Peptides 34 (2012) 266–273
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Peptides
journal homepage: www.elsevier.com/locate/peptides
Biostable and PEG polymer-conjugated insect pyrokinin analogs demonstrate
antifeedant activity and induce high mortality in the pea aphid Acyrthosiphon
pisum (Hemiptera: Aphidae)
Ronald J. Nachman a,∗, Mohamad Hamshou b, Krzysztof Kaczmarek a,c, Janusz Zabrocki a,c, Guy Smagghe b,∗∗
a
Areawide Pest Management Research, Southern Plains Agricultural Research Center, USDA, 2881 F/B Road, College Station, TX 77845, USA
Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium
c
Institute of Organic Chemistry, Technical University of Lodz, 90-924 Lodz, Poland
b
a r t i c l e
i n f o
Article history:
Received 11 October 2011
Received in revised form 7 November 2011
Accepted 7 November 2011
Available online 15 November 2011
Keywords:
Aphicide
Hindgut myotropic
Peptidase resistant
a b s t r a c t
The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, including
the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs based
on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid
over a period of three days and evaluated for antifeedant and aphicidal activity. The analogs contained
either modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance to
tissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugated
with 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 in
honeydew formation and very high mortality after 1 day. In contrast, a number of unmodified, natural
pyrokinins and several other analogs containing some of the same structural components that promote
biostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH2 ) and
PEGylated analog PK-dF-PEG8 [(P8 )-YF[dF]PRL-NH2 ], featured aphicidal activity calculated at LC50 ’s of
0.042 nmol/␮l [0.029 ␮g/␮l] (LT50 of 1.0 day) and 0.126 nmol/␮l (LT50 of 1.3 days), respectively, matching
the 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 aphicidal
effects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though the
aphicidal activity of the biostable analogs may result from disruption of digestive processes by interfering
with gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PK
analogs represent potential leads in the development of selective, environmentally friendly aphid pest
control agents.
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) peptides represent a multifunctional family that
plays a significant role in the physiology of insects. Leucopyrokinin
(LPK), isolated from the cockroach Leucophaea maderae in 1986
[13], was the first member of the family to be discovered. Since
that time, over 30 peptides have been identified. They include PKs,
myotropins (MTs), PBAN, melanization and reddish coloration hormone (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.be
(G. Smagghe).
0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2011.11.009
all of which share the common C-terminal pentapeptide FXPRLamide (X = S, T, G or V) [3,12,32,33]. Functions of the PK/PBAN
family include stimulation of sex pheromone biosynthesis in moths
[3,32–34], and mediation of key aspects of feeding (gut muscle
contractions) [19,38], development (embryonic diapause, pupal
diapause and pupariation) [14,20,30,45,46] and defense (melanin
biosynthesis) [5,18] in a variety of insects (cockroaches, flies,
locusts and moths). All of the above functions can be stimulated
by more than one peptide, and they demonstrate considerable
cross-activity between various PK/PBAN assays, thereby lacking
any 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 significance for many physiological functions elicited by the PK/PBAN
family 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
HO
267
O
O
O
O
O
(P4)
O
N
Hyp
O
N
Pro
N
O
O
O
corn earworm Helicoverpa zea) [11,23,24], oviduct contractile
(cockroach L. maderae) [21], egg diapause induction (silk worm
B. mori) [11,24,29], pupal diapause termination (corn earworm
budworm H. zea) [47], and pupariation (flesh fly Neobellieria
bullata) [46] assay systems.
Due to the susceptibility of PK/PBANs to both exo- and endopeptidases in the insect hemolymph and gut, these peptides cannot
be directly used as pest control agents and/or research tools
by insect neuroendocrinologists. Members of the PK/PBAN family are hydrolyzed, and therefore inactivated, by tissue-bound
peptidases of insects. The primary site within the C-terminal pentapeptide (Phe1 -Xxx2 -Pro3 -Arg4 -Leu5 -NH2 ) is between Pro3 and
the Arg4 residue [27]. To overcome the limitations inherent in the
physicochemical characteristics of peptides, the development of
peptidomimetic analogs has been used as a strategy to enhance
their biological effects. It has been proposed that blocking or overstimulating the receptors of insect neuropeptides could lead to
reduction of pest fitness or even increased mortality [17,26]. Peptidomimetics is a broader term used to refer to pseudopeptides
and non-peptides designed to perform the functions of a peptide. Generally these peptidomimetics are derived by the structural
modification of the lead peptide sequence to overcome a number of
metabolic limitations, such as proteolytic degradation that restrict
the use of peptides as agents capable of modulating aspects of insect
physiology [26,43].
One peptidomimetic approach employed with the PK/PBAN
class of neuropeptides is the replacement of the critical Pro residue
with such sterically hindered Pro analogs as octahydroindole-2carboxyl (Oic) and hydroxyprolyl (Hyp) [27] moieties (Fig. 1), as
well as the introduction of d-amino acids. This former approach
has been used to develop biostable Oic and Hyp analogs of the
PK/PBAN neuropeptide family that have demonstrated markedly
enhanced resistance to hydrolysis by tissue-bound peptidases.
These analogs also demonstrated activity in an in vivo adult female
Heliothis virescens moth pheromonotropic assay when delivered
orally under conditions in which the native PBAN peptide and/or
fragments do not [27]. A Hyp-containing PK/PBAN analog proved
to be more than 5-fold more potent than the native hormone DH, a
member of the PK/PBAN family in terminating diapause in pupae of
H. zea [47], presumably because of a longer hemolymph residence
time of the Hyp analog over the native hormone.
Another approach to the stabilization of peptides and/or proteins to enzymatic degradation in the digestive system as well
as the enhancement of penetration across cell membranes of the
gut into the hemolymph (blood) of insects is the conjugation of
polyethylene glycol (PEG) polymers (Fig. 2) to the N-terminus
[7,15,40]. Although not previously applied to neuropeptides of the
PK/PBAN class, conjugation of PEG polymers to the insect peptide
trypsin modulating oostatic factor (TMOF) enhanced the resistance
to degradation by the digestive enzyme leucine aminopeptidase,
leading to accumulation of the peptide in hemolymph of insects
and ticks [7,15,40].
About 250 of the 4000 aphid species that have been described
are serious pests to various crops around the world, causing
O
O
O
O
O
O
(P8)
Oic
Fig. 1. Structures of modified analogs of Pro (middle): hydroxyprolyl (Hyp; left) and
the bulky octahydroindole-2-carboxyl residues (Oic; right).
O
Fig. 2. Structures of two lengths of PEG polymer that was conjugated to the Nterminus 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 transmitting viruses that can devastate agricultural crops [2]. In particular,
the pea aphid Acyrthosiphon pisum causes hundreds of millions of
dollars of crop damage every year, and many populations have
already acquired resistance toward multiple conventional and
modern insecticides, making a search for alternative strategies
urgent [9]. Furthermore aphids are not sensitive to the toxins
from the bacterium Bacillus thuringiensis (Bt) [39]. Interestingly,
the 525 Mb genome of A. pisum has recently been sequenced by
the International Aphid Genomic Consortium providing a resource
for comparative genomics and the tools to identify targets for control (AphidBase; http://www.aphidbase.com; [35]). We identified
the sequences of the two native PKs as SPPYSPPFSPRL-NH2 and
GGTTQSSNGIWFGPRL-NH2 , as well as related PRLamide peptides
QAVMAQPQVPRL-NH2 and pQAVMAQPQVPRL-NH2 .
In this paper, we expanded on earlier synthetic work to prepare
a series of biostable analogs of the PK/PBAN class of neuropeptides containing the sterically hindered Pro residues Oic and Hyp, a
d-Phe amino acid and/or the attachment of hydrophobic hydrocarbon moiety onto the N-terminus. These PK/PBAN analogs are listed
below in the first group of structures:
PK-Oic-1:
PK-Oic-2:
PK-Oic-3:
PK-Oic-4:
PK-Hyp-1:
PK-Hyp-2:
PK-Hyp-3:
PK-Hyp-4:
PK-Hyp-5:
PK-2Abf:
PPK-AA:
FT[Oic]RL-NH2
Hex-FT[Oic]RL-NH2
SPPYSPPFS[Oic]RL-NH2
Ac-SPPYSPPFS[Oic]RL-NH2
Hex-FT[Hyp]RL-NH2
Ac-FT[Hyp]RL-NH2
Ahx-FT[Hyp]RL-NH2
pQFT[Hyp]RL-NH2
pQYFT[Hyp]RL-NH2
2Abf-Suc-FTPRL-NH2
Hex-Suc-A[dF]PRL-NH2
These biostable analogs of the PK/PBAN family were fed in solutions of artificial diet to the pea aphid A. pisum over a period of
three days and evaluated for antifeedant and aphicidal activity. A
comparison of the biostable analogs was made to unmodified, natural PK/PBAN peptides and/or the C-terminal pentapeptide fragment
that represents the active core of this large family of peptides listed
in the second group of structures below.
PK core fragment:
Acypi PK-1:
Acypi PK-2:
Acypi PRLamide:
Rhopr CAPA-2:
FTPRL-NH2
SPPYSPPFSPRL-NH2
GGTTQSSNGIWFGPRL-NH2
pQAVMAQPQVPRL-NH2
EGGFISFPRV-NH2
In addition, the first PEG polymer conjugates of the PK/PBAN
family were synthesized (see list of polymer-conjugate analogs in
the third list of structures below) and evaluated in the aphid bioassay.
PK-PEG4 :
PK-PEG8 :
DH-PEG4 :
PK-dF-PEG4 :
PK-dF-PEG8 :
PK-Oic-1-PEG4 :
PK-Oic-1-PEG8 :
(P4 )-YFTPRL-NH2
(P8 )-YFTPRL-NH2
(P4 )-LWFGPRL-NH2
(P4 )-YF[dF]PRL-NH2
(P8 )-YF[dF]PRL-NH2
(P4 )-FT[Oic]RL-NH2
(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
268
R.J. Nachman et al. / Peptides 34 (2012) 266–273
been previously shown to exhibit potent antifeedant and aphicidal
effects [22,42]. These four analogs are listed in the fourth and final
group of structures below.
IK-Aib-PEG4 :
IK-Aib-PEG8 :
TRP-Aib-PEG4 :
TRP-Aib-PEG8 :
(P4 )-R[Aib]FF[Aib]WG-NH2
(P8 )-R[Aib]FF[Aib]WG-NH2
(P4 )-A[Aib]SGFL[Aib]VR-NH2
(P8 )-A[Aib]SGFL[Aib]VR-NH2
2. Materials and methods
2.1. Synthesis and characterization of PK analogs
The PK analogs PK-Oic-2 [27], PK-Hyp-1 [27], PK-2Abf
[44], PPK-AA [28], Rhopr CAPA-2 [31] and the PK core
fragment [FTPRL-NH2 ] [19] were synthesized as previously
described. The Aib-containing TRP analogs Leuma-TRPAib-1 (pEA[Aib]SGFL[Aib]VR-NH2 ) and Leuma-TRP-Aib-2
(pEA[Aib]S[Aib]FL[Aib]VR-NH2 ) and the natural, unmodified
TRP analogs Leuma-TRP-1 (APSGFLGVR-NH2 ), Acypi-TRP-1
(ASMGFMGMR-NH2 ), and Acypi-TRP-2 (VPSADAFYGVR-NH2 )
were synthesized, purified and quantified by adoption of procedures that have been previously described by Nachman et al.
[21,48]. In brief, analogs were synthesized on an ABI 433A
peptide synthesizer with a modified FastMoc 0.25 procedure
using an Fmoc-strategy starting from Rink amide resin (Novabiochem, San Diego, CA; 0.5 mmol/g). The Fmoc protecting
group was removed by 20% 4-methyl piperidine (NMP) in
dimethyl formamide (DMF). A fourfold excess of the respective
Fmoc-amino acids was activated in situ using 2-(1h-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
(1 equivalent [eq.]/1-hydroxybenzotriazole) (1 eq.) in NMP (Nmethylpyrrolidone) (HOBt). The coupling reactions were base
catalyzed with N,N-diisopropylethylamine (DIPEA) (4 eq.). The
analogs were cleaved from the resin with side-chain deprotection
by treatment with trifluoroacetic acid (TFA):H2 O:triisopropylsilane
(TIS) (95.5:2.5:2.5, v/v/v) for 1.5 h. The PEG polymer conjugations
were accomplished as follows: after transferring peptidyl resin
with the completed peptide sequence into an 8 ml polypropylene syringe, a 1.2 molar equivalent of MS(PEG)4 or MS(PEG)8
reagent was added as a 10% solution in NMP (100 mg of viscous
reagent was reconstituted with 900 mg NMP). Both reagents are
commercially available (Thermo Scientific, Waltham, MA) and
they are N-hydroxysuccinimide esters of O-methyl-tetra- and
octa-ethyleneglycolcarboxylic acid, respectively. The syringes
were shaken over night at RT and, following a positive Kaiser
test, EDC was added (0.5 eq.) and shaken for one additional day.
After washing with DCM (3×) and methanol (3×) and drying the
PEGylated peptide analogs, cleavage from the resin was accomplished with a cocktail composed of TFA/DMB/TIS (92.5:5:2.5) and
precipitated with ether.
All analogs were desalted on a Waters C18 Sep Pak cartridge
(Milford, MA) in preparation for purification by HPLC. The analogs
were purified on a Waters Delta-Pak C18 reverse-phase column
(8 mm × 100 mm, 15 ␮m particle size, 100 Å pore size) with a
Waters 510 HPLC system with detection at 214 nm at ambient
temperature. Solvent A = 0.1% aqueous TFA; Solvent B = 80% aqueous acetonitrile containing 0.1% TFA. Initial conditions were 10%
B followed by a linear increase to 90% B over 40 min; flow rate,
2 ml/min. Delta-Pak C18 retention times: PK-Oic-3: 7.5 min, PKOic-4: 6.0 min, PK-PEG4 : 9.0 min, PK-PEG8 : 7.5 min, DH-PEG4 :
14.0 min, PK-dF-PEG4 : 9.0 min, PK-dF-PEG8 : 12.0 min, PK-OicPEG4 : 9.0 min, PK-Oic-PEG8 : 7.5 min, IK-Aib-PEG4 : 12.0 min,
IK-Aib-PEG8 : 12.0 min, TRP-Aib-PEG4 : 9.5 min, TRP-Aib-PEG8 :
9.0 min, Acypi PK-1: 8.0 min, Acypi PK-2: 7.5 min, Acypi
PRLamide: 5.0 min, PK-Oic-1: 12.0 min, PK-Hyp-2: 6.0 min, PKHyp-4: 6.0 min, and PK-Hyp-3: 7.5 min. The analogs were further
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: PKOic-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-AibPEG4 : 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 analysis
was carried out under previously reported conditions [21,45] to
quantify 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]; PKPEG8 : 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]; PKOic-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]; PKHyp-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 identity of the analogs was also confirmed by MALDI-MS on a Kratos
Kompact 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-OicPEG8 : 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. pisum
was maintained on young broad bean (Vicia faba) plants in the
Laboratory of Agrozoology at Ghent University, Belgium, under
standardized conditions of 23–25 ◦ C, a 16 h light photoperiod and
60–65% relative humidity. Mature aphids were put on plants,
resulting in synchronized offspring, i.e., neonate nymphs with an
age of 0–12 h, that were used throughout the experiments [36].
2.3. Bioassay with pea aphid in a feeding apparatus with an
artificial assay to determine antifeedant and aphicidal activity of
peptide analogs
For all aphid bioassays, neonates with an age of 0–12 h were
collected from a continuous colony of the pea aphid A. pisum as
described [37,42].
As food for the aphids, a standard diet previously developed for
A. pisum [10] was used as the basal diet to which the peptide analogs
were added. The feeding apparatus was prepared using plexiglass
cylinders (3 cm high and 3 cm diameter). The food sachet was made
under sterile conditions and consists of two layers of parafilm membrane on top of the container. About 200 ␮l of the artificial diet was
sandwiched between the two layers [37].
R.J. Nachman et al. / Peptides 34 (2012) 266–273
To challenge aphids to the insect peptide analogs or a combination of peptide analogs, a stock solution was prepared in the
solvent aqueous 80% acetone/0.01% TFA, and then diluted in the
artificial diet to prepare different concentrations between 0.001
and 0.500 nmol/␮l (=mM). In the treatments, 200 ␮l of each concentration was used to make a food sachet. A volume of 20 ␮l of
the analogs dissolved in aqueous 80% acetone/0.01% TFA was then
diluted up to 200 ␮l with distilled water. In the solvent-controls the
diet was supplemented with an equivalent amount of the solvent
80% acetone/0.01% TFA, and in the blank-controls with only distilled water. At day 0, 15 neonate nymphs (aged 0–12 h), obtained
from a synchronized population reared on V. faba plants, were
transferred onto the artificial diet. For each concentration, three
replicates were carried out and aphids were checked daily during 3
days for honeydew formation to determine antifeedant effects and
also for numbers of dead aphids to determine aphicidal effects. The
experiment was performed two times independently.
To determine antifeedant effects, the amounts of honeydew produced by the aphids in the treatments as compared to controls were
measured using the Ninhydrin test as described by Kanrar et al. [16].
In brief, a 3.6 cm-diameter petri dish, as described above in the feeding apparatus, was lined with a Whatman No. 3 filter paper. This
filter paper (with the honeydew) was removed and sprayed with
0.1% ninhydrin reagent to detect the presence of honeydew spots.
The aphid mortality percentages were analyzed using nonlinear sigmoid curve fitting, and the toxicity of each treatment was
evaluated on the basis of time-response curves and concentrationresponse curves using the GraphPad Prism 4.0 software (La Jolla,
CA). We calculated the median LT50 and LC50 values with their corresponding 95% confidence interval, which is the time period of
feeding on treated diet needed to kill 50% of the aphids and the
concentration of the kinin analog needed to kill 50% of the aphids,
respectively [41]. The mortality data were corrected according to
Abbott’s formula based on the mortality seen in the control groups;
in all experiments, mortality in the control groups averaged at a low
level of <10%.
3. Results
269
Fig. 3. Induction of aphid mortality by the insect PK peptide analog PK-Oic-1 in the
pea aphid Acyrthosiphon pisum. (A) Time-response over the three days of feeding
of aphids on treated diet with 0.500 nmol/␮l of PK-Oic-1, and (B) concentrationresponse curve for mortality of aphids by different concentrations of PK-Oic-1 when
fed for 3 days via treated diet. Mortality percentages are based on two repeated
experiments, each consisting of 3 groups of 15 nymphs each; a total of 90 aphids
were tested per concentration. Statistical analysis and graphs were generated with
the 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 incorporated into the native Acypi PK-1, the resulting analog PK-Oic-3
(53.3 ± 9.5%) did induce some significant mortality, but with less
potency than either PK-Oic-1 or PK-Oic-2. When the N-terminus
was acetylated in a bid to enhance resistance to aminopeptidases,
the resulting analog PK-Oic-3-Ac (17 ± 5%) lost all activity.
3.1. Effect of biostable PK analogs compared to natural PKs on
pea aphids
3.2. Effect of PEG polymer conjugated PK analogs on pea aphids
All of the PK analogs and natural PK peptides were fed to aphids
at an initial high concentration of 0.500 nmol/␮l (=mM) over a
three day period to distinguish active from inactive analogs. An
analog was deemed inactive if mortality was equal to or less than
30%. The native PK peptides Aphid PK-1 (0.0 ± 0.0%) and PK-2
(6.7 ± 0.0%) were completely inactive, and the PK pentapeptide
core fragment (33.3 ± 18.9%) demonstrated little or no activity.
A related natural, assassin bug CAP2b analog Rhopr CAPA-2
(0.0 ± 0.0%), that terminates in the sequence PRVamide, also
proved to be completely inactive in the aphid feeding assay.
Biostable PK analogs that were determined to be essentially inactive included PK-Hyp-1 (7 ± 9%), PK-Hyp-2 (3.3 ± 4.7%), PK-Hyp-3
(10.0 ± 14.1%), PK-Hyp-4 (3.3 ± 4.7%), PK-Hyp-5 (10.0 ± 4.7%),
PK-2Abf (20.0 ± 9.4%), and PPK-AA (3.3 ± 4.7%). However, the
Oic-containing analogs did induce significant mortality in the
aphid feeding assay. Analogs PK-Oic-1 (100.0 ± 0.0%) and PKOic-2 (100.0 ± 0.0%) showed relatively potent activity and were
selected to conduct a more extensive dose–response study. The
dose–response curves (see Fig. 3 for a dose–response curve for
PK-Oic-1) allowed for the calculation of LC50 values: 0.042 nmol/␮l
(95%CL: 0.022–0.076; R2 = 0.91) [0.029 ␮g/␮l] for PK-Oic-1 and
0.121 nmol/␮l (95%CL: 0.063–0.233; R2 = 0.93) [0.095 ␮g/␮l] for
PK-Oic-2 (Table 1) In addition, LT50 values were calculated and
Most of the PEG polymer-conjugated PK analogs were determined to be essentially inactive. At a high concentration of
0.5 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 activity, the PEG8 analog PK-dF-PEG8 (90.0 ± 4.7%) elicited a relatively
potent response. Analog PK-dF-PEG8 was selected to conduct a
more extensive dose-response study. The resulting dose–response
curve (Fig. 4) allowed for the calculation of an LC50 value of
0.126 nmol/␮l (95%CL: 0.111–0.143; R2 = 0.98) and an LT50 value
of 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 to
aphids, which led to a toxicity of 43.3 ± 23.3%. When fed separately
to 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 and
PK-dF-PEG4 (0.5 nmol/␮l) alone were not statistically significant
(p = 0.31). In contrast, the toxicity percentages observed for the
combined analogs and PK-Oic-1 (0.1 nmol/␮l) alone were statistically significant at the 90% confidence level (p = 0.07).
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Table 1
Aphid 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 peptides
against the pea aphid Acyrthosiphon pisum.
Name
PK-Oic-1
PK-Oic-2
PK-Hyp-1
PK-dF-PEG8
Pyrokinin fragment
Acypi PK-1
Acypi PK-2
a
Sequence
FT[Oic]RL-NH2
Hex-FT[Oic]RL-NH2
Hex-FT[Hyp]RL-NH2
(P8 )-YF[dF]PRL-NH2
FTPRL-NH2
SPPYSPPFSPRL-NH2
GGTTQSSNGIWFGPRL-NH2
LC50 in diet
␮g/␮l
nmol/␮l (=mM)
0.029
0.095
Inactive
0.155
Inactive
Inactive
Inactive
0.042
0.122
Inactive (7%/0.5 nmol/␮l)a
0.126
Inactive (33%/0.5 nmol/␮l)a
Inactive (0%/0.5 nmol/␮l)a
Inactive (0%/0.5 nmol/␮l)a
% Toxicity with highest concentration tested (given between brackets).
3.3. Effect of PEG polymer conjugated, biostable IK and TRP
analogs on pea aphids
As biostable Aib analogs of both the insect kinin (IK) and
tachykinin-related peptide (TRP) families had been previously
shown to exhibit potent antifeedant and aphicidal activity, PEG
polymer was conjugated to the N-terminus of the most active of
these analogs in an attempt to improve their oral bioavailability
in the aphid feeding assay. Unfortunately, two of the analogs were
essentially inactive; and whereas the other two analogs did retain
activity, the level of potency was much less than that observed for
the parent Aib analogs when evaluated at a high concentration of
0.500 nmol/␮l. While analog IK-Aib-PEG8 (30.0 ± 14.1%) had little
or no activity, the related PEG4 analog IK-Aib-PEG4 (56.7 ± 14.1%)
did show activity, although at a much lower level than the parent
Aib peptide K-Aib-1 (LC50 = 0.063 nmol/␮l). Similarly, the analog
TRP-Aib-PEG4 was inactive (13.3 ± 9.4%), whereas the related PEG8
analog TRP-Aib-PEG8 (63.3 ± 4.7%) was active at 0.500 nmol/␮l,
although it was considerably less potent than the highly active
parent Aib analog TRP-Aib-1 (LC50 = 0.0087 nmol/␮l).
Fig. 4. Induction of aphid mortality by the insect PK peptide analog PK-dF-PEG8 in
the pea aphid Acyrthosiphon pisum. (A) Time-response over the three days of feeding
of aphids on treated diet with 0.500 nmol/␮l of PK-dF-PEG8 , and (B) concentrationresponse curve for mortality of aphids by different concentrations of PK-dF-PEG8
when fed for 3 days via treated diet. Mortality percentages are based on two repeated
experiments, each consisting of 3 groups of 15 nymphs each; a total of 90 aphids
were tested per concentration. Statistical analysis and graphs were generated with
the GraphPad Prism 4.0 software.
4. Discussion
The two native pyrokinins (SPPYSPPFSPRL-NH2 and
GGTTQSSNGIWFGPRL-NH2 ) failed to demonstrate antifeedant
or aphicidal activity when fed to the pea aphid A. pisum. Similarly, a related native PRLamide peptide (QAVMAQPQVPRL-NH2 )
proved inactive in the aphid feeding assay. Down et al. [8] have
reported that peptides of the myosuppressin family are degraded
by aphid gut enzymes. As the two PK and two PRLamide peptides
native to aphids are likely inactivated by peptidases in the gut,
tissues and hemolymph of the aphid [8], analogs containing
modifications that could enhance resistance to peptidases that
inactivate the native peptides were evaluated in the feeding
assay. For instance, the primary tissue-bound peptidase hydrolysis
site of the pyrokinins has been identified as the peptide bond
between the Pro and Arg of the core region FXPRLamide [27].
Previous studies have shown that hindered Pro analogs, such
as octahydroindole-2-carboxylic acid (Oic) and hydroxyproline
(Hyp) (Fig. 1), can greatly enhance resistance to the tissue-bound
peptidases that inactivate native pyrokinins when they replace
the Pro of the pyrokinin C-terminal active core [27]. In particular, 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 at
least a 2 h period when exposed to peptidases bound to corn
earworm (H. zea) Malpighian tubule tissue under conditions in
which an unmodified, natural pyrokinin peptide was completely
hydrolyzed within 30 min or less. Furthermore, these two analogs
were shown to demonstrate oral pheromonotropic activity when
fed to adult female H. virescens moths [27]. Therefore, a series of
biostable analogs containing either Oic or Hyp in the C-terminal
pentapeptide core of the pyrokinins was evaluated in the aphid
feeding assay. All five of the PK analogs incorporating Hyp proved
to be essentially inactive, perhaps an indication that Hyp in
this position is incompatible with successful interaction with
the aphid PK receptor. Nonetheless, the original Oic-containing
analog PK-Oic-2 and the novel analog PK-Oic-1 (FT[Oic]RL-NH2 )
demonstrated a potent antifeedant and aphicidal effect on the
pea aphid, with LC50 values of 0.121 nmol/␮l and 0.042 nmol/␮l,
respectively. The LC50 value of the latter compound indicated that
its potency in the aphid feeding assay was intermediate between
biostable analogs of two other insect neuropeptide classes, the
insect kinins (IK) and tachykinin-related peptides (TRP). The
LC50 values for the antifeedant/aphicidal activity of the IK analog
K-Aib-1 and TRP analog Leuma-TRP-Aib-1 were reported to be
0.063 nmol/␮l and 0.0085 nmol/␮l, respectively [22,42]. However,
the LT50 value (1.0) was shorter than that observed for IK analog
K-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-1
incorporating an Oic in the core region transformed an inactive
peptide 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
R.J. Nachman et al. / Peptides 34 (2012) 266–273
less potency than Oic analogs PK-Oic-1 and PK-Oic-2. Attachment
of an acetyl group to the N-terminus of PK-Oic-3 leads to a complete loss of activity, even though this modification enhances the
resistance to aminopeptidases. Presumably, analogs must not only
be biostable, but also capable of interacting with the PK receptor
of the aphid. Two other PK analogs (PK-2Abf and PPK-AA) that
feature attachment of hydrophobic groups onto the N-terminus
also demonstrated no activity in the aphid feeding assay.
One strategy to enhance the oral bioavailability and resistance
to peptidase degradation is to conjugate a polyethylene glycol polymer (PEG) to the N-terminus of peptides [15,40]. Unfortunately, the
attachment of PEG polymer of two different lengths (Fig. 2) to the
active PK analogs PK-Oic-1 led to inactive analogs (PK-Oic-1-PEG4
and PK-Oic-1-PEG8 ). Three other PEGylated PK analogs (PK-PEG4 ,
PK-PEG8 , and DH-PEG4 ) proved to be inactive as well.
PEGylation of active, biostable analogs of two other
classes of insect neuropeptides also drastically reduced their
antifeedant/aphicidal activity. PEGylation of the IK analog K-Aib-1
(LC50 = 0.063 nmol/␮l), led to weakly active analog IK-Aib-PEG4
(56.7 ± 14.1% at 0.5 nmol/␮l) and an essentially inactive analog
IK-Aib-PEG8 (30.1 ± 14.1% at 0.5 nmol/␮l). And, PEGylation of the
highly potent TRP analog Leuma-TRP-1 (LC50 = 0.0085 nmol/␮l),
led to inactive analog TRP-Aib-PEG4 (13.3 ± 9.4% at 0.5 nmol/␮l)
and weakly active analog IK-Aib-PEG8 (63.3 ± 4.7% at 0.5 nmol/␮l).
One exception to the poor performance observed for PEGylated
analogs in this study was noted in the case of a PK analog containing a d-Phe residue. While the analog PK-dF-PEG4 proved to
have little or no activity, the related analog PK-dF-PEG8 demonstrated strong antifeedant and aphicidal activity with a relatively
potent LC50 value of 0.126 nmol/␮l (LT50 = 1.3 days) (see Fig. 4).
Whereas the other PEGylated analogs in this study involved PEG
polymer conjugation of neuropeptide agonists, PK-dF-PEG8 is a PEG
polymer conjugate of an antagonist of the PK neuropeptide class in
lepidopteran pheromonotropic and melanization assays [4,28]. The
reason for the potent aphicidal activity of PK-dF-PEG8 is unknown
but may be a consequence of this distinction. The addition of the
PEG polymers to the N-terminus of the PK agonists may impede
interaction with the aphid PK receptor, particularly receptor activation. The PK antagonist sequence is capable of binding to certain
PK receptors in other insects but is incapable of activating those
receptors; thus the addition of the PEG polymer cannot further
harm this aspect of the receptor interaction. Presumably, the PEG
polymer does not impede binding to the aphid PK receptor. This
hypothesis raises the intriguing possibility that PK-dF-PEG8 may
exhibit antifeedant/aphicidal activity by acting as an antagonist at
the putative PK receptor of aphids. To further test this possibility, the weakly active PEG analog of a PK antagonist, PK-dF-PEG4
(0.5 nmol/␮l), was added to the highly active agonist analog PKOic-1 (0.1 nmol/␮l) to see if it could block its potent oral aphicidal
effects. Indeed, the toxicity observed for the combined analog treatment was reduced by 55% in comparison with the aphid mortality
percentage observed for the agonist analog alone. This effect was
statistically significant (p = 0.07) at greater than a 90% confidence
level. Interestingly, the aphid toxicity for the combined treatment
was not statistically different than the antagonist analog alone
(p = 0.31). Importantly, the results suggest that the agonist and
antagonist analogs act at the same receptor and that this receptor
is likely a PK receptor. They further suggest that the potent aphicidal activity of PK-dF-PEG8 , a dF-containing cousin of PK-dF-PEG4 ,
arises via an antagonist response.
Two reference aphidicides that are currently used in the marketplace for selective IPM control against aphids in agriculture
are pymetrozine and flonicamid. Both compounds act specifically against aphids as feeding inhibitors, although their exact
mechanism(s) remain unidentified. Flonicamid [N-(cyanomethyl)4-(trifluoromethyl)-3-pyridinecarboxamide] is a novel insecticide;
271
its LC50 as determined in an experimental setup similar to that
used for the kinin analogs was 0.144 nmol/␮l with a typical loss of
honey dew formation followed by death, and its LT50 was 1.1 days
to 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-methyl4-[(3-pyridinylmethylene)amino]], Sadeghi et al. [36] calculated
with use of a similar feeding apparatus with a diet sachet an LC50
of 0.01 ␮g/ml [37]. The latter authors also tested imidacloprid and
found that 50% of aphids were killed with 0.03 ␮g/ml after 3 days
of feeding. Imidacloprid is a very active broad-spectrum neonicotinoid insecticide with the nicotinic acetylcholine receptor (nAChR)
as target, and to date it is used against a large variety of pest insects
and due to its high systemic activity, it is also highly active against
sucking pest insects like aphids and whiteflies. But intensive use
of imidacloprid has stimulated outbreaks of resistance and cross
resistance in many cases [9]. In the group of insect growth regulator (IGRs), azadirachtin (Neem), flufenoxuron and pyriproxyfen are
also commercially used in the selective control of aphids and they
have a respective LC50 of 7.9, 8.7 and 9.3 ␮g/ml artificial diet against
pea aphids [37]. Here typical phenotypic symptoms of aphid mortality were disruption of nymphal molt and abortion of molting. In
the field of insecticidal proteins, mannose-binding lectins received
much attention in the last decade because the Galanthus nivalis
agglutinin (GNA, homotetrameric protein composed of 12 kDa subunits) is highly detrimental to aphids [9,36,37] and can be delivered
via transgenic plants. Sadeghi et al. [36] reported an LC50 of 350
and 700 ␮g/ml for two mannose-binding lectins GNA and ASA after
feeding for 3 days on treated diet.
Thus, the fact that the stabilized PK analogs of this study, and
especially the analogs PK-Oic-1 and PK-dF-PEG8 , show rapid and
high activities against A. pisum aphids in the same order of magnitude as some commercial aphicides tested under comparable
conditions in the laboratory, suggests that they represent potentially valuable leads for alternative agents in the control of aphids
and in the struggle against insecticide resistance. In addition, we
believe that testing other sucking pest insects would also be of
interest. But before making firm conclusions on their potential
value as practical antifeedants, we believe more testing on a larger
scale and under more field-related conditions is required.
The aphicidal activity of the PK analogs is associated with
the presence of components that enhance the resistance of the
C-terminal core region to peptidases, as the unmodified PKs
demonstrate no activity. The mechanism of the aphid antifeedant
activity and high induction of mortality demonstrated by the
biostable PK analogs cannot be clearly identified at this point,
but it may be associated with disruption of the physiological
processes that this important neuropeptide family regulates in
insects, particularly hindgut and midgut contractile activity that
may impede normal digestive processes. For this to happen, the
biostable analogs with aphicidal activity would necessarily need to
interact with a native aphid PK receptor(s).
Finally, it is possible that activation of taste receptors by the
presence of the analogs may cause the aphids to avoid ingestion
of the diet altogether, leading to starvation. While honeydew formation is depressed in the aphids exposed to the active analogs,
the observations of normal piercing behavior and the presence
of at least some honeydew suggest that ingestion is nonetheless
taking place. Impairment of normal physiological patterns in the
aphids ingesting the active analogs may lead to a reduction in
subsequent feeding and, in turn, to the observed reduction in levels of honeydew formation. Furthermore, the unmodified peptides
are readily ingested by the aphids, as are the essentially inactive
analogs containing Hyp or many of those that are conjugated with
PEG polymer, which also contain unnatural amino acids found in
the active analogs. Thus, the fact that a number of the PK analogs
do not trigger avoidance of diet ingestion would seem to suggest
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R.J. Nachman et al. / Peptides 34 (2012) 266–273
that some ingestion of the three active biostable PK analogs may
also be taking place.
In summary, the presence of three biostable PK core analogs
PK-Oic-2, PK-dF-PEG8 , and particularly PK-Oic-1, in the diet
demonstrate significant antifeedant activity and induction of high
mortality in the pea aphid A. pisum that matches that of some
commercially available aphicides. Unmodified natural PK peptides
and some analogs containing some of the same structural components that promote biostability are inactive. The potent aphicidal
effects of PK-Oic-1 can be blocked by over 55% when combined
with a PEG polymer analog of a PK antagonist, a PEGylated cousin
of PK-dF-PEG8 , suggesting that the aphicidal effects are achieved
via interaction with a PK receptor. Analogs PK-Oic-1 and PK-Oic-2
demonstrate activity via an agonist response, whereas PK-dF-PEG8
acts via an antagonist response. The active biostable PK analogs
described in this study and/or 2nd generation analogs, either in
isolation or in combination with biostable analogs of other neuropeptide classes that also regulate aspects of diuretic, antidiuretic,
digestive, reproductive and/or developmental processes, represent
potential leads in the development of selective, environmentally
friendly pest aphid control 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
assistance from a grant from the USDA/DOD DWFP Initiative
(#0500-32000-001-01R) (RJN), a USDA-NIFA Grant No. 201167013-30199 (RJN) and a grant from the US-Israel Binational
Agricultural Research and Development Fund (BARD) (IS-420509C) (RJN, KK, and JZ), and support from the Fund of Scientific
Research (FWO-Vlaanderen, Belgium) and the Special Research
Fund of Ghent University (BOF-UGent) to GS.
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