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Research Article

Received: 7 May 2013

Revised: 2 August 2013

Accepted article published: 13 August 2013

Published online in Wiley Online Library: 7 October 2013

( DOI 10.1002/ps.3626

Electrical penetration graph studies to

investigate the effects of cyantraniliprole on
feeding behavior of Myzus persicae (Hemiptera:
Aphididae) on Capsicum annuum
Alana L. Jacobson and George G. Kennedy
BACKGROUND: The anthranilic diamide insecticide cyantraniliprole has been shown to suppress aphid and whitefly populations
as well as reduce transmission of plant viruses by thrips and whiteflies when taken up systemically by the plant. In this study,
electrical penetration graphing (EPG) was used to compare effects of cyantraniliprole on feeding behavior of Myzus persicae
with those of the neonicotinoid insecticide imidacloprid applied as a soil drench to pepper plants two-, six-, and ten-days
RESULTS: Significant reductions in the total amount of time spent probing, mean number of phloem feeding events, and
mean number of intracellular punctures were observed on both cyantraniliprole- and imidacloprid-treated plants, compared
to aphids that fed on plants treated only with water. Imidacloprid treatment also caused a significant reduction in the total
number of probes relative to the water treated control. The effects of cyantraniliprole were statistically significant only in
assays conducted at ten-days post-treatment, whereas the effects of imidacloprid on aphid feeding were significant in assays
conducted at two-, six-, and ten-days post-treatment.
CONCLUSION: These findings document significant effects of cyantraniliprole on feeding by Myzus persicae.
c 2013 Society of Chemical Industry

Keywords: Myzus persicae; electrical penetration graph; cyantraniliprole; anthranilic diamide; feeding behavior



Aphids are phytophagous insects that include many economically

important pests of agricultural and horticultural crops. These
pest species damage crops by feeding on the vascular bundles
of plants, and some are vectors of plant viruses.1 Anthranilic
diamide insecticides are a new class of compounds that affect
ryanodine receptors in insect muscle cells, and whose chemistries
exhibit varying degrees of antifeedant activities in different
groups of insects.2 4 A second generation anthranilic diamide,
cyantraniliprole, is active against sucking insects, including
aphids.4 6 Cyantraniliprole has systemic activity and is readily
taken up by plant roots and translocated via the xylem. It also has
translaminar activity when applied to the foliage.6 In laboratory
bioassays involving systemic uptake by excised leaves through
the petiole or stem of cabbage and cotton seedlings, there was
no evidence that common mechanisms of resistance to other
insecticides affected response of Myzus persicae (Sulzer) and Aphis
gossypii (Glover) to cyantraniliprole.6 Moreover, the LC50 values
observed for Myzus persicae in that study were comparable to
those reported for the widely used aphicide imidacloprid in
separate studies using a similar bioassay.7 Several field studies have
shown that cyantraniliprole applied as a soil drench application,
through drip irrigation, or as a soil injection significantly suppresses
Pest Manag Sci 2014; 70: 836840

Myzus persicae populations on pepper and cabbage8 10 and

Macrosiphum euphorbiae (Thomas) populations on tomato.11
Currently, the effects of cyantraniliprole on aphid feeding
behavior are not known. However, any such effects, if significant,
could impact aphid establishment and subsequent population
growth on treated plants, and potentially affect the transmission
of plant viruses by affected aphid vectors.12 14 Although we have
found no reports documenting suppression of aphid-transmitted
plant viruses in cyantraniliprole treated plants, cyantraniliprole
has been reported to reduce incidence of whitefly- and thripstransmitted viruses in melon and pepper.10,12,15,16
The development of techniques such as the electrical penetration graph (EPG)17 19 has greatly improved our ability to study
feeding behaviors of piercing sucking insects, including aphids,
associated with plant damage, virus transmission and the alterations of normal feeding caused by insecticide treatments.20 23
The EPG method incorporates an insect and a feeding substrate

Correspondence to: George G. Kennedy, Department of Entomology, North

Carolina State University, Box 7630, Raleigh, NC 276957630, USA. E-mail:
Department of Entomology, North Carolina State University, Box 7630, Raleigh,
NC, 27695-7630, USA

c 2013 Society of Chemical Industry

Effects of cyantraniliprole on Myzus persicae feeding behavior

into an electric circuit and records changes in voltage and electrical

resistance that occur when the insect begins feeding and completes the circuit. The voltage and resistance fluctuations produce
waveform signals that correspond to specific plant penetration
and feeding behaviors such as salivation, ingestion, cell punctures,
and stylet location within plant tissue. EPG studies of aphid
feeding on plants treated with insecticides have identified specific
changes in probing and feeding resulting from their applications,
and provided information related to mode of action that have
implications for virus control.7,24 27 This study was undertaken to
characterize effects of cyantraniliprole on the feeding behavior of
Myzus persicae on systemically treated pepper plants.

Table 1. Table of reported EPG parameters per four hour recording of

Myzus persicae that fed on water-, cyantraniliprole-, and imidaclopridtreated plants.


Average total number of probes.

Total amount of time spent probing.
This includes all types of described
feeding behaviors.
The average number of phloem feeding
Total duration (sum) of phloem feeding.
Mean number of intracellular
Mean duration of intracellular


2.1 Insects
A colony of Myzuspersicae originally collected from and maintained
on tobacco in North Carolina was transferred to Capsicum annuum
(L.) plants and reared for at least five generations before use in this
study. The colony was maintained in the glasshouse under natural
day length (1214 hours of daylight) and 18.936 C (average low
and high temperatures).
2.2 Plants and insecticide treatments
Capsicum annuum seeds were germinated in an insect-free
glasshouse. Four true-leaf stage plants were transplanted to
individual 296 mL plastic cups (Solo Cup Company, Lake Forest,
IL, USA) with a 25 mm diameter, round, fine mesh screen on
the bottom. After transplant 50 mL drench applications of either
distilled water, 10 mg ai/plant of cyantraniliprole (Cyazypyr 200
SC; DuPont, Delaware, USA), or 13.2 mg ai/plant of imidacloprid
(Admire Pro; Bayer, Kansas City, MO, USA) were made. Both
insecticides were applied at the currently (imidacloprid) or
anticipated (cyantraniliprole) labeled rates for aphids on pepper.
Plants were then held at room temperature, under grow lights,
and in insect-proof cages until they were used in EPG experiments.

Pest Manag Sci 2014; 70: 836840


Both cyantraniliprole and imidacloprid altered the feeding

behavior of Myzus persicae compared to the water-treated control.
Overall, when averaged over all post-treatment dates, M. persicae
that fed on cyantraniliprole-treated plants or imidacloprid-treated
plants showed significant reductions in the total amount of time
spent probing (TotPrbTm), mean number of phloem feeding
events (NumE), and mean number of intracellular punctures
(NumPD) compared to aphids that fed on plants treated only
with water. However, unlike cyantraniliprole, imidacloprid also
caused a significant reduction in the total number of probes
(TotNumPrbs) relative to the water-treated control (Table 2).
When the data were analyzed by days after treatment, the
results indicated that effects of imidacloprid on feeding behavior
were manifest sooner and were generally of greater magnitude
that those of cyantraniliprole (Table 2). The effects of imidacloprid
on each of the affected EPG parameters were significant in assays
conducted at two-, six- and ten-days post-treatment, whereas
significant effects of the cyantraniliprole treatment were observed
only in the EPG assays conducted at ten-days post-treatment
(Table 2). At ten-days post-treatment the mean total time spent
probing (TotPrbTm) and the mean duration of intracellular probes
(MnDurPD) were significantly less on imidacloprid-treated than
on cyantraniliprole-treated plants. In addition, the reduction in
the number of phloem feeding events (NumE) and the number
of intracellular punctures, although not significantly different
between the cyantraniliprole and imidacloprid treatments, were
proportionally greater relative to the water treatment in the
imidacloprid than the cyantraniliprole treatment (reduction in
NumE: imidacloprid versus water 79%, cyantraniliprole versus
water 64%; reduction in NumPD: imidacloprid versus water 73%,
cyantraniliprole versus water 55%).

c 2013 Society of Chemical Industry


2.3 EPG experiments

The effect of cyantraniliprole and imidacloprid on the probing
behavior of Myzus persicae compared to water-treated plants was
examined two-, six-, and ten-days post-treatment. Adult, apterous
M. persicae were tethered to a 2 cm long, 0.19 micron diameter
gold wire (EPG Systems, Wageningen, The Netherlands) with silver
paint (Pelco colloidal silver liquid, Ted Pella, Inc., Redding, CA,
USA). Aphids were held in a Petri dish without access to leaf
tissue for 30 minutes before the start of the EPG. Whole plants
were placed into Faraday cages, insects placed onto the youngest
fully expanded leaf, and EPGs were recorded for four hours under
ambient laboratory conditions. A completely randomized design
was used for these experiments. One Giga-4 DC-EPG system and
one Giga-8 DC EPG system with a 1 G of input resistance (EPG
Systems, Wageningen, The Netherlands) were used to record EPGs.
EPGs were recorded and analyzed using Stylet+ Software (EPG
Systems, Wageningen, The Netherlands).
SAS 9.2 Software (SAS Institute, Cary, NC, USA) was used to
compile the numerous output data files (.ANA) from Stylet+
analysis into one SAS dataset. SAS Proc GLIMMIX procedure with
mean separation by LSMeans was then used to calculate and
analyze sequential and non-sequential EPG feeding waveforms
previously described.19,28 Two analyses were conducted. The first
examined the significance of treatment effects averaged over all

post-treatment times (two-, six-, and ten-days post-treatment).

In this analysis, treatment was included in the model as a
fixed effect and date and post-treatment times were included
as random effects because these all represent independent
observations (repeated measures were not taken on the plants).
In the second analysis, data for the two-, six-, and ten-day posttreatment times were analyzed separately to examine the effect
of treatments on EPG parameters at each post-treatment time.
The number and total duration of waveform events during the
four hour recordings are reported for the waveforms that have
the most relevance to antifeedant effects and virus transmission
(Table 1).

AL Jacobson, GG Kennedy

Table 2. Mean (standard error in parentheses) EPG feeding parameters recorded over a four hour period for Myzus persicae that fed on water-,
cyantraniliprole-, and imidacloprid-treated Capsicum annuum during each of three post-treatment time intervals.
Feeding behavior












Two days

Six days

N1 = 24
N = 18
N = 11
N = 24
N = 18
N = 11
PN2 = 12/24
PN = 16/18
PN = 1/11
PN = 12/24
PN = 16/18
PN = 1/11
N = 24
N = 18
N = 11
N = 24
N = 18
N = 11

N = 25
N = 28
N = 17
N = 25
N = 28
N = 17
PN = 16/25
PN = 12/28
PN = 1/17
PN = 16/25
PN = 12/28
PN = 1/17
N = 25
N = 28
N = 17
N = 25
N = 28
N = 17

Ten days
N = 15
N = 16
N = 11
N = 15
N = 16
N = 11
PN = 15/15
PN = 7/16
PN = 1/11
PN = 15/15
PN = 7/16
PN = 1/11
N = 15
N = 16
N = 11
N = 15
N = 16
N = 11

N = 64
N = 62
N = 39
N = 64
N = 62
N = 39
PN = 43/64
PN = 35/62
PN = 3/39
PN = 43/64
PN = 35/62
PN = 3/39
N = 64
N = 62
N = 39
N = 64
N = 62
N = 39

*Mean separation vertical by LS Means at P 0.05. Means separation were performed on variables within each post-treatment time interval.
1 Total number of aphids recorded per treatment where all insects exhibited the reported waveform.
Number of aphids that produced the waveform over the total number of aphids recorded.


Although significant differences between the cyantraniliprole

and water treatments were not observed until ten days after
treatment, the magnitude of the numerical differences between
the cyantraniliprole and water treatments generally increased with
number of days post-treatment, suggesting that the aphids may
have received greater exposure to cyantraniliprole when feeding
on plants at six- and ten- than at two-days post-treatment, which
may reflect a higher concentration or a more effective distribution
of cyantraniliprole in the leaf tissue over time. The significant
effect of imidacloprid on feeding behavior observed at two-days
post-treatment is consistent with previous research documenting
strong antifeedant effects of imidacloprid on Myzus persicae at
low, sub-lethal concentrations.7
Cyantraniliprole acts in insects by binding with ryanodine receptors that regulate calcium release from intracellular stores in the
sarcoplasmic reticulum; leading to a depletion of those stores,
gradual muscle contraction, and paralysis.42 However, our results
do not indicate whether the effects of cyantraniliprole on feeding
behavior that we documented are a consequence of altered con-

traction of muscles associated with probing and feeding or are

an independent behavioral response to encountering cyantraniliprole.
In general, the use of insecticides to suppress spread of nonpersistently transmitted aphid-borne viruses has had very limited success. Such viruses are acquired from and inoculated into plants during probes lasting a matter of seconds. Few insecticides act quickly
enough to prevent brief intracellular probes of epidermal and mesophyll cells, and virus acquisition or inoculation.13,14 This is especially true for systemic insecticides that are translocated throughout the plant following uptake by the roots. In the absence of insecticide residue on the leaf surface, the presence of such insecticides
can be detected by aphids only during probing of plant tissues, and
transmission of nonpersistently transmitted viruses occurs during
brief, intracellular probes of epidermal or mesophyll cells, which are
manifest as potential drops (PDs) in EPG recordings.21 23,29 Hence,
although our results show that imidacloprid and cyantraniliprole
significantly reduced the number of intracellular punctures, this

c 2013 Society of Chemical Industry

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Effects of cyantraniliprole on Myzus persicae feeding behavior

decrease would not be expected to reduce transmission of nonpersistently transmitted viruses. Previous findings show that when
used as a systemic insecticide in potato, imidacloprid does not
reduce transmission of nonpersistently transmitted viruses.30
Imidacloprid has been shown to reduce incidence of persistently transmitted, aphid-borne viruses, the transmission of which
has been associated with phloem feeding (waveform E in EPG
recordings).13,31 41 For example, a reduction in feeding time and
percentage of individuals that reached the phloem is believed to
be responsible for reduced transmission of Barley yellow dwarf
virus, a phloem-borne virus, by Schizaphis graminum (Rondani).27
Imidacloprid has proven effective in reducing spread of Potato
leafroll virus (PLRV) in potato; this likely reflects both a reduction in
phloem feeding as well as the control of resident aphid vector populations within the field, which if not controlled, would contribute
to secondary spread of the virus.13,14,37,40 Our findings indicate
that both imidacloprid and cyantraniliprole significantly reduced
the number of phloem feeding events (NumE) relative to the water
treatment, but the effect of the imidacloprid treatment was greater
than that of cyantraniliprole. Additionally, the proportion of aphids
that fed on the phloem during EPG recording was much lower
on imidacloprid-treated plants than on cyantraniliprole-treated
plants. Field experiments will be necessary to determine the
extent to which cyantraniliprole suppresses spread of persistently
transmitted, aphid-borne viruses such as PLRV. Additional field and
laboratory studies will likely be required to determine the extent to
which any observed reductions in virus spread are due to effects of
cyantraniliprole on virus acquisition and/or inoculation, or to suppression of resident populations of aphid vectors within the crop.

The authors would like to thank Carol Berger and Damon
DAmbrosio for technical assistance, and Robert Williams of DuPont
for providing the Cyazypyr used in these experiments. The
authors would also like to thank Elaine Backus, Tim Ebert, Joy
Smith and Tom Chappell for their guidance and assistance with
managing and analyzing EPG data. Last, the authors appreciate
the financial support from DuPont that enabled the EPG analyses
reported in this paper.


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Pest Manag Sci 2014; 70: 836840