ala3] pELAAFPRVa 33(10–105) 100 Manse-CAP2b[Ala5] pELYAAPRVa 115(27–485) 70 Manse-CAP2b[Ala6] pELYAFARVa 89(62–129) 100 Manse-CAP2b[Ala7] pELYAFPAVa Inactive Manse-CAP2b[Ala8] pELYAFPRAa 3500(2250–5500) 45 a Valuesarederivedfromdose–responsecurvesbasedondatapointsderivedfromsixreplicates. truncated analogs, and a Manse-CAP2b Ala scan series of 4. Discussion analogs on housefly Malpighian tubules is presented in Table 1. The fluid secretion activity of Manduca CAP2b TheevaluationoftheCAP2bsequencefromM.sexta,Manse- (EC =53nM) lies midway between that of the native CAP2b, in heterologous Malpighian tubule fluid secretion 50 Musdo-CAP2b/PVK-1 (EC =9nM) and Musdo-CAP2b/PVK- assays in fruit flies and houseflies [3,5,18] established that 50 2(EC =102nM);andthuscanserveasareasonablemodel this family of peptides demonstrated diuretic activity in 50 for a structure-activity relationship study of CAP2b/PVK addition to cardioacceleratory activity. Seven of the eight peptides in the housefly. Interestingly, removal of the pE residuesofManse-CAP2bareidenticalwiththemostpotent residue on the N-terminus of Manse-CAP2b leads to the C- of the native housefly CAP2b/PVK peptides, Musdo-CAP2b/ terminal heptapeptide sequence common to both Manse- PVK-1. As can be seen in Table 1, the EC for housefly 50 CAP2bandMusdo-CAP2b-1;andthediureticactivityofthis Malpighian tubule fluid secretion of Manse-CAP2b is mid- fragment Manse-CAP2b[2-8] (EC =8nM) matches that of waybetweenthatofthetwoCAP2b/PVKpeptidesnativeto 50 Musdo-CAP2b-1 (EC =9nM). The increase in activity the housefly; and thus can serve as a relevant model 50 observed with the loss of pGlu1 likely arises from the fact sequence to determine a structure-activity relationship thatitnowmorecloselymatchesthesequenceofthenative profile. The C-terminal fragment Manse-CAP2b[3-8] retains peptide.RemovalofanotheraminoacidresiduefromtheN- asignificantportionoftheMalpighiantubulefluidsecretion terminus,asinManse-CAP2b[3-8]leadstoalargedropofan activity of the parent peptide (Table 1). The active core, order of magnitude from the parent Manduca CAP2b the minimum sequence required to retain significant peptide.Removalsofathird(Manse-CAP2b[4-8])andfourth diureticactivityinthehousefly,isthereforetheC-terminal (Manse-CAP2b[5-8])residueleadtoanalogsthatretainonly hexapeptide. However, full activity requires theC-terminal traceactivity.InthecaseoftheAlascanseries,replacement heptapeptide fragment (Manse-CAP2b[2-8], which is more of the Arg7 (Manse-CAP2b[Ala7]) led to an analog with no active than the parent Manse-CAP2b and equipotent significant activity, indicating that Arg7 is therefore the with the native Musdo-CAP2b-1 (Table 1). The results of most critical of residues. Replacement of Val8 (Manse- the Ala-replacement series identify two residues, Arg7 and CAP2b[Ala8]; EC =3500nM) led to a large drop in activity Val8, as critical for diuretic activity. The residue Leu2 is 50 (70-fold) in comparison with the parent peptide, and semicritical; whereas all others are not critical. Not demonstrated retention of only 45% of the maximal surprisingly, the critical (R7 and V8) and semicritical (L2) responseofManse-CAP2bandthenativehouseflypeptides. residuesareamongthemostconservedamongtheCAP2b/ Residues pGlu1 (Manse-CAP2b[Ala1]; EC =23nM), Tyr3 PVK family throughout arthropods. The most conserved 50 (Manse-CAP2b[Ala3]; EC =33nM), Phe5 (Manse-CAP2- residues among the CAP2b/PVKs are the C-terminal PRVa- 50 b[Ala5]; EC =115nM), and Pro6 (Manse-CAP2b[Ala6]; mide as well as a Leu at position 7 (equivalent to Leu2 in 50 EC =89nM) would appear not to be critical for housefly Manse-CAP2b) from the C-terminus [27]. The structure- 50 diureticactivityastheresultinganalogswereeitheralittle activityrelationshipprofiledeterminedinthisstudycanaid more or a little less active. While Manse-CAP2b[Ala5] in the design and development of peptidomimetic agonist/ demonstrated a maximal response of 70%, this value is antagonist analogs of this diuretic peptide family with not statistically different from that of the parent peptide. enhanced biostability and bioavailability as tools for Leu2 would appear to be a semicritical residue, as Manse- arthropod endocrinologists and as potential pest manage- CAP2b[Ala2](EC =373nM),provedtobeaboutsix-foldless ment agents capable of disrupting the water balance in 50 active. target insects. 60 peptides 28 (2007) 57–61 Acknowledgments [14] LoiPK,TublitzNJ.SequenceandexpressionoftheCAPA/ CAP2bgeneinthetobaccohawkmoth,Manducasexta..JExp Biol2004;207:3681–91. ThisstudywassupportedwithaCollaborativeResearchGrant [15] MengX,Wahlstro¨mG,ImmonenT,KolmerM,TirronenM, (#LST.CLG.979226) from the North Atlantic Treaty Organiza- PredelR,etal.TheDrosophilahugingenecodesfor tion(NATO)(RJN)andaBinationalAgriculturalResearchand myostimulatoryandecdysismodifyingneuropeptides. Development Grant (BARD #IS3356-02) (RJN), and a grant MechDev2002;117:5–13. providedbytheUSDA/DODDWFPResearchInitiative(#0500- [16] NachmanRJ,CoastGM,TichySE,RussellDH,MillerJA, 32000-001-01R)(RJN).Inaddition,weacknowledgethecapable PredelR.Occurrenceofinsectkininsinthefleshfly,stable flyandhornfly–massspectrometricidentificationfrom technicalassistanceofAllisonStrey,PawelZubrzakandNan singlenervesanddiureticactivity.Peptides2002;23: PryoroftheUSDAAreawidePestManagementResearchUnit, 1885–94. SouthernPlainsAgriculturalResearchCenter. [17] NachmanRJ,IsaacRE,CoastGM,HolmanGM.Aib- containinganaloguesoftheinsectkininneuropeptide references familydemonstrateresistancetoaninsectangiotensin- convertingenzymeandpotentdiureticactivity.Peptides 1997;18:53–7. [18] NachmanRJ,RussellWK,CoastGM,RussellDH,PredelR. [1] BeyenbachKW.Transportmechanismsofdiuresisin MassspectrometricassignmentofLeu/Ileinneuropeptides Malpighiantubulesofinsects.JExpBiol2003;206:3845–56. fromsingleneurohemalorganpreparationsofinsects. [2] CazzamaliG,TorpM,HauserF,WilliamsonM, Peptides2005;26:2151–6. GrimmelikhuijzenCJP.TheDrosophilageneCG9918codes [19] NachmanRJ,RussellWK,CoastGM,RussellDH,MillerJA, forapyrokinin-1receptor.BiochemBiophysResCommun PredelR.IdentificationofPVK/CAP2bneuropeptidesfrom 2005;335:14–9. singleneurohemalorganpreparationsofthestableflyand [3] CoastGM.Theneuroendocrineregulationofsaltandwater hornflyviaMALDI-TOF/TOFtandemmassspectrometry. balanceininsects.Zoology(ZACS)2001;103:179–88. Peptides2006;27:521–6. [4] CoastGM,OrchardI,PhillipsJE,SchooleyDA.Insect [20] NasselDR,OhlssonLG,CanteraR.Metamorphosisof diureticandantidiuretichormones.In:EvansPD,editor. identifiedneuronsinnervatingthoracicneurohemalorgans AdvancesinInsectPhysiology,London:AcademicPress; intheblowfly—transformationofcholecystokininlike 2002.p.279–409. immunoreactiveneurons.JCompNeurol1988;267:343–56. [5] DaviesSA,HuesmannGR,MaddrellSH,O’DonnellMJ,Skaer [21] NeupertS,PredelR,RussellWK,DaviesR,PietrantonioPV, NJV,DowJAT,etal.CAP2b,acardioacceleratorypeptide,is NachmanRJ.Identificationoftickperiviscerokinin,thefirst presentinDrosophilaandstimulatesfluidsecretionvia neurohormoneofIxodidae:singlecellanalysisbymeansof cGMP.AmJPhysiol1995;269:R1321–6. MALDI-TOF/TOFmassspectrometry.BiochemBiophysRes [6] DavisNT,HombergU,DircksenH,LevineRB,Hildebrand Commun2005;338:1860–4. JG.Crustaceancardioactivepeptide-immunoreactive [22] O’BrienMA,TaghertPH.Aperitrachealneuropeptide neuronsinthehawkmothManducasextaandchangesin systemininsects:releaseofmyomodulin-likepeptidesand theirimmunoreactivityduringpostembryonic ecdysis.JExpBiol1998;201:193–209. development.JCompNeurol1993;338:612–27. [23] PaluzziJ-P,OrchardI.Distribution,activityandevidence [7] EckertM,HerbertZ,Polla´kE,Molna´rL,PredelR.Identical forthereleaseofananti-diureticpeptideinthekissingbug cellulardistributionofallabundantneuropeptidesinthe Rhodniusprolixus.JExpBiol2006;209:907–15. majorabdominalneurohemalsystemofaninsect [24] ParkY,KimYJ,AdamsME.IdentificationofGprotein- (Periplanetaamericana).JCompNeurol2002;452: coupledreceptorsforDrosophilaPRXamidepeptides, 264–75. CCAP,corazonin,andAKHsupportsatheoryofligand- [8] EckertM,PredelR,GundelM.Periviscerokinin-like receptorcoevolution.ProcNatlAcadSciUSA immunoreactivityinthenervoussystemoftheAmerican 2002;99:11423–8. cockroach.CellTissueRes1999;295:159–70. [25] PredelR,KellnerR,BaggermanG,SteinmetzerT,SchoofsL. [9] Ga¨deG,AuerswaldL,PredelR,MarcoHG.Substrateusage Identificationofnovelperiviscerokininsfromsingle anditsregulationduringflightandswimminginthe neurohaemalreleasesitesininsectsMS/MSfragmentation backswimmer,Notonectaglauca.PhysiolEntomol complementedbyEdmandegradation.EurJBiochem 2004;29:84–93. 2000;267:3869–74. [10] HolmanGM,NachmanRJ,CoastGM.Isolation, [26] PredelR,RussellWK,TichySE,RussellDH,NachmanRJ. characterizationandbiologicalactivityofadiuretic Capa-geneproductsinMuscadomesticaandNeobellieria myokininneuropeptidefromthehousefly,Muscadomestica. bullata.Peptides2003;24:1487–91. Peptides1999;20:1–10. [27] PredelR,WegenerC.Periviscerokinins/CAPAgenerelated [11] HuesmannGR,CheungCC,LoiPK,LeeTD,SwiderekK, peptidesinarthropods.CMLS2006;63:2477–90. TublitzN.AminoacidsequenceofCAP2b,aninsect [28] PredelR,WegenerC,RussellWK,TichySE,RussellDH, cardioacceleretorypeptidefromthetobaccohornworm NachmanRJ.PeptidomicsofCNS-associatedneurohemal Manducasexta.FEBSLett1995;371:311–4. systemsofadultDrosophilamelanogaster:amass [12] IversenA,CazzamaliG,WilliamsonM,HauserF, spectrometricsurveyofpeptidesfromindividualflies.J GrimmelikhuijzenCJP.Molecularcloningandfunctional CompNeurol2004;474:379–92. expressionofaDrosophilareceptorfortheneuropeptides [29] QuinlanMC,TublitzNJ,O’DonnellMJ.Anti-diuresisinthe CAPA-1and-2.BiochemBiophysResCommun blood-feedinginsectRhodniusprolixusSta˚l:thepeptide 2002;299:628–33. CAP2bandcyclicGMPinhibitMalpighiantubulefluid [13] KeanL,CazenaveW,CostesL,BoderickKE,GrahamS, secretion.JExpBiol1997;200:2363–7. PollockVP,etal.Twonitridergicpeptidesareencoded [30] SantosJG,PollakE,RexerKH,MolnarL,WegenerC. bythegenecapabilityinDrosophilamelanogaster.AmJ MorphologyandmetamorphosisofthepeptidergicVa PhysiolRegulIntegrCompPhysiol2002;282: neuronsandthemediannervesystemofthefruitfly, 1297–307. Drosophilamelanogaster.CellTissueRes2006;326:187–99. peptides 28 (2007) 57–61 61 [31] SatoY,OguchiM,MenjoN,ImaiK,SaitoH,IkedaM,etal. [32] WegenerC,ReinlT,Ja¨nschL,PredelR.Directmass Precursorpolyproteinformultipleneuropeptidessecreted spectrometricpeptideprofilingandfragmentationof fromthesuboesophagealganglionofthesilkwormBombyx larvalpeptidehormonereleasesitesinDrosophila mori:characterizationofthecDNAencodingthediapause melanogasterrevealstagma-specificpeptideexpression hormoneprecursorandidentificationofadditional anddifferentialprocessing.JNeurochem2006;96: peptides.ProcNatlAcadSciUSA1993;90:3251–5. 1362–74.