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Peptides 34 (2012) 266–273

Contents lists available at SciVerse ScienceDirect

Peptides

j our na l ho me p age : www.elsev ier .com/ locate /pept ides

iostable and PEG polymer-conjugated insect pyrokinin analogs demonstratentifeedant activity and induce high mortality in the pea aphid Acyrthosiphonisum (Hemiptera: Aphidae)

onald J. Nachmana,∗, Mohamad Hamshoub, Krzysztof Kaczmareka,c, Janusz Zabrockia,c, 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, BelgiumInstitute of Organic Chemistry, Technical University of Lodz, 90-924 Lodz, Poland

r t i c l e i n f o

rticle history:eceived 11 October 2011eceived in revised form 7 November 2011ccepted 7 November 2011vailable online 15 November 2011

eywords:phicideindgut myotropiceptidase resistant

a b s t r a c t

The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, includingthe pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs basedon the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphidover a period of three days and evaluated for antifeedant and aphicidal activity. The analogs containedeither modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance totissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugatedwith two lengths of polyethyleneglycol (PEG) polymers were also evaluated in the aphid feeding assay.Three of the biostable PK analogs demonstrated potent antifeedant activity, with a marked reduction inhoneydew formation and very high mortality after 1 day. In contrast, a number of unmodified, naturalpyrokinins and several other analogs containing some of the same structural components that promotebiostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH2) andPEGylated analog PK-dF-PEG8 [(P8)-YF[dF]PRL-NH2], featured aphicidal activity calculated at LC50’s of0.042 nmol/�l [0.029 �g/�l] (LT50 of 1.0 day) and 0.126 nmol/�l (LT50 of 1.3 days), respectively, matchingthe potency of some commercially available aphicides. Notably, a PEGylated analog of a PK antagonist can

block over 55% of the aphicidal effects of the potent PK agonist PK-Oic-1, suggesting that the aphicidaleffects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though theaphicidal activity of the biostable analogs may result from disruption of digestive processes by interferingwith gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PKanalogs represent potential leads in the development of selective, environmentally friendly aphid pest control agents.

. Introduction

The pyrokinin/pheromone biosynthesis activating neuropep-ide (PK/PBAN) peptides represent a multifunctional family thatlays a significant role in the physiology of insects. LeucopyrokininLPK), isolated from the cockroach Leucophaea maderae in 198613], was the first member of the family to be discovered. Since

hat time, over 30 peptides have been identified. They include PKs,

yotropins (MTs), PBAN, melanization and reddish coloration hor-one (MRCH), diapause hormone (DH), pheromonotropin (PT),

∗ Corresponding author. Tel.: +1 979 260 9315; fax: +1 979 260 9377.∗∗ Corresponding author. Tel.: +32 9 2646150; fax: +32 9 2646239.

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

196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2011.11.009

© 2011 Elsevier Inc. All rights reserved.

all of which share the common C-terminal pentapeptide FXPRL-amide (X = S, T, G or V) [3,12,32,33]. Functions of the PK/PBANfamily include stimulation of sex pheromone biosynthesis in moths[3,32–34], and mediation of key aspects of feeding (gut musclecontractions) [19,38], development (embryonic diapause, pupaldiapause and pupariation) [14,20,30,45,46] and defense (melaninbiosynthesis) [5,18] in a variety of insects (cockroaches, flies,locusts and moths). All of the above functions can be stimulatedby more than one peptide, and they demonstrate considerablecross-activity between various PK/PBAN assays, thereby lackingany species-specific behavior [1,6,8,32,33].

Previous work has established that a trans oriented Pro as

an integral part of a type I �-turn structure holds broad signifi-cance for many physiological functions elicited by the PK/PBANfamily of peptides, including hindgut contractile (cockroach L.maderae) [19,25], pheromonotropic (silk worm Bombyx mori and

R.J. Nachman et al. / Peptides 34 (2012) 266–273 267

N ON O

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a

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Fig. 2. Structures of two lengths of PEG polymer that was conjugated to the N-

ig. 1. Structures of modified analogs of Pro (middle): hydroxyprolyl (Hyp; left) andhe bulky octahydroindole-2-carboxyl residues (Oic; right).

orn earworm Helicoverpa zea) [11,23,24], oviduct contractilecockroach L. maderae) [21], egg diapause induction (silk worm. mori) [11,24,29], pupal diapause termination (corn earwormudworm H. zea) [47], and pupariation (flesh fly Neobellieriaullata) [46] assay systems.

Due to the susceptibility of PK/PBANs to both exo- and endopep-idases in the insect hemolymph and gut, these peptides cannote directly used as pest control agents and/or research toolsy insect neuroendocrinologists. Members of the PK/PBAN fam-

ly are hydrolyzed, and therefore inactivated, by tissue-boundeptidases of insects. The primary site within the C-terminal pen-apeptide (Phe1-Xxx2-Pro3-Arg4-Leu5-NH2) is between Pro3 andhe Arg4 residue [27]. To overcome the limitations inherent in thehysicochemical characteristics of peptides, the development ofeptidomimetic analogs has been used as a strategy to enhanceheir biological effects. It has been proposed that blocking or over-timulating the receptors of insect neuropeptides could lead toeduction of pest fitness or even increased mortality [17,26]. Pep-idomimetics is a broader term used to refer to pseudopeptidesnd non-peptides designed to perform the functions of a pep-ide. Generally these peptidomimetics are derived by the structural

odification of the lead peptide sequence to overcome a number ofetabolic limitations, such as proteolytic degradation that restrict

he use of peptides as agents capable of modulating aspects of insecthysiology [26,43].

One peptidomimetic approach employed with the PK/PBANlass of neuropeptides is the replacement of the critical Pro residueith such sterically hindered Pro analogs as octahydroindole-2-

arboxyl (Oic) and hydroxyprolyl (Hyp) [27] moieties (Fig. 1), asell as the introduction of d-amino acids. This former approachas been used to develop biostable Oic and Hyp analogs of theK/PBAN neuropeptide family that have demonstrated markedlynhanced resistance to hydrolysis by tissue-bound peptidases.hese analogs also demonstrated activity in an in vivo adult femaleeliothis virescens moth pheromonotropic assay when deliveredrally under conditions in which the native PBAN peptide and/orragments do not [27]. A Hyp-containing PK/PBAN analog provedo be more than 5-fold more potent than the native hormone DH, a

ember of the PK/PBAN family in terminating diapause in pupae of. zea [47], presumably because of a longer hemolymph residence

ime of the Hyp analog over the native hormone.Another approach to the stabilization of peptides and/or pro-

eins to enzymatic degradation in the digestive system as wells the enhancement of penetration across cell membranes of theut into the hemolymph (blood) of insects is the conjugation ofolyethylene glycol (PEG) polymers (Fig. 2) to the N-terminus7,15,40]. Although not previously applied to neuropeptides of theK/PBAN class, conjugation of PEG polymers to the insect peptiderypsin modulating oostatic factor (TMOF) enhanced the resistanceo degradation by the digestive enzyme leucine aminopeptidase,eading to accumulation of the peptide in hemolymph of insects

nd ticks [7,15,40].

About 250 of the 4000 aphid species that have been describedre serious pests to various crops around the world, causing

terminus of PK peptide analogs in this study: O-Methyl-tetra-glycolcarboxyl- (PEG4,top) and O-Methyl-octa-ethyleneglycolcarboxyl- (PEG8, bottom).

both direct damage to plants and indirect damage by transmit-ting viruses that can devastate agricultural crops [2]. In particular,the pea aphid Acyrthosiphon pisum causes hundreds of millions ofdollars of crop damage every year, and many populations havealready acquired resistance toward multiple conventional andmodern insecticides, making a search for alternative strategiesurgent [9]. Furthermore aphids are not sensitive to the toxinsfrom the bacterium Bacillus thuringiensis (Bt) [39]. Interestingly,the 525 Mb genome of A. pisum has recently been sequenced bythe International Aphid Genomic Consortium providing a resourcefor comparative genomics and the tools to identify targets for con-trol (AphidBase; http://www.aphidbase.com; [35]). We identifiedthe sequences of the two native PKs as SPPYSPPFSPRL-NH2 andGGTTQSSNGIWFGPRL-NH2, as well as related PRLamide peptidesQAVMAQPQVPRL-NH2 and pQAVMAQPQVPRL-NH2.

In this paper, we expanded on earlier synthetic work to preparea series of biostable analogs of the PK/PBAN class of neuropep-tides containing the sterically hindered Pro residues Oic and Hyp, ad-Phe amino acid and/or the attachment of hydrophobic hydrocar-bon moiety onto the N-terminus. These PK/PBAN analogs are listedbelow in the first group of structures:PK-Oic-1: FT[Oic]RL-NH2

PK-Oic-2: Hex-FT[Oic]RL-NH2

PK-Oic-3: SPPYSPPFS[Oic]RL-NH2

PK-Oic-4: Ac-SPPYSPPFS[Oic]RL-NH2

PK-Hyp-1: Hex-FT[Hyp]RL-NH2

PK-Hyp-2: Ac-FT[Hyp]RL-NH2

PK-Hyp-3: Ahx-FT[Hyp]RL-NH2

PK-Hyp-4: pQFT[Hyp]RL-NH2

PK-Hyp-5: pQYFT[Hyp]RL-NH2

PK-2Abf: 2Abf-Suc-FTPRL-NH2

PPK-AA: Hex-Suc-A[dF]PRL-NH2

These biostable analogs of the PK/PBAN family were fed in solu-tions of artificial diet to the pea aphid A. pisum over a period ofthree days and evaluated for antifeedant and aphicidal activity. Acomparison of the biostable analogs was made to unmodified, natu-ral PK/PBAN peptides and/or the C-terminal pentapeptide fragmentthat represents the active core of this large family of peptides listedin the second group of structures below.PK core fragment: FTPRL-NH2

Acypi PK-1: SPPYSPPFSPRL-NH2

Acypi PK-2: GGTTQSSNGIWFGPRL-NH2

Acypi PRLamide: pQAVMAQPQVPRL-NH2

Rhopr CAPA-2: EGGFISFPRV-NH2

In addition, the first PEG polymer conjugates of the PK/PBANfamily were synthesized (see list of polymer-conjugate analogs inthe third list of structures below) and evaluated in the aphid bioas-say.PK-PEG4: (P4)-YFTPRL-NH2

PK-PEG8: (P8)-YFTPRL-NH2

DH-PEG4: (P4)-LWFGPRL-NH2

PK-dF-PEG4: (P4)-YF[dF]PRL-NH2

PK-dF-PEG8: (P8)-YF[dF]PRL-NH2

PK-Oic-1-PEG4: (P4)-FT[Oic]RL-NH2

PK-Oic-1-PEG8: (P8)-FT[Oic]RL-NH2

Finally, the first PEG polymer conjugates of the insect kinin (IK)and tachykinin-related peptide (TRP) families were also prepared,specifically PEG conjugates of biostable IK and TRP analogs that had

2 eptide

begIITT

2

2

[fdA(T(wc[puagdF1(mcab(wwlrrctowtAPpp

(w(WtoB2O1PI9PH

68 R.J. Nachman et al. / P

een previously shown to exhibit potent antifeedant and aphicidalffects [22,42]. These four analogs are listed in the fourth and finalroup of structures below.

K-Aib-PEG4: (P4)-R[Aib]FF[Aib]WG-NH2

K-Aib-PEG8: (P8)-R[Aib]FF[Aib]WG-NH2

RP-Aib-PEG4: (P4)-A[Aib]SGFL[Aib]VR-NH2

RP-Aib-PEG8: (P8)-A[Aib]SGFL[Aib]VR-NH2

. Materials and methods

.1. Synthesis and characterization of PK analogs

The PK analogs PK-Oic-2 [27], PK-Hyp-1 [27], PK-2Abf44], PPK-AA [28], Rhopr CAPA-2 [31] and the PK coreragment [FTPRL-NH2] [19] were synthesized as previouslyescribed. The Aib-containing TRP analogs Leuma-TRP-ib-1 (pEA[Aib]SGFL[Aib]VR-NH2) and Leuma-TRP-Aib-2

pEA[Aib]S[Aib]FL[Aib]VR-NH2) and the natural, unmodifiedRP analogs Leuma-TRP-1 (APSGFLGVR-NH2), Acypi-TRP-1ASMGFMGMR-NH2), and Acypi-TRP-2 (VPSADAFYGVR-NH2)ere synthesized, purified and quantified by adoption of pro-

edures that have been previously described by Nachman et al.21,48]. In brief, analogs were synthesized on an ABI 433Aeptide synthesizer with a modified FastMoc 0.25 proceduresing an Fmoc-strategy starting from Rink amide resin (Nov-biochem, San Diego, CA; 0.5 mmol/g). The Fmoc protectingroup was removed by 20% 4-methyl piperidine (NMP) inimethyl formamide (DMF). A fourfold excess of the respectivemoc-amino acids was activated in situ using 2-(1h-benzotriazol--yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)1 equivalent [eq.]/1-hydroxybenzotriazole) (1 eq.) in NMP (N-

ethylpyrrolidone) (HOBt). The coupling reactions were baseatalyzed with N,N-diisopropylethylamine (DIPEA) (4 eq.). Thenalogs were cleaved from the resin with side-chain deprotectiony treatment with trifluoroacetic acid (TFA):H2O:triisopropylsilaneTIS) (95.5:2.5:2.5, v/v/v) for 1.5 h. The PEG polymer conjugationsere accomplished as follows: after transferring peptidyl resinith the completed peptide sequence into an 8 ml polypropy-

ene syringe, a 1.2 molar equivalent of MS(PEG)4 or MS(PEG)8eagent was added as a 10% solution in NMP (100 mg of viscouseagent was reconstituted with 900 mg NMP). Both reagents areommercially available (Thermo Scientific, Waltham, MA) andhey are N-hydroxysuccinimide esters of O-methyl-tetra- andcta-ethyleneglycolcarboxylic acid, respectively. The syringesere shaken over night at RT and, following a positive Kaiser

est, EDC was added (0.5 eq.) and shaken for one additional day.fter washing with DCM (3×) and methanol (3×) and drying theEGylated peptide analogs, cleavage from the resin was accom-lished with a cocktail composed of TFA/DMB/TIS (92.5:5:2.5) andrecipitated with ether.

All analogs were desalted on a Waters C18 Sep Pak cartridgeMilford, MA) in preparation for purification by HPLC. The analogsere purified on a Waters Delta-Pak C18 reverse-phase column

8 mm × 100 mm, 15 �m particle size, 100 A pore size) with aaters 510 HPLC system with detection at 214 nm at ambient

emperature. Solvent A = 0.1% aqueous TFA; Solvent B = 80% aque-us acetonitrile containing 0.1% TFA. Initial conditions were 10%

followed by a linear increase to 90% B over 40 min; flow rate, ml/min. Delta-Pak C18 retention times: PK-Oic-3: 7.5 min, PK-ic-4: 6.0 min, PK-PEG4: 9.0 min, PK-PEG8: 7.5 min, DH-PEG4:4.0 min, PK-dF-PEG4: 9.0 min, PK-dF-PEG8: 12.0 min, PK-Oic-EG4: 9.0 min, PK-Oic-PEG8: 7.5 min, IK-Aib-PEG4: 12.0 min,

K-Aib-PEG8: 12.0 min, TRP-Aib-PEG4: 9.5 min, TRP-Aib-PEG8:.0 min, Acypi PK-1: 8.0 min, Acypi PK-2: 7.5 min, AcypiRLamide: 5.0 min, PK-Oic-1: 12.0 min, PK-Hyp-2: 6.0 min, PK-yp-4: 6.0 min, and PK-Hyp-3: 7.5 min. The analogs were further

s 34 (2012) 266–273

purified on a Waters Protein Pak I 125 column (7.8 mm × 300 mm).Conditions: isocratic using 80% acetonitrile containing 0.1% TFA;flow rate, 2 ml/min. Waters Protein Pak retention times: PK-Oic-3: 6.5 min, PK-Oic-4: 7.5 min, PK-PEG4: 6.0 min, PK-PEG8:5.5 min, DH-PEG4: 6.0 min, PK-dF-PEG4: 5.9 min, PK-dF-PEG8:6.0 min, PK-Oic-PEG4: 6.0 min, PK-Oic-PEG8: 7.5 min, IK-Aib-PEG4: 6.0 min, IK-Aib-PEG8: 5.5 min, TRP-Aib-PEG4: 6.0 min,TRP-Aib-PEG8: 6.0 min, Acypi PK-1: 9.0 min, Acypi PK-2: 13.5 min,Acypi PRLamide: 10.5 min, PK-Oic-1: 7.5 min, PK-Hyp-2: 6.0 min,PK-Hyp-4: 8.5 min, and PK-Hyp-3: 8.0 min. Amino acid analysiswas carried out under previously reported conditions [21,45] toquantify the analogs and to confirm identity: PK-Oic-3: F[1.0],L[1.0], P[0.9], S[1.0], Y[0.9]; PK-Oic-4: F[1.0], L[1.0], P[1.0], S[1.0],Y[1.0]; PK-PEG4: F[1.0], L[1.0], P[1.0], R[1.0], T[1.0], Y[1.0]; PK-PEG8: F[1.0], L[1.0], P[1.0], R[1.0], T[1.0], Y[1.0]; DH-PEG4: F[1.0],G[1.0], L[1.0], P[1.0], R[1.0]; PK-dF-PEG4: F[1.0], L[1.0], P[1.0],R[1.0], Y[1.0]; PK-dF-PEG8: F[1.0], L[0.9], P[1.0], R[0.9], Y[0.9]; PK-Oic-PEG4: F[1.0], L[0.9], R[0.9], T[0.9]; PK-Oic-PEG8: F[1.0], L[0.9],R[0.9], T[0.9]; IK-Aib-PEG4: F[1.0], G[0.9], R[1.0]; IK-Aib-PEG8:F[1.0], G[0.9], R[0.9]; TRP-Aib-PEG4: A[1.0], F[1.0], G[1.0], L[1.0],R[0.9], S[0.9], V[0.9]; TRP-Aib-PEG8: A[1.0], F[1.0], G[0.9], L[1.0],R[0.9], S[0.9], V[0.9]; Acypi PK-1: F[1.0], L[1.0], P[0.9], R[1.0], S[1.0],Y[1.0]; Acypi PK-2: F[1.0], G[0.9], I[0.6], L[1.0], N[0.9], P[0.8], Q[1.0],R[0.9], S[0.9], T[0.8]; Acypi PRLamide: A[1.0], L[1.0], M[1.0], P[1.0],Q[1.0], R[1.0], V[1.0]; PK-Oic-1: F[1.0], L[1.0], R[1.0], T[1.0]; PK-Hyp-2: F[1.0], L[1.0], R[1.0], T[1.0]; PK-Hyp-4: F[1.0], L[1.0], Q[0.9],R[1.0], T[1.0]; and PK-Hyp-3: F[1.0], L[1.0], R[1.0], T[1.0]. The iden-tity of the analogs was also confirmed by MALDI-MS on a KratosKompact Probe MALDI-MS instrument (Shimadzu, Columbia, MD).The following molecular ions (MH+) were observed: PK-Oic-3:1397.4 (calc. 1397.6), PK-Oic-4: 1440.8 (calc. 1440.6), PK-PEG4:1015 (calc. 1014.3), PK-PEG8: 1190.8 (calc. 1190.5), DH-PEG4:1106 (calc. 1106.2), PK-dF-PEG4: 1061 (calc. 1060.4), PK-dF-PEG8:1237 (calc. 1236.6), PK-Oic-PEG4: 905.5 (calc. 905.3), PK-Oic-PEG8: 1081.8 (calc. 1081.2), IK-Aib-PEG4: 1100.1 (calc. 1100.2),IK-Aib-PEG8: 1276.9 (calc. 1276.5), TRP-Aib-PEG4: 1137.4 (calc.1137.3), TRP-Aib-PEG8: 1313 (calc. 1313.5), Acypi PK-1: 1344.5(calc. 1344.5), Acypi PK-2: 1734.5 (calc. 1734.9), Acypi PRLamide:1319.9 (calc. 1319.5), PK-Oic-1: 686.9 (calc. 686.0), PK-Hyp-2:691.2 (calc. 691.3), PK-Hyp-4: 759 (calc. 759.4), and PK-Hyp-3:762.6 (calc. 762.3).

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 23–25 ◦C, a 16 h light photoperiod and60–65% relative humidity. Mature aphids were put on plants,resulting in synchronized offspring, i.e., neonate nymphs with anage of 0–12 h, that were used throughout the experiments [36].

2.3. Bioassay with pea aphid in a feeding apparatus with anartificial assay to determine antifeedant and aphicidal activity ofpeptide analogs

For all aphid bioassays, neonates with an age of 0–12 h werecollected from a continuous colony of the pea aphid A. pisum asdescribed [37,42].

As food for the aphids, a standard diet previously developed forA. pisum [10] was used as the basal diet to which the peptide analogswere added. The feeding apparatus was prepared using plexiglass

cylinders (3 cm high and 3 cm diameter). The food sachet was madeunder sterile conditions and consists of two layers of parafilm mem-brane on top of the container. About 200 �l of the artificial diet wassandwiched between the two layers [37].

eptides 34 (2012) 266–273 269

nsaactdd8tftrdae

dmIifi0

lerCrfcrAil

3

3p

ata3(cA(pBt(POaOsdP(0P

Fig. 3. Induction of aphid mortality by the insect PK peptide analog PK-Oic-1 in thepea aphid Acyrthosiphon pisum. (A) Time-response over the three days of feedingof aphids on treated diet with 0.500 nmol/�l of PK-Oic-1, and (B) concentration-response curve for mortality of aphids by different concentrations of PK-Oic-1 whenfed for 3 days via treated diet. Mortality percentages are based on two repeatedexperiments, each consisting of 3 groups of 15 nymphs each; a total of 90 aphids

R.J. Nachman et al. / P

To challenge aphids to the insect peptide analogs or a combi-ation of peptide analogs, a stock solution was prepared in theolvent aqueous 80% acetone/0.01% TFA, and then diluted in thertificial diet to prepare different concentrations between 0.001nd 0.500 nmol/�l (=mM). In the treatments, 200 �l of each con-entration was used to make a food sachet. A volume of 20 �l ofhe analogs dissolved in aqueous 80% acetone/0.01% TFA was theniluted up to 200 �l with distilled water. In the solvent-controls theiet was supplemented with an equivalent amount of the solvent0% acetone/0.01% TFA, and in the blank-controls with only dis-illed water. At day 0, 15 neonate nymphs (aged 0–12 h), obtainedrom a synchronized population reared on V. faba plants, wereransferred onto the artificial diet. For each concentration, threeeplicates were carried out and aphids were checked daily during 3ays for honeydew formation to determine antifeedant effects andlso for numbers of dead aphids to determine aphicidal effects. Thexperiment was performed two times independently.

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. [16].

n brief, a 3.6 cm-diameter petri dish, as described above in the feed-ng apparatus, was lined with a Whatman No. 3 filter paper. Thislter paper (with the honeydew) was removed and sprayed with.1% ninhydrin reagent to detect the presence of honeydew spots.

The aphid mortality percentages were analyzed using non-inear sigmoid curve fitting, and the toxicity of each treatment wasvaluated on the basis of time-response curves and concentration-esponse curves using the GraphPad Prism 4.0 software (La Jolla,A). We calculated the median LT50 and LC50 values with their cor-esponding 95% confidence interval, which is the time period ofeeding on treated diet needed to kill 50% of the aphids and theoncentration of the kinin analog needed to kill 50% of the aphids,espectively [41]. 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 <10%.

. Results

.1. Effect of biostable PK analogs compared to natural PKs onea aphids

All of the PK analogs and natural PK peptides were fed to aphidst an initial high concentration of 0.500 nmol/�l (=mM) over ahree day period to distinguish active from inactive analogs. Annalog was deemed inactive if mortality was equal to or less than0%. The native PK peptides Aphid PK-1 (0.0 ± 0.0%) and PK-26.7 ± 0.0%) were completely inactive, and the PK pentapeptideore fragment (33.3 ± 18.9%) demonstrated little or no activity.

related natural, assassin bug CAP2b analog Rhopr CAPA-20.0 ± 0.0%), that terminates in the sequence PRVamide, alsoroved to be completely inactive in the aphid feeding assay.iostable PK analogs that were determined to be essentially inac-ive included PK-Hyp-1 (7 ± 9%), PK-Hyp-2 (3.3 ± 4.7%), PK-Hyp-310.0 ± 14.1%), PK-Hyp-4 (3.3 ± 4.7%), PK-Hyp-5 (10.0 ± 4.7%),K-2Abf (20.0 ± 9.4%), and PPK-AA (3.3 ± 4.7%). However, theic-containing analogs did induce significant mortality in thephid feeding assay. Analogs PK-Oic-1 (100.0 ± 0.0%) and PK-ic-2 (100.0 ± 0.0%) showed relatively potent activity and were

elected to conduct a more extensive dose–response study. Theose–response curves (see Fig. 3 for a dose–response curve for

K-Oic-1) allowed for the calculation of LC50 values: 0.042 nmol/�l95%CL: 0.022–0.076; R2 = 0.91) [0.029 �g/�l] for PK-Oic-1 and.121 nmol/�l (95%CL: 0.063–0.233; R2 = 0.93) [0.095 �g/�l] forK-Oic-2 (Table 1) In addition, LT50 values were calculated and

were tested per concentration. Statistical analysis and graphs were generated withthe GraphPad Prism 4.0 software.

determined to be relatively brief: 1.0 (R2 = 0.98) and 1.1 (R2 = 0.98)days, respectively. When the hindered Oic residue was incorpo-rated into the native Acypi PK-1, the resulting analog PK-Oic-3(53.3 ± 9.5%) did induce some significant mortality, but with lesspotency than either PK-Oic-1 or PK-Oic-2. When the N-terminuswas acetylated in a bid to enhance resistance to aminopeptidases,the resulting analog PK-Oic-3-Ac (17 ± 5%) lost all activity.

3.2. Effect of PEG polymer conjugated PK analogs on pea aphids

Most of the PEG polymer-conjugated PK analogs were deter-mined to be essentially inactive. At a high concentration of0.5 nmol/�l, the inactive analogs included PK-PEG4 (23.3 ± 4.7),PK-PEG8 (0.0 ± 0.0%), DH-PEG4 (13.3 ± 9.4%), PK-Oic-1-PEG4(10 ± 14%), PK-Oic-1-PEG8 (7 ± 0%) and PK-dF-PEG4 (30.0 ± 4.7%).Interestingly, while PK-dF-PEG4 demonstrated little or no activ-ity, the PEG8 analog PK-dF-PEG8 (90.0 ± 4.7%) elicited a relativelypotent response. Analog PK-dF-PEG8 was selected to conduct amore extensive dose-response study. The resulting dose–responsecurve (Fig. 4) allowed for the calculation of an LC50 value of0.126 nmol/�l (95%CL: 0.111–0.143; R2 = 0.98) and an LT50 valueof 1.3 days (95%CL: 1.0–1.6; R2 = 0.95).

In a combination experiment, the weakly active PK-dF-PEG4(0.5 nmol/�l) was added to PK-Oic-1 (0.1 nmol/�l) and fed toaphids, which led to a toxicity of 43.3 ± 23.3%. When fed separatelyto aphids, PK-dF-PEG4 (0.5 nmol/�l) and PK-Oic-1 (0.1 nmol/�l)caused aphid mortality of 30.0 ± 4.7% and 96.7 ± 4.7%, respectively.The toxicity percentages observed for the combined analogs andPK-dF-PEG4 (0.5 nmol/�l) alone were not statistically significant

(p = 0.31). In contrast, the toxicity percentages observed for thecombined analogs and PK-Oic-1 (0.1 nmol/�l) alone were statis-tically significant at the 90% confidence level (p = 0.07).

270 R.J. Nachman et al. / Peptides 34 (2012) 266–273

Table 1Aphid mortality LC50 values, expressed as �g/�l and nmol/�l (=mM), in the artificial diet for 4 biostable PK analogs, a PK core fragment, and two natural aphid PK peptidesagainst the pea aphid Acyrthosiphon pisum.

Name Sequence LC50 in diet

�g/�l nmol/�l (=mM)

PK-Oic-1 FT[Oic]RL-NH2 0.029 0.042PK-Oic-2 Hex-FT[Oic]RL-NH2 0.095 0.122PK-Hyp-1 Hex-FT[Hyp]RL-NH2 Inactive Inactive (7%/0.5 nmol/�l)a

PK-dF-PEG8 (P8)-YF[dF]PRL-NH2 0.155 0.126Pyrokinin fragment FTPRL-NH2 Inactive Inactive (33%/0.5 nmol/�l)a

Acypi PK-1 SPPYSPPFSPRL-NH Inactive Inactive (0%/0.5 nmol/�l)a

3a

tsptieat0odATaap

Ftorwewt

2

Acypi PK-2 GGTTQSSNGIWFGPRL-NH2

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

.3. Effect of PEG polymer conjugated, biostable IK and TRPnalogs on pea aphids

As biostable Aib analogs of both the insect kinin (IK) andachykinin-related peptide (TRP) families had been previouslyhown to exhibit potent antifeedant and aphicidal activity, PEGolymer was conjugated to the N-terminus of the most active ofhese analogs in an attempt to improve their oral bioavailabilityn the aphid feeding assay. Unfortunately, two of the analogs weressentially inactive; and whereas the other two analogs did retainctivity, the level of potency was much less than that observed forhe parent Aib analogs when evaluated at a high concentration of.500 nmol/�l. While analog IK-Aib-PEG8 (30.0 ± 14.1%) had littler no activity, the related PEG4 analog IK-Aib-PEG4 (56.7 ± 14.1%)id show activity, although at a much lower level than the parentib peptide K-Aib-1 (LC50 = 0.063 nmol/�l). Similarly, the analog

RP-Aib-PEG4 was inactive (13.3 ± 9.4%), whereas the related PEG8nalog TRP-Aib-PEG8 (63.3 ± 4.7%) was active at 0.500 nmol/�l,lthough it was considerably less potent than the highly activearent Aib analog TRP-Aib-1 (LC50 = 0.0087 nmol/�l).

ig. 4. Induction of aphid mortality by the insect PK peptide analog PK-dF-PEG8 inhe pea aphid Acyrthosiphon pisum. (A) Time-response over the three days of feedingf aphids on treated diet with 0.500 nmol/�l of PK-dF-PEG8 , and (B) concentration-esponse curve for mortality of aphids by different concentrations of PK-dF-PEG8

hen fed for 3 days via treated diet. Mortality percentages are based on two repeatedxperiments, each consisting of 3 groups of 15 nymphs each; a total of 90 aphidsere tested per concentration. Statistical analysis and graphs were generated with

he GraphPad Prism 4.0 software.

Inactive Inactive (0%/0.5 nmol/�l)a

4. Discussion

The two native pyrokinins (SPPYSPPFSPRL-NH2 andGGTTQSSNGIWFGPRL-NH2) failed to demonstrate antifeedantor aphicidal activity when fed to the pea aphid A. pisum. Simi-larly, a related native PRLamide peptide (QAVMAQPQVPRL-NH2)proved inactive in the aphid feeding assay. Down et al. [8] havereported that peptides of the myosuppressin family are degradedby aphid gut enzymes. As the two PK and two PRLamide peptidesnative to aphids are likely inactivated by peptidases in the gut,tissues and hemolymph of the aphid [8], analogs containingmodifications that could enhance resistance to peptidases thatinactivate the native peptides were evaluated in the feedingassay. For instance, the primary tissue-bound peptidase hydrolysissite of the pyrokinins has been identified as the peptide bondbetween the Pro and Arg of the core region FXPRLamide [27].Previous studies have shown that hindered Pro analogs, suchas octahydroindole-2-carboxylic acid (Oic) and hydroxyproline(Hyp) (Fig. 1), can greatly enhance resistance to the tissue-boundpeptidases that inactivate native pyrokinins when they replacethe Pro of the pyrokinin C-terminal active core [27]. In particu-lar, the analogs PK-Oic-2 (Hex-FT[Oic]RL-NH2) and PK-Hyp-1(Hex-FT[Hyp]RL-NH2) were shown to survive intact over atleast a 2 h period when exposed to peptidases bound to cornearworm (H. zea) Malpighian tubule tissue under conditions inwhich an unmodified, natural pyrokinin peptide was completelyhydrolyzed within 30 min or less. Furthermore, these two analogswere shown to demonstrate oral pheromonotropic activity whenfed to adult female H. virescens moths [27]. Therefore, a series ofbiostable analogs containing either Oic or Hyp in the C-terminalpentapeptide core of the pyrokinins was evaluated in the aphidfeeding assay. All five of the PK analogs incorporating Hyp provedto be essentially inactive, perhaps an indication that Hyp inthis position is incompatible with successful interaction withthe aphid PK receptor. Nonetheless, the original Oic-containinganalog PK-Oic-2 and the novel analog PK-Oic-1 (FT[Oic]RL-NH2)demonstrated a potent antifeedant and aphicidal effect on thepea aphid, with LC50 values of 0.121 nmol/�l and 0.042 nmol/�l,respectively. The LC50 value of the latter compound indicated thatits potency in the aphid feeding assay was intermediate betweenbiostable analogs of two other insect neuropeptide classes, theinsect kinins (IK) and tachykinin-related peptides (TRP). TheLC50 values for the antifeedant/aphicidal activity of the IK analogK-Aib-1 and TRP analog Leuma-TRP-Aib-1 were reported to be0.063 nmol/�l and 0.0085 nmol/�l, respectively [22,42]. However,the LT50 value (1.0) was shorter than that observed for IK analogK-Aib-1 (1.7) [42] and TRP analog Leuma-TRP-Aib-1 (1.4) [22],although this difference was not significant in the comparison with

the latter analog. An analog of the native PK peptide Acypi PK-1incorporating an Oic in the core region transformed an inactivepeptide into an active, biostable analog in the aphid feeding assay(PK-Oic-3: 53.3 ± 9.5% at 0.5 nmol/�l), although with considerably

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ess potency than Oic analogs PK-Oic-1 and PK-Oic-2. Attachmentf an acetyl group to the N-terminus of PK-Oic-3 leads to a com-lete loss of activity, even though this modification enhances theesistance to aminopeptidases. Presumably, analogs must not onlye biostable, but also capable of interacting with the PK receptorf the aphid. Two other PK analogs (PK-2Abf and PPK-AA) thateature attachment of hydrophobic groups onto the N-terminuslso demonstrated no activity in the aphid feeding assay.

One strategy to enhance the oral bioavailability and resistanceo peptidase degradation is to conjugate a polyethylene glycol poly-

er (PEG) to the N-terminus of peptides [15,40]. Unfortunately, thettachment of PEG polymer of two different lengths (Fig. 2) to thective PK analogs PK-Oic-1 led to inactive analogs (PK-Oic-1-PEG4nd PK-Oic-1-PEG8). Three other PEGylated PK analogs (PK-PEG4,K-PEG8, and DH-PEG4) proved to be inactive as well.

PEGylation of active, biostable analogs of two otherlasses of insect neuropeptides also drastically reduced theirntifeedant/aphicidal activity. PEGylation of the IK analog K-Aib-1LC50 = 0.063 nmol/�l), led to weakly active analog IK-Aib-PEG456.7 ± 14.1% at 0.5 nmol/�l) and an essentially inactive analogK-Aib-PEG8 (30.1 ± 14.1% at 0.5 nmol/�l). And, PEGylation of theighly potent TRP analog Leuma-TRP-1 (LC50 = 0.0085 nmol/�l),

ed to inactive analog TRP-Aib-PEG4 (13.3 ± 9.4% at 0.5 nmol/�l)nd weakly active analog IK-Aib-PEG8 (63.3 ± 4.7% at 0.5 nmol/�l).

One exception to the poor performance observed for PEGylatednalogs in this study was noted in the case of a PK analog con-aining a d-Phe residue. While the analog PK-dF-PEG4 proved toave little or no activity, the related analog PK-dF-PEG8 demon-trated strong antifeedant and aphicidal activity with a relativelyotent LC50 value of 0.126 nmol/�l (LT50 = 1.3 days) (see Fig. 4).hereas the other PEGylated analogs in this study involved PEG

olymer conjugation of neuropeptide agonists, PK-dF-PEG8 is a PEGolymer conjugate of an antagonist of the PK neuropeptide class in

epidopteran pheromonotropic and melanization assays [4,28]. Theeason for the potent aphicidal activity of PK-dF-PEG8 is unknownut may be a consequence of this distinction. The addition of theEG polymers to the N-terminus of the PK agonists may impedenteraction with the aphid PK receptor, particularly receptor acti-ation. The PK antagonist sequence is capable of binding to certainK receptors in other insects but is incapable of activating thoseeceptors; thus the addition of the PEG polymer cannot furtherarm this aspect of the receptor interaction. Presumably, the PEGolymer does not impede binding to the aphid PK receptor. Thisypothesis raises the intriguing possibility that PK-dF-PEG8 mayxhibit antifeedant/aphicidal activity by acting as an antagonist athe putative PK receptor of aphids. To further test this possibil-ty, the weakly active PEG analog of a PK antagonist, PK-dF-PEG40.5 nmol/�l), was added to the highly active agonist analog PK-ic-1 (0.1 nmol/�l) to see if it could block its potent oral aphicidalffects. Indeed, the toxicity observed for the combined analog treat-ent was reduced by 55% in comparison with the aphid mortality

ercentage observed for the agonist analog alone. This effect wastatistically significant (p = 0.07) at greater than a 90% confidenceevel. Interestingly, the aphid toxicity for the combined treatment

as not statistically different than the antagonist analog alonep = 0.31). Importantly, the results suggest that the agonist andntagonist analogs act at the same receptor and that this receptors likely a PK receptor. They further suggest that the potent aphici-al activity of PK-dF-PEG8, a dF-containing cousin of PK-dF-PEG4,rises via an antagonist response.

Two reference aphidicides that are currently used in the mar-etplace for selective IPM control against aphids in agriculture

re pymetrozine and flonicamid. Both compounds act specifi-ally against aphids as feeding inhibitors, although their exactechanism(s) remain unidentified. Flonicamid [N-(cyanomethyl)-

-(trifluoromethyl)-3-pyridinecarboxamide] is a novel insecticide;

s 34 (2012) 266–273 271

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 [36].For pymetrozine [1,2,4-triazin-3(2H)-one,4,5-dihydro-6-methyl-4-[(3-pyridinylmethylene)amino]], Sadeghi et al. [36] calculatedwith use of a similar feeding apparatus with a diet sachet an LC50of 0.01 �g/ml [37]. 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 [9]. In the group of insect growth regula-tor (IGRs), azadirachtin (Neem), flufenoxuron and pyriproxyfen arealso commercially used in the selective control of aphids and theyhave a respective LC50 of 7.9, 8.7 and 9.3 �g/ml artificial diet againstpea aphids [37]. Here typical phenotypic symptoms of aphid mor-tality were disruption of nymphal molt and abortion of molting. Inthe field of insecticidal proteins, mannose-binding lectins receivedmuch attention in the last decade because the Galanthus nivalisagglutinin (GNA, homotetrameric protein composed of 12 kDa sub-units) is highly detrimental to aphids [9,36,37] and can be deliveredvia transgenic plants. Sadeghi et al. [36] reported an LC50 of 350and 700 �g/ml for two mannose-binding lectins GNA and ASA afterfeeding for 3 days on treated diet.

Thus, the fact that the stabilized PK analogs of this study, andespecially the analogs PK-Oic-1 and PK-dF-PEG8, show rapid andhigh activities against A. pisum aphids in the same order of mag-nitude as some commercial aphicides tested under comparableconditions in the laboratory, suggests that they represent poten-tially valuable leads for alternative agents in the control of aphidsand in the struggle against insecticide resistance. In addition, webelieve that testing other sucking pest insects would also be ofinterest. 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 PK analogs is associated withthe presence of components that enhance the resistance of theC-terminal core region to peptidases, as the unmodified PKsdemonstrate no activity. The mechanism of the aphid antifeedantactivity and high induction of mortality demonstrated by thebiostable PK analogs cannot be clearly identified at this point,but it may be associated with disruption of the physiologicalprocesses that this important neuropeptide family regulates ininsects, particularly hindgut and midgut contractile activity thatmay impede normal digestive processes. For this to happen, thebiostable analogs with aphicidal activity would necessarily need tointeract with a native aphid PK receptor(s).

Finally, it is possible that activation of taste receptors by thepresence of the analogs may cause the aphids to avoid ingestionof the diet altogether, leading to starvation. While honeydew for-mation is depressed in the aphids exposed to the active analogs,the observations of normal piercing behavior and the presenceof at least some honeydew suggest that ingestion is nonethelesstaking place. Impairment of normal physiological patterns in theaphids ingesting the active analogs may lead to a reduction insubsequent feeding and, in turn, to the observed reduction in lev-els of honeydew formation. Furthermore, the unmodified peptidesare readily ingested by the aphids, as are the essentially inactive

analogs containing Hyp or many of those that are conjugated withPEG polymer, which also contain unnatural amino acids found inthe active analogs. Thus, the fact that a number of the PK analogsdo not trigger avoidance of diet ingestion would seem to suggest

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hat some ingestion of the three active biostable PK analogs maylso be taking place.

In summary, the presence of three biostable PK core analogsK-Oic-2, PK-dF-PEG8, and particularly PK-Oic-1, in the dietemonstrate significant antifeedant activity and induction of highortality in the pea aphid A. pisum that matches that of some

ommercially available aphicides. Unmodified natural PK peptidesnd some analogs containing some of the same structural compo-ents that promote biostability are inactive. The potent aphicidalffects of PK-Oic-1 can be blocked by over 55% when combinedith a PEG polymer analog of a PK antagonist, a PEGylated cousin

f PK-dF-PEG8, suggesting that the aphicidal effects are achievedia interaction with a PK receptor. Analogs PK-Oic-1 and PK-Oic-2emonstrate activity via an agonist response, whereas PK-dF-PEG8cts via an antagonist response. The active biostable PK analogsescribed in this study and/or 2nd generation analogs, either in

solation or in combination with biostable analogs of other neu-opeptide classes that also regulate aspects of diuretic, antidiuretic,igestive, reproductive and/or developmental processes, representotential leads in the development of selective, environmentallyriendly pest aphid control agents capable of disrupting those crit-cal processes.

cknowledgements

The authors wish to thank Allison Strey (USDA, College Sta-ion) for able technical assistance. We also acknowledge financialssistance from a grant from the USDA/DOD DWFP Initiative#0500-32000-001-01R) (RJN), a USDA-NIFA Grant No. 2011-7013-30199 (RJN) and a grant from the US-Israel Binationalgricultural Research and Development Fund (BARD) (IS-4205-9C) (RJN, KK, and JZ), and support from the Fund of Scientificesearch (FWO-Vlaanderen, Belgium) and the Special Researchund of Ghent University (BOF-UGent) to GS.

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