Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

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Peptides 32 (2011) 587–594

Contents lists available at ScienceDirect

Peptides

journa l homepage: www.e lsev ier .com/ locate /pept ides

iostable multi-Aib analogs of tachykinin-related peptides demonstrate potentral aphicidal activity in the pea aphid Acyrthosiphon pisumHemiptera: Aphidae)

onald J. Nachmana,∗, Kamran Mahdianb, Dick R. Nässel c, R. Elwyn Isaacd, Nan Pryora, Guy Smaggheb,∗∗

Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USALaboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, BelgiumDepartment of Zoology, Stockholm University, SE-106 91 Stockholm, SwedenInstitute of Integrative and Comparative Biology, University of Leeds, Leeds LS2 9JT, UK

r t i c l e i n f o

rticle history:eceived 10 August 2010eceived in revised form4 September 2010ccepted 14 September 2010vailable online 1 October 2010

eywords:phicideindgut myotropiciuresiseptidase resistant

a b s t r a c t

The tachykinin-related peptides (TRPs) are multifunctional neuropeptides found in a variety of arthro-pod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). Two new biostableTRP analogs containing multiple, sterically hindered Aib residues were synthesized and found toexhibit significantly enhanced resistance to hydrolysis by angiotensin converting enzyme and neprilysin,membrane-bound enzymes that degrade and inactivate natural TRPs. The two biostable analogs werealso found to retain significant myostimulatory activity in an isolated cockroach hindgut preparation,the bioassay used to isolate and identify the first members of the TRP family. Indeed one of the analogs(Leuma-TRP-Aib-1) matched the potency and efficacy of the natural, parent TRP peptide in this myotropicbioassay. The two biostable TRP analogs were further fed in solutions of artificial diet to the pea aphid overa period of 3 days and evaluated for antifeedant and aphicidal activity and compared with the effect oftreatment with three natural, unmodified TRPs. The two biostable multi-Aib TRP analogs were observedto elicit aphicidal effects within the first 24 h. In contrast natural, unmodified TRPs, including two thatare native to the pea aphid, demonstrated little or no activity. The most active analog, double-Aib ana-

log Leuma-TRP-Aib-1 (pEA[Aib]SGFL[Aib]VR-NH2), featured aphicidal activity calculated at an LC50 of0.0083 nmol/�l (0.0087 �g/�l) and an LT50 of 1.4 days, matching or exceeding the potency of commer-cially available aphicides. The mechanism of this activity has yet to be established. The aphicidal activityof the biostable TRP analogs may result from disruption of digestive processes by interfering with gutmotility patterns and/or with fluid cycling in the gut; processes shown to be regulated by the TRPs in other

analool age

insects. These active TRPfriendly pest aphid contr

. Introduction

The tachykinin-related peptides (TRPs; also designated ‘insec-atachykinins’) are multifunctional neuropeptides found in sev-ral arthropod and invertebrate groups [4,13,18,22,26,28,29,2,44,45,68,69,70]. They were first isolated from the locust, Locustaigratoria, according to their myostimulatory activity on the iso-

ated hindgut of the cockroach Leucophaea maderae. Following theiriscovery, TRPs were also shown to have myostimulatory activityn Locusta foregut and oviduct preparations [53,54]. Experimentsonducted with the tobacco budworm Heliothis virescens indicated

∗ Corresponding author. Tel.: +1 979 260 9315; fax: +1 979 260 9377.∗∗ Corresponding author. Tel.: +32 92646150; fax: +32 92646239.

E-mail addresses: nachman@tamu.edu (R.J. Nachman), guy.smagghe@ugent.beG. Smagghe).

196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2010.09.013

gs and/or second generation analogs offer potential as environmentallynts.

© 2010 Elsevier Inc. All rights reserved.

that native TRPs stimulate contractions of the isolated hindgut andthat the active core region or minimum sequence is the C-terminalhexapeptide (GFLGVRamide) [17,37,46]. TRPs have been shown tobe present in endocrine cells of the cockroach [35], locust andDrosophila melanogaster midgut [34,47,57,72], and evidence sug-gests that the midgut serves as a hormonal release site for the TRPs[72]. Malpighian tubules are located in proximity to the midgut,and it is of interest to note that TRPs have been shown to stimu-late Malpighian tubule writhing in the locust L. migratoria [5], andstimulate diuretic activity in tubules of the locusts L. migratoria andSchistocerca gregaria [23], as well as in tubules of the moth Mand-uca sexta [58]. More recently, evidence has also been reported that

TRPs modulate both olfactory sensitivity and locomotor activity inthe fruitfly D. melanogaster [19,25,71].

Most TRPs share the evolutionarily conserved C-terminal pen-tapeptide motif Phe-X1-Gly-X2-Arg-NH2, a consensus sequencethat has not been reported in vertebrates [69]. They do, however,

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

5 eptide

stt

flatmW4[nstrippcTnMtcfmvd

ddnaTCAwbs(hTo(P1

caerfibdtpsaOdaoakcM

88 R.J. Nachman et al. / P

hare limited sequence similarity (approx. 30%) to the vertebrateachykinin family, appearing more similar to fish and amphibianachykinins (up to 45%) than to mammalian members [53,54].

Two TRP receptor sub-types have been cloned from the fruit-y D. melanogaster and designated DTKR [32] and NKD [33]nd these share about 50% sequence homology. NKD is reportedo be expressed in endocrine cells and muscle fibers of the D.elanogaster midgut [48] and DTKR in the Malpighian tubules [2].ithin its transmembrane regions, DTKR shows approximately

0–48% amino acid identity to vertebrate tachykinin receptors32]. Despite this sequence similarity, vertebrate tachykinins showo activity on STKR from the stable fly Stomoxys calcitrans (80%equence similarity with DTKR of D. melanogaster) at concentra-ions up to 10 �M [66]. Conversely, when the C-terminal Metesidue of the tachykinins substance P and physalaemin (amphib-an origin) was replaced by the Arg residue found in the analogousosition of TRPs, these mixed vertebrate–invertebrate tachykinineptide sequences behaved as potent agonists on STKR-expressingells [64]. Similarly, replacing the C-terminal Arg residue of locustRPs with the Met residue found in vertebrate tachykinins sig-ificantly increased their activity on human tachykinin receptors.ammalian receptor activity could be further enhanced if the next-

o-last residue is replaced with its highly conserved vertebrateounterpart [64]. The data thus indicate major pharmacological dif-erences between TRPs from invertebrates and vertebrates that are

ainly attributable to differences in the C-terminal amino acid (Args. Met) likely resulting from ligand–receptor coevolution. Theseifferences could be explored for design of insect-specific ligands.

Due to the susceptibility of TRPs to both exo- and endopepti-ases in the insect hemolymph and gut, these peptides cannot beirectly used as pest control agents and/or research tools by insecteuroendocrinologists. Members of the TRP family are hydrolyzed,nd therefore inactivated, by tissue-bound peptidases of insects.wo susceptible hydrolysis sites in TRPs have been reported in the-terminal active core region sequence Gly1-Phe2-Tyr3-Gly4-Val5-rg6-NH2. The primary site is between Gly4 and the Val5 residue,ith a secondary site between the Gly1 and Phe2 residues. It has

een demonstrated experimentally that the primary hydrolysisite is susceptible to cleavage by angiotensin converting enzymeANCE) from the housefly and both the primary and the secondaryydrolysis sites can be cleaved by neprilysin (NEP) [20,21,39,57].he N-terminal region of TRPs are susceptible to degradation notnly by aminopeptidases but by the enzyme dipeptidyl peptidase IVDPP IV); the latter has been shown to hydrolyze the bond betweenro and Ser of the locust TRP sequence Lom-TK-1 (or Locma-TRP-)(GPSGFYGVRamide) [21] and Leuma-TRPs [43].

To overcome the limitations inherent in the physicochemicalharacteristics of peptides, the development of peptidomimeticnalogs has been used as a strategy to enhance their biologicalffects. It has been proposed that blocking or overstimulating theeceptors of insect neuropeptides could lead to reduction of pesttness or even increased mortality [14,40]. Peptidomimetics is aroader term used to refer to pseudopeptides and non-peptidesesigned to perform the functions of a peptide. Generally these pep-idomimetics are derived by the structural modification of the leadeptide sequence to overcome a number of metabolic limitations,uch as proteolytic degradation that restrict the use of peptides asgents capable of modulating aspects of insect physiology [40,61].ne peptidomimetic approach is the incorporation of sterically hin-ered �,�-disubstituted amino acids such as �-amino-isobutyriccid (Aib). This approach has been used to develop biostable analogs

f the insect kinin neuropeptide family that have demonstratedgonist responses that match or exceed the potency of native insectinins in in vitro Malpighian tubule fluid secretion assays of thericket Acheta domesticus and mosquito Aedes aegypti, an in vivousca domestica housefly diuretic assay, as well as on receptor

s 32 (2011) 587–594

expressing systems of the tick Boophilus microplus and mosquitoAedes aegypti [38,41,62,63]. They have also demonstrated enhancedresistance to the endopeptidases ANCE and NEP, enzymes thatdeactivate the natural insect kinins as well as evidence of longerhemolymph residence times in the housefly. Biostable Aib insectkinin analogs have demonstrated enhanced in vivo activity overnative peptides in diuretic assays of the housefly and have shownsignificantly greater inhibition of weight gain in treated larvaeof the corn earworm Helicoverpa zea [41]. Recently, biostable Aibanalogs of the insect kinins have demonstrated potent antifeedantand aphicidal effects in the pea aphid Acyrthosiphon pisum whendelivered orally [60]. Biostable analogs of another peptide family,the C-type allatostatins, have also demonstrated aphicidal proper-ties in this same species as well [10].

In earlier work we have synthesized an analog of the cock-roach TRP (Leuma-TRP-1 or Lem-TK-1) containing a single Aiband have shown that it demonstrates potent activity on theisolated hindgut of the cockroach L. maderae [39]. This analog,designated Aib-Lem-TRP-1 (pGlu-Ala-Pro-Ser-Gly-Phe-Leu-[Aib]-Val-Arg-NH2) demonstrated marked resistance to Drosophila ACE,but nonetheless remains vulnerable to additional hydrolysis bypeptidases NEP (between Gly and Phe) and DPP IV (between Proand Ser). The development of TRP analogs containing multiple Aibresidues to redress these limitations is one of the goals of this paper.

About 250 of the 4,000 aphid species that have been describedare serious pests to various crops around the world, causing bothdirect damage to plants and indirect damage by transmittingviruses that can devastate agricultural crops [1]. In particular, thepea aphid A. pisum causes hundreds of millions of dollars of cropdamage every year, and many populations have already acquiredresistance towards multiple conventional and modern insecticides,making a search for alternative strategies urgent [11]. Further-more aphids are not sensitive to the toxins from the bacteriumBacillus thuringiensis (Bt) [56]. Interestingly, the 525 Mb genomeof A. pisum has recently been sequenced by the InternationalAphid Genomic Consortium providing a resource for comparativegenomics and the tools to identify targets for control (AphidBase;http://www.aphidbase.com; [49]). We identified the sequences ofthe three native TRPs as VPSADAFYGVR-NH2, ASMGFMGMR-NH2,and DYYSNNKGSAAGFFGMR-NH2.

In this paper, we expand on earlier synthetic work on a TRPanalog containing an �,�-disubstituted amino acid, the singleAib TRP analog of Leuma-TRP-1 [39], to prepare the two newbiostable TRP analogs listed below that incorporate multiple Aibmodifications and evaluate their susceptibility to hydrolysis bythe peptidases NEP and ANCE as compared with the unmodifiedLeuma-TRP-1. In addition, the analogs are evaluated for myos-timulatory activity on in vitro preparations of the hindgut of thecockroach L. maderae, the bioassay used to isolate the first mem-bers of the TRP family. The bioactivity profile of the TRP familyreveals similarities to that of the insect kinins [6,18,23]; specifi-cally gut myotropic and diuretic activity. Because biostable analogsof the insect kinins have been shown to demonstrate aphici-dal properties [60], the biostable analogs of the TRPs were fedin solutions of artificial diet to the pea aphid A. pisum over aperiod of 3 days and evaluated for antifeedant and aphicidal activ-ity.

Leuma-TRP-Aib-1: pEA[Aib]SGFL[Aib]VR-NH2Leuma-TRP-Aib-2: pEA[Aib]S[Aib]FL[Aib]VR-NH2

[TRP stands for tachykinin-related peptide analog].The two analogs are also blocked at the N-terminus with

pGlu, which confers additional resistance to hydrolytic degrada-tion by another class of peptidases, the aminopeptidases [15].The following three unmodified natural TRP peptides served

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

eptide

aa

2

2

(SLNpbbtFSrma1((1(Ns(f(vtPH

psaBdoLLactPATptPMPGEwMm9

R.J. Nachman et al. / P

s a positive control, two of which are native to the peaphid.

Leuma-TRP-1: APSGFLGVR-NH2Acypi-TRP-1: ASMGFMGMR-NH2Acypi-TRP-2: VPSADAFYGVR-NH2

. Materials and methods

.1. Synthesis and characterization of Aib-containing TRP analogs

The Aib-containing TRP analogs Leuma-TRP-Aib-1pEA[Aib]SGFL[Aib]VR-NH2) and Leuma-TRP-Aib-2 (pEA[Aib][Aib]FL[Aib]VR-NH2) and the natural, unmodified TRP analogseuma-TRP-1 (APSGFLGVR-NH2), Acypi-TRP-1 (ASMGFMGMR-H2), and Acypi-TRP-2 (VPSADAFYGVR-NH2) were synthesized,urified and quantified by adoption of procedures that haveeen previously described by Nachman et al. [35,38,73]. Inrief, analogs were synthesized on an ABI 433A peptide syn-hesizer with a modified FastMoc 0.25 procedure using anmoc-strategy starting from Rink amide resin (Novabiochem,an Diego, CA, 0.5 mmol/g). The Fmoc protecting group wasemoved by 20% 4-methyl piperidine in DMF (dimethyl for-amide). A fourfold excess of the respective Fmoc-amino

cids was activated in situ using HBTU (2-(1h-benzotriazol--yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)1 equiv.)/HOBt (1-hydroxybenzotriazole) (1 equiv.) in NMPN-methylpyrrolidone) or HATU (2-(7-Aza-1H-benzotiazole--yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)1 equiv.)/HOAt (1-hydroxy-7-azabenzotriazole) (1 equiv.) inMP for Aib and the amino acid immediately following it in the

equence. The coupling reactions were base catalyzed with DIPEAN,N-diisopropylethylamine) (4 equiv.). The analogs were cleavedrom the resin with side-chain deprotection by treatment with TFAtrifluoroacetic acid):H2O:TIS (triisopropylsilane) (95.5:2.5:2.5,/v/v) for 1.5 h. The solvents were evaporated by vacuum cen-rifugation and the analogs were desalted on a Waters C18 Sepak cartridge (Milford, MA) in preparation for purification byPLC.

The analogs were purified on a Waters Delta-Pak C18 reverse-hase column (8 mm × 100 mm, 15 �m particle size, 100 A poreize) with a Waters 510 HPLC system with detection at 214 nmt ambient temperature. Solvent A = 0.1% aqueous TFA; Solvent= 80% aqueous acetonitrile containing 0.1% TFA. Initial con-itions were 10% B followed by a linear increase to 90% Bver 40 min; flow rate, 2 ml/min. Delta-Pak C18 retention times:euma-TRP-1: 4.5 min, Acypi-TRP-1: 5.0 min, Acypi-TRP-2: 5.0 min,euma-TRP-Aib-1: 7.5 min, and Leuma-TRP-Aib-2: 9.0 min. Thenalogs were further purified on a Waters Protein Pak I 125olumn (7.8 mm × 300 mm). Conditions: isocratic using 80% ace-onitrile containing 0.1% TFA; flow rate, 2 ml/min. Waters Proteinak retention times: Leuma-TRP-1: 7.5 min, Acypi-TRP-1: 6.5 min,cypi-TRP-2: 8.0 min., Leuma-TRP-Aib-1: 6.5 min, and Leuma-RP-Aib-2: 6.5 min. Amino acid analysis was carried out underreviously reported conditions [38] to quantify the analogs ando confirm identity: Leuma-TRP-1: A[1.0], F[1.0], G[0.9], L[1.0],[1.0], R[0.8], S[1.0], V[0.8]; Acypi-TRP-1: A[1.0], F[1.0], G[2.0],[2.9], R[1.0], S[1.0]; Acypi-TRP-2: A[1.0], F[1.0], G[.9], L[1.0],

[1.0], R[0.8], S[1.0], V[0.8]; Leuma-TRP-Aib-1: A[1.0], E[0.9], F[1.0],[1.0], L[1.0], R[0.9], S[1.0], V[0.9]; and Leuma-TRP-Aib-2: A[1.0],

[1.0], F[1.0], L[1.0], R[0.9], S[1.0], V[0.9]. The identity of the analogsas also confirmed by MALDI-MS on a Kratos Kompact ProbeALDI-MS instrument (Shimadzu, Columbia, MD). The followingolecular ions (MH+) were observed: Leuma-TRP-1: 903.6 (calc.

03.1), Acypi-TRP-1: 987.7 (calc. 987.2), Acypi-TRP-2: 1181.5 (calc.

s 32 (2011) 587–594 589

1181.32), Leuma-TRP-Aib-1: 1030.8 (calc. 1030.44), and Leuma-TRP-Aib-2: 1058.7 (calc. 1058.39).

2.2. Aphid rearing

A continuous colony with all stages of the pea aphid A. pisumwas maintained on young broad bean (Vicia faba) plants in theLaboratory of Agrozoology at Ghent University, Belgium, understandardized conditions of 25 ± 2 ◦C, a 16 h light photoperiod and65 ± 5% relative humidity. Mature aphids were put on plants for24 h, resulting in a synchronized offspring, i.e. neonate nymphswith an age of 0–24 h, that were used throughout the experiments[50].

2.3. Enzyme degradation assays

The enzyme degradation assays were performed on the two TRPAib analogs and compared with the unmodified, natural parentLeuma-TRP-1. The assays were performed as reported previously[8,30,31,63,73]. In short, peptide analogs (125 �M) were incubatedwith either angiotensin converting enzyme (ANCE) (5 ng stabilizedwith 2 �g of bovine serum albumen), or human NEP (100 ng) in0.1 M HEPES buffer, pH 7 (total volume, 20 �l) at 25 ◦C. Reactionswere terminated after 2 h by the addition 5 �l of 8% TFA and inpreparation for HPLC analysis the volume was increased to 260 �lby the addition of 0.1% TFA. Percent hydrolysis of each peptidewas determined using HPLC to measure the amount of the par-ent peak remaining after incubation. Results are the mean of threeindividual assays and expressed as the mean ± s.e.m., n = 4. HPLCwas performed using a Jupiter 5 �, column (C18, 250 mm in lengthx 4.5 mm, internal diameter) and a linear solvent gradient of 18%rising to 42% of acetonitrile in 0.1% TFA over 15 min at flow rate of1 ml/min.

2.4. Isolated hindgut myotropic bioassay

The isolated hindgut of the L. maderae cockroach was used forbioassay as described in detail elsewhere [7,16,36]. The hindgutwas suspended in a 5 ml glass chamber (Radnoti Glass, Monrovia,CA) connected with a cotton thread in one end to the lever of anisotonic force transducer, coupled to an amplifier (both from KentScientific, Litchfield, CT). We used a cockroach saline described byCook and Holman [6]. Recordings were made on a flatbed recorder(Goertz, Model 120, Neudorf, Austria). Known concentrations ofLeuma-TRP-1 and the Aib analogs were added to the test chamberand data for dose–response plots and the concentration at which ahalf-maximal response (EC50 value) was obtained by testing bothpeptides on seven different intestines using Prism software. A graphof the activity of Leuma-TRP-Aib-1 compared with Leuma-TRP-1was prepared using GraphPad Prism 5.02 (GraphPad Software Inc,La Jolla, CA) and appropriate statistical tests were performed usingGraphPad InStat 3.06 with p < 0.05 accepted as significant. The max-imal response of Leuma-TRP-Aib-2 and Leuma-TRP-1 at 1 �M on 5separate hindgut preparations was compared and the data analyzedvia Student’s t-test.

In the different set of experiments we also determined the timecourse of the action of the most biostable of the two Aib TRP analogs(Leuma-TRP-Aib-2) and the natural Leuma-TRP-1 on the isolatedhindgut preparation. The Aib peptide analog was applied at 1 �Mfor 5 min, followed by 5 min washes with saline on 5 hindgut prepa-

rations. The same was undertaken with Leuma-TRP-1 after thehindgut had returned to a flat baseline. The duration of the inducedcontractile response was measured for the Aib peptide analog andcompared with that of Leuma-TRP-1. The data was analyzed via aStudent’s t-test (with p = 0.05).

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

5 eptides 32 (2011) 587–594

2a

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ttt2Iqb(Vccami

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3

3T

woitLcohA

L TRP 1

75

100Leuma-TRP-1

EC50 = 7.2 nM

CL 5.1 - 10.3 nM

50

75

25 Leuma-TRP-Aib-1

ma

xim

um

re

sp

on

se

0

EC50 = 10.5 nM

CL 6.9 - 16.0 nM

%

-10 -9 -8 -7

Log concentration (M)

Fig. 1. Dose–response curves obtained by testing the naturally occurring Leuma-TRP-1 (©) and the double-Aib TRP analog Leuma-TRP-Aib-1 (�) in the L. maderaecockroach hindgut contraction assay. The relative activity is given as a percent ofthe maximal response (amplitude of contraction) obtained for each intestine. Eachpoint represents seven determinations ±SE. The half-maximal response (EC50) is

90 R.J. Nachman et al. / P

.5. Bioassay with pea aphid to determine antifeedant andphicide activity by TRP analogs

For all aphid bioassays, neonates with an age of 0–24 h wereollected from a continuous colony of the pea aphid A. pisum thatas maintained under standardized conditions of 25 ± 2 ◦C, a 16 h

ight photoperiod, and 65 ± 5% relative humidity [51,60].As food for the aphids, an artificial liquid diet previously devel-

ped for A. pisum [12] was used as the basal diet to which theeptide analogs were added. The feeding apparatus was preparedsing plexiglass cylinders (3 cm high and 3 cm diameter). The foodachet was made under sterile conditions and consists of two layersf parafilm membrane on top of the container. About 200 �l of thertificial diet was sandwiched between the two layers [51].

To challenge aphids to the insect peptide analogs, a stock solu-ion was prepared in the solvent 80% acetone/0.01% TFA, andhen diluted in the artificial diet to prepare different concentra-ions between 0.001 and 0.500 nmol/�l (=mM). In the treatments,00 �l of each concentration was used to make a food sachet.n the solvent-controls the diet was supplemented with an ade-uate amount of the solvent 80% acetone/0.01% TFA, and in thelank-controls with distilled water. At day 0, 20 neonate nymphsaged 0–24 h), obtained from a synchronized population reared on. faba plants, were transferred onto the artificial diet. For eachoncentration, three replicates were carried out and aphids werehecked daily during 3 days for honeydew formation to determinentifeedant effects and also for numbers of dead aphids to deter-ine aphicidal effects. The experiment was performed two times

ndependently from each other.To determine antifeedant effects, the amounts of honeydew pro-

uced by the aphids in the treatments as compared to controls wereeasured using the Ninhydrin test as described by Kanrar et al. [27].

n brief, a 3.6 cm-diameter petri dish, as described above in theeeding apparatus, was lined with a Whatman No. 3 filter paper.his filter paper (with the honeydew) was removed every 24 h,nd sprayed with 0.1% ninhydrin reagent to detect the presencef honeydew spots.

The aphid mortality percentages were analyzed using non-inear sigmoid curve fitting, and the toxicity of each treatment

as evaluated on the basis of time–response curves andoncentrations–response curves using the GraphPad Prism 4.0 soft-are. We estimated the median LT50 and LC50 values with their

orresponding 95% confidence interval, which is the time periodf feeding on treated diet needed to kill 50% of the aphids and theoncentration of the kinin analog needed to kill 50% of the aphids,espectively [59]. The mortality data were corrected according tobbott’s formula based on the mortality seen in the control groups;

n all experiments, mortality in the control groups averaged at a lowevel of 7%.

. Results

.1. Relative susceptibility of Leuma-TRP-Aib analogs and naturalRP to enzyme degradation

The two multiple-Aib TRP analogs and natural Leuma-TRP-1ere incubated with both angiotensin converting enzyme (ANCE)

r human NEP, both tissue-bound peptidases that degrade andnactivate unmodified TRPs. Under conditions that led to essen-ially complete hydrolysis (98%) of the new multi-Aib TRP analogs

euma-TRP-Aib-1 and Leuma-TRP-Aib-2 proved to be essentiallyompletely resistant to degradation by ANCE, with 0.0% hydrolysisbserved for both. The natural Leuma-TRP-1 was also completelyydrolyzed by the enzyme NEP (98.7 ± 0.5%). Both of the multi-ib TRP analogs demonstrated significant resistance to degradation

indicated on the figure for each analog; the difference between the two EC50’s isnot statistically significant (CL overlap). The responses of the two analogs at 0.1 �Msuggest desensitization of the receptor [23,39].

by NEP, though the protection afforded to Leuma-TRP-Aib-2(1.1 ± 0.7% hydrolysis) was much greater than that to Leuma-TRP-Aib-1 (46.7 ± 2.4% hydrolysis). This difference is likely due to thepresence in Leuma-TRP-Aib-2 of the extra Aib residue which pro-tects the secondary NEP hydrolysis site between the Gly1 and Phe2

residues of the TRP C-terminal hexapeptide core sequence.

3.2. Myostimulatory activity of Leuma-TRP-Aib analogs andnatural TRP on isolated cockroach hindgut preparations

The two biostable Aib analogs were evaluated for myostimula-tory activity on an in vitro hindgut preparation from the cockroach L.maderae [7,16,36], the heterologous bioassay originally employedto isolate the first members of the TRP family from the locust L.migratoria [53,54]. Both of the Aib analogs demonstrated myos-timulatory activity, but Leuma-TRP-Aib-1 was considerably morepotent than Leuma-TRP-Aib-2. As shown in Fig. 1, the EC50 ofLeuma-TRP-Aib-1 is 10.5 nM (CL 6.9–16.0) as compared with anEC50 of 7.2 nM (CL 5.1–10.3) for the parent natural cockroach TRP,Leuma-TRP-1. This difference in potency is not statistically sig-nificant, so the two are essentially equivalent in myostimulatoryactivity. Both achieve a maximal response. Leuma-TRP-Aib-2 wasnot potent enough to develop a full dose–response curve, but itdid demonstrate significant activity at 1 �M, achieving 48 ± 21%of the maximal response of the parent TRP, Leuma-TRP-1. Clearly,the additional Aib residue in Leuma-TRP-Aib-1 confers an advan-tage over Leuma-TRP-Aib-2 in resistance to the enzyme NEP, butalso leads to a drop in activity in the hindgut myotropic assay.The duration of activity of the most biostable of the two analogs,Leuma-TRP-Aib-2, was compared with the unmodified Leuma-TRP-1 at 1 �M. The activity of Leuma-TRP-Aib-2 elicited a stimulatoryresponse (an increase in tonus) for 1.48 ± 0.11 min as comparedwith 0.74 ± 0.15 min for the natural Leuma-TRP-1, representinga twofold greater time of response. Enzymatic stability clearlyincreases the duration of the response, and this suggests that pep-tide degradation is likely to account, at least in part, for the transient

myostimulatory action of the TRPs on the isolated hindgut prepa-ration. Receptor desensitization is likely to be a major factor inthe observed diminishing response of the hindgut to the peptideanalogs.

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

R.J. Nachman et al. / Peptide

100

50

100 A

50

% m

ort

ali

ty

LT50 = 1.4 days

CL 0.6 - 2.1 days

100

0 1 2 3 4

Days of feeding

100 B

50

% m

ort

ali

ty

LC50 = 0.0085 mM

CL 0.0037 - 0.0196 nM

-4 -3 -2 -1 0 1 2 3 4 50

Log concentration (µM)

0

Fig. 2. Induction of aphid mortality by the double Aib-containing insect peptideanalog Leuma-TRP-Aib-1 in the pea aphid Acyrthosiphon pisum. (A) Time–responseover the 3 days of feeding of aphids on treated diet with 0.5 nmol/�l of Leuma-TRP-Aib-1, and (B) concentration–response curve for mortality of aphids by differentconcentrations of Leuma-TRP-Aib-1 when fed for 3 days via treated diet. Mortalitypoa

3n

TaeaaLttLTti

the N-terminal region of TRPs is susceptible to degradation not

TAA

ercentages are based on two repeated experiments, each consisting of 3 groupsf 20 nymphs each; a total of 120 aphids were tested per concentration. Statisticalnalysis and graphs were generated with the GraphPad Prism 4.0 software.

.3. Effect of biostable Leuma-TRP-Aib analogs compared toatural TRP on pea aphids

Different concentrations of the biostable TRP analogs Leuma-RP-Aib-1–2 were added to artificial diet and tested for antifeedantnd aphicide activity. Interestingly, it was clear that the aphicidalffects of these two peptide analogs occurred rapidly. The extent ofphid mortality was marked, with a 30–40% kill observed to occurlready in the first day of treatment with Leuma-TRP-Aib-1 andeuma-TRP-Aib-2 at 0.5 nmol/�l. With the latter concentration,he LT50 or the time of feeding on the treated diet treated neededo kill 50% of the aphids was estimated to be about 1.4 days for

euma-TRP-Aib-1 (Fig. 2A). This trend was also visible for Leuma-RP-Aib-2, but to a somewhat lower extent (LT50 = 2.2 days). Inhese conditions we observed normal piercing and probing behav-or of the aphids on the food sachets with treated diet, suggesting

able 1phid mortality LC50 values, expressed as �g/�l and nmol/�l (=mM), in the artificial diecyrthosiphon pisum.

Name Sequence MW (g/mol) L

�

Leuma-TRP-Aib-1 pEA[Aib]SGFL[Aib]VR-NH2 1028 0Leuma-TRP-Aib-2 pEA[Aib]S[Aib]FL[Aib]VR-NH2 1056 0Leuma-TRP-1 APSGFLGVR-NH2 902 InAcypi-TRP-1 ASMGFMGMR-NH2 986 InAcypi-TRP-2 VPSADAFYGVR-NH2 1180 In

a % toxicity with highest concentration tested (given between brackets).

s 32 (2011) 587–594 591

that ingestion is taking place. Further on, there was no recovery ofthe feeding during the experiment, and all the intoxicated aphidsdied.

From the concentration series tested, it was clear that thebiostable analog Leuma-TRP-Aib-1 (pEA[Aib]SGFL[Aib]VR-NH2)was the most toxic for the pea aphids as at 0.5 nmol/�l nearly allaphids were dead (80–100%) (Fig. 2B). With the use of sigmoidcurve analysis, an LC50 value of 0.0085 (0.0037–0.0196) nmol/�l(=mM) was calculated for Leuma-TRP-Aib-1 (Table 1). For the otherbiostabilized Leuma-TRP-Aib-2 activity was also observed but waslower than Leuma-TRP-Aib-1; its LC50 was calculated at 0.165(0.081–0.337) nmol/�l (Table 1) with Prism software.

In great contrast, the natural tachykinin of A. pisum (Acypi-TRP-1) was inactive as there was only 14% of aphid mortality with0.5 nmol/�l (the highest concentration tested), and with the othernative pea aphid peptide Acypi-TRP-2 the aphicidal effects wereessentially non-existent with only 20% mortality with 0.5 nmol/�l(the highest concentration tested). To a similar extent, feeding ofaphids on diet with the unmodified natural peptide Leuma-TRP-1(APSGFLGVR-NH2) elicited little or no activity with only 32% mor-tality at the highest concentration tested (=0.500 nmol/�l). In theseconditions, the honeydew formation with the three unmodifiedinsect peptides was similar to the controls (data not shown).

4. Discussion

In the current study, the most impressive result is that twobiostable TRP analogs containing multiple Aib residues elicitedstrong activity abbreviating the lifespan of aphid insects, whereasunmodified, natural TRPs demonstrated comparatively little or noactivity. In previous work, the replacement of Gly4 within theC-terminal hexapeptide L. maderae cockroach TRP core Gly1-Phe2-Leu3-Gly4-Val5-Arg6-NH2 with a sterically hindered Aib residuehas been proposed to enhance resistance to cleavage by tissue-bound peptidases (including ANCE) between Gly4 and the Val5

residue [39]. The single Aib TRP analog pGlu-Ala-Pro-Ser-Gly-Phe-Leu-[Aib]-Val-Arg-NH2 demonstrates complete resistance toDrosophila ANCE, an enzyme that degrades and inactivates thenatural TRP. This single Aib TRP analog proved to be nearly asactive as the native TRP Leuma-TRP-1 in the elicitation of myos-timulatory activity on the isolated L. maderae cockroach hindgut,the bioassay used to isolate and identify the first members of theTRP family. The peptidase neprilyisin (NEP) also degrades TRPs atthe same primary hydrolysis site, and the single Aib analog fea-tures protection against the attack of this specific peptide bondby NEP as well. Nonetheless, while the single Aib analog protectsthe primary hydrolysis-susceptible site, it cannot protect the sec-ondary site Gly1-Phe2, a site which NEP can also attack. In addition,

only by aminopeptidases but also by the enzyme dipeptidyl pep-tidase IV (DPP IV); the latter has been shown to hydrolyze thebond between Pro and Ser of the locust TRP sequence Lom-TK-1(or Locma-TRP-1)(GPSGFYGVRamide) [21]. For these reasons, other

t for two biostable TRP analogs and three natural insect TRP against the pea aphid

C50 in diet (95% CL)

g/�l nmol/�l (=mM)

.0087 (0.0038–0.020) 0.0085 (0.0037–0.0196)

.174 (0.086–0.356) 0.165 (0.081–0.337)active (32 ± 10% with 0.451 �g/�l)a Inactive (32 ± 10% with 0.500 mM)a

active (14 ± 8% with 0.493 �g/�l)a Inactive (14 ± 8% with 0.500 mM)a

active (20 ± 10% with 0.590 �g/�l)a Inactive (20 ± 10 with 0.500 mM)a

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

5 eptide

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ib-containing analogs that incorporate either a second and/or ahird sterically hindered Aib residue were synthesized in this studyo protect both the secondary hydrolysis susceptible site and the-terminal region. The multi-Aib TRP analogs Leuma-TRP-Aib-1nd Leuma-TRP-Aib-2 used in this study demonstrated a highlyignificant increase in resistance to hydrolysis by the peptidasesNCE and NEP as compared with the parent, natural TRP Leuma-RP-1. Both biostable Aib TRP analogs showed complete resistanceo Drosophila ANCE and enhanced resistance to NEP over a 2 heriod, whereas natural Leuma-TRP-1 degraded to the extent of8–99%. Nonetheless, the virtually complete protection afforded toeuma-TRP-Aib-2 (0.0% hydrolysis) was much greater than that toeuma-TRP-Aib-1 (47% hydrolysis) during exposure to NEP. Thisifference is likely due to the presence in Leuma-TRP-Aib-2 of thextra Aib residue which protects the secondary NEP hydrolysis siteetween the Gly1 and Phe2 residues of the TRP C-terminal hexapep-ide core sequence.

Although data on the activity of natural and/or modified TRPnalogs have not been previously collected on in vitro bioassays ofhe pea aphid, data on the evaluations of the natural and biostableRP analogs used in this study have been obtained for the isolatedockroach hindgut myotropic bioassay to determine and comparehe extent of retention of biological activity. Both TRP Aib analogsetained significant myostimulatory activity in the hindgut bioas-ay, although the extent of this activity retention was different.he hindgut myostimulatory activity of analog Leuma-TRP-Aib-was much greater than that of Leuma-TRP-Aib-1. As shown in

ig. 1, the EC50 of Leuma-TRP-Aib-1 for stimulation of hindgutontractility was 10.5 nM (6.9–16.0) as compared with an EC50 of.2 nM (5.1–10.3) for the parent natural cockroach TRP, Leuma-RP-1. The difference in activity is not statistically significant, sohe two are essentially equivalent in myostimulatory activity, andoth achieve a maximal response. Leuma-TRP-Aib-2 was consider-bly less potent, but did demonstrate significant activity at 1 �M,chieving 48 ± 21% of the maximal response of the parent naturalRP. While the additional Aib residue in Leuma-TRP-Aib-1 confersn advantage over Leuma-TRP-Aib-2 in resistance to the enzymeEP, it also leads to a considerable drop in activity in the hindgutyotropic assay.The duration of activity of the most biostable of the two analogs,

euma-TRP-Aib-2, was compared with the unmodified Leuma-RP-1 at 1 �M. The activity of Leuma-TRP-Aib-2 elicited at leasttwofold greater time of continuous stimulatory response (an

ncrease in tonus) before returning to baseline as compared withhe natural Leuma-TRP-1. Enzymatic stability clearly increases theuration of the response, and this suggests that peptide degradation

s likely to account, at least in part, for the transient myostimulatoryction observed for the TRPs on the isolated hindgut preparation.eceptor desensitization is likely to be another major factor inhe observed diminishing response of the hindgut to the peptidenalogs [23,39].

The biostability afforded the two Aib analogs would also appearo greatly enhance the physiological effects of TRP peptides whened to the pea aphid A. pisum. While the natural TRP peptides,ncluding two that are native to the aphid, showed little or no aphi-idal activity, high mortality appeared within the first 24 h for thosereatment groups that received the two biostable analogs. Aftereeding for 3 days, an LC50 value of 0.0085 (0.0037–0.0196) nmol/�las calculated for Leuma-TRP-Aib-1 (Table 1), matching or exceed-

ng the potency of commercially available aphicides. For the otheriostable Leuma-TRP-Aib-2, aphicidal activity was also observed

ut was lower than Leuma-TRP-Aib-1; its LC50 was calculated at.165 (0.081–0.337) nmol/�l (Table 1) with Prism software.

Two reference aphidicides that are currently used in the mar-etplace for selective IPM control against aphids in agriculturere pymetrozine and flonicamid. Both compounds act specifi-

s 32 (2011) 587–594

cally against aphids as feeding inhibitors, although their exactmechanism(s) remain unidentified. Flonicamid [N-(cyanomethyl)-4-(trifluoromethyl)-3-pyridinecarboxamide] is a new insecticide;its LC50 as determined in an experimental setup similar to thatused for the kinin analogs was 0.144 nmol/�l with a typical loss ofhoney dew formation followed by death, and its LT50 was 1.1 daysto kill 50% of aphids feeding on diet containing 0.44 nmol/�l [50].For pymetrozine [1,2,4-triazin-3(2H)-one,4,5-dihydro-6-methyl-4-[(3-pyridinylmethylene)amino]], Sadeghi et al. [50] calculatedwith use of a similar feeding apparatus with a diet sachet an LC50of 0.01 �g/ml [51]. The latter authors also tested imidacloprid andfound that 50% of aphids were killed with 0.03 �g/ml after 3 daysof feeding. Imidacloprid is a very active broad-spectrum neonicoti-noid insecticide with the nicotinic acetylcholine receptor (nAChR)as target, and to date it is used against a large variety of pest insectsand due to its high systemic activity, it is also highly active againstsucking pest insects like aphids and whiteflies. But intensive useof imidacloprid has stimulated outbreaks of resistance and crossresistance in many cases [11]. In the group of insect growth reg-ulator (IGRs), azadirachtin (Neem), flufenoxuron and pyriproxyfenare also commercially used in the selective control of aphids andthey have a respective LC50 of 7.9, 8.7 and 9.3 �g per ml artificial dietagainst pea aphids [51]. Here typical phenotypic symptoms of aphidmortality were disruption of nymphal molt and abortion of molting.In the field of insecticidal proteins, mannose-binding lectins havereceived a lot of attention in the last decade, because the Galan-thus nivalis agglutinin (GNA, homotetrameric protein composed of12 kDa subunits) is highly detrimental to aphids [9,52,67] and canbe delivered via transgenic plants. Sadeghi et al. [50] reported anLC50 of 350 and 700 �g/ml for two mannose-binding lectins GNAand ASA after feeding for 3 days on treated diet. In addition, itshould be mentioned that aphids are not sensitive to the insectici-dal toxins of Bt [56]. Thus, the fact that the stabilized TRP analogsof this study, and especially the di-Aib analog Leuma-TRP-Aib-1,show rapid and high activities against A. pisum aphids in the sameorder of magnitude as some commercial aphicides tested undercomparable conditions in the laboratory, suggests that they repre-sent potentially valuable leads for alternative agents in the controlof aphids and in the fight against insecticide resistance. In addition,we believe that testing other sucking pest insects would also beof interest. But before making firm conclusions on their potentialvalue as practical antifeedants, we believe more testing on a largerscale and under more field-related conditions is required.

The aphicidal activity of the biostable TRP analog Leuma-TRP-Aib-1 is the most potent observed for that of any otherstabilized insect neuropeptide analog; eightfold more potent thanbiostable insect kinin analogs [60] and over 20-fold more potentthan biostable analogs of the C-type allatostatin family [10]. Theaphicidal activity of the TRP and insect kinin analogs is clearly asso-ciated with the presence of components that enhance resistanceto degradative peptidases, as the unmodified and/or natural neu-ropeptides demonstrate little or no activity. However, the presenceof structural components that protect susceptible regions of insectpeptides is not in itself sufficient for the observed aphicidal activ-ity, as we have previously reported that an analog of the insectkinin neuropeptide family (labeled K-Aib-4) demonstrates little orno aphicidal effects even though its sterically hindered Aib residueis also present at the same position of the active insect kinin analogsK-Aib-1 and K-Aib-2 [60].

The mechanism of the aphid antifeedant activity and highinduction of mortality demonstrated by the biostable TRP analogs

cannot be clearly identified at this point, but it may be associatedwith disruption of the physiological processes that this importantneuropeptide family regulates in insects. For this to happen, thebiostable analogs with aphicidal activity would necessarily needto interact with a native aphid TRP receptor(s). In this respect, it

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

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s interesting to note that the absolute order of activity of the twoctive biostable TRP analogs in the aphid assay correlates with thatf the order of activity in the myotropic hindgut bioassay. The aphi-idal activity of Leuma-TRP-Aib-1 is more than 19-fold more potenthan Leuma-TRP-Aib-2. In the hindgut myotropic assay, EC50 val-es could only be obtained for one of the two biostable TRP analogs,aking a precise activity comparison difficult. Nonetheless, analog

euma-TRP-Aib-1 is also more active than Leuma-TRP-Aib-2 in theindgut myotropic assay by a similar magnitude.

The TRPs and analogs, like the insect kinins [6,17,60,65],ave been shown to stimulate both contractions of the hindgut46,53,54,69] as well as in vitro Malpighian tubule writhing [5]nd fluid secretion in insects [23]. The aphicidal activity of theiostable TRP analogs may therefore result from a disruption ofhe digestive process by interfering with normal gut motility pat-erns and/or interference with normal fluid cycling in the gut. Thesmotic pressure of plant phloem sap is generally higher thanhat of insect body fluids [55]. Pea aphids feature no Malpighianubules. Nonetheless, water cycling along the length of the gutumen is believed to contribute to the osmoregulation of aphidsnd mediated by a membrane-associated aquaporin. The pea aphidquaporin gene ApAqp1 expressed in the gut is the ortholog of therip gene (CG9023) that is expressed in the Malpighian tubulesf Drosophila [55]. Whether the TRP analogs may interfere withhis water cycling process in the aphid clearly remains to be estab-ished. It should also be noted that this is a chronic effect lasting forours (energy wasting), and chronic stimulation of water secretionr even water retention could be lethal.

Finally, is it possible that activation of taste receptors by theresence of the analogs may cause the aphids to avoid ingestion ofhe diet altogether, leading to starvation? While honeydew forma-ion is depressed in the aphids exposed to the active analogs, thebservations of normal piercing behavior and the presence of ateast some honeydew suggest that ingestion is nonetheless takinglace. Impairment of normal physiological patterns in the aphids

ngesting the active analogs may lead to a reduction in subsequenteeding and, in turn, to the observed reduction in levels of honey-ew formation. Furthermore, the unmodified native TRP peptidecypi-TRP-1 is readily ingested by the aphids, as is the inactive

nsect kinin analog K-Aib-4 (Ac-Arg-�3Phe-Phe-Phe-Aib-Trp-Gly-H2) [60] which also contains the same unnatural Aib residue found

n the active analogs. Thus, the fact that these insect neuropeptidenalogs do not trigger avoidance of diet ingestion would seem touggest that some ingestion of the two active biostable analogs maylso be taking place. The observation of high mortality within therst 24 h after exposure to the sugar water solutions containing theiostable TRP analogs also strongly suggests that feeding is takinglace, as death due to starvation would be expected to require a

onger time frame.The effectiveness of Leuma-TRP-Aib-1 as an aphicide could be

otentially enhanced by incorporating a moiety/motif other than anib (as used in the less active Leuma-TRP-Aib-2), such as a �-aminocid [3,24,73], to stabilize the secondary hydrolysis site of the pep-idase NEP and that might lead to greater retention of bioactivity.s nothing is presently known about the susceptibility of unmodi-ed or biostable TRP analogs to digestive enzymes in the aphid gut,

uture investigations into this question are warranted. While theresence of the sterically hindered, �,�-disubstituted Aib aminocids likely enhance resistance to digestive peptidases at least toome degree, a better knowledge of the degradation of TRP analogsy gut enzymes would facilitate the design of a second generation

f antifeedant analogs.

In summary, the presence of two biostable TRP analogseuma-TRP-Aib-2 and particularly Leuma-TRP-Aib-1 in the dietemonstrates significant aphicidal activity in the pea aphid A. pisumhat matches or exceeds that of commercially available aphicides.

[

s 32 (2011) 587–594 593

Conversely, unmodified natural TRPs demonstrate little or no aphi-cidal activity. The active biostable TRP analogs described in thisstudy and/or 2nd generation analogs, either in isolation or in com-bination with biostable analogs of other neuropeptide classes thatalso regulate aspects of diuretic, antidiuretic, digestive, reproduc-tive and/or developmental processes, represent potential leads inthe development of selective, environmentally friendly pest aphidcontrol agents capable of disrupting those critical processes.

Acknowledgements

The authors wish to thank Allison Strey (USDA, College Station)for able technical assistance. We also acknowledge financial assis-tance from the North Atlantic Treaty Organization (NATO) (RJN)Collaborative Research Grant (#LST.CLG.979226), a grant from theUSDA/DOD DWFP Initiative (#0500-32000-001-01R) (RJN), andsupport from the Fund of Scientific Research (FWO-Vlaanderen,Belgium) and the Special Research Fund of Ghent University (BOF-UGent) to GS.

References

[1] Alford DV. Pest and disease management handbook. Oxford, UK: BlackwellScience Ltd.; 2000.

[2] Birse RT, Johnson EC, Taghert PH, Nässel DR. Widely distributed DrosophilaG-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides. J Neurobiol 2006;66(1):33–46.

[3] Cheng RP, Gellman SH, De Grado WF. Beta-peptides: from structure to function.Chem Rev 2001;101(10):3219–32.

[4] Christie AE, Lundquist CT, Nässel DR, Nusbaum MP. Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis. J ExpBiol 1997;200(Pt 17):2279–94.

[5] Coast GM. The influence of neuropeptides on Malpighian tubule writhing andits significance for excretion. Peptides 1998;19:469–80.

[6] Coast GM, Orchard I, Phillips JE, Schooley DA. Insect diuretic and antidiuretichormones. Adv Insect Physiol 2002;29:279–341.

[7] Cook BJ, Holman GM. Comparative pharmacological properties of muscle func-tion in the foregut and the hindgut of the cockroach Leucophaea maderae. CompBiochem Physiol 1978;61C:291–5.

[8] Cornell MJ, Williams TA, Lamango NS, Coates D, Corvol P, Soubier F, et al. Cloningand expression of an evolutionary conserved single-domain angiotensin con-verting enzyme from Drosophila melanogaster. J Biol Chem 1995;270:13613–9.

[9] Down RE, Gatehouse AMR, Hamilton WDO, Gatehouse JA. Snowdrop lectininhibits development and fecundity of the glasshouse potato aphid (Aula-corthum solani) when administered in vitro and via transgenic plants, both inlaboratory and glasshouse trials. J Insect Physiol 1996;42:1035–45.

10] Down RE, Matthews J, Audsley N. Effects of Manduca sexta allatostatin andan analog on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) anddegradation by enzymes from the aphid gut. Peptides 2010;31:489–97.

11] Elbert A, Haas M, Springer B, Thielert W, Nauen R. Applied aspects of neonicoti-noid uses in crop protection. Pest Manag Sci 2008;64:1099–105.

12] Febvay G, Delobel B, Rahbé Y. Influence of amino acid balance on the improve-ment of an artificial diet for a biotype of Acyrthosiphon pisum (Homoptera:Aphididae). Can J Zool 1988;66:2449–53.

13] Fujisawa J, Muneoka Y, Takahashi T, Takao T, Shimonishi Y, Kubota I, et al.An invertebrate-type tachykinin isolated from the freshwater bivalve molluscAnodonta cygnea. In: Okada Y, editor. Peptide chemistry. Osaka Protein ResearchFoundation; 1994. p. 161–4.

14] Gäde G, Goldsworthy GJ. Insect peptide hormones: a selective review oftheir physiology and potential application for pest control. Pest Manag Sci2003;59:1063–75.

15] Gregory H, Hardy PM, Jones PM, Kenner DS, Sheppard RC. Antral hormonegastrin. Structure of gastrin. Nature 1964;204:931–3.

16] Holman GM, Nachman RJ, Schoofs L, Hayes TK, Wright MS, De Loof A. TheLeucophaea maderae hindgut preparation: a rapid and sensitive bioassay toolfor the isolation of insect myotropins of other insect species. Insect Biochem1991;21:107–12.

17] Holman GM, Nachman RJ, Wright MS. Comparative aspects of insect myotropicpeptides. In: Epple A, Scanes CG, Stetson MH, editors. Progress in comparativeendocrinology. New York: Wiley-Liss; 1990. p. 35–9.

18] Holman GM, Nachman RJ, Wright MS. Insect neuropeptides. Annu Rev Entomol1990;35:201–17.

19] Ignell R, Root CM, Birse RT, Wang JW, Nässel DR, Winther ÅM. Presynaptic

peptidergic modulation of olfactory receptor neurons in Drosophila. Proc NatlAcad Sci USA 2009;106(31):13070–5.

20] Isaac RE, Nassel DR. Identification and localization of a neprilysin-likeactivity that degrades tachykinin-related peptides in the brain of the cock-roach, Leucophaea maderae, and locust, Locusta migratoria. J Comp Neurol2003;457:57–66.

Journal Identification = PEP Article Identification = 68177 Date: February 8, 2011 Time: 6:51 pm

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[

[

[

[

[

[

[

[

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[

[

[

[

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94 R.J. Nachman et al. / P

21] Isaac RE, Parkin ET, Keen JN, Nassel DR, Siviter RJ, Shirras AD. Inactivation ofa tachykinin-related peptide: identification of four neuropeptide-degradingenzymes in neuronal membranes of insects from four different orders. Peptides2002;23:725–33.

22] Ikeda T, Minakata H, Nomoto K, Kubota I, Muneoka Y. Two novel tachykinin-related neuropeptides in the echiuroid worm, Urechis unicinctus. BiochemBiophys Res Commun 1993;192(1):1–6.

23] Johard HAD, Coast GM, Mordue W, Nassel DR. Diuretic action of the pep-tide locustatachykinin I: cellular localization and effects on fluid secretion inMalpighian tubules of locusts. Peptides 2003;24:1571–9.

24] Juaristi E, Soloshonok VA, editors. Enantioselective synthesis of �-amino acids.2nd ed. New York: Wiley; 2005.

25] Kahsai L, Martin JR, Winther AM. Neuropeptides in the Drosophila central com-plex in modulation of locomotor behavior. J Exp Biol 2010;213(Pt 13):2256–65.

26] Kanda A, Iwakoshi-Ukena E, Takuwa-Kuroda K, Minakata H. Isolation andcharacterization of novel tachykinins from the posterior salivary gland of thecommon octopus Octopus vulgaris. Peptides 2003;24(1):35–43.

27] Kanrar S, Venkateswari J, Kirti PB, Chopra VL. Transgenic Indian mustard (Bras-sica juncea) with resistance to the mustard aphid (Lipaphis erysimi Kalt.). PlantCell Rep 2002;20:976–81.

28] Kawada T, Masuda K, Satake H, Minakata H, Muneoka Y, Nomoto K.Identification of multiple urechistachykinin peptides, gene expression, phar-macological activity, and detection using mass spectrometric analyses.Peptides 2000;21(12):1777–83.

29] Kawada T, Satake H, Minakata H, Muneoka Y, Nomoto K. Characterizationof a novel cDNA sequence encoding invertebrate tachykinin-related peptidesisolated from the echiuroid worm, Urechis unicinctus. Biochem Biophys ResCommun 1999;263(3):848–52.

30] Lamango NS, Nachman RJ, Hayes TK, Strey A, Isaac RE. Hydrolysis of insectneuropeptides by an angiotensin converting enzyme from the housefly, Muscadomestica. Peptides 1997;18:47–52.

31] Lamango NS, Sajid M, Isaac RE. The endopeptidase activity and the activation byCl− of angiotensin-converting enzyme is evolutionarily conserved: purificationand properties of an angiotensin-converting enzyme from the housefly, Muscadomestica. Biochem J 1996;314:639–46.

32] Li XJ, Wolfgang W, Wu YN, North RA, Forte M. Cloning, heterologous expres-sion and developmental regulation of a Drosophila receptor for tachykinin-likepeptides. EMBO J 1991;10(11):3221–9.

33] Monnier D, Colas JF, Rosay P, Hen R, Borrelli E, Maroteaux L. NKD, adevelopmentally regulated tachykinin receptor in Drosophila. J Biol Chem1992;267(2):1298–302.

34] Montuenga LM, Zudaire E, Prado MA, Audsley N, Burrell MA, Coast GM. Pres-ence of Locusta diuretic hormone in endocrine cells of the ampullae of locustMalpighian tubules. Cell Tissue Res 1996;285:331–9.

35] Muren JE, Lundquist CT, Nässel DR. Abundant distribution of tachykinin-likepeptide in the nervous system and intestine of the cockroach Leucophaeamaderae. Phil Trans R Soc Lond B 1995;348:423–44.

36] Muren JE, Nassel DR. Seven tachykinin-related peptides isolated from the brainof the Madeira cockroach: evidence for tissue specific expression of isoforms.Peptides 1996;18:5–15.

37] Nachman RJ, Holman GM. Myotropic insect neuropeptide families from thecockroach Leucophaea maderae: structure–activity relationships. In: Menn JJ,Masler EP, editors. Insect neuropeptides: chemistry, biology, and action. Wash-ington, DC: American Chemical Society; 1991. p. 194–214.

38] Nachman RJ, Isaac RE, Coast GM, Holman GM. Aib-containing analoguesof the insect kinin neuropeptide family demonstrate resistance to aninsect angiotensin-converting enzyme and potent diuretic activity. Peptides1997;18:53–7.

39] Nachman RJ, Muren JE, Isaac RE, Lundquist CT, Karlsson A, Nassel DR.An aminoisobutyric acid-containing analogue of the cockroach tachykinin-related peptide, LemTRP-1, with potent bioactivity and resistance to an insectangiotensin-converting enzyme. Regul Pept 1998;74:61–6.

40] Nachman RJ, Roberts VA, Holman GM, Haddon WF. Leads for insect neuropep-tide mimetic development. Arch Insect Biochem Physiol 1993;22:181–97.

41] Nachman RJ, Strey A, Isaac E, Pryor N, Lopez JD, Deng JG, et al. Enhanced in vivoactivity of peptidase-resistant analogs of the insect kinin neuropeptide family.Peptides 2002;23:735–45.

42] Nassel DR. Neuropeptides in the nervous system of Drosophila and otherinsects: multiple roles as neuromodulators and neurohormones. Prog Neuro-biol 1996;48:325–30.

43] Nässel DR, Mentlein R, Bollner T, Karlsson A. Proline-specific dipeptidyl pep-tidase activity in the cockroach brain and intestine: partial characterization,distribution, and inactivation of tachykinin-related peptides. J Comp Neurol2000;418(1):81–92.

44] Nathoo AN, Moeller RA, Westlund BA, Hart AC. Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc NatlAcad Sci USA 2001;98(24):14000–5.

45] Nieto J, Veelaert D, Derua R, Waelkens E, Cerstiaens A, Coast G, et al. Iden-tification of one tachykinin and two kinin-related peptides in the brainof the white shrimp, Penaeus vannamei. Biochem Biophys Res Commun

1998;248(2):406–11.

46] Oeh U, Antonicek H, Nauen R. Myotropic effect of helicokinins, tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens(Lepidoptera: Noctuidae). J Insect Physiol 2003;49:323–37.

47] Pabla N, Lange AB. The distribution and myotropic activity of locustatachykinin-like peptides in locust midgut. Peptides 1999;20:1159–67.

[

[

s 32 (2011) 587–594

48] Poels J, Birse RT, Nachman RJ, Fichna J, Janecka A, Vanden Broeck J, et al. Charac-terization and distribution of NKD, a receptor for Drosophila tachykinin relatedpeptide 6. Peptides 2009;30(3):545–56.

49] Richards S, Gibbs RA, Gerardo NM, Moran N, Nakabachi A, Stern D, et al. Genomesequence of the pea aphid Acyrthosiphon pisum. PLoS Biol 2010;8:e1000313,doi:10.1371/journal.pbio.1000313.

50] Sadeghi A, Van Damme EJM, Michiels K, Kabera A, Smagghe G. Acute andchronic insecticidal activity of a new mannose-binding lectin from Alliumporrum against Acyrthosiphon pisum via an artificial diet. Can Entomol2009;141:95–101.

51] Sadeghi A, Van Damme EJM, Smagghe G. Evaluation of susceptibilityof pea aphid, Acyrthosiphon pisum, to a selection of novel biorationalinsecticides via artificial diet. J Insect Sci 2009;9:65. Available online:http://www.insectscience.org/9.65.

52] Sauvion N, Rahbé Y, Peumans WJ, Van Damme EJM, Gatehouse JA, GatehouseAMR. Effects of GNA and other mannose binding lectins on developmentand fecundity of the peach potato aphid Myzus persicae. Entomol Exp Appl1996;79:285–93.

53] Schoofs L, Holman GM, Hayes TK, Kochansky JP, Nachman RJ, De Loof A. Locus-tatachykinin III and IV: two additional insect neuropeptides with homologyto peptides of the vertebrate tachykinin family. Regul Pept 1990;31(3):199–212.

54] Schoofs L, Holman GM, Hayes TK, Nachman RJ, De Loof A. LocustatachykininI and II, two novel insect neuropeptides with homology to peptides of thevertebrate tachykinin family. FEBS Lett 1990;261(2):397–401.

55] Shakesby AJ, Wallace IS, Isaacs HV, Pritchard J, Roberts DM, Douglas AE. A water-specific aquaporin involved in aphid osmoregulation. Insect Biochem Mol Biol2009;39:1–10.

56] Sharma HC, Sharma KK, Crouch JH. Genetic transformation of crops for insectresistance: potentials and limitations. Crit Rev Plant Sci 2004;23:47–72.

57] Siviter RJ, Nachman RJ, Paulina Dani M, Keen JN, Shirras AD, Isaac RE. Peptidyldipeptidases (Ance and Acer) of Drosophila melanogaster: major differences inthe substrate specificity of two homologs of human angiotensin I-convertingenzyme. Peptides 2002;23:2025–34.

58] Skaer NJV, Nässel DR, Maddrell SHP, Tublitz NJ. Neurochemical fine tuning ofa peripheral tissue: peptidergic and aminergic regulation of fluid secretionby Malpighian tubules in the tobacco hawkmoth Manduca sexta. J Exp Biol2002;205:1869–80.

59] Smagghe G, Degheele D. Action of a novel nonsteroidal ecdysteroidmimic, tebufenozide (RH-5992), on insects of different orders. Pestic Sci1994;42:85–92.

60] Smagghe G, Mahdian K, Zubrzak P, Nachman RJ. Antifeedant activity and highmortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) inducedby biostable insect kinin analogs. Peptides 2010;31:498–505.

61] Steer DL, Lew RA, Perlmutter P, Smith AI, Aguilar MI. �-Amino acids: versatilepeptidomimetics. Curr Med Chem 2002;2:811–22.

62] Taneja-Bageshwar S, Strey A, Zubrzak P, Pietrantonio PV, Nachman RJ. Compar-ative structure-activity analysis of insect kinin core analogs on recombinantkinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae)and mosquito Aedes aegypti (Diptera: Culicidae). Arch Insect Biochem Physiol2006;62(3):128–40.

63] Taneja-Bageshwar S, Strey A, Isaac RE, Coast GM, Zubrzak P, PietrantonioP, et al. Biostable agonists that match or exceed activity of native insectkinins on recombinant arthropod GPCRs. Gen Comp Endocrinol 2009;162:122–8.

64] Torfs H, Detheux M, Oonk HB, Akerman KE, Poels J, Van Loy T, et al. Analysis of C-terminally substituted tachykinin-like peptide agonists by means of aequorin-based luminescent assays for human and insect neurokinin receptors. BiochemPharmacol 2002;63(9):1675–82.

65] Torfs P, Nieto J, Veelaert D, Boon D, van de Water G, Waelkens E, et al. The kininpeptide family in invertebrates. Ann NY Acad Sci 1999;897:361–73.

66] Torfs H, Shariatmadari R, Guerrero F, Parmentier M, Poels J, Van Poyer W, etal. Characterization of a receptor for insect tachykinin-like peptide agonists byfunctional expression in a stable Drosophila Schneider 2 cell line. J Neurochem2000;74(5):2182–9.

67] Michiels K, Van Damme EJM, Smagghe G. Plant–insect interactions: whatcan we learn from plant lectins? Arch Insect Biochem Physiol 2010;73:193–212.

68] Vanden Broeck J. Neuropeptides and their precursors in the fruitfly, Drosophilamelanogaster. Peptides 2001;22(2):241–54.

69] Van Loy T, Vandersmissen HP, Poels J, Van Hiel MB, Verlinden H, Vanden BroeckJ. Tachykinin-related peptides and their receptors in invertebrates: a currentview. Peptides 2010;31:520–4.

70] Veelaert D, Baggerman G, Derua R, Waelkens E, Meeusen T, Vande Water G,et al. Identification of a new tachykinin from the midgut of the desert locust,Schistocerca gregaria, by ESI-Qq-oa-TOF mass spectrometry. Biochem BiophysRes Commun 1999;266(1):237–42.

71] Winther AM, Acebes A, Ferrus A. Tachykinin-related peptides modulateodor perception and locomotor activity in Drosophila. Mol Cell Neurosci2006;31(3):399–406.

72] Winther AME, Nässel DR. Intestinal peptides as circulating hormones: releaseof tachykinin-related peptide from the locust and cockroach midgut. J Exp Biol2001;204:1269–80.

73] Zubrzak P, Williams H, Coast GM, Isaac RE, Reyes-Rangel G, Juaristi E, et al.Beta-amino acid analogs of an insect neuropeptide feature potent bioactivityand resistance to peptidase hydrolysis. Biopolymers 2007;88(1):76–82.